WO2017004609A1 - Thiadiazole modulators of s1p and methods of making and using - Google Patents

Thiadiazole modulators of s1p and methods of making and using Download PDF

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Publication number
WO2017004609A1
WO2017004609A1 PCT/US2016/040895 US2016040895W WO2017004609A1 WO 2017004609 A1 WO2017004609 A1 WO 2017004609A1 US 2016040895 W US2016040895 W US 2016040895W WO 2017004609 A1 WO2017004609 A1 WO 2017004609A1
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Prior art keywords
chloro
oxy
amino
thiadiazol
compound
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PCT/US2016/040895
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French (fr)
Inventor
Wei Xu
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Exelixis, Inc.
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Publication of WO2017004609A1 publication Critical patent/WO2017004609A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
    • C07D285/12—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles
    • C07D285/125—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles with oxygen, sulfur or nitrogen atoms, directly attached to ring carbon atoms, the nitrogen atoms not forming part of a nitro radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • This invention relates to the field of agonists of Sphingosine 1 -Phosphate Type 1 Receptor (S1P1R or SlPl) and/or Type 5 Receptor (S1P5R or S1P5), and methods of their use.
  • Sphingosine 1 -phosphate is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Circulation of mature lymphocytes between blood and secondary lymphoid tissues plays an important role in the immune system. Agonism of S1P1R has been shown to lead to the sequestration of peripheral lymphocytes into secondary lymphoid tissue. Such sequestration of lymphocytes has been shown to result in immunosuppressive activity in animal models. Known SlPl receptor agonists, such as FTY720, have been shown to markedly decrease peripheral blood lymphocytes through the sequestration of lymphocytes into secondary lymphoid tissues.
  • Potent agonists of the SlPl receptor are thought to induce long-term down-regulation of SlPl on lymphocytes, thereby inhibiting the migration of lymphocytes toward SIP.
  • the consequential decrease in trafficking and infiltration of antigen-specific T cells provides a means of immunomodulating activity that can be useful in the treatment of various immune- related conditions such as graft versus host disease and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosis. Therefore, agonists of S1P1R are potentially useful immunosuppressants for the treatment of a variety of autoimmune conditions.
  • the myelin sheath that surrounds neural cell axons is required to insulate neural axons and allow rapid movement of electrical impulses through the myelinated nerve fiber. Demyelination, or loss of the integrity of the myelin sheath is the hallmark of autoimmune neurodegenerative diseases, including multiple sclerosis.
  • the myelin sheath in the central nervous system is produced by oligodendrocytes. Mature, myelin-producing oligodendrocytes express SIP receptor transcripts in relative abundance of S1P5>S1P3>S1P1, with undetectable levels of S1P4.
  • Fingolimod Fingolimod (FTY720), a sphingosine- 1 -phosphate (SIP) analogue that has been used successfully in clinical trials as a systemic immunomodulatory therapy for multiple sclerosis, readily accesses the central nervous system.
  • SIP sphingosine- 1 -phosphate
  • Sphingosine-1 -phosphate has been shown to regulate the migration of osteoclast precursors, demonstrating a role in bone mineral homeostasis and suggesting a role in treating bone-destroying disorders such as rheumatoid arthritis and osteoporosis ⁇ Nature 2009, 458(7237), 524-528).
  • S1P3 has been associated with acute toxicity and bradycardia in rodents (Hale, et. al. Bioorganic & Med Chem Lett, 2004, 14(13), 3501-3505; J. Pharmacol. Exp. Ther. 2004, 309(2), 758-768; J. Med Chem 2005, 48(20), 6168-6173; J. Biol. Chem. 2004, 279(14), 13839-13848). Therefore agonists which are selective for S1P1 and/or S1P5, without being active for S1P3, are desirable.
  • the invention provides compounds that are agonists of SI PI and/or S1P5 and that are useful in the treatment of graft versus host disease and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and systemic lupus erythematosis, in mammals.
  • This invention also provides methods of making the compound, methods of using such compounds in the treatment of graft versus host disease and autoimmune diseases, especially in humans, and to pharmaceutical compositions containing such compounds.
  • Embodiment (1) of the first aspect of the invention provides a compound of Formula I:
  • R is heterocycloalkyl optionally substituted with one or two groups which groups are
  • R is according to formula (a)
  • n 1 or 2;
  • R 7d is -OR 1 or halo
  • R 1 is hydrogen or -P(0)(OR 6 ) 2 ;
  • R 2 and R 2a are independently hydrogen, cyano, halo, alkyl, haloalkyl, alkoxy, or haloalkoxy;
  • Ring A is phenyl, pyrimidinyl, or pyridinyl
  • R 3 is alkylamino, alkoxy, alkyl, or heterocycloalkyloxy
  • R 4 is halo, alkyl, alkoxy, alkoxycarbonayl, cyano, or hydroxy
  • R 3 is alkylamino, alkoxy, or alkyl
  • R 4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano
  • R 3 when R 3 is heterocycloalkyloxy, then R 4 is hydroxy;
  • R 5 is hydrogen or alkyl
  • R 5a is hydrogen, alkyl, formyl, alkylcarbonyl, or alkoxycarbonyl
  • each R 6 is independently hydrogen or alkyl
  • each R 7 and R 7a is independently hydrogen or alkyl;
  • R and R c are independently hydrogen or alkyl;
  • R is hydrogen, alkyl, or hydroxyalkyl
  • the invention is directed to a pharmaceutical composition which comprises 1) a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt thereof and 2) a pharmaceutically acceptable carrier, excipient, or diluent.
  • the invention provides a method for treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the Invention is directed to a method of making a Compound of the Invention which method comprises:
  • Ring A, R 3 , and R 4 are as defined in the Summary of the Invention for a Compound of Formula I, followed by treatment with Lawesson's reagent, to yield an intermediate of formula 118:
  • the symbol "-" means a single bond
  • " ⁇ ” means a triple bond
  • the symbol ' ⁇ /" refers to a group on a double-bond as occupying either position on the terminus of a double bond to which the symbol is attached; that is, the geometry, E- or Z-, of the double bond is ambiguous.
  • the " ' ⁇ " symbol will be used at the end of the bond which was theoretically cleaved in order to separate the group from its parent structural formula.
  • a substituent "R” may reside on any atom of the ring system, assuming replacement of a depicted, implied, or expressly defined hydrogen from one of the ring atoms, so long as a stable structure is formed.
  • the "R” group may reside on either the 5-membered or the 6-membered ring of the fused ring system.
  • alkyl can refer to a monovalent alkyl radical or a divalent radical (i.e., alkylene).
  • administering and variants thereof (e.g., “administering” a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment.
  • a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., surgery, radiation, and chemotherapy, etc.)
  • “administration” and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.
  • Alkyl means a linear saturated hydrocarbon radical of one to six carbon atoms or a branched saturated hydrocarbon radical of three to 6 carbon atoms, e.g., methyl, ethyl, propyl, 2-propyl, butyl (including all isomeric forms), or pentyl (including all isomeric forms), and the like.
  • Alkenyl means a linear hydrocarbon radical of two to six carbon atoms or a branched hydrocarbon radical of three to 6 carbon atoms which radical contains at least one double bond, e.g., ethenyl, propenyl, l-but-3-enyl, and l-pent-3-enyl, and the like.
  • Alkoxy means an -OR group where R is alkyl group as defined herein. Examples include methoxy, ethoxy, propoxy, isopropoxy, and the like.
  • Alkoxyalkyl means an alkyl group, as defined herein, substituted with at least one, specifically one, two, or three, alkoxy groups as defined herein. Representative examples include methoxymethyl and the like.
  • Alkoxyalkyloxy means an -OR group where R is alkoxyalkyl as defined herein.
  • Alkoxycarbonyl means a -C(0)R group where R is alkoxy, as defined herein.
  • Alkoxycarbonylamino means an -NHR group where R is alkoxycarbonyl as defined herien.
  • Alkylamino means an -NHR group where R is alkyl, as defined herein.
  • Alkylaminoalkyl means an alkyl group substituted with one or two alkylamino groups, as defined herein.
  • Alkylcarbonyl means a -C(0)R group where R is alkyl, as defined herein.
  • Alkylcarbonylamino means an -NHR group where R is alkylcarbonyl, as defined herein.
  • Alkylsulfonyl means an -S(0) 2 R group where R is alkyl, as defined herein, e.g. methylsulfonyl, isopropylsulfonyl .
  • Alkynyl means a linear hydrocarbon radical of two to six carbon atoms or a branched hydrocarbon radical of three to 6 carbon atoms which radical contains at least one triple bond, e.g., ethynyl, propynyl, butynyl, pentyn-2-yl and the like.
  • Amino means -NH 2 .
  • Aminocarbonyl means a -C(0)NH 2 group.
  • Aryl means a six- to fourteen-membered, mono- or bi-carbocyclic ring, wherein the monocyclic ring is aromatic and at least one of the rings in the bicyclic ring is aromatic. Unless stated otherwise, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. Representative examples include phenyl, naphthyl, and indanyl, and the like.
  • Arylalkyl means an alkyl radical, as defined herein, substituted with one or two aryl groups, as defined herein, e.g., benzyl and phenethyl, and the like.
  • Carboxy means a -C(0)OH group.
  • Cycloalkyl means a monocyclic or fused bicyclic, saturated or partially unsaturated (but not aromatic), hydrocarbon radical of three to ten carbon ring atoms.
  • Fused bicyclic hydrocarbon radical includes bridged ring systems.
  • the valency of the group may be located on any atom of any ring within the radical, valency rules permitting.
  • cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl, or cyclohex-3-enyl, and the like.
  • Cycloalkyloxy means an -OR group where R is cycloalkyl as defined herein.
  • Dialkylamino means a -NRR' radical where R and R' are alkyl as defined herein, or an N-oxide derivative, or a protected derivative thereof, e.g., dimethylamino, diethylamino, N,N-methylpropylamino or N,7V-methylethylamino, and the like.
  • Forml means an -C(0)H group.
  • fused ring means a polycyclic ring that contains bridged or fused rings; that is, where two rings have more than one shared atom in their ring structures.
  • fused ring systems are not necessarily all aromatic ring systems.
  • fused rings share a vicinal set of atoms, for example naphthalene or 1,2,3,4- tetrahydro-naphthalene.
  • a spiro ring system is not a fused ring system by this definition, but fused ring systems of the invention may themselves have spiro rings attached thereto via a single ring atom of the fused ring system.
  • two adjacent groups on an aromatic system may be fused together to form a ring structure.
  • the fused ring structure may contain heteroatoms and may be optionally substituted with one or more groups.
  • saturated carbons of such fused groups i.e. saturated ring structures
  • Halogen or "halo” refers to fluorine, chlorine, bromine and iodine.
  • Haloalkoxy means an -OR' group where R' is haloalkyl as defined herein, e.g., trifluoromethoxy or 2,2,2-trifluoroethoxy, and the like.
  • Haloalkyl mean an alkyl group substituted with one or more halogens, specifically one to five halo atoms, e.g., trifiuoromethyl, 2-chloroethyl, and 2,2-difluoroethyl, and the like.
  • Heteroaryl means a monocyclic, fused bicyclic, or fused tricyclic, radical of 5 to 14 ring atoms containing one or more, specifically one, two, three, or four ring heteroatoms independently selected from -0-, -S(0) consult- (n is 0, 1, or 2), -N-, -N(R X )-, and the remaining ring atoms being carbon, wherein the ring comprising a monocyclic radical is aromatic and wherein at least one of the fused rings comprising a bicyclic or tricyclic radical is aromatic.
  • R x is hydrogen, alkyl, hydroxy, alkoxy, acyl, or alkyl sulfonyl.
  • Fused bicyclic radical includes bridged ring systems. Unless stated otherwise, the valency may be located on any atom of any ring of the heteroaryl group, valency rules permitting. When the point of valency is located on the nitrogen, R x is absent.
  • heteroaryl includes, but is not limited to, 1 ,2,4-triazolyl, 1,3,5-triazolyl, phthalimidyl, pyridinyl, pyrrolyl, imidazolyl, thienyl, furanyl, indolyl, 2,3-dihydro-lH-indolyl (including, for example, 2,3-dihydro-lH-indol-2-yl or 2,3-dihydro- lH-indol-5-yl, and the like), isoindolyl, indolinyl, isoindolinyl, benzimidazolyl, benzodioxol- 4-yl, benzofuranyl, cinnolinyl, indolizinyl, naphthyridin-3-yl, phthalazin-3-yl, phthalazin-4- yl, pteridinyl, purinyl, quina
  • Heteroatom refers to O, S, N, and P.
  • Heterocycloalkyl means a saturated or partially unsaturated (but not aromatic) monocyclic group of 3 to 8 ring atoms or a saturated or partially unsaturated (but not aromatic) fused bicyclic group of 5 to 12 ring atoms in which one or more, specifically one, two, three, or four ring heteroatoms independently selected from O, S(0) n (n is 0, 1, or 2), N, N(R y ) (where R y is hydrogen, alkyl, hydroxy, alkoxy, acyl, or alkylsulfonyl), the remaining ring atoms being carbon.
  • Fused bicyclic radical includes bridged ring systems. Unless otherwise stated, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. When the point of valency is located on a nitrogen atom, R y is absent.
  • heterocycloalkyl includes, but is not limited to, azetidinyl, pyrrolidinyl, 2-oxopyrrolidinyl, 2,5-dihydro-lH-pyrrolyl, piperidinyl, 4-piperidonyl, morpholinyl, piperazinyl, 2-oxopiperazinyl, tetrahydropyranyl, 2-oxopiperidinyl, thiomorpholinyl, thiamorpholinyl, perhydroazepinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, dihydropyridinyl, tetrahydropyridinyl, oxazolinyl, oxazolidinyl, isoxazolidinyl, thiazolinyl, thiazolidinyl, quinuclidinyl, isothiazolidinyl, octahydroind
  • Heterocycloalkyloxy means an -OR group where R is hetero cycloalkyl as defined herein.
  • Heterocycloalkyloxy means an alkyl group substituted with at least one, in another example with one, two, or three, hydroxy groups.
  • Spirocyclyl or "spirocyclic ring” refers to a ring originating from a particular annular carbon of another ring.
  • a ring atom of a saturated bridged ring system (rings B and B'), but not a bridgehead atom, can be a shared atom between the saturated bridged ring system and a spirocyclyl (ring A) attached thereto.
  • a spirocyclyl can be carbocyclic or hete
  • Methodabolite refers to the break-down or end product of a compound or its salt produced by metabolism or biotransformation in the animal or human body; for example, biotransformation to a more polar molecule such as by oxidation, reduction, or hydrolysis, or to a conjugate (see Goodman and Gilman, "The Pharmacological Basis of Therapeutics” 8.sup.th Ed., Pergamon Press, Gilman et al. (eds), 1990 for a discussion of biotransformation).
  • the metabolite of a compound of the invention or its salt may be the biologically active form of the compound in the body.
  • a prodrug may be used such that the biologically active form, a metabolite, is released in vivo.
  • a biologically active metabolite is discovered serendipitously, that is, no prodrug design per se was undertaken.
  • An assay for activity of a metabolite of a compound of the present invention is known to one of skill in the art in light of the present disclosure.
  • Patient for the purposes of the present invention includes humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In a specific embodiment the patient is a mammal, and in a more specific embodiment the patient is human.
  • a "pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington 's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference or S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66: 1-19 both of which are incorporated herein by reference.
  • Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 2-
  • Examples of a pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Specific salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins.
  • organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine, N-methylglucamine, polyamine resins, and the like.
  • Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.”
  • Platinum(s)," and “platin-containing agent(s)” include, for example, cisplatin, carboplatin, and oxaliplatin.
  • Prodrug refers to compounds that are transformed (typically rapidly) in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood.
  • Common examples include, but are not limited to, ester and amide forms of a compound having an active form bearing a carboxylic acid moiety.
  • Examples of pharmaceutically acceptable esters of the compounds of this invention include, but are not limited to, alkyl esters (for example with between about one and about six carbons) the alkyl group is a straight or branched chain. Acceptable esters also include cycloalkyl esters and arylalkyl esters such as, but not limited to benzyl.
  • Examples of pharmaceutically acceptable amides of the compounds of this invention include, but are not limited to, primary amides, and secondary and tertiary alkyl amides (for example with between about one and about six carbons).
  • Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.
  • Stepoisomer means any of two or more isomers containing the same atoms bonded to each other in an identical manner but differing from each other in the spatial arrangement of the atoms or groups of atoms.
  • Stepreoisomer includes, for example, an enantiomer, a geometric isomer, a diastereomer, a rotamer, cis-isomer, trans-isomer, and conformational isomer.
  • the names and illustration used in this application to describe compounds of the invention, unless indicated otherwise, are meant to encompass all possible stereoisomers and any mixture, racemic or otherwise, thereof.
  • “Therapeutically effective amount” is an amount of a compound of the invention, that when administered to a patient, ameliorates a symptom of the disease.
  • the amount of a compound of the invention which constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the age of the patient to be treated, and the like.
  • the therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their knowledge and to this disclosure.
  • Treating" or "treatment” of a disease, disorder, or syndrome includes (i) preventing the disease, disorder, or syndrome from occurring in a human, i.e. causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e. , arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome.
  • Yield for each of the reactions described herein is expressed as a percentage of the theoretical yield.
  • the invention further comprises subgenera of embodiment (1) in which the substituents are selected as any and all combinations of structural formula (I), R, R 1 , R 2 , R 2a , R 3 , R 4 , R 5 , R 5a , R 7 , R 7a , R 7b , R 7c , and R 8 as defined herein, including without limitation, the following:
  • Structural Formula I is one of formulae (I-a) - (l-l). (II-a) - (II-c ⁇ . (III-a) - (III- g). qV-a - (IV-e), (V-a) - (V-e). and (Vl-a) - (Vl-g):
  • R 1 is selected from one of the following groups (la) - (lg):
  • R 2 and R 2a are selected from one of the following groups (2a) - (2m):
  • R 2 and R 2a are independently cyano, halo, or alkyl.
  • R 2 and R 2a are independently cyano, chloro, fluoro, or methyl.
  • R 2 and R 2a are independently halo or alkyl.
  • R 2 and R 2a are each independently halo.
  • R 2 and R 2a are each chloro.
  • R 2 is chloro or fluoro and R 2a is chloro or fluoro.
  • R 2 is chloro and R 2a is fluoro.
  • R 2 and R 2a are each independently alkyl.
  • R 2 and R 2a are each methyl.
  • R 2 is alkyl and R 2a is halo.
  • R 2 is methyl and R 2a is halo.
  • R 2 is methyl and R 2a is chloro or fluoro.
  • R 2 is methyl and R 2a is fluoro.
  • R 2 is methyl and R 2a is chloro.
  • R 3 and R 4 together are selected from one of the following groups (3a) - (3r):
  • R 3 is alkylamino, alkoxy, or alkyl and R 4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
  • R 3 is alkylamino and R 4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
  • R 3 is alkylamino and R 4 is alkyl, alkoxycarbonyl, or cyano.
  • R 3 is isopropylamino and R 4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
  • R 3 is isopropylamino and R 4 is alkyl, alkoxycarbonyl, or cyano.
  • R 3 is alkoxy and R 4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
  • R 3 is ethoxy or isopropoxy and R 4 is halo, alkyl, alkoxy, alkoxycarbonyl, or
  • R 3 is alkoxy and R 4 is alkoxy.
  • R 3 is ethoxy and R 4 is ethoxy.
  • R 3 is alkyl and R 4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
  • R 3 is isobutyl and R 4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
  • R 3 is alkyl and R 4 is alkyl.
  • R 3 is isobutyl and R 4 is methyl.
  • R 3 is alkylamino, alkoxy, or alkyl and R 4 is alkyl, alkoxy, cyano, or halo.
  • R is alkylamino, alkoxy, or alkyl and R is methyl, methoxy, ethoxy,
  • R 3 is ethoxy, isopropoxy, isopropylamino, or isobutyl and R 4 is methyl, methoxy, ethoxy, ethoxycarbonyl, cyano, or chloro.
  • R 3 is heterocycloalkyloxy and R 4 is hydroxy.
  • R 3 is heterocycloalkyloxy and R 4 is hydroxy or R 3 and R 4 are any one of groups (3a) - (3p).
  • R 3 is alkylamino and R 4 is alkyl, alkoxycarbonyl, or cyano, or R 3 is
  • heterocycloalkyloxy and R is hydroxy.
  • R 5 and R 5a are selected from one of the following groups (5a) - (5d):
  • R 5 is hydrogen and R 5a is hydrogen, alkyl, alkylcarbonyl, or alkoxycarbonyl.
  • R 5 is hydrogen and R 5a is alkylcarbonyl or alkoxycarbonyl.
  • R 5 is hydrogen and R 5a is hydrogen or alkyl.
  • R 5 is hydrogen; R 5a is hydrogen.
  • R is selected from one of the following groups (6a) - (6ii):
  • R any of groups (6q) - (6cc), wherein R 7c is hydrogen.
  • R is heterocycloalkyl optionally substituted with one or two groups which groups are hydrox l, tert-butyl-dimethylsilyloxy or alkoxycarbonyl; or R is according to
  • R 7 , R 7A , and R 7C are hydrogen; R 7B is hydrogen or alkyl; and R is hydrogen.
  • R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl-dimethylsilyloxy, or alkoxycarbonyl; or R is according to
  • R , R a , and R 0 are hydrogen; R is hydrogen or alkyl; and R is hydrogen.
  • R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl-dimethylsilyloxy, or alkoxycarbonyl.
  • R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl-dimethylsilyloxy, or alkoxycarbonyl or R is any one of groups (6a) - (6gg)
  • Particular embodiments of this aspect of the invention include compounds of any one of the formulae (I), (I-a) - (1-1), (Il-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), each as defined in each of the following rows, wherein each entry is a group number as defined above (e.g., (la) refers to R 1 is hydrogen), and a dash "-" indicates that the variable is as defined for formula (I) or defined according to any one of the applicable variable definitions (la)-(6kk) [e.g., when R 1 is a dash, it can be either as defined for Formula (I) or any one of definitions (la)-(lg)]:
  • Another aspect of the invention provides a pharmaceutical composition which comprises a compound of any one of Formulae (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier, excipient, or diluent.
  • a pharmaceutical composition which comprises a compound of any one of Formulae (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table
  • Another aspect of the invention is a method of treating disease, disorder, or syndrome where the disease is associated with uncontrolled, abnormal, and/or unwanted cellular activities effected directly or indirectly by S1P1 and/or S1P5 which method comprises administering to a human in need thereof a therapeutically effective amount of a compound of Formula (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or selected from any of the above embodiments, or a pharmaceutically acceptable salt or pharmaceutical composition thereof.
  • a compound of Formula (I) (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e),
  • Another aspect of the invention is directed to a method of treating an autoimmune disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound of Formula (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or selected from any of the above embodiments, optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof.
  • the autoimmune disease is multiple sclerosis.
  • the disease is psoriasis.
  • the disease is inflammatory bowel disease.
  • the autoimmune disease is graft-versus-host disease.
  • the disease is inflammation caused by an autoimmune disease.
  • Another aspect of the invention is directed to composition for treating an autoimmune disease, disorder, or syndrome which comprises a therapeutically effective amount of a compound of Formula (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or selected from any of the above embodiments, optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof.
  • the autoimmune disease is multiple sclerosis.
  • the disease is psoriasis.
  • the disease is inflammatory bowel disease.
  • the autoimmune disease is graft-versus-host disease.
  • the disease is inflammation caused by an autoimmune disease.
  • the invention is directed to the use of a compount of Formula (I), (I- a) - (1-1), (Il-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or selected from any of the above embodiments, optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof for the preparation of a medicament for treating an autoimmune disease, disorder, or syndrome.
  • the autoimmune disease is multiple sclerosis.
  • the disease is psoriasis.
  • the disease is inflammatory bowel disease.
  • the autoimmune disease is graft-versus-host disease.
  • the disease is inflammation caused by an autoimmune disease.
  • the invention provides pharmaceutical compositions comprising an inhibitor of S1P1 and/or S1P5 according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent.
  • administration is by the oral route.
  • Administration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration or agents for serving similar utilities.
  • administration can be, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, specifically in unit dosage forms suitable for simple administration of precise dosages.
  • compositions will include a conventional pharmaceutical carrier or excipient and a compound of the invention as the/an active agent, and, in addition, may include carriers and adjuvants, etc.
  • Adjuvants include preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. ]
  • a pharmaceutical composition of the invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylalted hydroxytoluene, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylalted hydroxytoluene, etc.
  • formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills or capsules) and the bioavailability of the drug substance.
  • pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size.
  • U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a crosslinked matrix of macromolecules.
  • 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • One specific route of administration is oral, using a convenient daily dosage regimen that can be adjusted according to the degree of severity of the disease-state to be treated.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
  • fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid
  • binders as for example, cellulose derivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia
  • humectants as for example, glycerol
  • disintegrating agents as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate
  • solution retarders as for example paraffin
  • absorption accelerators as for example,
  • Solid dosage forms as described above can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain pacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • Such dosage forms are prepared, for example, by dissolving, dispersing, etc., a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions for rectal administrations are, for example, suppositories that can be prepared by mixing the compounds of the present invention with for example suitable non- irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
  • suitable non- irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required.
  • Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.
  • Compressed gases may be used to disperse a compound of this invention in aerosol form.
  • Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc.
  • the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a suitable pharmaceutical excipient.
  • the composition will be between about 5% and about 75% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.
  • composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease-state in accordance with the teachings of this invention.
  • the compounds of the invention are administered in a therapeutically effective amount which will vary depending upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease-states, and the host undergoing therapy.
  • the compounds of the present invention can be administered to a patient at dosage levels in the range of about 0.1 to about 1,000 mg per day. For a normal human adult having a body weight of about 70 kilograms, a dosage in the range of about 0.01 to about 100 mg per kilogram of body weight per day is an example. The specific dosage used, however, can vary.
  • the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used.
  • the determination of optimum dosages for a particular patient is well known to one of ordinary skill in the art.
  • Compounds of this invention can be made by the synthetic procedures described below.
  • the starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis.), or Bachem (Torrance, Calif.), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Todd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4 th Edition) and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
  • the reactions described herein take place at atmospheric pressure and over a temperature range from about -78 °C to about 150 °C, more specifically from about 0 °C to about 125 °C and more specifically at about room (or ambient) temperature, e.g., about 20 °C. Unless otherwise stated (as in the case of an hydrogenation), all reactions are performed under an atmosphere of nitrogen.
  • Prodrugs can be prepared by techniques known to one skilled in the art. These techniques generally modify appropriate functional groups in a given compound. These modified functional groups regenerate original functional groups by routine manipulation or in vivo. Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Z?oche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.
  • the compounds of the invention may have asymmetric carbon atoms or quaternized nitrogen atoms in their structure.
  • Compounds of the invention that may be prepared through the syntheses described herein may exist as single stereoisomers, racemates, and as mixtures of enantiomers and diastereomers.
  • the compounds may also exist as geometric isomers. All such single stereoisomers, racemates and mixtures thereof, and geometric isomers are intended to be within the scope of this invention.
  • Some of the compounds of the invention may exist as tautomers.
  • the molecule may exist in the enol form; where an amide is present, the molecule may exist as the imidic acid; and where an enamine is present, the molecule may exist as an imine. All such tautomers are within the scope of the invention.
  • the present invention also includes isotopically-labeled compounds, which are identical to those recited in Formula I, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 0, 17 0, 31 P, 32 P, 35 S, 18 F, and 36 C1, respectively.
  • isotopically-labeled compounds of the present invention for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon- 14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability.
  • Isotopically- labeled compounds of Formula I of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples and Preparations below, by substituting a readily available isotopically-labeled reagent for a non- isotopically-labeled reagent.
  • the present invention also includes N-oxide derivatives and protected derivatives of compounds of the Invention.
  • compounds of the Invention when compounds of the Invention contain an oxidizable nitrogen atom, the nitrogen atom can be converted to an N-oxide by methods well known in the art.
  • compounds of the Invention contain groups such as hydroxy, carboxy, thiol or any group containing a nitrogen atom(s), these groups can be protected with a suitable "protecting group” or "protective group”.
  • a comprehensive list of suitable protective groups can be found in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc. 1991, the disclosure of which is incorporated herein by reference in its entirety.
  • nitrogen protecting groups include, but are not limited to Boc, Fmoc, benzyl, trityl, and the like.
  • the protected derivatives of compounds of the Invention can be prepared by methods well known in the art. [0103] Methods for the preparation and/or separation and isolation of single stereoisomers from racemic mixtures or non-racemic mixtures of stereoisomers are well known in the art. For example, optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • Enantiomers may be resolved by methods known to one of ordinary skill in the art, for example by: formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallization; via formation of diastereoisomeric derivatives which may be separated, for example, by crystallization, selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent.
  • enantiomer may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents or by converting on enantiomer to the other by asymmetric transformation.
  • enantiomer enriched in a particular enantiomer, the major component enantiomer may be further enriched (with concomitant loss in yield) by recrystallization.
  • the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
  • the invention additionally provides methods for making compounds of formula I.
  • the compounds of the invention may be prepared by the general methods and examples presented below, and methods known to those of ordinary skill in the art and reported in the chemical literature. Unless otherwise specified, solvents, temperatures, pressures, and other reaction conditions may be readily selected by one of ordinary skill in the art. Specific procedures are provided in the Synthetic Examples section. Intermediate substituted benzoic acids and their precursors (benzoic acids esters, benzamides or benzonitriles) are commercially available or are prepared by methods known to those skilled in the art (see WO20101 1316). Intermediate pyridyl carboxylic acids are commercially available or are prepared by methods known to those skilled in the art (see WO2009024905 and WO200802937). Intermediate substituted acyl hydrazines of formula III and VI are prepared from the corresponding substituted carboxylic acid esters by methods known to those skilled in the art.
  • R 2 , R 2a , R 3 , R 4 , R 7c , ring A, and n shall have the meanings defined in the detailed description of formula I, and optionally separating individual isomers; and optionally modifying any of the R 2 , R 2a , R 3 , R 4 , and R 7c groups; and optionally forming a pharmaceutically acceptable salt, hydrate, solvate or combination thereof.
  • a carboxylic acid of formula II is reacted with an acyl hydrazine of formula III under coupling conditions well known in the art such as by treatment with S0C1 2 or, N-iS-dimethylaminopropy -TV-ethylcarbodiimide hydrochloride (EDCI) in the presence of 1 -hydroxylbenzotriazole (HOBt) or, benzotriazol-1- yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP) or, O-(7-azabenzotriazol-l- yl)-N,N,N N'-tetramethyluronium hexafluorophosphate (HATU) or, O-(benzotriazol-l-yl- N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence or absence of a base such as Et 3 N or
  • a carboxylic acid of formula II is reacted with an acyl hydazine of formula VI, where X is a halogen such as fluorine, under coupling conditions well known in the art such as by treatment with SOCl 2 or, N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride in the presence of 1 -hydroxylbenzotriazole (HOBt) or, benzotriazol-l-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP) or, O-(7- azabenzotriazol-l-yl)-NN,N',jV'-tetramethyluronium hexafluorophosphate (HATU) or, O- (benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HB
  • Intermediate VII for this example is reacted with an alcohol VIII (where PG is a nitrogen protecting group such as a BOC group) in the presence of a base such as sodium hydride or lithium diisopropylamide (LDA), or potassium bis(trimethylsilyl)amide (KHMDS) in a suitable solvent such as DMF or THF to provide a compound of formula IX.
  • a base such as sodium hydride or lithium diisopropylamide (LDA), or potassium bis(trimethylsilyl)amide (KHMDS) in a suitable solvent such as DMF or THF
  • a suitable acid such as HC1
  • a suitable solvent such as dioxane, THF or methanol
  • Step 1 (2R,3R)-Methyl 2-amino-3-hydroxybutanoate (4).
  • MeOH 120 mL
  • acetyl chloride 20 mL, 282 mmol
  • D-allothreonine 3 1 1.31 g, 94.9 mmol
  • the reaction mixture was removed from the ice bath and heated to reflux for several hours. After cooling to RT, the reaction mixture was concentrated in vacuo. The crude product was used as is in the next reaction.
  • Step 2 (2R,3R)-Methyl 2-(feri-butoxycarbonylamino)-3-hydroxybutanoate (5).
  • Crude 4 (94.9 mmol), THF (300 mL) and triethylamine (21.6 g,) were combined and cooled in an ice bath.
  • Boc anhydride (31.9 g, 146 mmol) was added and the reaction mixture was allowed to warm to RT and stirred for several days. The reaction mixture was concentrated in vacuo and the resulting residue was partitioned between sat'd NaHC0 3 and diethyl ether. The phases were separated and the aqueous phase was further extracted with diethyl ether (2x).
  • Step 3 (4R,5R)-3- rt-Butyl 4-methyl 2,2,5-trimethyloxazoIidine-3,4- dicarboxylate (6).
  • Compound 5 (12.68 g, 54.4 mmol), toluene (150 mL), dimethoxypropane (75 mL, 610 mmol) and p-toluenesulfonic acid monohydrate (1.4 g, 7.4 mmol) were combined and stirred at 80 °C for 6 h. After cooling to RT, the reaction mixture was concentrated in vacuo and the resulting oil was partitioned between sat'd NaHC0 3 and diethyl ether.
  • Step 4 (4S,5R)-terf-Butyl 4-(hydroxymethyl)-2,2,5-trimethyloxazolidine-3- carboxylate (7).
  • Compound 6 (1 1.7 g, 42.8 mmol) was dissolved in THF (80 mL) and cooled in an ice bath.
  • Lithium borohydride (1.87 g, 85.8 mmol) was added followed by the dropwise addition of a 1 : 1 solution of THF:MeOH (22 mL) with stirring. After addition was complete, the reaction mixture was removed from the ice bath and stirred at RT overnight. The reaction was followed by LC-MS and NMR and additional lithium borohydride was added in 500 mg aliquots as needed until the reaction was complete.
  • reaction mixture was cooled in an ice bath then slowly added to a cold solution of 7.5% aqueous citric acid solution. The mixture was stirred for 30 min then extracted with EtOAc (3x). The combined EtOAc extractions were washed with sat'd NaCl (lx), dried (Na 2 S0 4 ) and concentrated in vacuo. The resulting crude material was purified by flash chromatography (20% EtOAc in hexanes) to give compound 7 as a colorless oil (6.9 g, 66%).
  • Step 1 Ethyl 2-(isopropylamino)-6-methylisonicotinate (9).
  • Ethyl 2-chloro-6- methylpyridine-4-carboxylate 8 550 mg, 2.75 mmol
  • Pd(OAc) 2 28 mg, 0.13 mmol
  • BINAP 156 mg, 0.25 mmol
  • Cs 2 C0 3 2.4 g, 7.5 mmol
  • 1 ,4-dioxane 25 mL
  • N 2 was bubbled into the mixture for a few minutes and isopropylamine (885 mg, 15 mmol) was added.
  • the sealed mixture was heated to 85 °C for 20 h.
  • the mixture was filtered through Celite and washed with EtOAc.
  • the solvents were removed in vacuo. Purification by flash column chromatography gave 9 (530 mg, 86%).
  • Step 2 2-(Isopropylamino)-6-methylisonicotinic acid (10).
  • Ethyl 2- (isopropylamino)-6-methylisonicotinate 9 (530 mg, 2.38 mmol) was dissolved in a mixture of MeOH (5 mL) and water (5 mL) and then treated with NaOH (480 mg, 1 1.9 mmol) at RT for 4 h.
  • MeOH was removed in vacuo and the resulting aqueous mixture was neutralized to pH 6 by addition of 20% aqueous HC1 solution.
  • the resulting solids were filtered and dried to give 10 (384 mg, 83%).
  • [0123] 1-3 To a chilled (-78°C) soln of methyl p-tolyl sulfoxide (15.4 g, 99.9 mmol) in 100 mL of THF was added a 1M soln of lithium bis(trimethylsilyl)amide in THF (105 mL, 105 mmol) over a 20 min period. The mixture was stirred for 5 min and then a soln of methyl trifluoroacetate (15.0 mL, 149 mmol) in 100 mL of THF was added over 20 min.
  • crude compound 1-150 can be purified by reverse phase HPLC using 20-100% ACN in water containing 0.1 % TFA as the gradient to afford compound 1-150 as a TFA salt.
  • Step 1 Ethyl 3,5-dichloro-4-fluorobenzoate (12).
  • a mixture of 3,5-dichloro-4- fluorobenzoate 11 (10.0 g, 47.8 mmol), TMS- CI (60 mL) and EtOH (60 mL) was heated to reflux overnight. After cooling to RT, the reaction mixture was concentrated in vacuo. The resulting oil was partitioned between sat'd NaHC0 3 and diethyl ether. The phases were separated and the aqueous phase was further extracted with diethyl ether (2x).
  • Step 4 (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-(2-(2-(isopropylamino)-6- methylisonicotinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (15).
  • Intermediate 10 (563 mg, 2.9 mmol), HOBT (378 mg, 2.8 mmol), EDCI (600 mg, 3.13 mmol) and DMA (6.0 mL) were combined and stirred at RT. After 10 min, compound 14 (1.1 1 g, 2.47 mmol) was added and the resulting mixture stirred at RT for 2 h.
  • Step 5 (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-1 ,4-thiadiazol-2-yl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (16).
  • Compound 15 (1.18 g, 1.9 mmol) dissolved in 2-methyltetrahydrofuran (10 mL) was heated to 80 °C in a sealed tube. Lawesson's reagent (482 mg, 1.19 mmol) was added and the resulting mixture was heated to 90 °C in a sealed tube with stirring overnight.
  • Step 6 (2R,3S)-3-Amino-4- ⁇ [2,6-dichloro-4-(5- ⁇ 2-methyl-6-[(l- methylethyl)amino]-pyridin-4-yl ⁇ -l,3,4-thiadiazol-2-yl)phenyl]oxy ⁇ butan-2-ol
  • Step 1 (S)-tert-Butyl 4-((2,6-dichloro-4-(ethoxycarbonyl)phenoxy)methyl)- 2,2-dimethyloxazolidine-3-carboxylate (17).
  • Compound 17 was synthesized using the same or an analogous synthetic procedure to that of compound 13 in Example 1, substituting intermediate 2 for intermediate 7.
  • Step 3 (S)-te/-/-Butyl 4-((2,6-dichloro-4-(2-(2-(isopropylamino)-6- methyIisonicotinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (19).
  • Compound 19 was synthesized from intermediates 18 and 10 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
  • Step 4 (S)-terf-Butyl 4-((2,6-dichloro-4-(5-(2-(isopropylamino)-6- methyIpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (20).
  • Compound 20 was synthesized using the same or an analogous synthetic procedure to that of compound 16 in Example 1.
  • Step 5 (R)-2-Amino-3-(2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)propan-l-ol.
  • the title compound was synthesized from intermediate 20 using the same or an analogous synthetic procedure to that of (2R,3S)-3-amino-4- ⁇ [2,6-dichloro-4-(5- ⁇ 2-methyl-6-[(l-methylethyl)amino]-pyridin-4- yl ⁇ -l,3,4-thiadiazol-2-yl)phenyl]oxy ⁇ butan-2-ol in Example 1.
  • Step 1 Ethyl 2-chloro-4,5-difluorobenzoate (22).
  • Compound 22 was synthesized using the same or an analogous synthetic procedure to that of compound 12 in Example 1.
  • Step 2 (4R,5R)-fer/-Butyl 4-((5-chloro-4-(ethoxycarbonyl)-2- fluorophenoxy)methyl)-2,2,5-trimethyloxazolidine-3-carboxyIate (24).
  • Intermediate 23 was made in an analogous manner to intermediate 7 using the appropriate enantiomerically pure starting material in place of compound 3.
  • Compound 24 was synthesized using the same or an analogous synthetic procedure to that of compound 13 in Example 1.
  • Step 4 (4R,5R)-terf-Butyl 4-((5-chloro-2-fluoro-4-(2-(2-(isopropylamino)-6- methylisonicotinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (26).
  • DMF dimethyl methyl
  • PyBop 3.82 g, 7.3 mmol
  • hydrazide 25 (2.65 g, 6.14 mmol
  • triethylamine (1.86 g, 18.42 mmol
  • Step 5 (4R,5R)-tert-Butyl 4-((5-chloro-2-fluoro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)methy ⁇
  • Step 6 (2R,3R)-3-Amino-4- ⁇ [5-chloro-2-fluoro-4-(5- ⁇ 2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl ⁇ -l,3,4-thiadiazol-2-yl)phenyl]oxy ⁇ butan-2-ol
  • Step 1 2-(Isopropylamino)-6-methylisonicotinohydrazide (33).
  • Compound 33 is made from intermediate 9 using the same or an analogous synthetic procedure to that of compound 14 in Example 1.
  • Step 2 2-Chloro-4-bromo-6-methylphenol (35).
  • NBS 6.2 g, 35 mmol
  • the solution was stirred at RT for 12 h.
  • AcOH was removed under reduced pressure.
  • the residue was diluted with EtOAc, washed with saturated Na 2 C0 3 solution and dried over Na 2 S0 4 . Removal of the solvents gave compound 35 (4 g, 52%) which was used in the next step without further purification.
  • Step 3 (S)-tert-Butyl 4-((4-bromo-2-chloro-6-methyIphenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (36).
  • intermediate 2 4.8 g, 21.7 mmol
  • triphenylphosphine 7.0 g, 27 mmol
  • diisopropylazodicarboxylate 5.4 g, 27 mmol
  • the reaction mixture was stirred at RT for 3 h. Water was added and the product was extracted with DCM. Purification by flash column chromatography gave compound 36 (5.8 g, 73%).
  • Step 4 (S)-tert-B ty ⁇ 4-((2-chloro-4-cyano-6-methyIphenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (37).
  • Compound 36 (5.68 g, 13 mmol), Zn(CN) 2 (920 mg, 7.8 mmol), Pd(dppf)Cl 2 DCM (475 mg, 0.65 mmol) and polymethylhydrosiloxane (390 mg) were dissolved in DMA/water (30 mL/0.3 mL). The reaction mixture was stirred at 90 °C for 6 h. Water was added and the product was extracted with EtOAc. Concentration and purification by flash column chromatography gave compound 37 (4.5 g, 91%).
  • Step 5 (S)-4-((3-(/ert-Butoxycarbonyl)-2,2-dimethyloxazoIidin-4-yl)methoxy)-
  • Step 6 (S)-/ert-Butyl 4-((2-chIoro-4-(2-(2-(isopropylamino)-6- methylisonicotinoyl)-hydrazinecarbonyl)-6-tnethylphenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (39).
  • Compound 39 was synthesized from intermediates 38 and 33 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
  • Step 7 (S)-te/"i-Butyl 4-((2-chloro-4-(5-(2-(isopropylamino)-6-methylpyridin-
  • Step 8 (R)-2-Amino-3-(2-chloro-4-(5-(2-(isopropyIamino)-6-methylpyridin-4- yl)-l,3,4-thiadiazol-2-yl)-6-methylphenoxy)propan-l-ol.
  • the title compound was synthesized from intermediate 40 using the same or an analogous synthetic procedure to that of (2R,3S)-3-amino-4- ⁇ [2,6-dichloro-4-(5- ⁇ 2-methyl-6-[(l-methylethyl)amino]-pyridin-4- yl ⁇ -l,3,4-thiadiazol-2-yl)phenyl]oxy ⁇ butan-2-ol in Example 1.
  • Step 1 tert-Butyl 4-bromo-2-chloro-5-fluorobenzoate (42).
  • 4-bromo-2-chloro-5-fluorobenzoic acid 41 (1.517 g, 5.99 mmol)
  • tert-butyl dicarbonate (2.07 g, 9.49 mmol)
  • DMAP 47 mg, 0.39 mmol
  • anhydrous chloroform 80 mL
  • Triethyl amine (0.92 mL, 6.64 mmol) was added at 0 °C and the mixture was allowed to stir at RT for 16 h. The mixture was poured into water and the organic layer was separated and collected.
  • Step 2 Benzyl 3-(4-(ter/-butoxycarbonyl)-5-chloro-2-fluorophenyl)-3- hydroxyazetidine-l-carboxylate (43).
  • a 100 mL two-neck round bottom flask was charged with tert-butyl 4-bromo-2-chloro-5-fluorobenzoate (42; 1.225 g, 3.96 mmol) and anhydrous THF (30 mL), under nitrogen atmosphere. After cooling the obtained solution to -18 °C (ice/salt bath), a 1.3 M solution of z ' PrMgCl LiCl (3.4 mL, 4.4 mmol) was added via cannula.
  • Step 3 4-[l-(Benzyloxycarbonyl)-3-hydroxyazetidin-3-yl]-2-chIoro-5- fluorobenzoic acid (44).
  • Benzyl 3-(4-(rert-butoxycarbonyl)-5-chloro-2-fluorophenyl)-3- hydroxyazetidine-l-carboxylate (43, 1.475 g, 3.38 mmol) was treated with TFA (9 mL) in chloroform (40 mL) at 0 °C for 90 min, then at RT for 16 h. The mixture was concentrated under reduced pressure, then azeotroped with toluene (twice).
  • Step 4 Benzyl 3-[5-chloro-2-fluoro-4-[2-[2-(isopropylamino)-6- methylisonicotinoyl]hydrazinecarbonyl]phenyl]-3-hydroxyazetidine-l-carboxylate (45).
  • Step 5 Benzyl 3-(tert-butyldimethylsilyloxy)-3-[5-chloro-2-fluoro-4-[2-[2- (isopropylamino)-6-methylisonicotinoyl]hydrazinecarbonyl]phenyl]azetidine-l- carboxylate (46).
  • compound 45 369 mg, 0.647 mmol
  • 2,6-lutidine 0.19 mL, 1.6 mmol
  • tert-butildimethylsilyltriflate 0.3 mL, 1.3 mmol
  • the residual crude material was twice purified by silica gel chromatography (85:15:0.5 to 70:30: 1 hexanes/ethyl acetate/methanol, first chromatography; 95:5 to 90: 10 CH 2 C1 2 /CH 3 0H, second chromatography) to afford 46 as an oil, partially contaminated by siloxane by-products. Pure fractions underwent full characterization as follows. The obtained material was subjected to the next step without further treatment.
  • Step 6 Benzyl 3-(teri-butyldimethylsilyloxy)-3-[5-chloro-2-fluoro-4-[5-[2- (isopropylamino)-6-methylpyridin-4-yl]-l,3,4-thiadiazol-2-yl]phenyl]azetidine-l- carboxylate (47).
  • Compound 47 was synthesized using the same or an analogous synthetic procedure to that of compound 16 in Example 1.
  • Step 7 Benzyl 3-[5-chloro-2-fluoro-4-[5-[2-(isopropylamino)-6- methylpyridin-4-yI]-l,3)4-thiadiazol-2-yI]phenyl]-3-hydroxyazetidine-l-carboxylate (48).
  • a solution of 47 (302 mg, 0.443 mmol) in anhydrous THF (16 mL) was treated with a 1 M solution of TBAF in THF (0.5 mL, 0.5 mmol) at RT for 20 h.
  • Step 8 3-(5-ChIoro-2-fluoro-4-(5-(2-(isopropylamino)-6-methylpyridin-4-yl)- l,3,4-thiadiazol-2-yl)phenyl)azetidin-3-ol.
  • Compound 48 140 mg, 0.246 mmol was suspended in methanesulfonic acid (5 mL) at RT. The mixture was stirred and gently heated at 45 °C until all material went into solution. Reaction was deemed completed within 3 h. It was poured onto ice; solid K 2 C0 3 was slowly added under vigorous stirring until pH was corrected to about 10.
  • the aqueous phase was extracted with EtOAc (2x); the combined organic layers were washed with brine and dried over MgS0 4 .
  • the aqueous phase was extracted with hot (60 °C) chloroform (3x); the combined organic layers were dried over MgS0 4 .
  • the residual crude material was purified by silica gel chromatography (90:9:1 CH 2 Cl 2 /CH 3 OH/28% (w/w) NH 4 OH) to afford the title compound as a yellow solid (96 mg, 0.222 mmol, 90%).
  • Step 1 6-Chloro-2-methylpyridin-3-amine (56).
  • 55 5 g, 29 mmol
  • EtOH 20 mL
  • cone HC1 20 mL
  • Fe powder 16.2 g, 289 mmole
  • the reaction mixture was stirred at RT for an additional 30 min.
  • the solvent was removed in vacuo and water was added to the residue which was then neutralized with NaHC0 3 and diluted with EtOAc.
  • the reaction mixture was filtered through Celite and washed with EtOAc. The filtrate was transferred to a separatory funnel and the phases separated.
  • the organic layer was washed with water and brine, dried over Na 2 S0 and concentrated to afford 56 (4.1 g, 99 %) as a yellow solid.
  • Step 2 6-ChIoro-N-isopropyl-2-methylpyridin-3-amine (57).
  • 56 (4.81 g, 33.75 mmol) and acetone (2.74 g, 47.2mmol) in dichloroethane (60 mL) was added NaBH(OAc) 3 (10.713 g, 50.53 mmol) and AcOH (3.44g, 57.2 mmol) at RT.
  • the reaction was stirred for 16 h and then diluted with IN NaOH.
  • the aqueous solution was extracted with DCM and the organic layer was washed with water and brine, dried over Na 2 S0 4 and concentrated to afford 57 (6.16 g, 98%).
  • Step 3 5-(Isopropylamino)-6-methylpicolinonitrile (58).
  • 57 6.2 g, 33.5 mmol
  • DMF 50 mL
  • Zn(CN) 2 5.5 g, 46.84 mmol
  • tetrakis 5.8 g, 5.01mmol
  • the reaction was then stirred at 130 °C for 16 h. After completion, the reaction mixture was absorbed on silical gel and chromatographed to obtain 58 (5 g, 85 %) as a thick liquid.
  • Step 4 5-(Isopropylamino)-6-methylpicolinic acid (59).
  • 58 5.0 g, 28.4 mmol
  • EtOH 40 mL
  • 20 % aqueous KOH 40 mL
  • the reaction mixture was refluxed for 12 h.
  • Volatiles were removed in vacuo and the resulting aqueous mixture was neutralized to pH 5 with citric acid solution which was then extracted with EtOAc.
  • the organic layer was dried, concentrated and washed with pentane to afford 59 (2.8 g, 50%) as a white solid.
  • Step 5 (4S,5R)-tert-Butyl 4-((5-chloro-2-fluoro-4-(2-(5-(isopropylamino)-6- methylpicolinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (61).
  • Intermediate 60 was made in an analogous manner to intermediate 14 using the appropriately substituted benzoic acid in place of compound 11.
  • Compound 61 was synthesized from intermediates 60 and 59 using the same or an analogous synthetic procedure to that of compound 26 in Example 3.
  • Step 6 (4S,5R)-ter/-Butyl 4-((5-chloro-2-fluoro-4-(5-(5-(isopropylamino)-6- methylpyridin-2-yl)-1 > 4-thiadiazol-2-yl)phenoxy)m
  • Step 7 (2R,3S)-3-Amino-4- ⁇ [5-chloro-2-fluoro-4-(5- ⁇ 6-methyl-5-[(l- methylethyl)amino]pyridin-2-yl ⁇ -l,3,4-thiadiazol-2-yl)phenyI]oxy ⁇ butan-2-ol
  • Step 1 6-Methyl-4-nitropicolinonitrile (64).
  • a mixture of 4-nitro-2-picoline-N- oxide 63 (6g, 38 mmol) and dimethyl sulfate (5.3 g, 42 mmol) was heated to 65-70 °C for 2 h. After cooling, the resulting solid was filtered and washed with 30 ml of n-hexane. The solid was then dissolved in water (80 mL) and cooled to -10 °C under an atmosphere of N 2 . A solution of sodium cyanide (7.7 g, 152 mmol) in water (55 mL) was added dropwise over 40 min with stirring. The mixture was then stirred for an additional 3 hours at the same temperature.
  • Step 2 6-Methyl-4-nitropicolinic acid (65).
  • Step 3 4-Bromo-6-methylpicolinic acid (66).
  • a solution of 6-Methyl-4-nitro- pyridine-2-carboxylic acid 65 (6 g, 32.7 mmol) in 48% hydrobromic acid (65 mL) was heated at 100 °C overnight and then allowed to cool to RT. The solution was then evaporated to dryness in vacuo to give crude 66 contaminated with inorganic salts (10 g) which was directly used in the next step.
  • Step 4 4-(Isopropylamino)-6-methylpicolinic acid (67).
  • 4-bromo-6-methyl-pyridine-2-carboxylic acid 66 (10 g, 46.5 mmol) in butanol (120 mL) was added isopropylamine (8.2 g, 139 mmol) and the mixture was heated to 1 17 °C for 2 d. The solvent was removed in vacuo and the resulting residue was purified by column chromatography to afford 67 (5.0 g, 78% yield over two steps from 65) as a brown solid.
  • Step 5 (4S,5S)-terf-Butyl 4-((5-chloro-2-fluoro-4-(2-(4-(isopropylamino)-6- methylpicolinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (69).
  • Intermediate 68 was made in an analogous manner to intermediate 14 using the appropriately substituted benzoic acid in place of compound 11 and the appropriate enantiomerically pure analog of intermediate 7.
  • Compound 69 was synthesized from intermediates 68 and 67 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
  • Step 6 (4S,5S)-terf-Butyl 4-((5-chloro-2-nuoro-4-(5-(4-(isopropylamino)-6- methylpyridin-2-yl)-1 ,4-thiadiazoI-2-yl)phenoxy)methyl)-2,2,5-trimethyloxazoIidine-3- carboxylate (70).
  • Compound 70 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
  • Step 7 (2S,3S)-3-Amino-4- ⁇ [5-chloro-2-fluoro-4-(5- ⁇ 6-methyI-4-[(l- methylethyl)amino]pyridin-2-yl ⁇ -l,3,4-thiadiazol-2-yl)phenyl]oxy ⁇ butan-2-ol
  • Step 1 Methyl 4-(alIyloxy)-3-chlorobenzoate (72).
  • methyl 3-chloro-4-hydroxybenzoate 71 (3.0 g, 16.12 mmol) in DMF (15 mL) was added allyl bromide (2.8 mL, 32.2 mmol) and K 2 C0 3 (4.4 g, 32.2 mmol) and the subsequent mixture heated to 80 °C for 3 h. After cooling to RT, the reaction mixture was diluted with water (200 mL) and extracted with EtOAc. The combined organic layers were dried over Na 2 S0 4 and concentrated to give 72 (3.4 g, 94.4%).
  • Step 2 4-(Allyloxy)-3-chlorobenzoic acid (73).
  • 72 3.4 g, 15 mmol
  • THF 15 mL
  • NaOH 0.66 g, 16.5 mmol
  • water 10 mL
  • the solvent was distilled off under reduced pressure.
  • the resulting residue was diluted with water (100 mL), acidified with 2N HC1 and extracted with EtOAc.
  • the organic phase was washed with water, brine, dried over Na 2 S0 4 and concentrated to afford 73 (2.6 g, 81.2 %).
  • Step 3 (S)-ferf-Butyl 4-((4-(2-(4-(allyloxy)-3- chlorobenzoyl)hydrazinecarbonyl)-5-chloro-2-fluorophenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (75).
  • Intermediate 74 was made in an analogous manner to intermediate 18 using the appropriately substituted benzoic acid in place of compound 12.
  • Compound 75 was synthesized from intermediates 74 and 73 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
  • Step 4 (S)-terf-Butyl 4-((4-(5-(4-(allyloxy)-3-chIorophenyl)-l,3,4-thiadiazoI-2- yl)-5-chloro-2-fluorophenoxy)methyI)-2,2-dimethyloxazolidine-3-carboxyIate (76).
  • Step 5 (S)-terf-Butyl 4-((5-chloro-4-(5-(3-chloro-4-hydroxyphenyl)-l,3,4- thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate (77).
  • Step 7 (2R)-2-Amino-3-[(5-chloro-4- ⁇ 5-[3-chloro-4-(oxetan-3-yloxy)phenyl]- l,3,4-thiadiazol-2-yl ⁇ -2-fluorophenyl)oxy]propan-l-ol trifluoroacetate salt.
  • Compound 78 (0.6 g, 0.95 mmol) was dissolved in DCM (4 mL) and cooled to 0 °C. TFA/DCM (1 :1, 5 mL) was added and the resulting mixture stirred at 0 °C for 10 min and then at RT for 1 h.
  • Step 1 2-(Allyloxy)-6-chloroisonicotinic acid (80).
  • KO'Bu 5.85 g, 52 mmol
  • Compound 79 5.0 g, 26 mmol
  • the reaction mixture was concentrated in vacuo.
  • the resulting residue was diluted with water, acidified with citric acid and extracted with diethyl ether. The organic layer was dried and concentrated to afford 80 (3.0 g, 54.5%) which was used as such for the next step.
  • Step 2 (S)-tert-Butyl 4-((4-(2-(2-(allyloxy)-6- chloroisonicotinoyl)hydrazinecarbonyl)-5-chloro-2-fluorophenoxy)methyl)-2,2- dimethyIoxazolidine-3-carboxylate (81).
  • Compound 81 was synthesized from intermediates 74 and 80 using the same or an analogous synthetic procedure to that of compound 26 in Example 3.
  • Step 3 (S)-fert-Butyl 4-((4-(5-(2-(allyloxy)-6-chloropyridin-4-yI)-l,3,4- thiadiazol-2-yl)-5-chloro-2-fluorophenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (82).
  • Compound 82 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
  • Step 4 (S)-ter/-ButyI 4-((5-chloro-4-(5-(2-chloro-6-hydroxypyridin-4-yl)- l,3 5 4-thiadiazol-2-yl)-2-fluorophenoxy)methyI)-2,2-dimethyloxazoIidine-3-carboxylate (83).
  • Compound 83 was synthesized using the same or an analogous synthetic procedure to that of compound 77 in Example 10.
  • Step 5 (S)-fert-Butyl 4-((5-chloro-4-(5-(2-chloro-6-(oxetan-3-yloxy)pyridin-4- yl)-l,3i4-thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (84).
  • Compound 84 was synthesized using the same or an analogous synthetic procedure to that of compound 78 in Example 10.
  • Step 6 (R)-2-Amino-3-(5-chloro-4-(5-(2-chloro-6-(oxetan-3-yloxy)pyridin-4- yl)-l,3,4-thiadiazol-2-yl)-2-fluorophenoxy)propan-l-ol trifluoroacetate salt (85).
  • Compound 85 was synthesized using the same or an analogous synthetic procedure to that of (2R)-2-amino-3 -[(5 -chloro-4- ⁇ 5 - [3 -chloro-4-(oxetan-3-yloxy)pheny 1]- 1 ,3 ,4-thiadiazol-2-yl ⁇ - 2-fluorophenyl)oxy]propan-l-ol trifluoroacetate salt in Example 10.
  • Step 7 4-[5-(4- ⁇ [(2R)-2-Amino-3-hydroxypropyl]oxy ⁇ -2-chloro-5- fluorophenyl)-l,3,4-thiadiazol-2-yl]-6-(oxetan-3-yloxy)pyridin-2-ol acetate salt.
  • the TFA salt 85 was found to slowly decompose to form the corresponding hydroxyl pyridine, which was isolated as follows. A solution of 85 in EtOAc was basified to pH 12 with IN NaOH aqueous solution. The organic phase was separated, washed with brine, dried with MgS0 4 , and concentrated in vacuo. The residue was purified by prep HPLC eluting with a
  • Step 1 Ethyl 2,6-dichloroisonicotinate (86). To an ice cold solution of 79 (10 g, 52 mmol) in EtOH (100 mL) was added cone H 2 S0 4 (5 mL) and the reaction mixture was heated to reflux for 12 h. Solvent was removed in vacuo. The resulting residue was cooled in an ice bath, neutralized with NaHC0 3 solution and extracted with EtOAc. The organic layer was dried and concentrated to afford 86 (1 1 g, 96%).
  • Step 2 Ethyl 2-chloro-6-(isopropylamino)isonicotinate (87).
  • ester 86 (6.5.0 g, 29.5 mmol) in dry dioxane (60 mL)
  • Cs 2 C0 3 (14.3 g, 44 mmol)
  • isopropylamine (6 mL, 70.8 mmol) was added.
  • BINAP 2.3 g, 3.6 mmol
  • Pd(II) acetate 0.8 g, 3.6 mmol
  • Step 3 2-Chloro-6-(isopropylamino)isonicotinic acid (88). To a stirred solution of 87 (5.7 g, 2.35 mmol) in a mixture of THF (15 mL) and water (10 mL) was added NaOH (2.4 g, 6.12 mmol), and the resulting mixture was stirred at RT for 1 h. The solvent was distilled off under reduced pressure. Water (100 mL) was added to the resulting residue and the mixture acidified with citric acid and extracted with EtOAc. The organic phase was washed with water and brine, dried over Na 2 S0 4 and concentrated to afford 88 (5 g, 100 %).
  • Step 4 (4S,5S)- rf-Butyl 4-((5-chloro-4-(2-(2-chloro-6- (isopropylamino)isonicotinoyl)-hydrazinecarbonyl)-2-fluorophenoxy)methyl)-2,2,5- trimethyloxazolidine-3-carboxylate (89).
  • Compound 89 was synthesized from intermediates 68 and 88 using the same or an analogous synthetic procedure to that of compound 26 in Example 3.
  • Step 5 (4S,5R)-terf-Butyl 4-((5-chloro-4-(5-(2-chloro-6- (isopropylamino)pyridin-4-yl)-l,3 > 4-thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2,5- trimethyIoxazolidine-3-carboxylate (90).
  • Compound 90 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
  • Step 6 (4S,5S)-ter/-Butyl 4-((5-chloro-4-(5-(2-cyano-6- (isopropylamino)pyridin-4-yl)-l,3,4-thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2,5- trimethyloxazolidine-3-carboxylate (91) and (4S,5S)-tert-Butyl 4-((5-cyano-4-(5-(2- cyano-6-(isopropylamino)pyridin-4-yl)-l,3,4-thiadiazol-2-yI)-2-fluorophenoxy)methyl)- 2,2,5-trimethyloxazolidine-3-carboxylate (92).
  • Step 7 4-[5-(4- ⁇ [(2S,3S)-2-Amino-3-hydroxybutyl]oxy ⁇ -2-chloro-5- fluorophenyl)-1 ,4-thiadiazoI-2-yl]-6-[(l-methylethyl)amino]pyridine-2-carbonitrile di- trifluoroacetate salt and Ethyl 4-[5-(4- ⁇ [(2S,3S)-2-amino-3-hydroxybutyl]oxy ⁇ -2-chloro- 5-fluorophenyl)-l,3,4-thiadiazol-2-yl]-6-[(l-methylethyl)amino]pyridine-2-carboxylate di-trifluoroacetate salt.
  • Step 8 4-[5-(4- ⁇ [(2S,3S)-2-Amino-3-hydroxybutyl]oxy ⁇ -2-cyano-5- fluorophenyl)-1 ,4-thiadiazol-2-yl]-6-[(l-methyIethyl)amino]pyridine-2-carbonitrile di- trifluoroacetate salt.
  • Compound 92 (0.2 g, 0.32 mmol) was dissolved in DCM (4 mL) and cooled to 0 °C. TFA/DCM (3:7, 5 mL) was added and the resulting mixture stirred at 0 °C for 10 min and at RT for 1 h.
  • Step 1 3-Chloro-4-isopropoxybenzonitrile (94).
  • a stirring suspension of 93 (20.0 g, 128 mmol) and NaH (6.17 g, 154 mmol) in THF (200 mL) was cooled to -10 °C and isopropyl alcohol (12.8 mL, 167 mmol) was added very slowly over a period of 1 h.
  • the temperature of the reaction mixture was allowed to warm to 10 °C and stirred at that temperature for 2 h.
  • the reaction mixture was then further cooled and quenched with ice.
  • THF was removed under reduced pressure.
  • the resulting residue was diluted with water and extracted with EtOAc.
  • the organic layer was dried and concentrated to afford 94 (24 g, 95%) which was used as such for the next step.
  • Step 2 3-Chloro-4-isopropoxybenzoic acid (95).
  • 94 57 g, 0.29 mol
  • EtOH 120 mL
  • 12 % aqueous KOH 300 mL
  • the reaction mixture was then cooled and acidified with HC1 to pH 5.
  • the resulting yellow solid was filtered, washed water and air-dried.
  • the resulting carboxylic acid was then suspended in hexane, stirred for 5 min, then the hexane was decanted off. The hexane wash was repeated, the resulting solids were filtered and washed with hexane to afford 95 (45 g, 72%) as a white solid.
  • Step 3 Methyl 3-chloro-4-isopropoxybenzoate (96). To an ice cold solution of 95 (20.0 g, 930 mmol) in MeOH (200 mL) was added cone H 2 S0 4 (20 mL) and the reaction mixture was heated to reflux for 3 h. Excess MeOH was removed in vacuo. The resulting residue was ice cooled and then neutralized with NaHC0 3 solution. The resulting aqueous mixture was extracted with EtOAc. The organic layer was dried and concentrated. The resulting crude compound was passed through a short column (silica 60-120, EtOAc:hexane 10:90) to obtain 96 (16.6 g, 78%).
  • Step 4 3-Chloro-4-isopropoxybenzohydraz.de (97).
  • Compound 97 is made from intermediate 96 using the same or an analogous synthetic procedure to that of compound 14 in Example 1.
  • Step 5 l-Bromo-2-chloro-5-fluoro-4-nitrobenzene (99).
  • 98 (19.5 g, 93.10 mmol) in cone H 2 S0 (160 mL) at 0 °C was added KN0 3 (10.32 g, 102.41 mmol) in portions over 30 min.
  • the resulting yellow solution was allowed to warm to RT and stirred overnight at RT.
  • the solution was poured into ice and extracted with EtOAc. The organic phase was washed with water and brine, dried and concentrated to afford 99 (23 g, 97.66 %) as a white solid.
  • Step 6 4-Bromo-5-chloro-2-fluoroaniline (100). To a stirred solution of 99 (23 g, 90.39 mmol) in EtOH (90 mL) and cone HC1 (90 mL) cooled to 0 °C was added Fe powder (47.52 g, 848.5 mmol) in small portions over 30 min. After addition was complete, the reaction mixture was stirred at RT for another 30 min. The solvent was distilled off under reduced pressure. Water was added to the resulting residue and the mixture neutralized with NaHC0 3 and diluted with EtOAc. The resulting biphasic reaction mixture was filtered through Celite and washed with EtOAc. The phases of the filtrate were separated and the organic layer was washed with water and brine solution, dried over Na 2 S0 4 and concentrated to afford 100 (18 g, 88.75 %) as a yellow solid.
  • Step 7 4-Amino-2-chloro-5-fluorobenzonitrile (101).
  • DMF 100 mL
  • cuprous cyanide 1 1.49 g, 128.2 mmol
  • the reaction mixture was cooled to RT and and DMF was evaporated under reduced pressure.
  • the resulting residue was partitioned between DCM and water and the resulting biphasic mixture was filtered. The phases of the filtrate were separated and the organic layer was washed with brine, dried over Na 2 S0 4 and concentrated in vacuo.
  • the crude compound was purified by column
  • Step 8 2-Chloro-4-(diallylamino)-5-fluorobenzonitrile (102).
  • 101 6.0 g, 35.17 mmol
  • DMF 60 mL
  • NaH 3.0 g, 73.86 mmol
  • Allyl bromide (6.38 g, 52.76 mmol) was added and reaction was further stirred at the same temperature for 2 h and was then quenched with ice with stirring for 15 min at 0 °C.
  • the resulting mixture was neutralized with citric acid solution and extracted with EtOAc, dried and concentrated to obtain 102 (6.0 g, 68 %) which was used as such in the next step.
  • Step 9 2-Chloro-4-(diallylamino)-5-fluorobenzoic acid (103).
  • 102 6.0 g, 23.93 mmol
  • EtOH 60 mL
  • 20 % aqueous KOH 60 mL
  • the reaction mixture was refluxed for 4 h.
  • Solvent was removed in vacuo and the resulting residue was neutralized to pH 5 with citric acid solution.
  • the resulting aqueous mixture was extracted with EtOAc.
  • the organic layer was dried and concentrated and the resulting solid washed with pentane to afford 103 (5.5 g, 85.27%) as a white solid.
  • Step 10 2-Chloro-N , -(3-chloro-4-isopropoxybenzoyl)-4-(diallylamino)-5- fluorobenzohydrazide (104).
  • Compound 104 was synthesized from intermediates 103 and 97 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
  • Step 11 N,N-Diallyl-5-chloro-4-(5-(3-chloro-4-isopropoxy pheny 1)-1 ,3,4- thiadiazol-2-yl)-2-fluoroaniline (105).
  • Compound 105 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
  • Step 12 5-Chloro-4-(5- ⁇ 3-chloro-4-[(l-methylethyl)oxy]phenyl ⁇ -l,3,4- thiadiazol-2-yl)-2-fluoroaniIine.
  • a stirred solution of 105 (3.0 g, 6.27 mmol), 1,3-dimethyl barbituric acid (3.92 g, 25.08 mmol), Pd(OAc) 2 (1.40 g, 6.27 mmol) and triphenylphosphine (1.64 g, 6.27 mmol) in EtOH (30 mL) was purged with argon for 20 min. The reaction mixture was then heated to 85 °C for 2 h.
  • Steps 1-3 5-Bromo-6-(isopropylamino)nicotinic acid (109).
  • Intermediate 109 can be prepared by one skilled in the art from commercially available 106 in three steps using the standard procedures outlined in the scheme above.
  • Step 5 (4S,5R)-ferf-Butyl 4-((4-(5-(5-bromo-6-(isopropylamino)pyridin-3-yl)- 1 j 4-thiadiazol-2-yl)-2,6-dimethylphenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (112).
  • Compound 112 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
  • Step 6 (4S,5R)-/er/-Butyl 4-((4-(5-(5-cyano-6-(isopropylamino)pyridin-3-yI)- l,3,4-thiadiazol-2-yl)-2,6-dimethylphenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (113).
  • Intermediate 113 can be prepared by one skilled in the art from intermediate 112 using standard procedures. Typically, intermediate 112 is dissolved in an appropriate solvent such as DMF and heated in the presence of a palladium catalyst and an appropriate cyanide donor such as KCN.
  • Step 7 5-(5-(4-((2S,3R)-2-Amino-3-hydroxybutoxy)-3,5-dimethylphenyl)- l,3,4-thiadiazol-2-yl)-2-(isopropylamino)nicotinonitrile ditrifluoroacetate salt.
  • Step 1 (R)- r/-Butyl l-(2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)-3-hydroxypropan-2-ylcarbamate (114).
  • Step 2 (S)-tert-Butyl l-(di-te/-/-butoxyphosphoryloxy)-3-(2,6-dichloro-4-(5-(2- (isopropylamino)-6-methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)propan-2- ylcarbamate (115).
  • Compound 114 (306 mg, 0.54 mmol) was dissolved in a 3% solution of tetrazole in acetonitrile (10.5 mL).
  • Di-/ert-butyl diethylphosphoramidite (602 mg, 2.41 mmol) was added and the resulting mixture was stirred at RT overnight.
  • reaction mixture was then cooled in an ice bath and w-chloroperbenzoic acid (461 mg, 2.67 mmol) was added with continued stirring in the ice bath for 2 h.
  • the reaction mixture was then diluted with EtOAc and washed with sat'd NaHC0 3 (3x), sat'd NaCl (lx), dried (Na 2 S0 4 ) and
  • Step 3 (S)-2-Amino-3-(2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)propyl dihydrogen phosphate.
  • HC1 (4.0 in 1,4-dioxane, 1.0 mL, 4.0 mmol) was added to the rbf containing compound 1-105 (57 mg, 0.092 mmol). The rxn mixture was stirred at RT for 2 h. The volatiles were evaporated under reduced pressure. The crude pdt was diluted with EtOAc (50 mL) and washed with sat'd NaHC0 3 aq soln (3x10 mL). The aq phase was extracted with DCM (5x35 mL). The combined organics were concentrated. The resulting crude pdt was dissolved in DMSO and purified by reverse phase HPLC using 5-95% ACN in water as the gradient.
  • the rxn mixture was cooled to 0°C again and quenched with slow addition of sat'd NH 4 C1 aq soln (6 mL).
  • the mixture was diluted with EtOAc (40 mL) and sat'd NaHC0 3 aq soln (5 mL).
  • the layers were separated and the organic phase was further washed with water (2x10 mL).
  • the organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated.
  • the resulting crude pdt was purified by normal phase flash chromatography using 0-10% MeOH/DCM as the gradient to afford compound 1-23.
  • the mixtue is diluted with DCM (50 mL) and washed with water (3x10 mL), then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.
  • the resulting crude pdt is purified by normal phase column chromatography using 1-10% MeOH in DCM as the gradient to afford the title compound.
  • the resulting crude pdt is purified by normal phase flash column chromatography using 0-10% MeOH in DCM as the gradient.
  • the resulting pdt is still not pure and purified again by reverse phase prepartive HPLC using 5-90% ACN/water containing 0.1% TFA as the gradient (20 min run).
  • the pdt fractions are combined and lyophilized overnight to afford a solid.
  • This solid is dissolved in MeOH (5 mL) and filtered through a PL-HCO3MP SPE cartriage (StratoSpheresTM SPE, Part No.: PL3540-C603) and washed with MeOH (3x5 mL). The filtrate is concentrated to afford the title compound.
  • Suitable in vitro assays for measuring SI PI and S 1P5 agonist activity are known in the art. All Compounds in Table 1 were tested in one or more of the following biological assays and were found to be agonists of S1P1 and/or S1P5. As such compounds of Formula I are useful for treating diseases, particularly autoimmune disease in which SI PI and/or S1P5 activity contributes to the pathology and/or symptomatology of the disease, for example, multiple sclerosis and graft-versus host disease. Suitable in vivo models for autoimmune diseases are known to those of ordinary skill in the art and are also described below, e.g.
  • HEK293 cells expressing the CNG channel and SI Pi are thawed and plated into the wells of a black, clear bottom, 384- well CellBind plate (Corning, Corning, NY) at 14,000 cells per well.
  • HEK293 cells expressing the CNG channel and CB1 are cultured and plated under the same conditions.
  • the cells are incubated for 16 h at 37 °C in complete DMEM medium (Invitrogen Carlsbad, CA) containing 10% FBS (HyClone Logan, UT), 250 ⁇ g/mL geneticin (Invitrogen), and 1 ⁇ g/mL puromycin (Sigma-Aldrich, St. Louis, MO).
  • FBS HyClone Logan, UT
  • GPI GPI-Aldrich
  • puromycin Sigma-Aldrich, St. Louis, MO
  • a membrane potential dye (BD Biosciences) is added and the plates are incubated for 2-2.5 h at room temperature.
  • Test compounds are tested at maximum concentrations of 10 ⁇ . Compounds are diluted in DMSO (10 concentration points, 3-fold each) and added to the assay plate at final DMSO concentrations of 1.8%. For each compound, there are duplicate assay plates and each assay plate have duplicate wells per concentration point. Test compounds are added to the cells in a DPBS solution containing 25 ⁇ Ro 20-1724 (Sigma-Aldrich), 500 nM of the A2b receptor agonist NEC A (Sigma-Aldrich) and 10 nM (EC 95 ) of SIP (Avanti Alabaster, AL) and incubated for 90 min.
  • the assay plate is read before compound addition (T 0 ) and after the 90 min incubation (T 90 ) using an EnVision plate reader (PerkinElmer, Waltham, MA) at an excitation wavelength of 350 nm and an emission wavelength of 590 nm.
  • the T 90 /T 0 ratio is determined for each concentration of the test compounds.
  • the percent agonist activity is determined as [(test compound - DMSO alone control) / (NECA alone control- DMSO alone control) * 100].
  • the percent activities are plotted against compound concentration to determine EC 50 using XLFit (IDBS, Alameda, CA).
  • the control used for calculating rEC50 in the SI Pi CNG agonist assay is DMSO.
  • the cytoplasmic C-terminus of SI Pi was tethered to the tTA transcriptional activator with a linker that contains a cleavage site for the Nla protease from tobacco etch virus (TEV protease).
  • TEV protease tobacco etch virus
  • the C-terminus of the human P-arrestin2 protein was fused to TEV protease. Binding of an agonist recruits the ⁇ -arrestin- TEV fusion protein to the receptor resulting in cleavage of the linker and released of tTA to enter the nucleus and subsequently activated a tTA-dependent luciferase reporter gene.
  • Assay 2a Frozen HEK293 cells transiently transfected with receptor cDNAs for SI Pi (Invitrogen) are thawed and suspended in 10 mL of Pro293a-CDM culture medium (Invitrogen) supplemented with 4 mM L-Glutamine (Invitrogen), IX Pen/Strep (100 units/mL penicillin and 100 g/mL streptomycin, Invitrogen) and 0.1% fatty acid free BSA (Sigma- Aldrich). Cells are added to the wells of a 384- well white opaque bottom assay plate (PerkinElmer) at 3,000-6,000 cells per well and the plate is incubated for approximately 4 h in a 37 °C incubator.
  • Pro293a-CDM culture medium Invitrogen
  • IX Pen/Strep 100 units/mL penicillin and 100 g/mL streptomycin, Invitrogen
  • 0.1% fatty acid free BSA Sigma- Aldrich
  • Test compounds are tested at maximum concentrations of 10 ⁇ for the agonist assays.
  • Compounds are diluted in DMSO (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration.
  • For each compound there are duplicate assay plates and each assay plate has duplicate wells per concentration point.
  • the plate is incubated at 37 °C for 30 min.
  • the efficacy control is 5 ⁇ SIP (Avanti).
  • the assay plates are incubated in a 37 °C incubator for 16-18 h. Luciferase assay reagent is added and luminescence measured in an EnVision plate reader (PerkinElmer).
  • percent activity is calculated as [(test compound - background) / (positive control- background) * 100], where background is the luminescence of the DMSO alone control and the positive control is the luminescence from cells incubated with the efficacy control 5 ⁇ SIP.
  • the percent activities are plotted against compound concentration to determine EC50 using XLFit (IDBS).
  • Assay 2b Alternatively, U20S cells expressing the reporter gene and SlPi (Invitrogen) were added to the wells of a 384-well white opaque bottom assay plate (PerkinElmer) at 0.3125 X 10 6 cells per well. The cells were serum starved for 48 h in Freestyle medium (Invitrogen). Test compounds were tested at maximum concentrations of 1 ⁇ for the agonist assay. Compounds were diluted in DMSO (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration. The efficacy control was 1 ⁇ SIP (Avanti). For each compound, there were duplicate assay plates and each assay plate had duplicate wells per concentration point.
  • the plate was incubated overnight at 37 °C.
  • the GeneBLAzer ⁇ -lactamase assay reagent (Invitrogen) was added and the plates were incubated for an additional 2 h at room temperature. Fluorescence was measured using an EnVision plate reader (PerkinElmer, Waltham, MA) at an excitation wavelength of 409 nm and emission wavelengths of 460 nm and 530 nm. The emission intensity at each wavelength was background subtracted against wells containing medium only and the F 460n m F 5 3 0 nm ratio determined for each concentration of the test compounds.
  • Percent activity was calculated as [(test compound ratio - DMSO ratio) / (positive control ratio - DMSO ratio) * 100], where the positive control and DMSO ratios are from cells incubated with the efficacy control 1 ⁇ SIP and 1% DMSO, respectively. The percent activities were plotted against compound concentration to determine EC 50 using XLFit (IDBS).
  • Assay 3 a The hSlPU? GTPyS binding assay was carried out at room temperature in 96 well non-binding surface assay plates. The reaction in each well contained 4 ⁇ g hSIPIR (hEdgl) membrane protein (Lonza), 30 ⁇ GDP, 0.1 nM [ 35 S]GTPyS, 0.25% fatty acid free BSA, and serially diluted hSIPIR agonist compound in 200 ⁇ assay buffer (25mM Tris- HC1 PH 7.9, 100 mM NaCl, 3 mM MgC12, and 0.2 mM EGTA).
  • hSIPIR hEdgl membrane protein
  • WGA Wood Germ Agglutinin
  • Assay 3b The DELFIA GTP-Eu binding assay (PerkinElmer) is a time-resolved fluorometric assay based on GDP-GTP exchange.
  • CHO cell membranes (Lonza) expressing human SI Pi are incubated in 96-well filter plates (Pall, East Hills, NY) in a final volume of 100 ⁇ buffer containing 40 ⁇ g/mL membrane, 50 mM HEPES, 2 ⁇ GDP, 10 mM MgCl 2 , 100 mM NaCl, 500 ⁇ / ⁇ Saponin and test compound. Test compounds are tested at maximum concentrations of 10 ⁇ .
  • Compounds are diluted (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration. For each compound, there are duplicate assay plates and each assay plate has duplicate wells per concentration point. The plates are incubated for 30 min at room temperature on a plate shaker at low speed. GTP-Eu is added to each well (10 ⁇ ,, 10 nM final concentration) and the plate is incubated for an additional 30 min with slow shaking. The wells are washed with ice cold GTP washing buffer (3 X 150 ⁇ _.) using a vacuum manifold and the assay plates read in an EnVision plate reader (PerkinElmer) at an excitation wavelength of 340 nm and an emission wavelength of 615 nm.
  • EnVision plate reader PerkinElmer
  • percent activity is calculated as [(test compound - background)/ (positive control- background) * 100], where background is the fluorescence in absence of compound and the positive control is the fluorescence from membranes incubated with 1 ⁇ SIP (Avanti). The percent activities are plotted against compound concentration to determine IC 50 or EC 50 using XLFit (IDBS).
  • the hSlPi?5 GTPyS binding assay was carried out at room temperature in 96-well non-binding surface assay plates.
  • the reaction in each well contained 5 ⁇ g hSlP/?5 (hEdg8) membrane protein from CHO cells expressing hSlPR5, 30 ⁇ GDP, 0.1 nM [ 35 S]GTPyS, 0.25% fatty acid free BSA, and serially-diluted Compound of the Invention in 200 ⁇ , assay buffer (25 mM Tris-HCl PH 7.9, 100 mM NaCl, 3 mM MgC12, and 0.2 mM EGTA).
  • Table 3 gives EC 50 data (unless otherwise indicated) for the compounds in Table 1 and Table 2 and are in nM units.
  • Assay 2b is the TangoTM ⁇ -arrestin Recruitment Assay in U20S cells, as described in Biological Example 2.
  • Assay 3a is the hS IP 17? GTPyS Binding Assay as described in Biological Example 3.
  • Assay 4 is the hSlP5R GTPyS Binding Assay as described in Biological Example 4.
  • Assay 5 is the TangoTM ⁇ -arrestin Recruitment Assay in HEK293 cells, as described in Biological Example 5.
  • F means the compound has an EC 50 or relative EC 50 of less than or equal to 250 nM and G means the compound has an EC 50 or relative EC 5 o of greater than 250 nM.
  • A means the compound has an EC 50 or relative EC 50 of less than or equal to 10 nM.
  • B means the compound has an EC 50 or relative EC 50 greater than 10 nM but less than or equal to 50 nM.
  • C means the compound has an EC 50 or relative EC 50 greater than 50 nM but less than or equal to 250 nM.
  • D means the compound has an EC50 or relative EC50 greater than 250 nM but less than or equal to 2800 nM.
  • E means the compound has an EC 50 or relative EC 50 greater than 2800 nM but less than 10000 nM.
  • nt means the Compound was not tested and "na” means the compound was tested but had no measurable activity under the assay conditions employed.
  • 35 isopropoxy-5 -methy lpyridin-3 -yl)- 1 ,3 ,4-thiadiazol-2- nt C nt nt yl)phenoxy)propan- 1 -ol

Abstract

The invention is directed to compounds of the formula: wherein each of the variables are defined herein, as well as methods of making and using the compounds as agonists of S1P1 and/or S1P5 for instance treating an autoimmune disease.

Description

THIADIAZOLE MODULATORS OF SIP AND METHODS
OF MAKING AND USING
PRIORITY CLAIM
[0001] This application claims priority to United States Provisional Application Serial No. 62/188,257, filed July 2, 2015. The entire contents of the aforementioned application are ncorporated herein by reference.
BACKGROUND OF THE INVENTION
Statement Regarding Joint Research Agreement
[0002] One or more inventions contained in this application were developed under a joint research agreement as defined in the Cooperative Research and Technology Enhancement Act of 2001 between Exelixis, Inc. and Boehringer Ingelheim International,GmbH.
Field of the Invention
[0003] This invention relates to the field of agonists of Sphingosine 1 -Phosphate Type 1 Receptor (S1P1R or SlPl) and/or Type 5 Receptor (S1P5R or S1P5), and methods of their use.
Summary of the Related Art
[0004] Sphingosine 1 -phosphate (SIP) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Circulation of mature lymphocytes between blood and secondary lymphoid tissues plays an important role in the immune system. Agonism of S1P1R has been shown to lead to the sequestration of peripheral lymphocytes into secondary lymphoid tissue. Such sequestration of lymphocytes has been shown to result in immunosuppressive activity in animal models. Known SlPl receptor agonists, such as FTY720, have been shown to markedly decrease peripheral blood lymphocytes through the sequestration of lymphocytes into secondary lymphoid tissues. Potent agonists of the SlPl receptor are thought to induce long-term down-regulation of SlPl on lymphocytes, thereby inhibiting the migration of lymphocytes toward SIP. The consequential decrease in trafficking and infiltration of antigen-specific T cells provides a means of immunomodulating activity that can be useful in the treatment of various immune- related conditions such as graft versus host disease and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosis. Therefore, agonists of S1P1R are potentially useful immunosuppressants for the treatment of a variety of autoimmune conditions. [0005] The myelin sheath that surrounds neural cell axons is required to insulate neural axons and allow rapid movement of electrical impulses through the myelinated nerve fiber. Demyelination, or loss of the integrity of the myelin sheath is the hallmark of autoimmune neurodegenerative diseases, including multiple sclerosis. The myelin sheath in the central nervous system is produced by oligodendrocytes. Mature, myelin-producing oligodendrocytes express SIP receptor transcripts in relative abundance of S1P5>S1P3>S1P1, with undetectable levels of S1P4. Fingolimod (FTY720), a sphingosine- 1 -phosphate (SIP) analogue that has been used successfully in clinical trials as a systemic immunomodulatory therapy for multiple sclerosis, readily accesses the central nervous system. In vitro studies using either mature adult human oligodendrocytes or oligodendrocyte precursor cells demonstrate that fingolomid or SIP has significant effects on oligodendrocyte process elongation and retraction as well as on cell survival and apoptosis. These studies further demonstrated that the effects seen were, at least in part, due to S1P5. These results indicate that S 1 P5 may play an important role in the beneficial effect observed with fingolomid treatment of multiple sclerosis.
[0006] Sphingosine-1 -phosphate (SIP) has been shown to regulate the migration of osteoclast precursors, demonstrating a role in bone mineral homeostasis and suggesting a role in treating bone-destroying disorders such as rheumatoid arthritis and osteoporosis {Nature 2009, 458(7237), 524-528).
[0007] S1P3 has been associated with acute toxicity and bradycardia in rodents (Hale, et. al. Bioorganic & Med Chem Lett, 2004, 14(13), 3501-3505; J. Pharmacol. Exp. Ther. 2004, 309(2), 758-768; J. Med Chem 2005, 48(20), 6168-6173; J. Biol. Chem. 2004, 279(14), 13839-13848). Therefore agonists which are selective for S1P1 and/or S1P5, without being active for S1P3, are desirable.
SUMMARY OF THE INVENTION
[0008] The following only summarizes certain aspects of the invention and is not intended to be limiting in nature. These aspects and other aspects and embodiments are described more fully below. All references cited in this specification are hereby incorporated by reference in their entirety. In the event of a discrepancy between the express disclosure of this specification and the references incorporated by reference, the express disclosure of this specification shall control.
[0009] The invention provides compounds that are agonists of SI PI and/or S1P5 and that are useful in the treatment of graft versus host disease and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and systemic lupus erythematosis, in mammals. This invention also provides methods of making the compound, methods of using such compounds in the treatment of graft versus host disease and autoimmune diseases, especially in humans, and to pharmaceutical compositions containing such compounds.
[0010] Embodiment (1) of the first aspect of the invention provides a compound of Formula I:
Figure imgf000004_0001
I
or a stereoisomer or mixture of isomers thereof and optionally as a pharmaceutically
acceptable salt thereof, where
R is heterocycloalkyl optionally substituted with one or two groups which groups are
hydroxyl, tert-butyl-dimethylsilyloxy, or alkoxycarbonyl; or R is according to formula (a)
Figure imgf000004_0002
(a)
m is 1 or 2;
R7d is -OR1 or halo;
R1 is hydrogen or -P(0)(OR6)2;
R2 and R2a are independently hydrogen, cyano, halo, alkyl, haloalkyl, alkoxy, or haloalkoxy;
Ring A is phenyl, pyrimidinyl, or pyridinyl;
R3 is alkylamino, alkoxy, alkyl, or heterocycloalkyloxy;
R4 is halo, alkyl, alkoxy, alkoxycarbonayl, cyano, or hydroxy;
provided that when R3 is alkylamino, alkoxy, or alkyl, then R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano; and
when R3 is heterocycloalkyloxy, then R4 is hydroxy;
R5 is hydrogen or alkyl;
R5a is hydrogen, alkyl, formyl, alkylcarbonyl, or alkoxycarbonyl;
each R6 is independently hydrogen or alkyl;
each R7 and R7a is independently hydrogen or alkyl; R and R c are independently hydrogen or alkyl; and
o
R is hydrogen, alkyl, or hydroxyalkyl;
provided that the compound of formula I is neither one of the following compounds nor a pharmaceutically acceptable salt of one of the following compounds,
(2i?)-2-amino-3- { [5-chloro-4-(5 - { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy } propan- 1 -ol;
(2£)-2-amino-3 - { [4-(5- { 3 -bromo-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluoropheny 1] oxy } propan- 1 -ol ;
2-amino-3- { [5-chloro-4-(5- { 5-chloro-6-[( 1 -methylethyl)oxy]pyridin-3-yl} - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy } propan- 1 -ol ;
(2S)-2-amino-3 - { [5 -chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluoropheny l]oxy } propan- 1 -ol ;
(2S)-2-amino-3- { [4-(5- { 3-bromo-4-[( 1 -methylethyl)oxy]phenyl} - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluorophenyl]oxy } propan- 1 -ol ;
(2tf)-2-amino-3 - { [4-(5- { 3 -bromo-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluorophenyl]oxy}propyl dihydrogen phosphate;
(25)-2-amino-3 - { [5 -chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy}propyl dihydrogen phosphate;
2-amino-3 - { [5 -chloro-4-(5- { 6-chloro-5 - [( 1 -methy lethyl)oxy]pyridin-2-yl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy}propan- 1 -ol;
(2i?)-2-amino-3 - { [5 -chloro-4-(5 - { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy} propyl dihydrogen phosphate;
(2i?)-2-amino-3 - [(5-chloro-4- { 5 -[3 -chloro-4-(propyloxy)pheny 1]- 1 ,3 ,4-thiadiazol-2-yl } -2- fluorophenyl)oxy]propan- 1 -ol ;
(2i?)-2-amino-3- { [5 -chloro-4-(5 - { 5-chloro-6-[( 1 -methylethyl)oxy]pyridin-3 -yl } - 1 ,3 ,4- thiadiazol -2-y l)-2-fluoropheny 1] oxy } propan- 1 -ol ;
(2/?)-2-amino-3-{[5-chloro-4-(5-{6-chloro-5-[(l-methylethyl)oxy]pyridin-2-yl}-l,3,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}propan-l-ol;
(2i?)-2-amino-3-[(5-chloro-4-{5-[3-chloro-4-(ethyloxy)phenyl]-l,3,4-thiadiazol-2-yl}-2- fluorophenyl)oxy]propan- 1 -ol ; (2^)-2-amino-3- { [5-chloro-4-(5- {3 ,5-dichloro-4-[( 1 -methylethy l)oxy]phenyl } - 1 ,3 ,4-thiadiazot- 2-yl)-2-fluorophenyl]oxy}propan-l-ol;
(27?)-2-amino-3- { [4-(5 - { 3 -bromo-5- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluoropheny 1] oxy } propan- 1 -ol ;
(25)-2-amino-3-{[4-(5-{3-bromo-4-[(l-methylethyl)oxy]phenyl}-l,3,4-thiadiazol-2-yl)-5- chloro-2-fluorophenyl]oxy} propyl dihydrogen phosphate;
(2S)-2-amino-3- { [5-chloro-4-(5- {5-chloro-6-[( 1 -methylethyl)oxy]pyridin-3-yl } - 1 ,3 ,4- thiadiazol-2-yl)-2-fluorophenyl]oxy } propan- 1 -ol ;
(2S)-2-amino-3 - { [5 -chloro-4-(5 - { 6-chloro-5-[( 1 -methylethyl)oxy]pyridin-2-yl } - 1 ,3 ,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}propan- 1 -ol;
(2^)-2-amino-3 - { [3 -chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)phenyl]oxy}propan-l-ol;
5-[5-(4-{[(2 ?)-2-amino-3-hydroxypropyl]oxy}-2-chloro-5-fluorophenyl)-l ,3,4-thiadiazol-2-yl]- 2-[(l-methylethyl)oxy]benzonitrile;
(2i?)-2-amino-3- { [2,6-dichloro-4-(5- { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-y l)phenyl] oxy } propan- 1 -ol ;
(2i?)-2-amino-3-{[2-chloro-4-(5-{3-chloro-4-[(l-methylethyl)oxy]phenyl}-l ,3,4-thiadiazol-2- yl)-6-fluoropheny l]oxy } propan- 1 -ol ;
(2i?)-2-amino-3-{[5-chloro-2-fluoro-4-(5-{3-fluoro-4-[(l -methylethyl)oxy]phenyl}-l ,3,4- thiadiazol-2-yl)phenyl]oxy } propan- 1 -ol ;
(2i?)-2-amino-3 - { [5 -chloro-4-(5 - { 3 -chloro-4-[( 1 -methylethyl)amino]phenyl } - 1 ,3 ,4-thiadiazol- 2-y l)-2-fluoropheny 1] oxy } propan- 1 -ol ;
(2S)-2-amino-3-{[5-chloro-4-(5-{3-chloro-4-[(l-methylethyl)amino]phenyl}-l,3,4-thiadiazol-2- yl)-2-fluorophenyl]oxy}propan-l-ol;
(2i?,35)-3-amino-4-{[5-chloro-4-(5-{5-chloro-6-[(l-methylethyl)amino]pyridin-3-yl}-l,3,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}butan-2-ol;
( 1 i?,2S)-2-amino-3 - { [5 -chloro-4-(5- { 3 -cyano-4- [( 1 -methylethyl)oxy]pheny 1 } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
( 1 i?,2S)-2-amino-3 - { [5 -chloro-4-(5- { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate; ( 1 S,2S)-2-amino-3 - { [5-chloro-4-(5- { 3 -cyano-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
5-[5-(4-{[(2 ?,3S)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2- yl]-2-[( 1 -methylethyl)oxy]benzonitrile;
(2R,3S)-3 -amino-4- { [5 -chloro-2-fluoro-4-(5- { 3 -methyl-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4- thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(25,35)-3 -amino-4- { [5-chloro-2-fluoro-4-(5- { 3 -methyl-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4- thiadiazol-2-yl)phenyl]oxy } butan-2-ol;
(2R,3S}-3 -amino-4- { [5 -chloro-2-fluoro-4-(5- { 2-methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl } - l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2S,3S)-3 -amino-4- { [5-chloro-2-fluoro-4-(5- { 2-methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol;
( lS,2 ?)-2-amino-3- { [5-chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
(2R,3S)-2 -amino-4- { [5-chloro-2-fluoro-4-(5- { 5-methyl-6-[( 1 -methylethyl)amino]pyridin-3-yl } - l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{5-methyl-6-[(l -methylethyl)amino]pyridin-3-yl}- 1 ,3,4-thiadiazol-2-yl)phenyl]oxy }butan-2-ol;
(2 ?,3i?)-3-amino-4- { [5-chloro-2-fluoro-4-(5- { 5-methyl-6-[( 1 -methylethyl)amino]pyridin-3-yl} - 1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol;
(2R,3R)-3 -amino-4- { [5-chloro-2-fluoro-4-(5- { 6-[( 1 -methylethyl)amino]pyridin-3 -yl } - 1 ,3 ,4- thiadiazol-2-yl)phenyl]oxy } butan-2-ol ;
( 1 S,2S)-2-amino-3- { [5 -chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
(l/?,2/?)-2-amino-3-{[5-chloro-4-(5-{3-chloro-4-[(l -methylethyl)oxy]phenyl}-l,3,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
(2S,3i?)-3-amino-4- { [5-chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}butan-2-ol;
(2S,35)-3 -amino-4- { [5-chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy } butan-2-ol ; (2R,3S)-3 -amino-4- { [5 -chloro-4-(5 - { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}butan-2-ol;
(2i?,3i?)-3-amino-4-{ [5-chloro-4-(5-{3-chloro-4-[(l -methylethyl)oxy]phenyl}-l ,3,4-thiadiazol- 2-y l)-2-fluoropheny 1] oxy } butan-2-ol ;
5-[5-(4-{[(27?)-2-amino-3-hydroxypropyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2-yl]- 2-(ethyloxy)benzonitrile;
(25)-2-amino-3 - { [5-chloro-4-(5- { 3 -cyano-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy} propyl dihydrogen phosphate;
(2i?)-2-amino-3- { [5-chloro-4-(5- { 5-chloro-6-[( 1 -methylethyl)amino]pyridin-3-yl} - 1 ,3 ,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}propan- 1 -ol;
5-[5-(4-{[(2S,3i?)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2- y 1] -2- [( 1 -methy lethy l)oxy]benzonitrile ;
5-[5-(4-{[(25',3S)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l ,3,4-thiadiazol-2- yl]-2-[(l-methylethyl)oxy]benzonitrile;
5-[5-(4-{[(2i?,3i?)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l ,3,4-thiadiazol-2- yl]-2-[(l-methylethyl)oxy]benzonitrile;
( 1 i?,25)-2-amino-3- { [5 -chloro-2-fluoro-4-(5- { 2-methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3,4-thiadiazol-2-yl)phenyl]oxy}-l -methylpropyl dihydrogen phosphate;
( 1 S,25)-2-amino-3- { [5-chloro-2-fluoro-4-(5- { 2-methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } - l,3,4-thiadiazol-2-yl)phenyl]oxy}-l -methylpropyl dihydrogen phosphate;
(2S,3i?)-3-amino-4- { [5-chloro-2-fluoro-4-(5 - { 5 -methyl-6- [( 1 -methylethyl)amino]pyridin-3 -yl } - l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2R,3R)-3 -amino-4- { [5 -chloro-2-fluoro-4-(5- {2-methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } - l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2S,3i?)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyridin-4-yl}- 1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol ;
(li?,2S)-2-amino-3-{[5-chloro-2-fluoro-4-(5-{3-methyl-4-[(l-methylethyl)oxy]phenyl}-l,3,4- thiadiazol-2-yl)phenyl]oxy}-l -methylpropyl dihydrogen phosphate;
( 1 i?,2i?)-2-amino-3 - { [5 -chloro-2-fluoro-4-(5 - { 2-methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3,4-thiadiazol-2-yl)phenyl]oxy}-l -methylpropyl dihydrogen phosphate; ( 1S,27?)-2-amino-3 - { [5 -chloro-2-fluoro-4-(5- { 2-methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3,4-thiadiazol-2-yl)phenyl]oxy }- 1 -methylpropyl dihydrogen phosphate;
(2i?,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{6-methyl-5-[(l-methylethyl)amino]pyridin-2-yl}- l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
( 1S,25)-2-amino-3 - { [5 -chloro-2-fluoro-4-(5- { 3 -methyl-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4- thiadiazol-2-yl)phenyl]oxy}-l -methylpropyl dihydrogen phosphate;
(2S,3S)-3-amino-4-(5-chloro-2-fluoro-4-{5-[6-methyl-5-(propan-2-ylamino)pyridin-2-yl]-l,3,4- thiadiazol-2-yl } phenoxy)butan-2-ol; and
(2S,3S)-3-amino-4-(5-chloro-2-fluoro-4-{5-[6-methyl-4-(propan-2-ylamino)pyridin-2-yl]-l,3,4- thiadiazol-2-yl } phenoxy)butan-2-ol .
[0011] All genera described herein expressly exclude all specific compounds disclosed in international application PCT/US 10/28702.
[0012] In a second aspect, the invention is directed to a pharmaceutical composition which comprises 1) a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt thereof and 2) a pharmaceutically acceptable carrier, excipient, or diluent.
[0013] In a third aspect, the invention provides a method for treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
[0014] In a fourth aspect, the Invention is directed to a method of making a Compound of the Invention which method comprises:
(a) reacting an intermediate of formula 116 or a salt thereof:
Figure imgf000009_0001
116 where PG is a nitrogen-protecting group, and R, R2, R2a, and R7c are as defined in the
Summary of the Invention for a Compound of Formula I; with an intermediate of formula 117:
Figure imgf000010_0001
117
where Ring A, R3, and R4 are as defined in the Summary of the Invention for a Compound of Formula I, followed by treatment with Lawesson's reagent, to yield an intermediate of formula 118:
Figure imgf000010_0002
118
and followed by deprotection and ring opening to yield a Compound of the Invention of Formula 121:
Figure imgf000010_0003
121
and optionally separating individual isomers; and optionally modifying any of the R2, R2a, R3, R4, and R7c groups; and optionally forming a pharmaceutically acceptable salt, hydrate, solvate or combination thereof; or
(b) reacting an intermediate of formula 119, or a salt thereof:
Figure imgf000010_0004
119
where PG is a nitrogen-protecting group, and R2, R2a, and R7c are as defined in the Summary of the Invention for a Compound of Formula I; with an intermediate of formula 10:
Figure imgf000010_0005
10 where n, Ring A, R3, and R4 are as defined in the Summary of the Invention for a Compound of Formula I, to yield an intermediate of formula 120:
Figure imgf000011_0001
120
followed by treatment with Lawesson's reagent, deprotection, and ring opening, to yield a Compound of formula 121
Figure imgf000011_0002
and optionally separating individual isomers; and optionally modifying any of the R2, R2a, R3, R4, and R7c groups; and optionally forming a pharmaceutically acceptable salt, hydrate, solvate or combination thereof.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Definitions
[0015] The following abbreviations and terms have the indicated meanings throughout:
Figure imgf000011_0003
Abbreviation Meanin
Figure imgf000012_0001
Abbreviation Meanin
Figure imgf000013_0001
[0016] The symbol "-" means a single bond, "=" means a double bond, "≡" means a triple bond, " " means a single or double bond. The symbol 'ΊΛΛΛ/" refers to a group on a double-bond as occupying either position on the terminus of a double bond to which the symbol is attached; that is, the geometry, E- or Z-, of the double bond is ambiguous. When a group is depicted removed from its parent formula, the " '^" symbol will be used at the end of the bond which was theoretically cleaved in order to separate the group from its parent structural formula. [0017] When chemical structures are depicted or described, unless explicitly stated otherwise, all carbons are assumed to have hydrogen substitution to conform to a valence of four. For example, in the structure on the left-hand side of the schematic below there are nine hydrogens implied. The nine hydrogens are depicted in the right-hand structure. Sometimes a particular atom in a structure is described in textual formula as having a hydrogen or hydrogens as substitution (expressly defined hydrogen), for example, -CH2CH2-. It is understood by one of ordinary skill in the art that the aforementioned descriptive techniques are common in the chemical arts to provide brevity and simplicity to description of otherwise complex structures.
Figure imgf000014_0001
[0018] If a group "R" is depicted as "floating" on a ring system, as for example in the formula:
Figure imgf000014_0002
then, unless otherwise defined, a substituent "R" may reside on any atom of the ring system, assuming replacement of a depicted, implied, or expressly defined hydrogen from one of the ring atoms, so long as a stable structure is formed.
[0019] If a group "R" is depicted as floating on a fused ring system, as for example in the formulae:
Figure imgf000014_0003
then, unless otherwise defined, a substituent "R" may reside on any atom of the fused ring system, assuming replacement of a depicted hydrogen (for example the -NH- in the formula above), implied hydrogen (for example as in the formula above, where the hydrogens are not shown but understood to be present), or expressly defined hydrogen (for example where in the formula above, "Z" equals =CH-) from one of the ring atoms, so long as a stable structure is formed. In the example depicted, the "R" group may reside on either the 5-membered or the 6-membered ring of the fused ring system.
[0020] When a group "R" is depicted as existing on a ring system containing saturated carbons, as for example in the formula:
Figure imgf000015_0001
where, in this example, "y" can be more than one, assuming each replaces a currently depicted, implied, or expressly defined hydrogen on the ring; then, unless otherwise defined, where the resulting structure is stable, two "R's" may reside on the same carbon. A simple example is when R is a methyl group; there can exist a geminal dimethyl on a carbon of the depicted ring (an "annular" carbon). In another example, two R's on the same carbon, including that carbon, may form a ring, thus creating a spirocyclic ring (a "spirocyclyl" group) structure with the depicted ring as for example in the formula:
Figure imgf000015_0002
[0021] Although all moieties are generally referred to as their monovalent form (e.g., alkyl, aryl), those skilled in the art will understand from the context and standard valence rules when di-, tri-, etc., valent radicals are intended. So, for example, alkyl can refer to a monovalent alkyl radical or a divalent radical (i.e., alkylene).
[0022] "Administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., surgery, radiation, and chemotherapy, etc.), "administration" and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.
[0023] "Alkyl" means a linear saturated hydrocarbon radical of one to six carbon atoms or a branched saturated hydrocarbon radical of three to 6 carbon atoms, e.g., methyl, ethyl, propyl, 2-propyl, butyl (including all isomeric forms), or pentyl (including all isomeric forms), and the like.
[0024] "Alkenyl" means a linear hydrocarbon radical of two to six carbon atoms or a branched hydrocarbon radical of three to 6 carbon atoms which radical contains at least one double bond, e.g., ethenyl, propenyl, l-but-3-enyl, and l-pent-3-enyl, and the like. [0025] "Alkoxy" means an -OR group where R is alkyl group as defined herein. Examples include methoxy, ethoxy, propoxy, isopropoxy, and the like.
[0026] "Alkoxyalkyl" means an alkyl group, as defined herein, substituted with at least one, specifically one, two, or three, alkoxy groups as defined herein. Representative examples include methoxymethyl and the like.
[0027] "Alkoxyalkyloxy" means an -OR group where R is alkoxyalkyl as defined herein.
[0028] "Alkoxycarbonyl" means a -C(0)R group where R is alkoxy, as defined herein.
[0029] "Alkoxycarbonylamino" means an -NHR group where R is alkoxycarbonyl as defined herien.
[0030] "Alkylamino" means an -NHR group where R is alkyl, as defined herein.
[0031] "Alkylaminoalkyl" means an alkyl group substituted with one or two alkylamino groups, as defined herein.
[0032] "Alkylcarbonyl" means a -C(0)R group where R is alkyl, as defined herein.
[0033] "Alkylcarbonylamino" means an -NHR group where R is alkylcarbonyl, as defined herein.
[0034] "Alkylsulfonyl" means an -S(0)2R group where R is alkyl, as defined herein, e.g. methylsulfonyl, isopropylsulfonyl .
[0035] "Alkynyl" means a linear hydrocarbon radical of two to six carbon atoms or a branched hydrocarbon radical of three to 6 carbon atoms which radical contains at least one triple bond, e.g., ethynyl, propynyl, butynyl, pentyn-2-yl and the like.
[0036] "Amino" means -NH2.
[0037] "Aminocarbonyl" means a -C(0)NH2 group.
[0038] "Aryl" means a six- to fourteen-membered, mono- or bi-carbocyclic ring, wherein the monocyclic ring is aromatic and at least one of the rings in the bicyclic ring is aromatic. Unless stated otherwise, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. Representative examples include phenyl, naphthyl, and indanyl, and the like.
[0039] "Arylalkyl" means an alkyl radical, as defined herein, substituted with one or two aryl groups, as defined herein, e.g., benzyl and phenethyl, and the like.
[0040] "Carboxy" means a -C(0)OH group.
[0041] "Cycloalkyl" means a monocyclic or fused bicyclic, saturated or partially unsaturated (but not aromatic), hydrocarbon radical of three to ten carbon ring atoms. Fused bicyclic hydrocarbon radical includes bridged ring systems. Unless stated otherwise, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. One or two ring carbon atoms may be replaced by a -C(O)-, -C(S)-, or -C(=NH)- group. More specifically, the term cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl, or cyclohex-3-enyl, and the like.
[0042] "Cycloalkyloxy" means an -OR group where R is cycloalkyl as defined herein.
[0043] "Dialkylamino" means a -NRR' radical where R and R' are alkyl as defined herein, or an N-oxide derivative, or a protected derivative thereof, e.g., dimethylamino, diethylamino, N,N-methylpropylamino or N,7V-methylethylamino, and the like.
[0044] "Formyl" means an -C(0)H group.
[0045] "Fused ring" means a polycyclic ring that contains bridged or fused rings; that is, where two rings have more than one shared atom in their ring structures. In this application, fused ring systems are not necessarily all aromatic ring systems. Typically, but not necessarily, fused rings share a vicinal set of atoms, for example naphthalene or 1,2,3,4- tetrahydro-naphthalene. A spiro ring system is not a fused ring system by this definition, but fused ring systems of the invention may themselves have spiro rings attached thereto via a single ring atom of the fused ring system. In some examples, as appreciated by one of ordinary skill in the art, two adjacent groups on an aromatic system may be fused together to form a ring structure. The fused ring structure may contain heteroatoms and may be optionally substituted with one or more groups. It should additionally be noted that saturated carbons of such fused groups (i.e. saturated ring structures) can contain two substitution groups.
[0046] "Halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.
[0047] "Haloalkoxy" means an -OR' group where R' is haloalkyl as defined herein, e.g., trifluoromethoxy or 2,2,2-trifluoroethoxy, and the like.
[0048] "Haloalkyl" mean an alkyl group substituted with one or more halogens, specifically one to five halo atoms, e.g., trifiuoromethyl, 2-chloroethyl, and 2,2-difluoroethyl, and the like.
[0049] "Heteroaryl" means a monocyclic, fused bicyclic, or fused tricyclic, radical of 5 to 14 ring atoms containing one or more, specifically one, two, three, or four ring heteroatoms independently selected from -0-, -S(0)„- (n is 0, 1, or 2), -N-, -N(RX)-, and the remaining ring atoms being carbon, wherein the ring comprising a monocyclic radical is aromatic and wherein at least one of the fused rings comprising a bicyclic or tricyclic radical is aromatic. One or two ring carbon atoms of any nonaromatic rings comprising a bicyclic or tricyclic radical may be replaced by a -C(O)-, -C(S)-, or -C(=NH)- group. Rx is hydrogen, alkyl, hydroxy, alkoxy, acyl, or alkyl sulfonyl. Fused bicyclic radical includes bridged ring systems. Unless stated otherwise, the valency may be located on any atom of any ring of the heteroaryl group, valency rules permitting. When the point of valency is located on the nitrogen, Rx is absent. More specifically, the term heteroaryl includes, but is not limited to, 1 ,2,4-triazolyl, 1,3,5-triazolyl, phthalimidyl, pyridinyl, pyrrolyl, imidazolyl, thienyl, furanyl, indolyl, 2,3-dihydro-lH-indolyl (including, for example, 2,3-dihydro-lH-indol-2-yl or 2,3-dihydro- lH-indol-5-yl, and the like), isoindolyl, indolinyl, isoindolinyl, benzimidazolyl, benzodioxol- 4-yl, benzofuranyl, cinnolinyl, indolizinyl, naphthyridin-3-yl, phthalazin-3-yl, phthalazin-4- yl, pteridinyl, purinyl, quinazolinyl, quinoxalinyl, tetrazoyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isooxazolyl, oxadiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl (including, for example, tetrahydroisoquinolin-4-yl or tetrahydroisoquinolin-6-yl, and the like), pyrrolo[3,2-c]pyridinyl (including, for example, pyrrolo[3,2-c]pyridin-2-yl or pyrrolo[3,2-c]pyridin-7-yl, and the like), benzopyranyl, thiazolyl, isothiazolyl, thiadiazolyl, benzothiazolyl, benzothienyl, and the derivatives thereof, or N-oxide or a protected derivative thereof.
[0050] "Heteroatom" refers to O, S, N, and P.
[0051] "Heterocycloalkyl" means a saturated or partially unsaturated (but not aromatic) monocyclic group of 3 to 8 ring atoms or a saturated or partially unsaturated (but not aromatic) fused bicyclic group of 5 to 12 ring atoms in which one or more, specifically one, two, three, or four ring heteroatoms independently selected from O, S(0)n (n is 0, 1, or 2), N, N(Ry) (where Ry is hydrogen, alkyl, hydroxy, alkoxy, acyl, or alkylsulfonyl), the remaining ring atoms being carbon. One or two ring carbon atoms may be replaced by a -C(O)-, -C(S)-, or -C(=NH)- group. Fused bicyclic radical includes bridged ring systems. Unless otherwise stated, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. When the point of valency is located on a nitrogen atom, Ry is absent. More specifically the term heterocycloalkyl includes, but is not limited to, azetidinyl, pyrrolidinyl, 2-oxopyrrolidinyl, 2,5-dihydro-lH-pyrrolyl, piperidinyl, 4-piperidonyl, morpholinyl, piperazinyl, 2-oxopiperazinyl, tetrahydropyranyl, 2-oxopiperidinyl, thiomorpholinyl, thiamorpholinyl, perhydroazepinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, dihydropyridinyl, tetrahydropyridinyl, oxazolinyl, oxazolidinyl, isoxazolidinyl, thiazolinyl, thiazolidinyl, quinuclidinyl, isothiazolidinyl, octahydroindolyl, octahydroisoindolyl, decahydroisoquinolyl, tetrahydrofuryl, and tetrahydropyranyl, and the derivatives thereof and N-oxide or a protected derivative thereof.
[0052] "Heterocycloalkyloxy" means an -OR group where R is hetero cycloalkyl as defined herein. [0053] "Hydroxyalkyl" means an alkyl group substituted with at least one, in another example with one, two, or three, hydroxy groups.
[0054] "Spirocyclyl" or "spirocyclic ring" refers to a ring originating from a particular annular carbon of another ring. For example, as depicted below, a ring atom of a saturated bridged ring system (rings B and B'), but not a bridgehead atom, can be a shared atom between the saturated bridged ring system and a spirocyclyl (ring A) attached thereto. A spirocyclyl can be carbocyclic or hete
Figure imgf000019_0001
[0055] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. One of ordinary skill in the art would understand that with respect to any molecule described as containing one or more optional substituents, only sterically practical and/or synthetically feasible compounds are meant to be included. "Optionally substituted" refers to all subsequent modifiers in a term. So, for example, in the term "optionally substituted arylCi-8 alkyl," optional substitution may occur on both the "Ci-8 alkyl" portion and the "aryl" portion of the molecule may or may not be substituted.
[0056] "Metabolite" refers to the break-down or end product of a compound or its salt produced by metabolism or biotransformation in the animal or human body; for example, biotransformation to a more polar molecule such as by oxidation, reduction, or hydrolysis, or to a conjugate (see Goodman and Gilman, "The Pharmacological Basis of Therapeutics" 8.sup.th Ed., Pergamon Press, Gilman et al. (eds), 1990 for a discussion of biotransformation). As used herein, the metabolite of a compound of the invention or its salt may be the biologically active form of the compound in the body. In one example, a prodrug may be used such that the biologically active form, a metabolite, is released in vivo. In another example, a biologically active metabolite is discovered serendipitously, that is, no prodrug design per se was undertaken. An assay for activity of a metabolite of a compound of the present invention is known to one of skill in the art in light of the present disclosure.
[0057] "Patient" for the purposes of the present invention includes humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In a specific embodiment the patient is a mammal, and in a more specific embodiment the patient is human.
[0058] A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference or S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66: 1-19 both of which are incorporated herein by reference.
[0059] Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid, and salicylic acid and the like.
[0060] Examples of a pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Specific salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine, N-methylglucamine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine."Platin(s)," and "platin-containing agent(s)" include, for example, cisplatin, carboplatin, and oxaliplatin.
[0061] "Prodrug" refers to compounds that are transformed (typically rapidly) in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. Common examples include, but are not limited to, ester and amide forms of a compound having an active form bearing a carboxylic acid moiety. Examples of pharmaceutically acceptable esters of the compounds of this invention include, but are not limited to, alkyl esters (for example with between about one and about six carbons) the alkyl group is a straight or branched chain. Acceptable esters also include cycloalkyl esters and arylalkyl esters such as, but not limited to benzyl. Examples of pharmaceutically acceptable amides of the compounds of this invention include, but are not limited to, primary amides, and secondary and tertiary alkyl amides (for example with between about one and about six carbons). Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.
[0062] "Stereoisomer" means any of two or more isomers containing the same atoms bonded to each other in an identical manner but differing from each other in the spatial arrangement of the atoms or groups of atoms. "Stereoisomer" includes, for example, an enantiomer, a geometric isomer, a diastereomer, a rotamer, cis-isomer, trans-isomer, and conformational isomer. The names and illustration used in this application to describe compounds of the invention, unless indicated otherwise, are meant to encompass all possible stereoisomers and any mixture, racemic or otherwise, thereof.
[0063] "Therapeutically effective amount" is an amount of a compound of the invention, that when administered to a patient, ameliorates a symptom of the disease. The amount of a compound of the invention which constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the age of the patient to be treated, and the like. The therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their knowledge and to this disclosure.
[0064] "Treating" or "treatment" of a disease, disorder, or syndrome, as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a human, i.e. causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e. , arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by one of ordinary skill in the art.
[0065] "Yield" for each of the reactions described herein is expressed as a percentage of the theoretical yield.
Embodiments of the Invention
[0066] The following paragraphs present a number of embodiments of compounds of the invention. In each instance the embodiment includes the recited compounds, as well as a single stereoisomer or mixture of stereoisomers thereof, as well as a pharmaceutically acceptable salt thereof.
[0067] The invention further comprises subgenera of embodiment (1) in which the substituents are selected as any and all combinations of structural formula (I), R, R1, R2, R2a, R3, R4, R5, R5a, R7, R7a, R7b, R7c, and R8 as defined herein, including without limitation, the following:
[0068] Structural Formula I is one of formulae (I-a) - (l-l). (II-a) - (II-c^. (III-a) - (III- g). qV-a - (IV-e), (V-a) - (V-e). and (Vl-a) - (Vl-g):
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000025_0002
9] R1 is selected from one of the following groups (la) - (lg):
(la) hydrogen,
(lb) -P(0)(OR6)2.
(lc) -P(0)(OR6)2, wherein each R6 is independently alkyl.
(Id) -P(0)(OR6)2, wherein each R6 is independently hydrogen or t-butyl.
(le) -P(0)(OR6)2, wherein each R6 is independently t-butyl.
(If) hydrogen or -P(0)(OH)2. (lg) -P(0)(OH)2.
[0070] R2 and R2a are selected from one of the following groups (2a) - (2m):
(2a) R2 and R2a are independently cyano, halo, or alkyl.
(2b) R2 and R2a are independently cyano, chloro, fluoro, or methyl.
(2c) R2 and R2a are independently halo or alkyl.
(2d) R2 and R2a are each independently halo.
(2e) R2 and R2a are each chloro.
(2f) R2 is chloro or fluoro and R2a is chloro or fluoro.
(2g) R2 is chloro and R2a is fluoro.
(2h) R2 and R2a are each independently alkyl.
(2i) R2 and R2a are each methyl.
(2j) R2 is alkyl and R2a is halo.
(2k) R2 is methyl and R2a is halo.
(21) R2 is methyl and R2a is chloro or fluoro.
(2m) R2 is methyl and R2a is fluoro.
(2n) R2 is methyl and R2a is chloro.
[0071] R3 and R4 together are selected from one of the following groups (3a) - (3r):
(3a) R3 is alkylamino, alkoxy, or alkyl and R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
(3b) R3 is alkylamino and R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
(3c) R3 is alkylamino and R4 is alkyl, alkoxycarbonyl, or cyano.
(3d) R3 is isopropylamino and R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano. (3e) R3 is isopropylamino and R4 is alkyl, alkoxycarbonyl, or cyano.
(3f) R3 is alkoxy and R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
(3g) R3 is ethoxy or isopropoxy and R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or
cyano.
(3h) R3 is alkoxy and R4 is alkoxy.
(3i) R3 is ethoxy and R4 is ethoxy.
(3j) R3 is alkyl and R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
(3k) R3 is isobutyl and R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano.
(31) R3 is alkyl and R4 is alkyl.
(3m) R3 is isobutyl and R4 is methyl.
(3n) R3 is alkylamino, alkoxy, or alkyl and R4 is alkyl, alkoxy, cyano, or halo. 00) R is alkylamino, alkoxy, or alkyl and R is methyl, methoxy, ethoxy,
ethoxy carbonyl, cyano, or chloro.
(3p) R3 is ethoxy, isopropoxy, isopropylamino, or isobutyl and R4 is methyl, methoxy, ethoxy, ethoxycarbonyl, cyano, or chloro.
(3q) R3 is heterocycloalkyloxy and R4 is hydroxy.
(3r) R3 is heterocycloalkyloxy and R4 is hydroxy or R3 and R4 are any one of groups (3a) - (3p).
(3s) R3 is alkylamino and R4 is alkyl, alkoxycarbonyl, or cyano, or R3 is
heterocycloalkyloxy and R is hydroxy.
[0072] R5 and R5a are selected from one of the following groups (5a) - (5d):
(5a) R5 is hydrogen and R5a is hydrogen, alkyl, alkylcarbonyl, or alkoxycarbonyl.
(5b) R5 is hydrogen and R5a is alkylcarbonyl or alkoxycarbonyl.
(5c) R5 is hydrogen and R5a is hydrogen or alkyl.
(5d) R5 is hydrogen; R5a is hydrogen.
[0073] R is selected from one of the following groups (6a) - (6ii):
Figure imgf000027_0001
(6i) any of groups (6a) - (6h), wherein each R7 and R7a is hydrogen.
(6j) any of groups (6a) - (6i), wherein R7b is hydrogen.
(6k) any of groups (6a) - (6j), wherein R8 is hydrogen or alkyl.
(61) any of groups (6a) - (6k), wherein R is hydrogen.
(6m) any of groups (6a) - (61), wherein R c is hydrogen or alkyl (6n) any of groups (6a) - (61), wherein R7c is hydrogen or methyl, (60) any of groups (6a) - (61), wherein R7c is methyl,
(6p) any of roups (6a) - (61), wherein R7c is hydrogen.
Figure imgf000028_0001
(6cc) any of groups (6q) - (6bb), wherein each R7 and R7a is hydrogen.
(6dd) any of groups (6q) - (6cc), wherein R7c is hydrogen or alkyl.
(6ee) any of groups (6q) - (6cc), wherein R7c is hydrogen or methyl.
(6ff) any of groups (6q) - (6cc), wherein R7c is methyl.
(6gg) any of groups (6q) - (6cc), wherein R7c is hydrogen. R is heterocycloalkyl optionally substituted with one or two groups which groups are hydrox l, tert-butyl-dimethylsilyloxy or alkoxycarbonyl; or R is according to
formula
Figure imgf000029_0001
, wherein R7, R7A, and R7C are hydrogen; R7B is hydrogen or alkyl; and R is hydrogen.
(6ii) R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl-dimethylsilyloxy, or alkoxycarbonyl; or R is according to
Figure imgf000029_0002
the formula , wherein R , R a, and R 0 are hydrogen; R is hydrogen or alkyl; and R is hydrogen..
(6jj) R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl-dimethylsilyloxy, or alkoxycarbonyl.
(6kk) R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl-dimethylsilyloxy, or alkoxycarbonyl or R is any one of groups (6a) - (6gg)
[0074] Particular embodiments of this aspect of the invention include compounds of any one of the formulae (I), (I-a) - (1-1), (Il-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), each as defined in each of the following rows, wherein each entry is a group number as defined above (e.g., (la) refers to R1 is hydrogen), and a dash "-" indicates that the variable is as defined for formula (I) or defined according to any one of the applicable variable definitions (la)-(6kk) [e.g., when R1 is a dash, it can be either as defined for Formula (I) or any one of definitions (la)-(lg)]:
Figure imgf000029_0003
Figure imgf000029_0004
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
[0075] Another aspect of the invention provides a pharmaceutical composition which comprises a compound of any one of Formulae (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier, excipient, or diluent.
[0076] Another aspect of the invention is a method of treating disease, disorder, or syndrome where the disease is associated with uncontrolled, abnormal, and/or unwanted cellular activities effected directly or indirectly by S1P1 and/or S1P5 which method comprises administering to a human in need thereof a therapeutically effective amount of a compound of Formula (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or selected from any of the above embodiments, or a pharmaceutically acceptable salt or pharmaceutical composition thereof.
[0077] Another aspect of the invention is directed to a method of treating an autoimmune disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound of Formula (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or selected from any of the above embodiments, optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof. In another embodiment the autoimmune disease is multiple sclerosis. In another embodiment, the disease is psoriasis. In another embodiment, the disease is inflammatory bowel disease. In another embodiment the autoimmune disease is graft-versus-host disease. In another embodiment, the disease is inflammation caused by an autoimmune disease.
[0078] Another aspect of the invention is directed to composition for treating an autoimmune disease, disorder, or syndrome which comprises a therapeutically effective amount of a compound of Formula (I), (I-a) - (1-1), (II-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or selected from any of the above embodiments, optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof. In an embodiment the autoimmune disease is multiple sclerosis. In another embodiment, the disease is psoriasis. In another embodiment, the disease is inflammatory bowel disease. In another embodiment the autoimmune disease is graft-versus-host disease. In another embodiment, the disease is inflammation caused by an autoimmune disease.
[0079] In another aspect, the invention is directed to the use of a compount of Formula (I), (I- a) - (1-1), (Il-a) - (II-c), (Ill-a) - (Ill-g), (IV-a) - (IV-e), (V-a) - (V-e), and (Vl-a) - (Vl-g), or a compound selected from Table 1 or Table 2 (below) or selected from any of the above embodiments, optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof for the preparation of a medicament for treating an autoimmune disease, disorder, or syndrome. In an embodiment the autoimmune disease is multiple sclerosis. In another embodiment, the disease is psoriasis. In another embodiment, the disease is inflammatory bowel disease. In another embodiment the autoimmune disease is graft-versus-host disease. In another embodiment, the disease is inflammation caused by an autoimmune disease.
Representative Compounds
[0080] Representative compounds of Formula I are depicted below. The examples are merely illustrative and do not limit the scope of the invention in any way. Compounds of the invention are named according to systematic application of the nomenclature rules agreed upon by the International Union of Pure and Applied Chemistry (IUPAC), International Union of Biochemistry and Molecular Biology (IUBMB), and the Chemical Abstracts Service (CAS). Names were generated using ACD/Labs naming software or ChemDraw Ultra 10.0.
Figure imgf000038_0001
Figure imgf000039_0001
y-NH F l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol
Figure imgf000040_0001
Figure imgf000041_0001
phosphate
Figure imgf000041_0002
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
General Administration
[0081] In one aspect, the invention provides pharmaceutical compositions comprising an inhibitor of S1P1 and/or S1P5 according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent. In certain other specific embodiments, administration is by the oral route. Administration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration or agents for serving similar utilities. Thus, administration can be, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, specifically in unit dosage forms suitable for simple administration of precise dosages.
[0082] The compositions will include a conventional pharmaceutical carrier or excipient and a compound of the invention as the/an active agent, and, in addition, may include carriers and adjuvants, etc.
[0083] Adjuvants include preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. ]
[0084] If desired, a pharmaceutical composition of the invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylalted hydroxytoluene, etc.
[0085] The choice of formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills or capsules) and the bioavailability of the drug substance. Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1,000 nm in which the active material is supported on a crosslinked matrix of macromolecules. U.S. Pat. No. 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability.
[0086] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
[0087] One specific route of administration is oral, using a convenient daily dosage regimen that can be adjusted according to the degree of severity of the disease-state to be treated.
[0088] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, cellulose derivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, magnesium stearate and the like (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.
[0089] Solid dosage forms as described above can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain pacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
[0090] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.
[0091] Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
[0092] Compositions for rectal administrations are, for example, suppositories that can be prepared by mixing the compounds of the present invention with for example suitable non- irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
[0093] Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.
[0094] Compressed gases may be used to disperse a compound of this invention in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc.
[0095] Generally, depending on the intended mode of administration, the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a suitable pharmaceutical excipient. In one example, the composition will be between about 5% and about 75% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.
[0096] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease-state in accordance with the teachings of this invention.
[0097] The compounds of the invention, or their pharmaceutically acceptable salts or solvates, are administered in a therapeutically effective amount which will vary depending upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease-states, and the host undergoing therapy. The compounds of the present invention can be administered to a patient at dosage levels in the range of about 0.1 to about 1,000 mg per day. For a normal human adult having a body weight of about 70 kilograms, a dosage in the range of about 0.01 to about 100 mg per kilogram of body weight per day is an example. The specific dosage used, however, can vary. For example, the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to one of ordinary skill in the art.
[0098] If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described above and the other pharmaceutically active agent(s) within its approved dosage range. Compounds of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a combination formulation is inappropriate.
General Synthesis
[0099] Compounds of this invention can be made by the synthetic procedures described below. The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis.), or Bachem (Torrance, Calif.), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Todd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition) and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). These schemes are merely illustrative of some methods by which the compounds of this invention can be synthesized, and various modifications to these schemes can be made and will be suggested to one skilled in the art having referred to this disclosure. The starting materials and the intermediates of the reaction may be isolated and purified if desired using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography and the like. Such materials may be characterized using conventional means, including physical constants and spectral data.
[00100] Unless specified to the contrary, the reactions described herein take place at atmospheric pressure and over a temperature range from about -78 °C to about 150 °C, more specifically from about 0 °C to about 125 °C and more specifically at about room (or ambient) temperature, e.g., about 20 °C. Unless otherwise stated (as in the case of an hydrogenation), all reactions are performed under an atmosphere of nitrogen.
[00101] Prodrugs can be prepared by techniques known to one skilled in the art. These techniques generally modify appropriate functional groups in a given compound. These modified functional groups regenerate original functional groups by routine manipulation or in vivo. Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Z?oche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.
[0100] The compounds of the invention, or their pharmaceutically acceptable salts, may have asymmetric carbon atoms or quaternized nitrogen atoms in their structure. Compounds of the invention that may be prepared through the syntheses described herein may exist as single stereoisomers, racemates, and as mixtures of enantiomers and diastereomers. The compounds may also exist as geometric isomers. All such single stereoisomers, racemates and mixtures thereof, and geometric isomers are intended to be within the scope of this invention. Some of the compounds of the invention may exist as tautomers. For example, where a ketone or aldehyde is present, the molecule may exist in the enol form; where an amide is present, the molecule may exist as the imidic acid; and where an enamine is present, the molecule may exist as an imine. All such tautomers are within the scope of the invention.
[0101] The present invention also includes isotopically-labeled compounds, which are identical to those recited in Formula I, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 180, 170, 31P, 32P, 35S, 18F, and 36C1, respectively. Compounds of the present invention, prodrugs thereof, and
pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon- 14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically- labeled compounds of Formula I of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples and Preparations below, by substituting a readily available isotopically-labeled reagent for a non- isotopically-labeled reagent.
[0102] The present invention also includes N-oxide derivatives and protected derivatives of compounds of the Invention. For example, when compounds of the Invention contain an oxidizable nitrogen atom, the nitrogen atom can be converted to an N-oxide by methods well known in the art. When compounds of the Invention contain groups such as hydroxy, carboxy, thiol or any group containing a nitrogen atom(s), these groups can be protected with a suitable "protecting group" or "protective group". A comprehensive list of suitable protective groups can be found in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc. 1991, the disclosure of which is incorporated herein by reference in its entirety. For example nitrogen protecting groups include, but are not limited to Boc, Fmoc, benzyl, trityl, and the like. The protected derivatives of compounds of the Invention can be prepared by methods well known in the art. [0103] Methods for the preparation and/or separation and isolation of single stereoisomers from racemic mixtures or non-racemic mixtures of stereoisomers are well known in the art. For example, optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. Enantiomers (R- and S-isomers) may be resolved by methods known to one of ordinary skill in the art, for example by: formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallization; via formation of diastereoisomeric derivatives which may be separated, for example, by crystallization, selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where a desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step may be required to liberate the desired enantiomeric form. Alternatively, specific enantiomer may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents or by converting on enantiomer to the other by asymmetric transformation. For a mixture of enantiomers, enriched in a particular enantiomer, the major component enantiomer may be further enriched (with concomitant loss in yield) by recrystallization.
[0104] In addition, the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
[0105] The chemistry for the preparation of the compounds of this invention is known to those skilled in the art. In fact, there may be more than one process to prepare the compounds of the invention. The following examples illustrate but do not limit the invention. All references cited herein are incorporated by reference in their entirety.
[0106] The compounds of formula I may be made using the general synthetic methods described below, which also constitute part of the invention.
GENERAL SYNTHETIC METHODS
[0107] The invention additionally provides methods for making compounds of formula I. The compounds of the invention may be prepared by the general methods and examples presented below, and methods known to those of ordinary skill in the art and reported in the chemical literature. Unless otherwise specified, solvents, temperatures, pressures, and other reaction conditions may be readily selected by one of ordinary skill in the art. Specific procedures are provided in the Synthetic Examples section. Intermediate substituted benzoic acids and their precursors (benzoic acids esters, benzamides or benzonitriles) are commercially available or are prepared by methods known to those skilled in the art (see WO20101 1316). Intermediate pyridyl carboxylic acids are commercially available or are prepared by methods known to those skilled in the art (see WO2009024905 and WO200802937). Intermediate substituted acyl hydrazines of formula III and VI are prepared from the corresponding substituted carboxylic acid esters by methods known to those skilled in the art.
[0108] The methods described below and in the Synthetic Examples section may be used to prepare compounds of formula I. In the Schemes below, R2, R2a, R3, R4, R7c, ring A, and n shall have the meanings defined in the detailed description of formula I, and optionally separating individual isomers; and optionally modifying any of the R2, R2a, R3, R4, and R7c groups; and optionally forming a pharmaceutically acceptable salt, hydrate, solvate or combination thereof.
Scheme I
Figure imgf000054_0001
I
[0109] As illustrated above, a carboxylic acid of formula II is reacted with an acyl hydrazine of formula III under coupling conditions well known in the art such as by treatment with S0C12 or, N-iS-dimethylaminopropy -TV-ethylcarbodiimide hydrochloride (EDCI) in the presence of 1 -hydroxylbenzotriazole (HOBt) or, benzotriazol-1- yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP) or, O-(7-azabenzotriazol-l- yl)-N,N,N N'-tetramethyluronium hexafluorophosphate (HATU) or, O-(benzotriazol-l-yl- N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence or absence of a base such as Et3N or N.N-diisopropylethylamine (DIPEA) in a suitable solvent such as DMF or dimethylacetamide to provide an intermediate of formula IV. Bis-acyl hydrazine IV is reacted with Lawesson's reagent in a suitable solvent such as dioxane at 80-100°C to provide an intermediate of formula V. Intermediate V where R is defined as a heterocycloalkyloxy and PG is a nitrogen protecting group such as a BOC group is reacted with a suitable acid such as HCl in a suitable solvent such as dioxane, THF or methanol to provide the desired compound of formula I.
[0110] An alternate approach that may be used to obtain compounds of formula I is illustrated in Scheme II.
Scheme II
Figure imgf000055_0001
[0111] As illustrated above, a carboxylic acid of formula II is reacted with an acyl hydazine of formula VI, where X is a halogen such as fluorine, under coupling conditions well known in the art such as by treatment with SOCl2 or, N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride in the presence of 1 -hydroxylbenzotriazole (HOBt) or, benzotriazol-l-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP) or, O-(7- azabenzotriazol-l-yl)-NN,N',jV'-tetramethyluronium hexafluorophosphate (HATU) or, O- (benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence or absence of a base such as Et3N or N,N-diisopropylethylamine (DIPEA) in a suitable solvent such as DMF or dimethylacetamide to provide an intermediate bis-acyl hydrazine which is treated with Lawesson's reagent in a suitable solvent such as dioxane at 80-100°C to provide a thiadiazole of formula VII. Intermediate VII for this example is reacted with an alcohol VIII (where PG is a nitrogen protecting group such as a BOC group) in the presence of a base such as sodium hydride or lithium diisopropylamide (LDA), or potassium bis(trimethylsilyl)amide (KHMDS) in a suitable solvent such as DMF or THF to provide a compound of formula IX. Treatment of intermediate IX with a suitable acid such as HC1 in a suitable solvent such as dioxane, THF or methanol provides the desired compound of formula I.
[0112] Another approach that may be used to obtain compounds of formula I is illustrated in Scheme III.
Scheme III
Figure imgf000056_0001
[0113] Reaction of intermediate VII with a base such as tetrabutylammonium hydroxide in a suitable solvent such as dioxane or aqueous NaOH or aqueous KOH in a suitable solvent such as acetonitrile provides a compound of formula X. Alkylation of intermediate X with a suitable electrophile (for example an alkyl halides or, an aziridines or, an epoxides) such as alkyl halide Villa (X is a I, Br, or CI) where PG is an amine protecting group such as BOC in the presence of a base such as NaH, LDA or KHMDS in a suitable solvent such as DMF, DMSO or THF provides a compound of formula IX. Alternately, reacting intermediate X and an alcohol Villa (X is OH) under Mitsunobu conditions, for example in the presence of PPh3 and di-tert-butyl azodicarboxylate in a suitable solvent such as THF provides intermediate IX. Deprotection of intermediate IX as shown in Scheme II with a suitable acid such as HCl in a suitable solvent such as dioxane, THF or methanol provides the desired compound of formula I.
Synthetic Examples
Intermediate 2
(S)-iert-Butyl 4-(hydroxymethyl)-2,2-dimethyloxazolidine-3-carboxylate
Figure imgf000057_0001
1 2
[0114] tert-Butyl 4-(hydroxymethyl)-2,2-dimethyloxazolidine-3-carboxylate (2). To an ice cold solution of commercially available 1 (35 g, 135 mmol) in THF/MeOH (500 mL (95:5)), was added LiBH4 (5.9 g, 271 mmol) portionwise and the suspension was stirred for 2 h at RT. The reaction mixture was cooled and quenched with ice. Solvent was removed under reduced pressure, and water was added. The aqueous layer was extracted with EtOAc, concentrated and chromatographed (EtOAc:hexanes, 3:7) to yield 2 (23 g, 74 % yield).
Intermediate 7
4S,5R -/i?/*i'-But I 4- h drox meth l)-2,2,5-trimeth loxazoIidine-3-carboxyIate ne
Figure imgf000057_0002
6 7
[0115] Step 1: (2R,3R)-Methyl 2-amino-3-hydroxybutanoate (4). To MeOH (120 mL) cooled in an ice bath was added acetyl chloride (20 mL, 282 mmol) dropwise with stirring. The mixture was stirred in the ice bath for 15 min after which time D-allothreonine 3 (1 1.31 g, 94.9 mmol) was added. The reaction mixture was removed from the ice bath and heated to reflux for several hours. After cooling to RT, the reaction mixture was concentrated in vacuo. The crude product was used as is in the next reaction.
[0116] Step 2: (2R,3R)-Methyl 2-(feri-butoxycarbonylamino)-3-hydroxybutanoate (5). Crude 4 (94.9 mmol), THF (300 mL) and triethylamine (21.6 g,) were combined and cooled in an ice bath. Boc anhydride (31.9 g, 146 mmol) was added and the reaction mixture was allowed to warm to RT and stirred for several days. The reaction mixture was concentrated in vacuo and the resulting residue was partitioned between sat'd NaHC03 and diethyl ether. The phases were separated and the aqueous phase was further extracted with diethyl ether (2x). The combined diethyl ether extractions were washed with sat'd NaHC03 (lx) and sat'd NaCl (lx), dried (Na2S04), concentrated and dried under high vacuum overnight. The resulting residue was sonicated in hexanes followed by stirring in hexanes for several hours. Stirring was stopped and the mixture allowed to settle. The hexanes were decanted off the resulting oil. The oil was dissolved in DCM and reconcentrated in vacuo followed by drying under high vacuum to give compound 5 (12.68 g, 57% over 2 steps from compound 3). Ή NMR (400 MHz, OMSO-d6) 6 7.02 (d, 1H), 4.92 (m, 1H), 3.88 (m, 1H), 3.80 (m, 1H), 3.60 (s, 3H), 1.35 (s, 9H), 1.06 (d, 3H).
[0117] Step 3: (4R,5R)-3- rt-Butyl 4-methyl 2,2,5-trimethyloxazoIidine-3,4- dicarboxylate (6). Compound 5 (12.68 g, 54.4 mmol), toluene (150 mL), dimethoxypropane (75 mL, 610 mmol) and p-toluenesulfonic acid monohydrate (1.4 g, 7.4 mmol) were combined and stirred at 80 °C for 6 h. After cooling to RT, the reaction mixture was concentrated in vacuo and the resulting oil was partitioned between sat'd NaHC03 and diethyl ether. Phases were separated and the aqueous phase was further extracted with diethyl ether (2x). The combined diethyl ether extractions were washed with sat'd NaCl (lx), dried (Na2S04), concentrated, and dried under high vacuum overnight to give compound 6 (1 1.7 g, 79%).). Ή NMR (400 MHz, OMSO-d6) 6 4.40 (m, 1H), 4.28 (m, 1H), 3.65 (s, 3H), 1.56 (d, 3H), 1.39 (s, 3H), 1.31 (s, 9H), 1.07 (d, 3H).
[0118] Step 4: (4S,5R)-terf-Butyl 4-(hydroxymethyl)-2,2,5-trimethyloxazolidine-3- carboxylate (7). Compound 6 (1 1.7 g, 42.8 mmol) was dissolved in THF (80 mL) and cooled in an ice bath. Lithium borohydride (1.87 g, 85.8 mmol) was added followed by the dropwise addition of a 1 : 1 solution of THF:MeOH (22 mL) with stirring. After addition was complete, the reaction mixture was removed from the ice bath and stirred at RT overnight. The reaction was followed by LC-MS and NMR and additional lithium borohydride was added in 500 mg aliquots as needed until the reaction was complete. Once reaction was complete, the reaction mixture was cooled in an ice bath then slowly added to a cold solution of 7.5% aqueous citric acid solution. The mixture was stirred for 30 min then extracted with EtOAc (3x). The combined EtOAc extractions were washed with sat'd NaCl (lx), dried (Na2S04) and concentrated in vacuo. The resulting crude material was purified by flash chromatography (20% EtOAc in hexanes) to give compound 7 as a colorless oil (6.9 g, 66%). Ή NMR (400 MHz, DMSO-i/, δ 4.65 (m, 1H), 4.20 (m, 1H), 3.70-3.60 (m, 1H), 3.50-3.35 (m, 2H), 1.41 (s, 9H), 1.40 (s, 6H), 1.22 (d, 3H).
Intermediate 10
2-(Isopropylamino)-6-methylisonicotinic acid
Figure imgf000059_0001
[0119] Step 1: Ethyl 2-(isopropylamino)-6-methylisonicotinate (9). Ethyl 2-chloro-6- methylpyridine-4-carboxylate 8 (550 mg, 2.75 mmol), Pd(OAc)2 (28 mg, 0.13 mmol), BINAP (156 mg, 0.25 mmol), Cs2C03 (2.4 g, 7.5 mmol) and 1 ,4-dioxane (25 mL) were combined in a sealed tube. N2 was bubbled into the mixture for a few minutes and isopropylamine (885 mg, 15 mmol) was added. The sealed mixture was heated to 85 °C for 20 h. The mixture was filtered through Celite and washed with EtOAc. The solvents were removed in vacuo. Purification by flash column chromatography gave 9 (530 mg, 86%).
[0120] Step 2: 2-(Isopropylamino)-6-methylisonicotinic acid (10). Ethyl 2- (isopropylamino)-6-methylisonicotinate 9 (530 mg, 2.38 mmol) was dissolved in a mixture of MeOH (5 mL) and water (5 mL) and then treated with NaOH (480 mg, 1 1.9 mmol) at RT for 4 h. MeOH was removed in vacuo and the resulting aqueous mixture was neutralized to pH 6 by addition of 20% aqueous HC1 solution. The resulting solids were filtered and dried to give 10 (384 mg, 83%).
Intermediate 1-2
Figure imgf000059_0002
1-1 I-2
[0121] 1-1: To stirred methanol (50 mL) was added acetyl chloride (2.0 mL, 28 mmol) dropwise to generate HC1 in situ. 2-Chloro-4,5-difluorobenzoic acid (2.60 g, 13.5 mmol) was then added and the mixture was warmed at reflux over the weekend (65 h). The mixture was then concentrated and diluted with sat'd aq sodium bicarbonate (50 mL) and extracted with ether (3x25 mL). The combined organic layers were washed with sat'd aq sodium bicarbonate (25 mL), brine (25 mL), dried over magnesium sulfate, filtered and concentrated to afford compound 1-1.
[0122] 1-2: To 60% sodium hydride in mineral oil (900 mg, 22.5 mmol) in DMSO (25 mL) was added 2-(methylsulfonyl)-ethanol (2.80 g, 22.6 mmol) in DMSO (10 msL). After hydrogen evolution ceases, compound 1-1 (4.00 g, 19.4 mmol) was added. After 1 h, the mixture was diluted with sat'd aq ammonium chloride (125 mL) and extracted with ethyl acetate (4x40 mL). The combined organic layers were washed with brine (3x25 mL), dried over magnesium sulfate, filtered and concentrated. To the residue was added DCM followed by heptane and the mixture was passed through a pad of silica gel eluting first with DCM - heptane (1 :1) and then ethyl acetate-heptane (1 : 1) to afford compound 1-2.
Intermediate 1-9
Figure imgf000060_0001
[0123] 1-3: To a chilled (-78°C) soln of methyl p-tolyl sulfoxide (15.4 g, 99.9 mmol) in 100 mL of THF was added a 1M soln of lithium bis(trimethylsilyl)amide in THF (105 mL, 105 mmol) over a 20 min period. The mixture was stirred for 5 min and then a soln of methyl trifluoroacetate (15.0 mL, 149 mmol) in 100 mL of THF was added over 20 min. After 20 min, the mixture was first quenched with sat'd aq ammonium chloride (100 mL) then warmed to RT, made acidic with 1 N aq HCl and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed in turn with brine (3x50 mL), sat'd sodium bicarbonate aq soln (2x50 mL), brine (50 mL), dried over magnesium sulfate, filtered and concentrated. The resulting solid was triturated with ether-hexanes to afford compound 1-3 which was used without further purification.
[0124] 1-4: To a soln of compound 1-3 (16.9 g, 67.5 mmol) in toluene (15 mL) was added l,l,l-trimethyl-N-(triphenylphosphoranylidene)silanamine (23.4 g, 67.0 mmol). The mixture was stirred over the weekend. The mixture was concentrated and the oil was dissolved in DCM -hexanes (1 :1) and passed through a pad of silica gel (600 mL funnel) eluting with 20% EtOAc in hexanes to afford compound 1-4.
[0125] 1-5: To a soln of compound 1-4 (14.7 g, 59.0 mmol) in THF (120 mL)/water (30 mL) was added sodium borohydride (5.20 g, 137 mmol) in several portions. The mixture was stirred overnight. The mixture was then concentrated to remove the excess THF and then diluted with ether (75 mL), sat'd aq ammonium chloride (25 mL), and then made acidic the IN aq HCl (50 mL). The acidic aq layer was separated and the organic layer was extracted with 1 N aq HCl (4x25 mL). The combined acidic aq layers were washed with ether (4x30 mL) and made basic with K2C03. The white solid was filtered to afford compound 1-5.
[0126] 1-6: To a chilled (0°C) soln of compound 1-5 (8.65 g, 34.4 mmol) in ACN (150 mL) was added neat TMSC1 (16.0 mL, 126 mmol). The mixture was stirred for 15 min, and then sodium iodide (32.0 g, 213 mmol) was added in several portions. The cold bath was then removed and the dark red mixture stirred over night. The mixture was then diluted with aq Na2S03 to destroy the excess iodine and then extracted with ether (3x40 mL). The combined organic layers were washed with brine (3x40 mL) and then extracted with IN aq HCl (3x40 mL). The combined acidic aq layers were made basic with potassium carbonate and extracted with EtOAc (3x40 mL). The combined organic layers were washed with brine (3x30 mL), dried over magnesium sulfate, filtered and concentrated. The crude residue was then passed through a pad of silica gel using 30% EtOAc in hexanes to afford compound 1-6.
[0127] 1-7: To a soln of compound 1-6 (5.00 g, 21.3 mmol) in pyridine (50 mL) was added 2- nitrobenzenesulfonyl chloride (7.60 g, 34.3 mmol). The mixture was warmed to 70°C for 18 h. The mixture was then cooled and diluted with IN HCl aq soln (40 mL) and extracted with ether (3x50 mL). The combined ether layers were washed with IN aq HCl (340 mL), brine (3x40 mL), sat'd aq sodium bicarbonate (3x40 mL), dried over magnesium sulfate, filtered and concentrated. The crude material was chromatographed twice on silica gel using DCM in hexanes (20-100% gradient) to afford compound 1-7.
[0128] 1-8: To a soln of compound 1-7 (4.00 g, 9.51 mmol) in DCM (25 mL) was added trimethyloxonium tetrafluoroborate salt (1.60 g, 10.8 mmol). The mixture was stirred 6 h, and then the solid was collected by filtration washing with ether to afford compound 1-8.
[0129] 1-9: To a soln of compound 1-8 (6.71 g, 15.4 mmol) in THF (100 mL) was added sodium hydride (1.52 g, 38.0 mmol). The mixture was stirred for 2 h and was then diluted with sat'd aq ammonium chloride (50 mL) and extracted with ether (3x40 mL). The combined organics were washed with brine (3x30 mL), dried over magnesium sulfate, filtered and concentrated. The residue was chromatographed on silica gel eluting with DCM in hexanes (25-75% gradient) to afford compound 1-9. -13
Figure imgf000062_0001
1-12
1-11
Figure imgf000062_0002
1-13 [0130] 1-10: To a soln of compound 1-2 (230 mg, 1.12 mmol) in DMSO (6 mL) was added 60% sodium hydride in mineral oil (50.0 mg, 1.25 mmol). The mixture stirred until hydrogen evolution ceased and then compound 1-9 (367 mg, 1.24 mmol) was added. After 1 h, the mixture was quenched with sat'd aq ammonium chloride (40 mL) and extracted with EtOAc (3x20 mL). The combined organics were washed with sat'd ammonium chloride aq soln (15 mL), brine (3x15 mL), dried over magnesium sulfate, filtered and concentrated. The crude material was purified on silica gel (using a gradient of 8-50% ethyl acetate in heptane) to afford compound 1-10.
[0131] 1-11: A mixture of compound 1-10 (380 mg, 0.760 mmol) and hydrazine (0.24 mL, 7.5 mmol) in methanol (9 mL) was warmed at reflux. After 24 h, the mixture was concentrated and diluted with water and the solid was collected. The filter cake was dissolved in DCM, dried over magnesium sulfate, filtered and concentrated to afford compound 1-11. The aq filtrate was extracted with EtOAc (4x10 mL). The combined organics were dried over magnesium sulfate, filtered, and concentrated to afford additional compound 1-11.
[0132] 1-12: To 3-chloro-4-isopropoxybenzoic acid (1 10 mg, 0.510 mmol) in DMF (3 mL) was added N-iS-dimethylaminoprop^-N'-ethylcarbondiimide hydrochloride (EDC1) (107 mg, 0.560 mmol) followed by 1 -hydroxylbenzotriazole (HOBt) (76 mg, 0.51 mmol). After 1.5 h, compound 1-11 (256 mg, 0.510 mmol) was added. After 18 h, the mixture was diluted with sat'd aq sodium bicarbonate (15 mL) and extracted with EtOAc (4x15 mL). The combined organics were washed with brine (3x10 mL), dried over magnesium sulfate, filtered and concentrated. The residue was purified on silica gel (using a solvent gradient from 10-60% EtOAc in heptane) to afford compound 1-12.
[0133] 1-13: A mixture of compound 1-12 (279 mg, 0.400 mmol) and 2,4-bis-(4- methoxyphenyl)-l,3-dithia-2,4-diphosphetane 2,4-disulfide (Lawesson's Reagent) (260 mg, 0.64 mmol) in 2-methyltetrahydrofuran (2 mL) was warmed at 90°C. After 18 h, the mixture was concentrated, diluted with sat'd ammonium chloride aq soln (10 mL) and extracted with EtOAc (3x10 mL). The combined organics were washed with sat'd ammonium chloride aq soln (2x10 mL), brine (10 mL), dried over magnesium sulfate, treated with carbon (Norit A), filtered through filter agent and concentrated. The residue was purified on silica gel (using a solvent gradient from 5-30% EtOAc in heptane) to afford compound 1-13. Intermediate 1-14
Figure imgf000064_0001
85°C
1-14
[0134] 1-14: Methyl 2-chloro-6-methylpyridine-4-carboxylate (10.0 g, 53.9 mmol), 9,9- dimethyl-4,5-bis(diphenylphosphino)xanthene (Xantphos) (9.35 g, 16.2 mmol), CsC03 (52.7 g, 162 mmol) and isopropylamine (27.5 mL, 323 mmol) were added to dry p-dioxane (100 mL) and degassed under argon for 15 min. Palladium(II) acetate (2.42 g, 10.8 mmol) was then added. The rxn vessel was sealed and heated at 85°C for 18 h. The mixture was filtered through celite and concentrated. The residue diluted with sat'd ammonium chloride aq soln (100 mL) and extracted with EtOAc (5x50 mL). The combined extracts were washed with brine (50 mL) and dried with MgS0 . The mixture was filtered, concentrated, and purified by silica gel chromatography (0-10% MeOH in DCM) to give compound 1-14.
Intermediate 1-15
Figure imgf000064_0002
[0135] 1-15: To the soln of compound 1-14 (3.00 g, 14.4 mmol) in ethanol (60 mL) was added hydrazine (4.19 mL, 134 mmol). The mixture was then heated to reflux for 8 h. The mixture was concentrated. The residue was triturated with ether to provide compound 1-15.
Intermediate 1-17
Figure imgf000064_0003
1-17
[0136] 1-17: A soln of methyl (R)-(+)-3-(/ert-butoxycarbonyl)-2,2-dimethyl-4- oxazolidinecarboxylate (15.0 g, 56.7 mmol) in dry THF (130 mL) in a 1-L rbf under N2 was cooled to 0°C. Lithium borohydride (2.74 g, 113 mmol) was added slowly to the rxn soln in a period of 3 min. The rxn mixture was stirred at 0°C for 1 hr and RT overnight. The mixture was cooled to 0°C and stirred for 5 min. Citric acid (22.3 g) dissolved in water (297 mL) was added slowly at 0°C. MeOH (100 mL) was added and the soln was continued to stir at 0°C for 30 min. The mixture was extracted with EtOAc (3x150 mL). The combined organics were washed with sat'd NaCl aq soln, dried over anhydrous sodium sulfate, filtered, and concentrated. MeOH (100 mL) was added and evaporated and this process was repeated twice. Anhydrous diethyl ether (100 mL) was added and evaporated. The resultant light yellow oil was further evaporated on high vacuum pump at 60°C for 1 h, on house vacuum for three days, then on high vacuum pump at 60°C for 1 h to afford compound 1-17.
-22
Figure imgf000065_0001
1-19 ,.2o
Figure imgf000065_0002
[0137] 1-19: To D-allo-threonine (500 mg, 4.20 mmol) in MeOH (5 mL) was added thionyl chloride (0.31 mL, 4.2 mmol) at 0°C. The rxn mixture was stirred at RT for 1.5 h, then heated to 80°C for 16 h. The solvent was removed under reduced pressure. MeOH was added and removed twice. The residue was triturated with ether, filtered, and dried under high vacuum to afford compound 1-19 as a HC1 salt.
[0138] 1-20: To compound 1-19 HC1 salt (712 mg, 4.20 mmol) in DCM (10 mL) were added Et3N (1.72 mL, 12.6 mmol) and Boc20 (1.01 g, 4.62 mmol) at 0°C. The rxn was warmed to RT and stirred over the weekend. The rxn was added sat'd NH4C1 aq soln, and extracted with DCM. The organics were washed in turn with water, sat'd NH4C1 aq soln, and brine, then dried and concentrated to afford compound 1-20.
[0139] 1-21: The mixture of compound 1-20 (42.1 g, 181 mmol), DMP (44.3 mL, 361 mmol), and TsOH · H20 (515 mg, 2.71 mmol) in toluene (420 mL) was heated to reflux for 2 h. Additional DMP (20 mL) was added. The rxn was refluxed for another 2 h. The rxn mixture was cooled to RT and diluted with EtOAc, washed with brine and concentrated in vacuo. The mixture was diluted again with ether and washed in turn with sat'd NaHC03, water, and brine, and filtered. The filtrate was concentrated to afford compound 1-21.
[0140] 1-22: Lithium borohydride (4.80 g, 198 mmol) was added in batches to compound I- 21 (27.1 g, 99.1 mmol) in dry THF (250 mL) in a 1-L rbf under N2 protection at 0°C. The mixture was stirred at 0°C for 1 h. The ice/water bath was removed and the rxn mixture was stirred at RT over the weekend. MeOH (15 mL) was added at 0°C and the rxn mixture was continued to stir at that temperature for 20 min. The mixture was stirred at RT for another 2 h. The rxn was quenched with 10% aq citric acid soln slowly until no more solid exists. The mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2S04, filtered and concentrated. The resulting crude pdt was purified by combiflash using 0-50% EtOAc in heptane as the gradient to afford compound 1-22. -30
Figure imgf000066_0001
[0141] 1-27: Absolute EtOH (126 mL, 2.16 mol) was cooled to 0°C on an ice/water bath. Acetyl chloride (21.5 mL, 300 mmol) was added dropwise to generate HC1 in situ. After the addition, the rxn was stirred at 0°C for an additional 30 min. D-Aspartic acid (10.0 g, 75.1 mmol) was added in one portion. The ice/water bath was removed. The suspension was stirred at RT for five min then heated slowly to reflux. Reflux was continued for 4 h. The rxn soln was further stirred at RT for 3 days. The solvent was removed under reduced pressure to afford crude compound 1-27 as a HC1 salt which was used in the next step without purification.
[0142] 1-28: Compound 1-27 HC1 salt (16.8 g, 74.5 mmol) was dissolved in water (17.3 mL) and p-dioxane (43.6 mL) and then cooled to 0°C. TEA (21.6 mL, 155 mmol) was added. BOC20 (21.9 g, 101 mmol) dissolved in p-dioxane (15 mL) was then added slowly. The resulting rxn mixture was heated to 50°C and continued to stir at that temperature for 3.5 h. Solvent was removed under reduced pressure. Aq 10% citric acid (40 mL) was added to adjust the pH to 2-3. The aq phase was extracted with EtOAc (4x75 mL). The combined organic layers were washed with sat'd NaCl soln (29 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was further evaporated on high vacuum pump at 50°C for 30 min then on house vacuum at RT for 60 h to afford crude compound 1-28 which was used in the next step without purification.
[0143] 1-29: A stirred soln of compound 1-28 (22.3 g, 77.0 mmol) in absolute EtOH (362 mL) was cooled to 0°C on an ice/water bath. Sodium borohydride (29.1 g, 770 mmol) was added portionwise. The rxn mixture was stirred at 0°C for 30 min. The ice/water bath was removed and the rxn mixture was continued to stir at RT for 30 min. Sat'd NaCl aq soln (210 mL) was added to the rxn mixture and the mixture was stirred for 5 min. The mixture was filtered and the filtrate was concentrated under reduced pressure to about 50 mL of volume. The mixture was then extracted with EtOAc (6x150 mL). The insoluble material was extracted by stirring in EtOAc (300 mL). The combined organics were washed with sat'd NaCl aq soln (10 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated. The resulting residue was further evaporated on high vacuum pump at 60°C for 1 h to afford crude compound 1-29 which was used in the next step without purification.
[0144] 1-30: Compound 1-29 (1 1.9 g, 53.9 mmol) was dissolved in DCM (246 mL). While stirring, DMP (74 mL, 591 mmol) and p-toluenesulfonic acid monohydrate (1.02 g, 5.30 mmol) were added. The rxn mixture was stirred at RT for 32 h. The rxn mixture was washed with aq 5% NaHC03 soln (2x50 mL) and sat'd NaCl aq soln (40 mL), dried over anhydrous MgS04, filtered, and concentrated under reduced pressure to afford an oil, which crystallizes upon standing. The solid was fully redissolved in heptane (300 mL) at 60°C on rotavap. Heptane was then evaporated on rotavap until around 70 mL of soln was left. The soln was then set in the refrigerator for 10 min. White crystals were formed. The crystals were filtered and washed with heptane. The crystals were further dried on high vacuum at RT overnight to afford compound 1-30. Intermediate 1-87
Figure imgf000068_0001
1-87
[0145] 1-87: Under nitrogen absolute EtOH (219 mL) in a 1-L rbf was cooled to 0°C on an ice/water bath. Stirred for 5 min. Acetyl chloride (37.1 mL, 519 mmol) was added slowly to generate HC1 in situ. After the addition, the rxn was stirred at 0°C for an additional 30 min. 2- Chloro-4,5-difluorobenzoic acid (25.0 g, 130 mmol) was added. The ice/water bath was removed and the rxn was stirred at RT for 84 h. The rxn was then heated to reflux for 6 h. The solvent was removed under reduced pressure. The crude oil was diluted with sat'd NaHC03 aq soln (62.5 mL). The mixture was extracted with diethyl ether (3x100 mL). The combined organics were washed with sat'd NaCl aq soln (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to afford compound 1-87 which was used as was in the next step.
-43
Figure imgf000068_0002
[0146] 1-147: 3-Cyano-4-isopropoxy-benzoic acid (120 mg, 0.573 mmol), TBTU (228 mg, 0.688 mmol), Et3N (0.20 mL, 1.4 mmol), tert-butyl carbazate (81.1 mg, 0.602 mmol) were combined in DMF (2.0 mL). The rxn mixture was stirred at RT overnight (18 h). The mixture was diluted with EtOAc (50 mL) and washed in turn with sat'd NaHC03 aq soln (5 mL), water (3x20 mL), and sat'd NaCl aq soln (5 mL). The organics were dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash column using 20-100% EtOAc in heptane as the gradient to afford compound I- 147.
[0147] 1-43: To a soln of compound 1-147 (142 mg, 0.445 mmol) in DCM (2.0 mL) was added 4.0 HC1 in 1,4-dioxane (1.11 mL, 4.44 mmol) slowly. The rxn mixture was stirred at RT overnight (19 h). A white solid was formed. Diethyl ether anhydrous (5 mL) was added and stirred overnight. The mixture was filtered and washed with diethyl ether anhydrous (5 mL). The resulting white solid was further evaporated to afford compound 1-43. -45
Figure imgf000069_0001
90°C 1.45
[0148] 1-44: To a soln of 2-chloro-4,5-difluorobenzoic acid (271 mg, 1.41 mmol) in DMF (9.0 mL) was added TBTU (587 mg, 1.58 mmol) followed by TEA (0.70 mL, 5.0 mmol). The rxn was allowed to stir for 1 h at RT. Compound 1-43 HC1 salt (403 mg, 1.58 mmol) was added and the resulting rxn mixture was maintained at RT for an additional 24 h. Finally, the rxn mixture was diluted with EtOAc and treated with sat'd aq NaHC03. Following the separation of the aq phase from the organic phase, the former was extracted with EtOAc three times. The combined organic extracts were washed with brine, dried (Na2S04), filtered and concentrated. The resulting crude solid was purified via trituration with DCM. The mother liquor was concentrated and the resulting solid was triturated with DCM to generate a second batch of the pdt. The combined pdt was compound 1-44.
[0149] 1-45: To a mixture of compound 1-44 (202 mg, 0.513 mmol) in 2- methyltetrahydrofuran (3.6 mL) was added Lawesson's reagent (152 mg, 0.365 mmol). The rxn mixture was heated at 90°C in an oil bath for 22 h. The rxn mixture was cooled to RT and the volatiles were concentrated. The resulting crude material was purified by normal phase column chromatography using 0-100% EtOAc in heptane as the gradient to afford compound 1-45. Intermediate 1-49
Figure imgf000070_0001
[0150] 1-47: A mixture of 2-bromo-4,5-difluorotoluene (4.00 g, 19.3 mmol), DIPEA (5.2 mL, 30.0 mmol), dichlorobis(benzonitrile)palladium(II) (269 mg, 0.700 mmol) and 1,1- bis(diphenylphosphino)ferrocene (dppf) (482 mg, 0.870 mmol) in absolute ethanol (50 mL) was placed in a sealed bomb with stirring and placed under 10 bars of carbon monoxide and heated to 135°C for 4 h. The mixture was then cooled to RT, returned to atmospheric pressure and opened. The rxn mixture was concentrated and diluted with brine (100 mL) and extracted with EtOAc (3x50 mL). The combined organic layers were washed with brine (2x50 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by normal phase flash column chromatography on silica gel using 5-20% EtOAc in heptane to afford compound 1-47.
[0151] 1-48: To a stirred soln of compound 1-47 (2.50 g, 12.5 mmol) and compound 1-17 (3.20 g, 14.0 mmol) in anhydrous DMF (20 mL) was added 60% NaH in mineral oil (560 mg, 14.0 mmol) at RT. After 3 h, water (100 mL) was added to quench the rxn. The mixture was extracted with EtOAc (2x150 mL), washed with brine (50 mL), dried over sodium sulfate and concentrated. The residue was purified by normal phase flash column chromatography on silica gel using 0-50% EtOAc in heptane to afford compound 1-48.
[0152] 1-49: A mixture of compound 1-48 (3.2 g, 7.8 mmol) and hydrazine (2.5 g, 77 mmol) in MeOH (25 mL) was heated to reflux. After 18 h, the rxn was cooled to RT and concentrated in vacuo, diluted with water (50 mL) and extracted with EtOAc (2x100 mL). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate and concentrated to afford compound 1-49.
[0153] The following compound was synthesized in a similar fashion from the appropriate intermediates:
Figure imgf000071_0002
-57
Figure imgf000071_0001
[0154] 1-55: To a stirred soln of 2-fluoro-3-methyl-pyridine-5-carboxylic acid (3.50 g, 22.6 mmol) in MeOH (20 mL) was added 2M trimethylsilyldiazomethane in ethyl ether soln (22.0 mL, 44.0 mmol) at RT. After 1 h, acetic acid (8.0 mL) was added to quench the rxn. Solvent was removed under reduced pressure to provide compound 1-55.
[0155] 1-56: To a stirred soln of compound 1-55 (2.0 g, 12 mmol) in anhydrous DMF (8.0 mL) was added iPrOH (0.90 g, 15 mmol) and 60% NaH in mineral oil (0.60 g, 15 mmol). After 3 h, water (100 mL) was added to quench the rxn. The mixture was extracted with EtOAc (2x150 mL), washed with brine, dried over sodium sulfate and concentrated. The residue was purified via Combi-flash column on silica gel using a solvent gradient from 0- 20% EtOAc in heptanes to afford compound 1-56.
[0156] 1-57: To a stirred soln of the compound 1-56 (1.6 g, 7.6 mmol) in MeOH (20 mL) was added 2M aq NaOH (7.5 mL, 15 mmol). The rxn was warmed to reflux. After 2 h, the rxn was cooled to RT and the solvent was removed under reduced pressure. The residue was acidified with 2N aq HC1 to pH = 4. The solid was filtered and evaporated. The solid was dissolved in MeOH (6 mL) and purified via a reverse phase CI 8 prep HPLC column using a solvent gradient from 5% MeCN in H20 to 100% MeCN to afford compound 1-57. -64
Figure imgf000072_0001
1-22 1-63 1-64
[0157] 1-63: Sodium hydride (0.40 g, 10 mmol) was added to a soln of compound 1-1 (2.0 g, 9.7 mmol) and compound 1-22 (2.4 g, 9.8 mmol) in DMF (10 mL) at RT under a stream of Argon. After 18 h, the rxn was quenched with sat'd aq ammonium chloride (25 mL) and extracted with EtOAc (3x50 mL). The combined organics were washed with brine (50 mL), dried over sodium sulfate and concentrated. The crude residue was purified by silica gel chromatography using a combiflash with a 0-10% EtOAc in heptanes gradient to afford compound 1-63.
[0158] 1-64: A mixture of compound 1-63 (1.0 g, 2.3 mmol) and hydrazine (0.74 g, 23 mmol) in MeOH (15 mL) was heated to reflux. After 18 h, the rxn mixture was concentrated. The resulting crude pdt was diluted with water (50 mL) and extracted with EtOAc (2x100 mL). The combined organics were washed with brine, dried over sodium sulfate and concentrated to afford compound 1-64.
Intermediate 1-69
Figure imgf000073_0001
1-66 1-67
Figure imgf000073_0002
1-68 1-69
[0159] 1-66: To a soln of 3-hydroxy-2-methylpyridine (7.0 g, 64 mmol) in DMF (50 mL) was added sodium hydride (2.6 g, 65 mmol) at RT under a stream of nitrogen. After 20 min 2- bromopropane (18.0 mL, 192 mmol) was added. After 2 h the rxn was quenched with sat'd ammonium chloride aq soln (40 mL) and extracted with EtOAc (4x20 mL). The combined organic were washed with brine (30 mL), dried over sodium sulfate and concentrated. The resulting crude residue was diluted with heptanes and filtered. The filtrate was concentrated to afford compound 1-66.
[0160] 1-67: To a soln of compound 1-66 (5.10 g, 33.7 mmol) in DCM (40 ml) was added MCPBA (10.0 g, 44.6 mmol) at RT. After 16 h the rxn mixture was diluted with DCM (20 mL) and water (10 mL). Layers were separated. The aq layer was extracted with DCM (10 mL). The combined organic layers were washed with sat'd NaHC03 aq soln (50 mL), brine (50 mL), dried over sodium sulfate, filtered and concentrated. The resulting crude pdt was purified by silica gel chromatography using 0-50% methanol in CH2CI2 as the gradient to afford compound 1-67.
[0161] 1-68: To a soln of compound 1-67 (3.90 g, 23.3 mmol) in ACN (50 mL) was added TMSCN (15.5 mL, 1 16 mmol) followed by DIPEA (21.0 mL, 1 17 mmol). The mixture was heated at reflux for 3 h. The mixture was then diluted with sat'd aq ammonium chloride (50 mL) and then extracted with EtOAc (3x30 mL). The extract was washed with brine (20 mL), dried over sodium sulfate and concentrated. The resulting crude pdt was filtered through a pad of silica gel using 20% EtOAc/heptanes to afford compound 1-68. [0162] 1-69: To a soln of 1-68 (0.56 g, 3.2 mmol) in EtOH (20 mL) was added potassium hydroxide (1.8 g, 32 mmol) and water (5 mL). The mixture was heated to reflux. After 18 h the mixture was concentrated, acidified with IN HCl and extracted with EtOAc (4x10 mL). The extract was washed with brine (2x20 mL), dried over sodium sulfate and concentrated to afford compound 1-69.
Intermediate 1-74
Figure imgf000074_0001
1-72 1-73 1-74
[0163] 1-71: To a soln of 2-hydroxy-6-methyl-isonicotinic acid ethyl ester (3.00 g, 16.6 mmol) in DMF (30 mL) was added N-phenyltrifluoromethanesulfonimide (7.22 g, 20.0 mmol) and DIPEA (3.52 mL, 20.0 mmol). After sirring for 20 h at RT, the mixture was diluted with sat'd ammonium chloride aq soln (50 mL), extracted with EtOAc (4x25 mL), washed with brine (30 mL) and dried over sodium sulfate. The organics were then concentrated to provide the crude which was purified by silica gel chromatography using a 0- 50% EtOAc in hexanes as the gradient to afford compound 1-71.
[0164] 1-72 and 1-73: Methylisopropylamine (3 mL) was added to compound 1-71 (500 mg, 1.60 mmol) and warmed to 50°C in a sealed tube. After stirring for 16 h, the mixture was concentrated to afford a mixture of compound 1-72 and compound 1-73.
[0165] 1-74: To a soln of the mixture of compound 1-72 and compound 1-73 in EtOH (10 mL) was added sodium hydroxide (0.40 g, 10 mmol) and water (3 mL). The mixture was heated to reflux. After 4 h, the mixture was concentrated, acidified with HCl aq soln and extracted with EtOAc (4x10 mL). The extracts were washed with brine, dried over sodium sulfate and concentrated to afford the crude pdt. The crude was taken up in diethyl ether and filtered to afford compound I- 74.
Intermediate 1-77
Figure imgf000075_0001
[0166] 1-76: To a soln of methyl 2-chloro-6-methylpyridine-4-carboxylate (0.50 g, 2.7 mmol) in DMF (3 mL) was added tetrakis(triphenylphosphine)palladium(0) (0.20 g, 0.17 mmol), potassium carbonate (1.00 g, 15.2 mmol), and water (1 mL). Argon was bubbled through the mixture for 5 min and the mixture was heated to 120°C in a microwave reactor. After 1 h the mixture was cooled to RT then diluted with sat'd ammonium chloride aq soln (10 mL), extracted with EtOAc (3x5 mL), dried over sodium sulfate, filtered, and concentrated. The resulting crude material was purified by silica gel chromatography using 0- 50% (20% MeOH in EtOAc) in heptanes as the gradient to afford compound 1-76.
[0167] 1-77: To a soln of compound 1-76 (520 mg, 2.72 mmol) in MeOH (25 mL) was added 10 wt.% Pd/C (200 mg, 0.192 mmol) and 5 drops of concentrated HC1. The mixture was hydrogenated overnight using a hydrogen balloon. After 16 h the mixture was filtered and concentrated to afford compound 1-77.
Intermediate 1-82
Figure imgf000075_0002
water
[0168] 1-80: To a soln of MeOH (5.00 mL, 123 mmol) in 100 mL of THF was added NaH (60% dispersion in mineral oil) (4.20 g, 105 mmol). When gas evolution ceases, methyl 2,6- dichloropyridine-4-carboxylate (20.0 g, 97.1 mmol) was added in one portion. After stirring for 2 h the rxn mixture was diluted with sat'd ammonium chloride aq soln and extracted with EtOAC (4x30 mL). The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated. The resulting crude pdt was washed with ether/hexanes to afford compound 1-80.
[0169] 1-81: Compound 1-80 (1.00 g, 4.96 mmol) was dissolved in DMF (15 mL). Tetrakis(triphenylphosphine)palladium(0) (400 mg, 0.346 mmol), potassium carbonate (2.10 g, 15.2 mmol), water (3 mL), and 2-methylpropenylboronic acid pinacol ester (1.07 mL, 5.20 mmol) were added. Argon was bubbled through the mixture for 5 min. The mixture was heated to 100°C overnight. After 19 h the mixture was cooled to RT. The mixture was diluted with sat'd ammonium chloride aq soln (10 mL) and acidified with HC1 then extracted with EtOAc (4x5 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. Upon standing the resulting oil begins to solidify. The material was then triturated with ether and filtered to afford compound 1-81.
[0170] 1-82: To a soln of compound 1-81 (0.723 g, 3.49 mmol) in MeOH (25 mL) was added 10 wt.% Pd/C (200 mg, 0.192 mmol) and 5 drops of concentrated HC1. The mixture was hydrogenated overnight using a hydrogen balloon. After 16 h the mixture was filtered and concentrated. The resulting crude material was purified by silica gel chromatography using 0- 50% (20% MeOH in EtOAc) in heptanes as the gradient to afford compound 1-82.
Intermediate 1-85
Figure imgf000076_0001
1-84 1-85
[0171] 1-84: NaH (0.475 g, 1 1.9 mmol) was added to 2-hydroxy-6-methylpyridine-4- carboxylic acid ethyl ester (2.0 g, 1 1 mmol) in DMF (40 mL) at RT under a stream of nitrogen. After 10 min 2-bromopropane (5.0 mL, 53 mmol) was added. After 2 h the rxn was quenched with sat'd aq ammonium chloride (40 mL) and extracted with EtOAc (4x20 mL). The combined organic was washed with brine (30 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was passed through a pad of silica using 20% EtOAc in heptanes to afford compound 1-84.
[0172] 1-85: To a soln of compound 1-84 (1.40 g, 6.27 mmol) in EtOH (20 mL) was added KOH (3.5 g, 62 mmol) and water (4 mL). The mixture was heated to reflux. After 18 h the mixture was concentrated, acidified with HC1 soln and extracted with EtOAc (4x10 mL). The extract was washed with brine, dried over sodium sulfate, filtered, and concentrated to afford compound 1-85.
Intermediate 1-88
Figure imgf000077_0001
1-87 1-30 1-88
[0173] 1-88: Compound 1-87 (1.00 g, 4.53 mmol) and compound 1-30 (1.22 g, 4.99 mmol) were dissolved in dry THF (40 mL) in a 250 mL rbf under N2. The flask was cooled in an ice/water bath. KHMDS (0.5 in toluene, 10.0 mL, 4.99 mmol) was added dropwise. The rxn mixture was stirred at 0°C for 15 min then RT for 1.5 h. The rxn mixture was cooled to 0°C and quenched with slow addition of sat'd NH4C1 aq soln (60 mL). The mixture was diluted with EtOAc (300 mL) and water (30 mL). The layers were separated and the organic layer was further washed with water (30 mL) and sat'd NaCl aq soln (30 mL). The organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash chromatography using 5-60% EtOAc in heptane as the gradient to afford compound 1-88.
Intermediate 1-89
Figure imgf000077_0002
1-88 1-89
[0174] 1-89: Compound 1-88 (200 mg, 0.449 mmol) was dissolved in THF (1 mL) in a 20- mL vial. To this soln was added 2 NaOH aq soln. The rxn mixture was heated at 65°C for 17 h. The mixture was neutralized with slow addition of IN HC1 (0.4 mL) to pH 7. Sat'd NaCl soln (10 mL) was added to the soln. Additional NaCl salt was also added until no more salt dissolved in the liquid phase. The mixture was extracted with EtOAc (3x50 mL). The combined organics was dried over anhydrous sodium sulfate, filtered, and concentrated to afford compound 1-89. -98
Figure imgf000078_0001
[0175] 1-94: 5-Bromo-6-chloronicotinic acid (2.00 g, 8.46 mmol) was heated in S0C12 (8.0 mL, 0.11 mol) at 80°C for 1 h. Excess of SOCl2 was removed under reduced pressure to provide compound 1-94.
[0176] 1-95: Compound 1-94 (2.20 g, 8.46 mmol) was heated in -PrOH (10 mL) at 80°C for 1 h. Excess of /-PrOH was removed under reduced pressure. The residue was purified by flash chromatography using 0-20% EtAOc in heptane as the gradient to afford compound I- 95.
[0177] 1-96: Compound 1-95 (2.5 g, 8.8 mmol) and /-PrOH (2.7 mL, 35 mmol) were dissolved in dry THF (25 mL) under N2. The flask was cooled to 0°C and l soln of KOtBu in THF (10.6 mL, 10.6 mmol) was added dropwise. The rxn was stirred for 1 h at 0°C. The rxn mixture were added sat'd aq NH4C1 soln (30 mL) and EtOAc (50 mL). The layers were separated and the aq phase was extracted with EtOAc (50 mL). The combined organics were washed with brine (10 mL), dried over Na2S04, filtered, and concentrated. The resulting residue was purified by flash chromatography using a 0-20% EtAOc in heptane as the gradient to provide compound 1-96.
[0178] 1-97: A mixture of 1-96 (1.00 g, 3.31 mmol), Zn(CN)2 (805 mg, 6.86 mmol), and Pd(PPh3)4 (115 mg, 0.100 mmol) in DMF (10 mL) was heated at 120°C for 3 h. The mixture was cooled to RT. The mixture was diluted with sat'd NaHC03 aq soln (15 mL) and extracted with EtOAc. The organic layer was washed with water and brine, dried over Na2S04, filtered and concentrated. The resulting residue was purified by flash chromatography using 0-30% EtAOc in heptane as the gradient to provide compound 1-97. [0179] 1-98: A soln of compound 1-97 (690 mg, 2.78 mmol), LiOH«H20 (233 mg, 5.56 mmol) in dioxane (15 mL) and H20 (5 mL) was stirred for 4 h. The solvent was removed under reduced pressure. Water and DCM were added. The mixture was acidified to pH = 2 with IN HCl aq soln. The layers were separated. The inorganic layer was extracted with DCM. The combined organics were washed with brine, dried over Na2S04, filtered and concentrated to afford compound 1-98. -103
Figure imgf000079_0001
1-88 1-103
[0180] 1-103: To the suspension of compound 1-88 (400 mg, 0.897 mmol) in dry MeOH (9 mL) in a dry 100-mL rbf was added hydrazine (0.29 mL, 9.0 mmol) under nitrogen. The rxn mixture was heated to 70°C and continued to stir at that temperature for 21 h. The temperature was then increased to 90°C and the rxn was continued to stir at that temperature for 24 h. The rxn mixture was concentrated. The crude pdt was dissolved in EtOAc (50 mL), washed with water (2x5 mL) and sat'd NaCl aq soln (5 mL), dried over anhydrous sodium sulfate, filtered, concentrated and further evaporated under high vacuum to afford compound 1-103.
Intermediate 1-107
Figure imgf000079_0002
1-107
[0181] 1-107: To a soln of ethyl 8-chloro-6-(trifluoromethyl)imidazo[l,2-a]pyridine-2- carboxylate (3.00 g, 10.3 mmol) in EtOH (20 mL) was added hydrazine (35% in H20, 4.6 mL, 51 mmol) at RT. The soln was heated under reflux for 16 h. The soln was then cooled down and water was added. The soln was extracted with EtOAc and the combined organics were dried over MgS04, filtered, and concentrated to afford compound 1-107. -109
Figure imgf000080_0001
[0182] 1-108: 2-Chloro-4,5-difluorobenzoic acid (1.59 g, 8.25 mmol), EDCI (1.58 g, 8.25 mmol), and HOBt (1.34 g, 9.91 mmol) were dissolved in DMF (30 mL). DIPEA was then added and the rxn was stirred for 15 min. Compound 1-107 (2.30 g, 8.26 mmol) was added and the rxn was stirred for 4 h. Water (5 mL) was added and stirred for 20 min. The mixture was filtered and the resulting solid was further washed with water and dried filter cake for 2 h on house vacuum to afford compound 1-108.
[0183] 1-109: Compound 1-108 (1.00 g, 2.21 mmol) and Lawesson's reagent (0.89 g, 2.21 mmol) were combined in DCE (10 mL) and heated at 150°C for 10 min in the microwave. DCM (100 mL) was added and the mixture was washed with sat'd ammonium chloride (2x100 mL). The organics was then concentrated. The resulting crude pdt was purified by normal phase flash column chromatography using 0-50% EtOAc in heptane as the gradient. Desired pdt was not observed in fractions. The filter cake on the loading cartridge was found to be compound 1-109.
Intermediate 1-112
Figure imgf000081_0001
[0184] 1-111: To a soln of compound 1-22 (2.21 g, 9.00 mmol) and 3,5-dichloro-4-hydroxy- benzoic acid methyl ester (2.00 g, 9.05 mmol) in THF (30 mL) was added PPh3 (5.24 g, 20.0 mmol) and di-tert-butylazo-dicarboxylate (4.15 g, 18.0 mmol) at RT. After 30 min the rxn was warmed up to 70°C. After 5 h the solvent was removed under reduced pressure. The residue was purified via Combi-flash column on silica gel (10-40% ethyl acetate in heptanes) to afford compound 1-111.
[0185] 1-112: A mixture of compound 1-111 (1.00 g, 2.23 mmol) and hydrazine (0.737 g, 23.0 mmol) in MeOH (10 mL) was heated to reflux and stirred at that temperature for 5 h. The rxn mixture was cooled to RT and concentrated. The resulting residue was diluted with water (50 mL) and extracted with EtOAc (2x100 mL). The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated to afford compound 1-112.
Intermediate 1-118
Figure imgf000081_0002
[0186] 1-116: 5-Bromo-4-methyl-pyridine-2-carboxylic acid methyl ester (800 mg, 3.48 mmol) was dissolved in DMF (16 mL). Tetrakis(triphenylphosphine)palladium(0) (462 mg, 0.400 mmol), potassium carbonate (2.07 g, 15.0 mmol), and 2,2-dimethylethenylboronic acid pinacol ester (0.82 mL, 4.0 mmol) were added. Argon was bubbled through the mixture for 5 min. The mixture was heated to 120°C in a microwave reactor for 1 h. The mixture was diluted with sat'd ammonium chloride (10 mL) then extracted with EtOAc (3x5 mL). The combined organics were dried over sodium sulfate and concentrated. The resulting crude material was purified by silica gel chromatagraphy using 0-40% EtOAc in heptanes as the gradient to provide compound 1-116.
[0187] 1-117: A soln of compound 1-116 (400 mg, 1.95 mmol) in MeOH (15 mL) was passed through the H-cube with Pd/C cartridge at 50°C, 50 bar for 2 h. The solvent was removed to afford compound 1-117.
[0188] 1-118: To a soln of compound 1-117 (320 mg, 1.54 mmol) in MeOH (8 mL) was added 2.0 NaOH aq soln (7.5 mL, 15 mmol) at RT. The rxn was heated to reflux for 1 h. The solvent was removed under reduced pressure and the resulting residue was acidified with 2N HCl to pH = 3. The solid was filtered and the filtrate was concentrated to afford compound 1-118.
Intermediate 1-129
Figure imgf000082_0001
1-128 "-1 29
[0189] 1-128: A degassed soln of methyl 2-chloro-6-methylpyrimidine-4-carboxylate (500 mg, 2.68 mmol), 2-methyl-l-propenylboronic acid pinacol ester (687 μί, 3.35 mmol), Pd(Ph3)4 (310 mg, 0.268 mmol), sodium carbonate (2 aq soln, 2.68 mL, 5.40 mmol) in DME (10 mL) was heated to 120°C under microwave radiation for 2 h. The rxn mixture was then cooled to RT and acidified with concentrated aq HCl to a final pH of 2. Minimal water was added (5 mL) and the mixture was extracted with EtOAc (3x25 mL). The combined organics were dried (Na2S04), decanted and concentrated. The resulting crude residue was purified via flash chromatography (Si02; 10-40% 5: 1 : 10 MeOH: AcOH: EtOAc in heptane) to afford compound 1-128. [0190] 1-129: Compound 1-128 (0.25 g, 1.3 mmol) was dissolved in MeOH (10 mL). To this soln were added 10 drops of AcOH followed by 10 wt.% Pd/C (40 mg, 0.38 mmol). The flask was evacuated and flushed three times with N2, then evacuated and filled with H2. The rxn was stirred under the H2 atmosphere for 2 h then filtered through a 1 cm thick pad of celite. The filtrate was concentrated to afford compound 1-129.
Intermediate 1-132
Figure imgf000083_0001
[0191] 1-131 and 1-132: Compound 518 (250 mg, 0.529 mmol) was dissolved in DMF (2 mL). To this soln was added imidazole (40 mg, 0.58 mmol) and TBSCI (88 mg, 0.58 mmol) and the rxn was stirred for 24 h. The rxn mixture was diluted with EtOAc (10 mL) and poured into sat'd aq ammonium chloride (10 mL). The aq phase was separated and extracted two more times with EtOAc. The organic layers were combined, dried (Na2S04), decanted and concentrated. The resulting residue was separated via flash chromatography (Si02, 10- 50% EtOAc-Heptane) to afford compound 1-131 and compound 1-132.
Intermediate 1-150
Figure imgf000084_0001
[0192] 1-16: DIPEA (6.3 mL, 36 mmol) was added to a soln of 2-chloro-4,5-difluoro-benzoic acid (3.50 g, 18.2 mmol) in DMF (30 mL) at RT. After 5 min HATU (7.60 g, 20.0 mmol) was added. After 5 min compound 1-15 (3.40 g, 16.3 mmol) was added and the rxn mixture was stired at RT for 20 h. The rxn was worked up and the crude was purified by combiflash, silica gel (120 g) eluting with 0-100% EtOAc in heptane to give compound 1-16.
[0193] 1-150: To a 150-mL dry sealable rxn tube was added Lawesson's reagent (2.32 g, 5.56 mmol). Under nitrogen, compound 1-16 (3.36 g, 4.85 mmol) dissolved in 2- methyltetrahydrofuran (60 mL) was added. The rxn soln was heated at 90°C for 20 h. The mixture was diluted with EtOAc (300 mL) and washed in turn with sat'd NaHC03 aq soln (50 mL), water (3x100 mL), and sat'd NaCl aq soln (10 mL). The organics were dried over anhydrous sodium sulfate, filtered, and concentrated to afford the crude pdt which was purified by normal phase flash chromatography using 5-25% EtOAc/heptanes as the gradient to afford compound 1-150 .
[0194] Alternatively, crude compound 1-150 can be purified by reverse phase HPLC using 20-100% ACN in water containing 0.1 % TFA as the gradient to afford compound 1-150 as a TFA salt.
Example 1
(2R,3S)-3-Amino-4-{[2,6-dichloro-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyridin-4- yl}-l,3,4-thiadiazol-2-yI)phenyl]oxy}butan-2-ol
Figure imgf000085_0001
[0195] Step 1: Ethyl 3,5-dichloro-4-fluorobenzoate (12). A mixture of 3,5-dichloro-4- fluorobenzoate 11 (10.0 g, 47.8 mmol), TMS- CI (60 mL) and EtOH (60 mL) was heated to reflux overnight. After cooling to RT, the reaction mixture was concentrated in vacuo. The resulting oil was partitioned between sat'd NaHC03 and diethyl ether. The phases were separated and the aqueous phase was further extracted with diethyl ether (2x). The combined diethyl ether extractions were washed with sat'd NaHC03, sat'd NaCl, dried (Na2S04), concentrated in vacuo, and dried under high vacuum overnight to give 12 (10.74 g, 95%). Ή NMR (400 MHz, OMSO-d6) δ 8.06 (d, 2H), 4.33(q, 2H), 1.33 (t, 3H).
[0196] Step 2: (4S,5R)-fert-Butyl 4-((2,6-dichloro-4-
(ethoxycarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3-carboxylate (13). To a mixture of compound 12 (1.45 g, 6.1 mmol), intermediate 7 (1.8 g, 7.3 mmol) and dry THF (18 mL) cooled in an ice bath was added 60% NaH (614 mg, 9.21 mmol) in portions over 30 min. Stirring in the ice bath was continued for another 30 min after which EtOH was added dropwise until bubbling ceased. The resulting reaction mixture was further diluted with sat'd NH4C1 and then extracted with EtOAc (3x). The combined EtOAc extractions were washed with water, dried (Na2S04) and concentrated in vacuo. The crude product was purified by flash chromatography (2% EtOAc in hexanes followed by 5% EtOAc in hexanes) to give compound 13 (1.44 g, 51%). Ή NMR (400 MHz, DMSO-i/6) δ 7.97 (d, 2H), 4.42-4.20 (comp m, 4H), 4.09 (m, 1H), 3.92 (m, 1H), 1.50-1.42 (comp m, 13H), 1.34-1.27 (comp m, 8H).
[0197] Step 3: (4S,5R)-ferf-ButyI 4-((2,6-dichloro-4-
(hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3-carboxylate (14). Compound 13 (1.44 g, 3.1 mmol) was dissolved in MeOH (10 mL) to which was added hydrazine (1.0 mL). The resulting mixture was heated to reflux for several hours until the reaction was complete as followed by LC-MS. The reaction mixture was allowed to cool to RT then concentrated in vacuo. The resulting oil was diluted with water and extracted with EtOAc (3x). The combined EtOAc extractions were washed with sat'd NaCl, dried (Na2S0 ), concentrated in vacuo and dried under high vacuum to give 14 (1.32 g, 95%). Ή NMR (400 MHz, DMSO-<¾ δ 9.95 (s, 1H), 7.94 (d, 2H), 4.57 (br s, 2H), 4.37 (m, 1H), 4.22 (m, 1H), 4.06 (m, 1H), 3.90 (m, 1H), 1.50-1.42 (comp m, 13H), 1.26 (s, 5H).
[0198] Step 4: (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-(2-(2-(isopropylamino)-6- methylisonicotinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (15). Intermediate 10 (563 mg, 2.9 mmol), HOBT (378 mg, 2.8 mmol), EDCI (600 mg, 3.13 mmol) and DMA (6.0 mL) were combined and stirred at RT. After 10 min, compound 14 (1.1 1 g, 2.47 mmol) was added and the resulting mixture stirred at RT for 2 h. The reaction mixture was diluted with sat'd NaHC03 and extracted with EtOAc (3x). The combined EtOAc extractions were washed with sat'd NaCl, dried (Na2S04) and concentrated in vacuo. The crude material was purified by flash chromatography (1 : 1 EtOAc:hexanes) to give compound 15 (1.08 g, 70%). Ή NMR (400 MHz, DMSO-<¾) 6 10.68 (s, 1H), 10.55 (s, 1H), 8.03 (d, 2H), 6.68 (d, 2H), 6.57 (d, 1H), 4.39 (m, 1H), 4.26 (m, 1H), 4.10-3.85 (comp m, 3H), 2.32 (s, 3H), 1.49-1.43 (comp m, 13H), 1.28 (s, 5H), 1.20-1.13 (comp m, 6H).
[0199] Step 5: (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-1 ,4-thiadiazol-2-yl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (16). Compound 15 (1.18 g, 1.9 mmol) dissolved in 2-methyltetrahydrofuran (10 mL) was heated to 80 °C in a sealed tube. Lawesson's reagent (482 mg, 1.19 mmol) was added and the resulting mixture was heated to 90 °C in a sealed tube with stirring overnight. Another aliquot of Lawesson's Reagent (186 mg) was added and the resulting mixture was stirred at 100 °C for another night. After cooling to RT, the reaction mixture was diluted with water and extracted with EtOAc (3x). The combined EtOAc extractions were washed with sat'd NaCl and concentrated in vacuo. The resulting crude material was purified by flash chromatography (20% EtOAc in hexanes followed by 20% EtOAc and 5% TEA in hexanes) to give compound 16 (703 mg, 60%). Ή NMR (400 MHz, DMSO-d6) δ 8.18 (d, 2H), 6.82 (d, 2H), 6.75 (d, 1H), 4.40 (m, 1H), 4.27 (m, 1H), 4.12-3.88 (comp m, 3H), 2.36 (s, 3H), 1.54- 1.43 (comp m, 13H), 1.30 (s, 6H), 1.16 (d, 6H).
[0200] Step 6: (2R,3S)-3-Amino-4-{[2,6-dichloro-4-(5-{2-methyl-6-[(l- methylethyl)amino]-pyridin-4-yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol
Compound 16 (703 mg, 1.13 mmol) was dissolved in a mixture of 4M HC1 in dioxane (30 mL) and MeOH (10 mL) and the resulting mixture was stirred at RT for 30 min. The reaction mixture was partitioned between water and diethyl ether. The phases were separated and the aqueous phase was further washed with diethyl ether. The aqueous phase was concentrated slightly in vacuo to remove any residual diethyl ether and then cooled in an ice bath. The pH was adjusted to 14 with the addition of 7.5N NaOH. The resulting basic aqueous mixture was extracted with EtOAc (3x). The combined EtOAc extractions were washed with sat'd NaCl, dried (Na2S04) and concentrated in vacuo. The resulting solid was suspended in a minimal amount of DCM to which was added hexanes with vigorous stirring. The resulting solids were filtered, washed with hexanes and dried under high vacuum to give the title compound (428 mg, 78%) as a yellow solid. Ή NMR (400 MHz, OMSO-d6) 6 8.14 (s, 2H), 6.81 (d, 2H), 6.74 (d, 1H), 4.61 (d, 1H), 4.20 (dd, 1H), 4.05 (m, 1H), 3.94 (t, 1H), 3.63 (m, 1H), 2.98 (m, 1H), 2.35 (s, 3H), 1.71 (br s, 2H), 1.16 (m, 9H); MS (EI) for C2iH25Cl2N502S, found 482.1 (MH+).
[0201] Using the same or analogous synthetic techniques as in Example 1 and substituting with appropriate reagents, the following compounds were prepared.
[0202] (2R,3S)-3-Amino-4-{[2-chloro-6-fluoro-4-(5-{2-methyl-6-[(l- methylethy aminolpyridin^-ylJ-ljS^-thiadiazol-l-ylJphenylJoxyJbutan- -ol
dihydrochloride salt. Ή NMR (400 MHz, OMSO-d6) δ 8.3 (bs, 2H), 8.1 (m, 2H), 7.4 (m, 1H), 7.2 (m, 1H), 4.5-4.4 (m, 2H), 4.2 (m, 1H), 4.1 (m, 1H), 3.5 (m, 1H), 2.6 (s, 3H), 1.2 (m, 9H); MS (EI) for C2iH25ClFN502S, found 466 (MH+).
[0203] (2R^S)-3-Amino-4-(4-(5-(2-(isopropylamino)-6-methylpyridin-4-yl)-l,3,4- thiadiazol-2-yl)-2,6-dimethylphenoxy)butan-2-ol dihydrochloride salt. Ή NMR (400 MHz, CD3OD) 6 7.82 (s, 2H), 7.45 (s, 1H), 7.28 (s, 1H), 4.20-4.00 (comp m, 4H), 3.65 (m, 1H), 2.61 (s, 3H), 2.42 (s, 6H), 1.40 (d, 6H), 1.30 (d, 3H); MS (EI) for C23H3iN502S, found 442.25 (MH+).
[0204] (2R,3S) -Amino-4<2-fluoro-4-(5-(2-(isopropylamino)-6-methylpyridin-4-yl)- l,3,4-thiadiazol-2-yl)-5-methylphenoxy)butan-2-ol. Ή NMR (400 MHz, CD3OD) δ 7.75 (d, 1H), 7.48 (s, 1H), 7.30 (m, 2H), 4.52 (m, 1H), 4.40 (m, 1H), 4.18 (m, 2H), 3.65 (m, 1H), 2.63 (s, 3H), 2.61 (s, 3H), 1.38 (m, 9H); MS (EI) for C22H28FN502S, found 446.15 (MH+).
Example 2
(R)-2-Amino-3-(2,6-dichIoro-4-(5-(2-(isopropylamino)-6-methylpyridin-4-yI)-l,3,4- -2-yI)phenoxy)propan-l-ol
Figure imgf000088_0001
[0205] Step 1: (S)-tert-Butyl 4-((2,6-dichloro-4-(ethoxycarbonyl)phenoxy)methyl)- 2,2-dimethyloxazolidine-3-carboxylate (17). Compound 17 was synthesized using the same or an analogous synthetic procedure to that of compound 13 in Example 1, substituting intermediate 2 for intermediate 7.
[0206] Step 2: (S)-fert-Butyl 4-((2,6-dichloro-4-
(hydrazinecarbonyl)phenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate (18).
Compound 18 was synthesized using the same or an analogous synthetic procedure to that of compound 14 in Example 1.
[0207] Step 3: (S)-te/-/-Butyl 4-((2,6-dichloro-4-(2-(2-(isopropylamino)-6- methyIisonicotinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (19). Compound 19 was synthesized from intermediates 18 and 10 using the same or an analogous synthetic procedure to that of compound 15 in Example 1. [0208] Step 4: (S)-terf-Butyl 4-((2,6-dichloro-4-(5-(2-(isopropylamino)-6- methyIpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (20). Compound 20 was synthesized using the same or an analogous synthetic procedure to that of compound 16 in Example 1.
[0209] Step 5: (R)-2-Amino-3-(2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)propan-l-ol. The title compound was synthesized from intermediate 20 using the same or an analogous synthetic procedure to that of (2R,3S)-3-amino-4-{[2,6-dichloro-4-(5-{2-methyl-6-[(l-methylethyl)amino]-pyridin-4- yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol in Example 1. Ή NMR (400 MHz, DMSO- d6) 6 8.13 (s, 2H), 6.82 (d, 2H), 6.74 (d, 1H), 4.67 (br s, 1H), 4.08 (m, 2H), 3.94 (m, 1H), 3.55-3.40 (comp m, 2H), 3.1 1 (m, 1H), 2.35 (s, 3H), 1.65 (br s, 2H), 1.16 (d, 6H); MS (EI) for C2oH23Cl2N502S, found 468.1 (MH+).
[0210] Using the same or analogous synthetic techniques as in Example 2 and substituting with appropriate reagents, the following compound was prepared.
[0211] (2R)-2-Amino-3-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}propan-l-ol. Ή NMR
(400 MHz, CD3OD) δ 8.3 (d, 1H), 7.6 (d, 1H), 7.5 (s, 1H), 7.3 (s, 1H), 4.5-4.4 (m, 2H), 4.1 (m, 1H), 4.0-3.8 (m, 2H), 3.8-3.7 (m, 1H), 2.6 (s, 3H), 1.4 (d, 6H); MS (EI) for C2oH23ClFN502S, found 452 (MH+).
Example 3
(2R,3R)-3-Amino-4- { [5-chloro-2-fluoro-4-(5- {2-methyl-6- [(1 - methylethyl)amino]pyridin-4-yI}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol
dihydrochloride salt
Figure imgf000089_0001
Figure imgf000090_0001
[0212] Step 1: Ethyl 2-chloro-4,5-difluorobenzoate (22). Compound 22 was synthesized using the same or an analogous synthetic procedure to that of compound 12 in Example 1.
[0213] Step 2: (4R,5R)-fer/-Butyl 4-((5-chloro-4-(ethoxycarbonyl)-2- fluorophenoxy)methyl)-2,2,5-trimethyloxazolidine-3-carboxyIate (24). Intermediate 23 was made in an analogous manner to intermediate 7 using the appropriate enantiomerically pure starting material in place of compound 3. Compound 24 was synthesized using the same or an analogous synthetic procedure to that of compound 13 in Example 1.
[0214] Step 3: (4R,5R)-terf-Butyl 4-((5-chloro-2-fluoro-4-
(hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3-carboxylate (25). Compound 25 was synthesized using the same or an analogous synthetic procedure to that of compound 14 in Example 1.
[0215] Step 4: (4R,5R)-terf-Butyl 4-((5-chloro-2-fluoro-4-(2-(2-(isopropylamino)-6- methylisonicotinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (26). To a stirred solution of compound 10 (1.7 g, 7.3 mmol) in DMF (10 mL) was added PyBop (3.82 g, 7.3 mmol) followed by addition of hydrazide 25 (2.65 g, 6.14 mmol) and triethylamine (1.86 g, 18.42 mmol) at RT. The reaction mixture was stirred at RT for 15 h. After completion, the reaction mixture was diluted with water (50 mL) and extracted with EtOAc. The combined organic layers were dried over anhydrous Na2S04 and concentrated in vacuo to afford crude 26 in quantitative yield which was used as such in the next step. [0216] Step 5: (4R,5R)-tert-Butyl 4-((5-chloro-2-fluoro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)methy^
carboxylate (27). To a stirred solution of 26 (4.25 g, 7.2 mmol) in toluene (30 mL) was added pyridine (1.1 mL) and Lawesson's reagent (4.078 g, 10.08 mmol) and the reaction mixture was stirred at 120 °C for 4 h. The reaction mixture was cooled to RT and solvent was removed in vacuo. To the resulting residue was added pyridine (20 mL) and phosphorous pentasulfide (1.59 g, 7.2 mmol) and the reaction mixture stirred at 120 °C for 3 h. Water was added and aqueous phase was extracted with EtOAc. The organic layer was dried, concentrated and purified by column chromatography to give 27 (1.3 g, 29.8%) which was used as such for the next step.
[0217] Step 6: (2R,3R)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol
dihydrochloride salt. To a stirred solution of 27 (1.3 g, 2.14 mmol) in ethanol (5 mL) was added ethanolic HC1 (15 mL) at 0 °C and reaction was stirred at RT for 1 h. The reaction mixture was concentrated in vacuo and the solid mass obtained was washed with diethyl ether and acetonitrile. The crude compound was purified by Prep HPLC and lyophilized. The resulting TFA salt was dissolved in ethanol to which was added ethanolic HC1 at 0 °C and the mixture stirred at RT for 45 min. The reaction mixture was concentrated in vacuo and the resulting solid was washed with diethyl ether and hexane to afford the title compound as the yellow dihydrochloride salt (0.9 g, 80.3%). Ή NMR (400 MHz, DMSO- 6) δ 8.4-8.2 (m, 3H), 7.7 (d, 1H), 7.5 (m, 1H), 7.2 (m, 1H), 4.5-4.3 (m, 2H), 4.2 (m, 1H), 4.0 (m, 1H), 3.4 (m, 1H), 2.6 (s, 3H), 1.2 (m, 9H); MS (EI) for C2iH25ClFN502S, found 466 (MH+).
Example 5
(R)-2-Amino-3-(2-chloro-4-(5-(2-(isopropylamino)-6-methylpyridin-4-yl)-l,3,4- -2-yl)-6-methylphenoxy)propan-l-ol
Figure imgf000091_0001
Figure imgf000092_0001
[0218] Step 1: 2-(Isopropylamino)-6-methylisonicotinohydrazide (33). Compound 33 is made from intermediate 9 using the same or an analogous synthetic procedure to that of compound 14 in Example 1.
[0219] Step 2: 2-Chloro-4-bromo-6-methylphenol (35). To a solution of 2-chloro-6- methylphenol 34 (5 g, 35 mmol) in AcOH (70 mL) was added NBS (6.2 g, 35 mmol). The solution was stirred at RT for 12 h. AcOH was removed under reduced pressure. The residue was diluted with EtOAc, washed with saturated Na2C03 solution and dried over Na2S04. Removal of the solvents gave compound 35 (4 g, 52%) which was used in the next step without further purification.
[0220] Step 3: (S)-tert-Butyl 4-((4-bromo-2-chloro-6-methyIphenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (36). To a solution of compound 35 (4 g, 18 mmol), intermediate 2 (4.8 g, 21.7 mmol) and triphenylphosphine (7.0 g, 27 mmol) in DCM (80mL) was added diisopropylazodicarboxylate (5.4 g, 27 mmol). The reaction mixture was stirred at RT for 3 h. Water was added and the product was extracted with DCM. Purification by flash column chromatography gave compound 36 (5.8 g, 73%).
[0221] Step 4: (S)-tert-B ty\ 4-((2-chloro-4-cyano-6-methyIphenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (37). Compound 36 (5.68 g, 13 mmol), Zn(CN)2 (920 mg, 7.8 mmol), Pd(dppf)Cl2 DCM (475 mg, 0.65 mmol) and polymethylhydrosiloxane (390 mg) were dissolved in DMA/water (30 mL/0.3 mL). The reaction mixture was stirred at 90 °C for 6 h. Water was added and the product was extracted with EtOAc. Concentration and purification by flash column chromatography gave compound 37 (4.5 g, 91%).
[0222] Step 5: (S)-4-((3-(/ert-Butoxycarbonyl)-2,2-dimethyloxazoIidin-4-yl)methoxy)-
3- chloro-5-methylbenzoic acid (38). To a solution of 37 (3.26 g, 8.6 mmol) in EtOH/water (15 mL/15 mL) was added NaOH (1.7 g, 42 mmol). The mixture was heated to 70 °C and stirred for 10 h. EtOH was removed in vacuo. The residue was dissolved in water (10 mL) and neutralized by the addition of concentrated HC1. The product was filtered and dried to give 38 (3 g, 87%).
[0223] Step 6: (S)-/ert-Butyl 4-((2-chIoro-4-(2-(2-(isopropylamino)-6- methylisonicotinoyl)-hydrazinecarbonyl)-6-tnethylphenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (39). Compound 39 was synthesized from intermediates 38 and 33 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
[0224] Step 7: (S)-te/"i-Butyl 4-((2-chloro-4-(5-(2-(isopropylamino)-6-methylpyridin-
4- yl)-l,354-thiadiazoI-2-yl)-6-methylphenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (40). Compound 40 was synthesized using the same or an analogous synthetic procedure to that of compound 16 in Example 1.
[0225] Step 8: (R)-2-Amino-3-(2-chloro-4-(5-(2-(isopropyIamino)-6-methylpyridin-4- yl)-l,3,4-thiadiazol-2-yl)-6-methylphenoxy)propan-l-ol. The title compound was synthesized from intermediate 40 using the same or an analogous synthetic procedure to that of (2R,3S)-3-amino-4-{[2,6-dichloro-4-(5-{2-methyl-6-[(l-methylethyl)amino]-pyridin-4- yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol in Example 1. Ή NMR (400 MHz, DMSO- d6) 6 7.95 (s, IH), 7.88 (s, IH), 6.83 (s, IH), 6.81 (s, IH), 6.72 (d, IH), 4.66 (t, IH), 4.06 (m, IH), 3.93 (m, IH), 3.81 (m, IH), 3.46 (m, 2H), 3.09 (m, IH), 2.39 (s, 3H), 2.35 (s, 3H), 1.77 (br s, 2H), 1.16 (d, 6H); MS (EI) for C2,H26C1N502S, found 447.9 (MH+).
Example 6
3-(5-Chloro-2-fluoro-4-(5-(2 isopropylamino)-6-methylpyridin-4-yl)-l,3,4-thiadiazol-2- yl)phenyl)azetidin-3-ol
Figure imgf000093_0001
Figure imgf000094_0001
[0226] Step 1: tert-Butyl 4-bromo-2-chloro-5-fluorobenzoate (42). A 200 niL round bottom flask was charged with 4-bromo-2-chloro-5-fluorobenzoic acid 41 (1.517 g, 5.99 mmol), tert-butyl dicarbonate (2.07 g, 9.49 mmol), DMAP (47 mg, 0.39 mmol) and anhydrous chloroform (80 mL). Triethyl amine (0.92 mL, 6.64 mmol) was added at 0 °C and the mixture was allowed to stir at RT for 16 h. The mixture was poured into water and the organic layer was separated and collected. The aqueous layer was extracted again with chloroform. The combined organic layers were dried over MgS04. After removal of solvents, the residual crude material was purified by silica gel chromatography (95:5 hexane/ethyl acetate) to afford 42 as a foamy solid (1.236 g, 4.00 mmol, 67%). Ή NMR (400 MHz, CDC13) δ 7.63 (d, J=6.1, 1H), 7.52 (d, J=8.5, 1H), 1.60 (s, 9H); MS (EI) for CnHnBrClFCb, found 251, 253 (MH-tBu+).
[0227] Step 2: Benzyl 3-(4-(ter/-butoxycarbonyl)-5-chloro-2-fluorophenyl)-3- hydroxyazetidine-l-carboxylate (43). A 100 mL two-neck round bottom flask was charged with tert-butyl 4-bromo-2-chloro-5-fluorobenzoate (42; 1.225 g, 3.96 mmol) and anhydrous THF (30 mL), under nitrogen atmosphere. After cooling the obtained solution to -18 °C (ice/salt bath), a 1.3 M solution of z'PrMgCl LiCl (3.4 mL, 4.4 mmol) was added via cannula. The mixture was allowed to stir at the same temperature for 90 min. A pre-cooled (-18 °C) solution of benzyl 3-oxoazetidine-l-carboxylate (freshly twice azeotroped in toluene; 871 mg, 4.24 mmol) in anhydrous THF (8 mL) was added via cannula. The cooling bath was removed within 5 min of completion of addition. The mixture was quenched 30 min later by pouring into a saturated solution of NH4C1 (70 mL). Extractions with ethyl acetate (2x 100 mL) were followed by a water (50 mL) and brine (40 mL) wash of the combined organic layers. The latter were dried over MgS04. After removal of solvents, the residual crude material was purified by silica gel chromatography (80:20 hexane/ethyl acetate to 70:30:0.5 20 hexane/ethyl acetate/methanol) to afford 43 as a foamy solid (1.475 g, 3.38 mmol, 85%). Ή NMR (400 MHz, CDC13) δ 7.50 (d, J=6.9, 1H), 7.43 (d, J=10.8, 1H), 7.37 - 7.24 (m, 5H), 5.07 (s, 2H), 4.67 (s, 1H), 4.42 (d, J=9.7, 2H), 4.19 (d, J=8.9, 2H), 1.59 (s, 9H); MS (EI) for C22H23C1FN05, found 436 (MH+).
[0228] Step 3: 4-[l-(Benzyloxycarbonyl)-3-hydroxyazetidin-3-yl]-2-chIoro-5- fluorobenzoic acid (44). Benzyl 3-(4-(rert-butoxycarbonyl)-5-chloro-2-fluorophenyl)-3- hydroxyazetidine-l-carboxylate (43, 1.475 g, 3.38 mmol) was treated with TFA (9 mL) in chloroform (40 mL) at 0 °C for 90 min, then at RT for 16 h. The mixture was concentrated under reduced pressure, then azeotroped with toluene (twice). The obtained solid material was dissolved in a 1 : 1 ACN/water mixture and freeze-dried to afford 44 as a fluffy, off-white solid (1.265 g, 3.33 mmol, 98%). Ή NMR (400 MHz, CD3OD) δ 7.63 (dd, J=18.3, 9.0, 2H), 7.41 - 7.30 (m, 5H), 5.13 (s, 2H), 4.48 (d, J=9.4, 2H), 4.20 (s, 2H); MS (EI) for Ci8H15ClFN05, found 378 ([M-H]").
[0229] Step 4: Benzyl 3-[5-chloro-2-fluoro-4-[2-[2-(isopropylamino)-6- methylisonicotinoyl]hydrazinecarbonyl]phenyl]-3-hydroxyazetidine-l-carboxylate (45).
[0230] Compound 39 was synthesized from intermediates 44 and 33 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
[0231] Step 5: Benzyl 3-(tert-butyldimethylsilyloxy)-3-[5-chloro-2-fluoro-4-[2-[2- (isopropylamino)-6-methylisonicotinoyl]hydrazinecarbonyl]phenyl]azetidine-l- carboxylate (46). To a solution of compound 45 (369 mg, 0.647 mmol) and 2,6-lutidine (0.19 mL, 1.6 mmol) in anhydrous DCM (25 mL), tert-butildimethylsilyltriflate (0.3 mL, 1.3 mmol) was added at 0 °C. The reaction mixture was allowed to gradually warm to RT. After 3 h, as reaction was not deemed complete, a second aliquot of reactants was added, at 0 °C, and then the mixture was stirred at RT. A third aliquot was added 16 h later, at 0 °C, for a total of 0.53 mL of lutidine (4.55 mmol) and 1.00 mL of triflate (4.35 mmol). After stirring for a further 16 h, the reaction was deemed complete by TLC. Upon removal of volatiles, the residual crude material was twice purified by silica gel chromatography (85:15:0.5 to 70:30: 1 hexanes/ethyl acetate/methanol, first chromatography; 95:5 to 90: 10 CH2C12/CH30H, second chromatography) to afford 46 as an oil, partially contaminated by siloxane by-products. Pure fractions underwent full characterization as follows. The obtained material was subjected to the next step without further treatment. Ή NMR (400 MHz, CD3OD) δ 7.55 - 7.50 (m, 2H), 7.40 - 7.30 (m, 5H), 6.84 (s, 1H), 6.72 (s, 1H), 5.13 (s, 2H), 4.51 (s, 2H), 4.28 (d, J=9.6, 2H), 3.87 (dt, J=12.8, 6.4, 1H), 2.41 (s, 3H), 1.26 (d, J=6.4, 6H), 0.91 - 0.89 (m, 9H), 0.02 (s, 6H); MS (EI) for C34H43ClFN505Si, found 684 (MH+).
[0232] Step 6: Benzyl 3-(teri-butyldimethylsilyloxy)-3-[5-chloro-2-fluoro-4-[5-[2- (isopropylamino)-6-methylpyridin-4-yl]-l,3,4-thiadiazol-2-yl]phenyl]azetidine-l- carboxylate (47). Compound 47 was synthesized using the same or an analogous synthetic procedure to that of compound 16 in Example 1.
[0233] Step 7: Benzyl 3-[5-chloro-2-fluoro-4-[5-[2-(isopropylamino)-6- methylpyridin-4-yI]-l,3)4-thiadiazol-2-yI]phenyl]-3-hydroxyazetidine-l-carboxylate (48). A solution of 47 (302 mg, 0.443 mmol) in anhydrous THF (16 mL) was treated with a 1 M solution of TBAF in THF (0.5 mL, 0.5 mmol) at RT for 20 h. After removal of solvents, the residual crude material was purified by silica gel chromatography (95:5 to 90: 10 CH2C12/CH30H) to afford 48 as a yellow solid (190 mg, 0.335 mmol, 76%). 1H NMR (400 MHz, CD3OD) δ 8.17 (d, J=1 1.6, 1H), 7.76 (d, J=7.1, 1H), 7.41 - 7.29 (m, 5H), 6.94 (d, J=0.7, 1H), 6.87 (s, 1H), 5.15 (s, 2H), 4.53 (d, J=9.4, 2H), 4.24 (s, 2H), 4.01 - 3.83 (m, 1H), 2.44 (s, 3H), 1.29 (d, J=6.4, 6H); MS (EI) for C28H27C1FN503S, found 568 (MH+).
[0234] Step 8: 3-(5-ChIoro-2-fluoro-4-(5-(2-(isopropylamino)-6-methylpyridin-4-yl)- l,3,4-thiadiazol-2-yl)phenyl)azetidin-3-ol. Compound 48 (140 mg, 0.246 mmol) was suspended in methanesulfonic acid (5 mL) at RT. The mixture was stirred and gently heated at 45 °C until all material went into solution. Reaction was deemed completed within 3 h. It was poured onto ice; solid K2C03 was slowly added under vigorous stirring until pH was corrected to about 10. The aqueous phase was extracted with EtOAc (2x); the combined organic layers were washed with brine and dried over MgS04. The aqueous phase was extracted with hot (60 °C) chloroform (3x); the combined organic layers were dried over MgS04. After removal of solvents, the residual crude material was purified by silica gel chromatography (90:9:1 CH2Cl2/CH3OH/28% (w/w) NH4OH) to afford the title compound as a yellow solid (96 mg, 0.222 mmol, 90%). Ή NMR (400 MHz, CD3OD) 6 8.17 (d, J=11.5, 1H), 7.69 (d, J=7.0, 1H), 6.95 (s, 1H), 6.87 (s, 1H), 4.23 (d, J=9.6, 2H), 3.99 - 3.89 (m, 1H), 3.86 (d, J=9.8, 2H), 3.35 (dt, J=3.3, 1.6, 1H), 2.44 (s, 3H), 1.29 (d, J=6.4, 6H); MS (EI) for C20H21C1FN5OS, found 434 (MH+). Example 8
(2R S)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{6-methyl-5-[(l-methylethyI)amino]pyridin- -yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol dihydrochloride salt
Figure imgf000097_0001
[0235] Step 1: 6-Chloro-2-methylpyridin-3-amine (56). To a stirred solution of 55 (5 g, 29 mmol) in EtOH (20 mL) and cone HC1 (20 mL) was added Fe powder (16.2 g, 289 mmole) in small portions at RT over 30 min. The resulting reaction mixture was stirred at RT for an additional 30 min. The solvent was removed in vacuo and water was added to the residue which was then neutralized with NaHC03 and diluted with EtOAc. The reaction mixture was filtered through Celite and washed with EtOAc. The filtrate was transferred to a separatory funnel and the phases separated. The organic layer was washed with water and brine, dried over Na2S0 and concentrated to afford 56 (4.1 g, 99 %) as a yellow solid.
[0236] Step 2: 6-ChIoro-N-isopropyl-2-methylpyridin-3-amine (57). To a stirred solution of 56 (4.81 g, 33.75 mmol) and acetone (2.74 g, 47.2mmol) in dichloroethane (60 mL) was added NaBH(OAc)3 (10.713 g, 50.53 mmol) and AcOH (3.44g, 57.2 mmol) at RT. The reaction was stirred for 16 h and then diluted with IN NaOH. The aqueous solution was extracted with DCM and the organic layer was washed with water and brine, dried over Na2S04 and concentrated to afford 57 (6.16 g, 98%).
[0237] Step 3: 5-(Isopropylamino)-6-methylpicolinonitrile (58). To a stirred solution of 57 (6.2 g, 33.5 mmol) in DMF (50 mL) was added Zn(CN)2 (5.5 g, 46.84 mmol) and tetrakis (5.8 g, 5.01mmol) at 25 °C. The reaction was then stirred at 130 °C for 16 h. After completion, the reaction mixture was absorbed on silical gel and chromatographed to obtain 58 (5 g, 85 %) as a thick liquid.
[0238] Step 4: 5-(Isopropylamino)-6-methylpicolinic acid (59). To a stirred solution of 58 (5.0 g, 28.4 mmol) in EtOH (40 mL) was added 20 % aqueous KOH (40 mL) and the reaction mixture was refluxed for 12 h. Volatiles were removed in vacuo and the resulting aqueous mixture was neutralized to pH 5 with citric acid solution which was then extracted with EtOAc. The organic layer was dried, concentrated and washed with pentane to afford 59 (2.8 g, 50%) as a white solid.
[0239] Step 5: (4S,5R)-tert-Butyl 4-((5-chloro-2-fluoro-4-(2-(5-(isopropylamino)-6- methylpicolinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (61). Intermediate 60 was made in an analogous manner to intermediate 14 using the appropriately substituted benzoic acid in place of compound 11. Compound 61 was synthesized from intermediates 60 and 59 using the same or an analogous synthetic procedure to that of compound 26 in Example 3.
[0240] Step 6: (4S,5R)-ter/-Butyl 4-((5-chloro-2-fluoro-4-(5-(5-(isopropylamino)-6- methylpyridin-2-yl)-1 >4-thiadiazol-2-yl)phenoxy)m
carboxylate (62). Compound 62 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
[0241] Step 7: (2R,3S)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{6-methyl-5-[(l- methylethyl)amino]pyridin-2-yl}-l,3,4-thiadiazol-2-yl)phenyI]oxy}butan-2-ol
dihydrochloride salt. The title compound was synthesized from intermediate 62 using the same or an analogous synthetic procedure to that of (2R,3R)-3-amino-4-{[5-chloro-2-fluoro- 4-(5- { 2-methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4-thiadiazol-2- yl)phenyl]oxy}butan-2-ol dihydrochloride salt in Example 3. Ή NMR (400 MHz, DMSO-<¾ 5 8.4 (bs, 2H), 8.1 (d, 1H), 8.0 (d, 1H), 7.7 (d, 1H), 7.1 (d, 1H), 4.5 (m, 1 H), 4.4 (m, 1H), 4.0 (m, 1H), 3.8 (m, 1H), 3.5 (m, 1H), 2.4 (s, 3H), 1.2 (m, 9H); MS (EI) for C2iH25ClFN502S, found 466 (MH+). Example 9
(2S,3S)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{6-methyl-4-[(l-methylethyl)amino]pyridin- 2- I}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol (..hydrochloride salt
Figure imgf000099_0001
reflux 48 67
Figure imgf000099_0002
[0242] Step 1: 6-Methyl-4-nitropicolinonitrile (64). A mixture of 4-nitro-2-picoline-N- oxide 63 (6g, 38 mmol) and dimethyl sulfate (5.3 g, 42 mmol) was heated to 65-70 °C for 2 h. After cooling, the resulting solid was filtered and washed with 30 ml of n-hexane. The solid was then dissolved in water (80 mL) and cooled to -10 °C under an atmosphere of N2. A solution of sodium cyanide (7.7 g, 152 mmol) in water (55 mL) was added dropwise over 40 min with stirring. The mixture was then stirred for an additional 3 hours at the same temperature. The reaction mixture was then diluted with a mixture of EtOAc (200 mL) and water (100 mL). After the mixture was stirred for 1 h, it was allowed to stand overnight. The organic layer was separated, washed with water, dried over Na2S04 and concentrated. The crude product was purified by column chromatography to afford 64 (7g, 99%) as a yellow solid. [0243] Step 2: 6-Methyl-4-nitropicolinic acid (65). A solution of 6-Methyl-4-nitro- pyridine-2-carbonitrile 64 (7 g, 42 mmol) in 90% sulfuric acid (40 mL) was heated at 120 °C for 2 h and then allowed to cool to RT. A solution of sodium nitrite (7.63 g, 113 mmol, 2.65 equiv.) in water (15 mL) was added dropwise over a period of 30 min maintaining the temperature between 12-15 °C. The reaction was stirred for 30 min at RT and then for 1 h at 80 °C. The solution was allowed to cool to RT and poured onto crushed ice. After stirring the yellow solution for 10 min, 200 ml of water was added. The resulting solid was filtered, washed with a small amount of water and dried in vacuo to yield 65 as a light yellow crystalline solid (7 g, 76%).
[0244] Step 3: 4-Bromo-6-methylpicolinic acid (66). A solution of 6-Methyl-4-nitro- pyridine-2-carboxylic acid 65 (6 g, 32.7 mmol) in 48% hydrobromic acid (65 mL) was heated at 100 °C overnight and then allowed to cool to RT. The solution was then evaporated to dryness in vacuo to give crude 66 contaminated with inorganic salts (10 g) which was directly used in the next step.
[0245] Step 4: 4-(Isopropylamino)-6-methylpicolinic acid (67). To a stirred solution of 4-bromo-6-methyl-pyridine-2-carboxylic acid 66 (10 g, 46.5 mmol) in butanol (120 mL) was added isopropylamine (8.2 g, 139 mmol) and the mixture was heated to 1 17 °C for 2 d. The solvent was removed in vacuo and the resulting residue was purified by column chromatography to afford 67 (5.0 g, 78% yield over two steps from 65) as a brown solid.
[0246] Step 5: (4S,5S)-terf-Butyl 4-((5-chloro-2-fluoro-4-(2-(4-(isopropylamino)-6- methylpicolinoyl)hydrazinecarbonyl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (69). Intermediate 68 was made in an analogous manner to intermediate 14 using the appropriately substituted benzoic acid in place of compound 11 and the appropriate enantiomerically pure analog of intermediate 7. Compound 69 was synthesized from intermediates 68 and 67 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
[0247] Step 6: (4S,5S)-terf-Butyl 4-((5-chloro-2-nuoro-4-(5-(4-(isopropylamino)-6- methylpyridin-2-yl)-1 ,4-thiadiazoI-2-yl)phenoxy)methyl)-2,2,5-trimethyloxazoIidine-3- carboxylate (70). Compound 70 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
[0248] Step 7: (2S,3S)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{6-methyI-4-[(l- methylethyl)amino]pyridin-2-yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol
dihydrochloride salt. The title compound was synthesized from intermediate 70 using the same or an analogous synthetic procedure to that of (2R,3R)-3-amino-4-{[5-chloro-2-fluoro- 4-(5- { 2-methyl-6- [( 1 -methy lethyl)amino]pyridin-4-yl } - 1 ,3 ,4-thiadiazol-2- yl)phenyl]oxy}butan-2-ol dihydrochloride salt in Example 3. Ή NMR (400 MHz, DMSO-<¾ 8 8.9 (m, IH), 8.4 (bs, 2H), 8.2 (d, IH), 7.7 (d, IH), 7.5 (m, IH), 6.8 (m, IH), 4.6-4.4 (m, 2H), 4.0 (m, 2H), 3.4 (m, IH), 2.6 (s, 3H), 1.2 (m, 9H); MS (EI) for C2iH25ClFN502S, found 466 (MH+).
[0249] Using the same or analogous synthetic techniques as in Example 9 and substituting with appropriate reagents, the following compound was prepared.
[0250] (2R,3S)-3-amino-4-{[5-chloro-2-nuoro-4-(5-{6-methyl-4-[(l- methylethyl)amino]pyridin-2-yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-oI
dihydrochloride salt. Ή NMR (400 MHz, CD3OD) δ 8.3 (d, IH), 7.6 (d, IH), 7.4-7.2 (m, IH), 6.9 (s, IH), 6.7 (s, IH, -NH), 4.6-4.4 (m, 2H), 4.2 (m, IH), 4.0 (m, IH), 3.7 (m, IH), 2.7-2.5 (m, 3H), 1.4 (m, 9H); MS (EI) for C2iH25ClFN502S, found 466 (MH+).
Example 10
(2R)-2-Amino-3-[(5-chloro-4-{5-[3-chloro-4-(oxetan-3-yloxy)phenyl]-l,3,4-thiadiazol-2- l}-2-fluorophenyl)oxy]propan-l-ol trifluoroacetate salt
Figure imgf000101_0001
Figure imgf000102_0001
[0251] Step 1: Methyl 4-(alIyloxy)-3-chlorobenzoate (72). To as stirred solution of methyl 3-chloro-4-hydroxybenzoate 71 (3.0 g, 16.12 mmol) in DMF (15 mL) was added allyl bromide (2.8 mL, 32.2 mmol) and K2C03 (4.4 g, 32.2 mmol) and the subsequent mixture heated to 80 °C for 3 h. After cooling to RT, the reaction mixture was diluted with water (200 mL) and extracted with EtOAc. The combined organic layers were dried over Na2S04 and concentrated to give 72 (3.4 g, 94.4%).
[0252] Step 2: 4-(Allyloxy)-3-chlorobenzoic acid (73). To a stirred solution of 72 (3.4 g, 15 mmol) in a mixture of ethanol (15 mL) and THF (15 mL) was added NaOH (0.66 g, 16.5 mmol) in water (10 mL), and the resulting mixture was stirred at RT for 1 h. The solvent was distilled off under reduced pressure. The resulting residue was diluted with water (100 mL), acidified with 2N HC1 and extracted with EtOAc. The organic phase was washed with water, brine, dried over Na2S04 and concentrated to afford 73 (2.6 g, 81.2 %).
[0253] Step 3: (S)-ferf-Butyl 4-((4-(2-(4-(allyloxy)-3- chlorobenzoyl)hydrazinecarbonyl)-5-chloro-2-fluorophenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (75). Intermediate 74 was made in an analogous manner to intermediate 18 using the appropriately substituted benzoic acid in place of compound 12. Compound 75 was synthesized from intermediates 74 and 73 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
[0254] Step 4: (S)-terf-Butyl 4-((4-(5-(4-(allyloxy)-3-chIorophenyl)-l,3,4-thiadiazoI-2- yl)-5-chloro-2-fluorophenoxy)methyI)-2,2-dimethyloxazolidine-3-carboxyIate (76).
Compound 76 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
[0255] Step 5: (S)-terf-Butyl 4-((5-chloro-4-(5-(3-chloro-4-hydroxyphenyl)-l,3,4- thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate (77).
To a solution of 76 (1.9 g, 3.1 mmol) in MeOH (30 mL) at RT was added Tetrakis (0.05 g, 0.046 mmol) and the reaction mixture purged with N2. K2C03 (1.3 g, 9.3 mmol) was added and again the reaction mixture was purged with N2 followed by stirring at 70 °C for 12 h. Solvent was removed in vacuo and the resulting residue was diluted with water and extracted with DCM. The organic layer was washed with brine, dried and concentrated to obtain 77 (1.0 g, 56%).
[0256] Step 6: (S)-tert-Butyl 4-((5-chloro-4-(5-(3-chIoro-4-(oxetan-3-yloxy)phenyl)- l,3,4-thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate (78). To as stirred solution of 77 (0.6 g, 1.0 mmol) in DMF (10 mL) was added 3- iodooxetane (0.3 g, 1.6 mmol) and K2C03 (0.3 g, 2.0 mmol) and the resulting mixture was heated to 90 °C for 14 h. After cooling to RT, the reaction mixture was diluted with water (75 mL) and extracted with DCM. The combined organic layers were dried over Na2S04 and concentrated to give 78 (0.6 g, 91%).
[0257] Step 7: (2R)-2-Amino-3-[(5-chloro-4-{5-[3-chloro-4-(oxetan-3-yloxy)phenyl]- l,3,4-thiadiazol-2-yl}-2-fluorophenyl)oxy]propan-l-ol trifluoroacetate salt. Compound 78 (0.6 g, 0.95 mmol) was dissolved in DCM (4 mL) and cooled to 0 °C. TFA/DCM (1 :1, 5 mL) was added and the resulting mixture stirred at 0 °C for 10 min and then at RT for 1 h. Solvent was removed under reduced pressure, and the compound was purified by preperative HPLC to give the title compound (0.15 g, 26%) as an off-white solid. Ή NMR (400 MHz, DMSO- ¾) δ 8.2 (m, 4H), 8.0 (d, 1H), 7.6 (d, 1H), 7.0 (d, 1H), 5.5 (m, 2H), 5.0 (m, 2H), 4.6 (m, 2H), 4.4 (m, 2H), 3.8-3.6 (m, 3H); MS (EI) for C20H,8Cl2FN3O4S, found 485.95 (MH+).
Example 11
4-[5-(4-{[(2R)-2-Amino-3-hydroxypropyl]oxy}-2-chloro-5-fluorophenyl)-l,3>4- thiadiazol-2-yI]-6-(oxetan-3-yloxy)pyridin-2-ol acetate salt
Figure imgf000103_0001
Figure imgf000104_0001
[0258] Step 1: 2-(Allyloxy)-6-chloroisonicotinic acid (80). KO'Bu (5.85 g, 52 mmol) was added to allyl alcohol (25 mL) and the resulting mixture was stirred for 5 min at RT. Compound 79 (5.0 g, 26 mmol) was added to the reaction mixture and it was heated for 20 h at 100 °C. After cooling to RT, the reaction mixture was concentrated in vacuo. The resulting residue was diluted with water, acidified with citric acid and extracted with diethyl ether. The organic layer was dried and concentrated to afford 80 (3.0 g, 54.5%) which was used as such for the next step.
[0259] Step 2: (S)-tert-Butyl 4-((4-(2-(2-(allyloxy)-6- chloroisonicotinoyl)hydrazinecarbonyl)-5-chloro-2-fluorophenoxy)methyl)-2,2- dimethyIoxazolidine-3-carboxylate (81). Compound 81 was synthesized from intermediates 74 and 80 using the same or an analogous synthetic procedure to that of compound 26 in Example 3.
[0260] Step 3: (S)-fert-Butyl 4-((4-(5-(2-(allyloxy)-6-chloropyridin-4-yI)-l,3,4- thiadiazol-2-yl)-5-chloro-2-fluorophenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (82). Compound 82 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
[0261] Step 4: (S)-ter/-ButyI 4-((5-chloro-4-(5-(2-chloro-6-hydroxypyridin-4-yl)- l,354-thiadiazol-2-yl)-2-fluorophenoxy)methyI)-2,2-dimethyloxazoIidine-3-carboxylate (83). Compound 83 was synthesized using the same or an analogous synthetic procedure to that of compound 77 in Example 10.
[0262] Step 5: (S)-fert-Butyl 4-((5-chloro-4-(5-(2-chloro-6-(oxetan-3-yloxy)pyridin-4- yl)-l,3i4-thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2-dimethyloxazolidine-3- carboxylate (84). Compound 84 was synthesized using the same or an analogous synthetic procedure to that of compound 78 in Example 10.
[0263] Step 6: (R)-2-Amino-3-(5-chloro-4-(5-(2-chloro-6-(oxetan-3-yloxy)pyridin-4- yl)-l,3,4-thiadiazol-2-yl)-2-fluorophenoxy)propan-l-ol trifluoroacetate salt (85).
Compound 85 was synthesized using the same or an analogous synthetic procedure to that of (2R)-2-amino-3 -[(5 -chloro-4- { 5 - [3 -chloro-4-(oxetan-3-yloxy)pheny 1]- 1 ,3 ,4-thiadiazol-2-yl } - 2-fluorophenyl)oxy]propan-l-ol trifluoroacetate salt in Example 10. Ή NMR (400 MHz, DMSO-i/6) δ 8.5 (s, 1H), 8.4 (s, 1 H), 8.2 (m, 3H), 7.7 (d, 1H), 5.7 (m, 1H), 5.0 (m, 2H), 4.8 (m, 2H), 4.4 (m, 2H), 4.0-3.6 (m, 4H); MS (EI) for C19Hi7Cl2FN404S, found 487 (MH+).
[0264] Step 7: 4-[5-(4-{[(2R)-2-Amino-3-hydroxypropyl]oxy}-2-chloro-5- fluorophenyl)-l,3,4-thiadiazol-2-yl]-6-(oxetan-3-yloxy)pyridin-2-ol acetate salt. The TFA salt 85 was found to slowly decompose to form the corresponding hydroxyl pyridine, which was isolated as follows. A solution of 85 in EtOAc was basified to pH 12 with IN NaOH aqueous solution. The organic phase was separated, washed with brine, dried with MgS04, and concentrated in vacuo. The residue was purified by prep HPLC eluting with a
MeCN/water/NH4OAc solvent system to give the title compound as a light yellow solid. Ή NMR (400 MHz, DMSO-^ plus a drop of D20) δ 8.08 (d, I S), 7.51 (d, 1H), 6.50 (d, 1H), 6.30 (d, 1H), 5.15 (m, 1H), 4.24 (dd, 1H), 4.14 (dd, 1H), 4.08-4.01 (m, 2H), 3.75 (m, 1H), 3.61 (dd, 1H), 3.51-3.41 (m, 2H), 3.14 (m, 1H), 1.81 (s, 3H, acetate); MS (EI) for
Ci9H18ClFN405S, found 469.1 (MH+).
Example 12
4-[5-(4-{[(2S,3S)-2-Amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4- thiadiazol-2-yl]-6-[(l-methylethyl)amino]pyridine-2-carbonitrile di-trifluoroacetate salt,
Ethyl 4-[5-(4-{[(2S,3S)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4- thiadiazol-2-yl]-6-[(l-methyIethyl)amino]pyridine-2-carboxylate di-trifluoroacetate salt, and 4-[5-(4-{[(2S,3S)-2-Amino-3-hydroxybutyl]oxy}-2-cyano-5-fluorophenyl)-l,3,4- thiadiazol-2-yl]-6-[(l-methyIethyl)amino]pyridine-2-carbonitrile di-trifluoroacetate salt
Figure imgf000106_0001
■\^NH 88
Figure imgf000106_0002
Figure imgf000107_0001
Figure imgf000107_0002
[0265] Step 1: Ethyl 2,6-dichloroisonicotinate (86). To an ice cold solution of 79 (10 g, 52 mmol) in EtOH (100 mL) was added cone H2S04 (5 mL) and the reaction mixture was heated to reflux for 12 h. Solvent was removed in vacuo. The resulting residue was cooled in an ice bath, neutralized with NaHC03 solution and extracted with EtOAc. The organic layer was dried and concentrated to afford 86 (1 1 g, 96%).
[0266] Step 2: Ethyl 2-chloro-6-(isopropylamino)isonicotinate (87). To a stirred solution of ester 86 (6.5.0 g, 29.5 mmol) in dry dioxane (60 mL), Cs2C03 (14.3 g, 44 mmol) and isopropylamine (6 mL, 70.8 mmol) was added. The mixture was degassed and kept under N2 atmosphere while BINAP (2.3 g, 3.6 mmol) and Pd(II) acetate (0.8 g, 3.6 mmol) were added. The reaction mixture was stirred in a sealed vessel at 80 °C for 3 h. The reaction mixture was cooled to RT, filtered and concentrated. The crude compound was purified by column chromatography to give ester 87 (5.5 g, 77%).
[0267] Step 3: 2-Chloro-6-(isopropylamino)isonicotinic acid (88). To a stirred solution of 87 (5.7 g, 2.35 mmol) in a mixture of THF (15 mL) and water (10 mL) was added NaOH (2.4 g, 6.12 mmol), and the resulting mixture was stirred at RT for 1 h. The solvent was distilled off under reduced pressure. Water (100 mL) was added to the resulting residue and the mixture acidified with citric acid and extracted with EtOAc. The organic phase was washed with water and brine, dried over Na2S04 and concentrated to afford 88 (5 g, 100 %).
[0268] Step 4: (4S,5S)- rf-Butyl 4-((5-chloro-4-(2-(2-chloro-6- (isopropylamino)isonicotinoyl)-hydrazinecarbonyl)-2-fluorophenoxy)methyl)-2,2,5- trimethyloxazolidine-3-carboxylate (89). Compound 89 was synthesized from intermediates 68 and 88 using the same or an analogous synthetic procedure to that of compound 26 in Example 3.
[0269] Step 5: (4S,5R)-terf-Butyl 4-((5-chloro-4-(5-(2-chloro-6- (isopropylamino)pyridin-4-yl)-l,3>4-thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2,5- trimethyIoxazolidine-3-carboxylate (90). Compound 90 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
[0270] Step 6: (4S,5S)-ter/-Butyl 4-((5-chloro-4-(5-(2-cyano-6- (isopropylamino)pyridin-4-yl)-l,3,4-thiadiazol-2-yl)-2-fluorophenoxy)methyl)-2,2,5- trimethyloxazolidine-3-carboxylate (91) and (4S,5S)-tert-Butyl 4-((5-cyano-4-(5-(2- cyano-6-(isopropylamino)pyridin-4-yl)-l,3,4-thiadiazol-2-yI)-2-fluorophenoxy)methyl)- 2,2,5-trimethyloxazolidine-3-carboxylate (92). To a stirred solution of 90 (0.2 g, 0.31 mmol) in DMF (2 mL) was added Zn(CN)2 (76 mg, 0.64 mmol) and tetrakis (46mg, 0.04 mmol) at 25 °C. The reaction was stirred at 100 °C for 1 h in a microwave. After completion, the reaction was cooled to RT and diluted with water. The aqueous layer was extracted with EtOAc, dried and concentrated to give a mixture of compounds 91 and 92. The crude mixture was purified by column chromatography to afford pure 91 (30mg, 15 %) and pure 92 (50 mg, 25%).
[0271] Step 7: 4-[5-(4-{[(2S,3S)-2-Amino-3-hydroxybutyl]oxy}-2-chloro-5- fluorophenyl)-1 ,4-thiadiazoI-2-yl]-6-[(l-methylethyl)amino]pyridine-2-carbonitrile di- trifluoroacetate salt and Ethyl 4-[5-(4-{[(2S,3S)-2-amino-3-hydroxybutyl]oxy}-2-chloro- 5-fluorophenyl)-l,3,4-thiadiazol-2-yl]-6-[(l-methylethyl)amino]pyridine-2-carboxylate di-trifluoroacetate salt. A ethanolic HC1 solution was added to compound 91 (0.15 g, 0.24 mmol) at 0 °C and reaction was stirred at 0 °C for 30 min. Solvent was removed in vacuo and the mixture was purified by Prep HPLC and lyophilized to afford the di-TFA salts of 4-[5-(4- {[(2S,3S)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l ,3,4-thiadiazol-2-yl]-6- [(l-methylethyl)amino]pyridine-2-carbonitrile (22 mg, 13%) and ethyl 4-[5-(4-{[(2S,3S)-2- amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2-yl]-6-[(l- methylethyl)amino]pyridine-2-carboxylate (30 mg, 16%) as yellow solids. 4-[5-(4-{[(2S,3S)- 2-Amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2-yl]-6-[(l- methylethyl)amino]pyridine-2-carbonitrile di-trifluoroacetate salt: Ή NMR (400 MHz, CD3OD) 6 8.2 (d, 1H), 7.5 (d, 1H), 7.4 (s, 1H), 7.2 (s, 1H), 4.5-4.3 (m, 2H), 4.1 (m, 2H), 3.5 (m, 1H), 1.4 (d, 3H), 1.2 (d, 6H); MS (EI) for C2,H22C1FN602S, found 477 (MH+). Ethyl 4- [5-(4-{[(2S,3S)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2- yl]-6-[(l-methylethyl)amino]pyridine-2-carboxylate di-trifluoroacetate salt: 1H NMR (400 MHz, CD3OD) δ 8.2 (d, IH), 7.8 (s, IH), 7.5 (d, IH), 7.3 (s, IH), 4.5-4.3 (m, 4H), 4.2 (m, IH), 4.1 (m, IH), 3.5 (m, IH), 1.5 (m, 3H), 1.4 (m, 3H), 1.3 (m, 6H); MS (EI) for
C23H27C1FN504S, found 524 (MH+).
[0272] Step 8: 4-[5-(4-{[(2S,3S)-2-Amino-3-hydroxybutyl]oxy}-2-cyano-5- fluorophenyl)-1 ,4-thiadiazol-2-yl]-6-[(l-methyIethyl)amino]pyridine-2-carbonitrile di- trifluoroacetate salt. Compound 92 (0.2 g, 0.32 mmol) was dissolved in DCM (4 mL) and cooled to 0 °C. TFA/DCM (3:7, 5 mL) was added and the resulting mixture stirred at 0 °C for 10 min and at RT for 1 h. Solvent was removed under reduced pressure, and the compound was purified by preperative HPLC to give the title compound (0.1 g, 44%) as a yellow solid. Ή NMR (400 MHz, CD3OD) δ 8.1 (d, IH), 7.8 (d, IH), 7.5 (s, IH), 7.3 (s, IH), 4.6-4.4 (m, 2H), 4.2-4.0 (m, 2H), 3.5 (m, IH), 1.4 (m, 3H), 1.2 (m, 6H); MS (EI) for C22H22FN702S, found 468 (MH+).
[0273] Using the same or analogous synthetic techniques as in Example 12 and substituting with appropriate reagents, the following compounds were prepared.
[0274] 4-[5-(4-{[(2S,3R)-2-Amino-3-hydroxybutyI]oxy}-2-chloro-5-fluorophenyl)- l,3,4-thiadiazol-2-yl]-6-[(l-methylethyl)amino]pyridine-2-carbonitrile. Ή NMR (400 MHz, CD3OD) δ 8.2 (d, IH), 7.5 (d, IH), 7.4 (s, IH), 7.2 (s, IH), 4.6-4.4 (m, 2H), 4.2-4.0 (m, 2H), 3.7 (m, IH), 1.4 (d, 3H), 1.2 (d, 6H); MS (EI) for C2,H22C1FN602S, found 477 (MH+).
[0275] 4-[5-(4-{[(2S,3R)-2-Amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)- l,3)4-thiadiazol-2-yl]-6-[(l-methylethyl)amino]pyridine-2-carbonitrile. Ή NMR (400 MHz, CD3OD) δ 8.1 (d, IH), 7.9 (d, IH), 7.5 (s, IH), 7.3 (s, IH), 4.6-4.4 (m, 2H), 4.2-4.1 (m, 2H), 3.7 (m, IH), 1.4 (m, 3H), 1.2 (m, 6H); MS (EI) for C22H22FN702S, found 468 (MH+). Example 13
5-Chloro-4-(5-{3-chloro-4-[(l-methylethyl)oxy]phenyl}-l,3,4-thiadiazol-2-yl)-2- fluoroaniline
Figure imgf000109_0001
Figure imgf000110_0001
[0276] Step 1: 3-Chloro-4-isopropoxybenzonitrile (94). A stirring suspension of 93 (20.0 g, 128 mmol) and NaH (6.17 g, 154 mmol) in THF (200 mL) was cooled to -10 °C and isopropyl alcohol (12.8 mL, 167 mmol) was added very slowly over a period of 1 h. The temperature of the reaction mixture was allowed to warm to 10 °C and stirred at that temperature for 2 h. The reaction mixture was then further cooled and quenched with ice. THF was removed under reduced pressure. The resulting residue was diluted with water and extracted with EtOAc. The organic layer was dried and concentrated to afford 94 (24 g, 95%) which was used as such for the next step.
[0277] Step 2: 3-Chloro-4-isopropoxybenzoic acid (95). To a stirring solution of 94 (57 g, 0.29 mol) in EtOH (120 mL) was added 12 % aqueous KOH (300 mL) and the reaction mixture was heated to 100 °C for 3 h. The reaction mixture was then cooled and acidified with HC1 to pH 5. The resulting yellow solid was filtered, washed water and air-dried. The resulting carboxylic acid was then suspended in hexane, stirred for 5 min, then the hexane was decanted off. The hexane wash was repeated, the resulting solids were filtered and washed with hexane to afford 95 (45 g, 72%) as a white solid.
[0278] Step 3: Methyl 3-chloro-4-isopropoxybenzoate (96). To an ice cold solution of 95 (20.0 g, 930 mmol) in MeOH (200 mL) was added cone H2S04 (20 mL) and the reaction mixture was heated to reflux for 3 h. Excess MeOH was removed in vacuo. The resulting residue was ice cooled and then neutralized with NaHC03 solution. The resulting aqueous mixture was extracted with EtOAc. The organic layer was dried and concentrated. The resulting crude compound was passed through a short column (silica 60-120, EtOAc:hexane 10:90) to obtain 96 (16.6 g, 78%).
[0279] Step 4: 3-Chloro-4-isopropoxybenzohydraz.de (97). Compound 97 is made from intermediate 96 using the same or an analogous synthetic procedure to that of compound 14 in Example 1.
[0280] Step 5: l-Bromo-2-chloro-5-fluoro-4-nitrobenzene (99). To a stirred solution of 98 (19.5 g, 93.10 mmol) in cone H2S0 (160 mL) at 0 °C was added KN03 (10.32 g, 102.41 mmol) in portions over 30 min. The resulting yellow solution was allowed to warm to RT and stirred overnight at RT. The solution was poured into ice and extracted with EtOAc. The organic phase was washed with water and brine, dried and concentrated to afford 99 (23 g, 97.66 %) as a white solid.
[0281] Step 6: 4-Bromo-5-chloro-2-fluoroaniline (100). To a stirred solution of 99 (23 g, 90.39 mmol) in EtOH (90 mL) and cone HC1 (90 mL) cooled to 0 °C was added Fe powder (47.52 g, 848.5 mmol) in small portions over 30 min. After addition was complete, the reaction mixture was stirred at RT for another 30 min. The solvent was distilled off under reduced pressure. Water was added to the resulting residue and the mixture neutralized with NaHC03 and diluted with EtOAc. The resulting biphasic reaction mixture was filtered through Celite and washed with EtOAc. The phases of the filtrate were separated and the organic layer was washed with water and brine solution, dried over Na2S04 and concentrated to afford 100 (18 g, 88.75 %) as a yellow solid.
[0282] Step 7: 4-Amino-2-chloro-5-fluorobenzonitrile (101). To a stirred solution of 100 (18 g, 80.19 mmol) in DMF (100 mL) was added cuprous cyanide (1 1.49 g, 128.2 mmol) and the resulting mixture was heated to 140 °C for 10 h. The reaction mixture was cooled to RT and and DMF was evaporated under reduced pressure. The resulting residue was partitioned between DCM and water and the resulting biphasic mixture was filtered. The phases of the filtrate were separated and the organic layer was washed with brine, dried over Na2S04 and concentrated in vacuo. The crude compound was purified by column
chromatography (4% EtOAc/hexane) to afford 101 (7 g, 51.20%) as a white solid.
[0283] Step 8: 2-Chloro-4-(diallylamino)-5-fluorobenzonitrile (102). To an ice cooled solution of 101 (6.0 g, 35.17 mmol) in DMF (60 mL) was added NaH (3.0 g, 73.86 mmol) and the reaction mixture was stirred at the same temperature for 30 min. Allyl bromide (6.38 g, 52.76 mmol) was added and reaction was further stirred at the same temperature for 2 h and was then quenched with ice with stirring for 15 min at 0 °C. The resulting mixture was neutralized with citric acid solution and extracted with EtOAc, dried and concentrated to obtain 102 (6.0 g, 68 %) which was used as such in the next step.
[0284] Step 9: 2-Chloro-4-(diallylamino)-5-fluorobenzoic acid (103). To a stirred solution of 102 (6.0 g, 23.93 mmol) in EtOH (60 mL) was added 20 % aqueous KOH (60 mL) and the reaction mixture was refluxed for 4 h. Solvent was removed in vacuo and the resulting residue was neutralized to pH 5 with citric acid solution. The resulting aqueous mixture was extracted with EtOAc. The organic layer was dried and concentrated and the resulting solid washed with pentane to afford 103 (5.5 g, 85.27%) as a white solid.
[0285] Step 10: 2-Chloro-N,-(3-chloro-4-isopropoxybenzoyl)-4-(diallylamino)-5- fluorobenzohydrazide (104). Compound 104 was synthesized from intermediates 103 and 97 using the same or an analogous synthetic procedure to that of compound 15 in Example 1.
[0286] Step 11 : N,N-Diallyl-5-chloro-4-(5-(3-chloro-4-isopropoxy pheny 1)-1 ,3,4- thiadiazol-2-yl)-2-fluoroaniline (105). Compound 105 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
[0287] Step 12: 5-Chloro-4-(5-{3-chloro-4-[(l-methylethyl)oxy]phenyl}-l,3,4- thiadiazol-2-yl)-2-fluoroaniIine. A stirred solution of 105 (3.0 g, 6.27 mmol), 1,3-dimethyl barbituric acid (3.92 g, 25.08 mmol), Pd(OAc)2 (1.40 g, 6.27 mmol) and triphenylphosphine (1.64 g, 6.27 mmol) in EtOH (30 mL) was purged with argon for 20 min. The reaction mixture was then heated to 85 °C for 2 h. After cooling, the reaction mixture was filtered through Celite. The filtrate was concentrated and the resulting residue was diluted water then extracted with EtOAc. The organic layer was dried, concentrated and the crude material obtained purified by column chromatography. The resulting yellow solid was washed with diethyl ether, filtered and dried to afford the title compound (0.320 g, 12.85%). Ή NMR (400 MHz, DMSO-i¾ δ 8.1 (s, 1H), 7.9 (m, 2H), 7.3 (d, 1H), 7.0 (d, 1H), 6.2 (m, 2H), 4.8 (m, 1H), 1.4 (m, 6H); MS (EI) for C17H14C12FN30S, found 397.90 (MH+).
[0288] Using the same or analogous synthetic techniques as in Example 13 and substituting with appropriate reagents, the following compound was prepared.
[0289] 2,5-Dichloro-4-(5-{3-chIoro-4-[(l-methylethyl)oxy]phenyl}-l,3,4-thiadiazol-2- yl)aniline. Ή NMR (400 MHz, OMSO-d6) δ 8.1 (m, 2H), 8.0 (d, 1H), 7.4 (d, 1H), 7.0 (s, 1H), 6.4 (bs, 2H), 4.8 (m, 1H), 1.4 (d, 6H); MS (EI) for C,7Hi4Cl3N3OS, found 413.85 (MH+).
Example 14 5-(5-(4-((2S,3R)-2-Amino-3-hydroxybutoxy)-3,5-dimethylphenyl)-l,354-thiadiazol-2-yl)- -(isopropylamino)nicotinonitrile
Figure imgf000113_0001
[0290] Steps 1-3: 5-Bromo-6-(isopropylamino)nicotinic acid (109). Intermediate 109 can be prepared by one skilled in the art from commercially available 106 in three steps using the standard procedures outlined in the scheme above.
[0291] Step 4: (4S,5R)-terf-Butyl 4-((4-(2-(5-bromo-6-
(isopropylamino)nicotinoyl)hydrazine-carbonyl)-2,6-dimethylphenoxy)methyl)-2,2,5- trimethyloxazolidine-3-carboxylate (111). Intermediate 110 was made in an analogous manner to intermediate 14 using the appropriately substituted benzoic acid in place of compound 11. Compound 111 was synthesized from intermediates 110 and 109 using the same or an analogous synthetic procedure to that of compound 26 in Example 3. [0292] Step 5: (4S,5R)-ferf-Butyl 4-((4-(5-(5-bromo-6-(isopropylamino)pyridin-3-yl)- 1 j4-thiadiazol-2-yl)-2,6-dimethylphenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (112). Compound 112 was synthesized using the same or an analogous synthetic procedure to that of compound 27 in Example 3.
[0293] Step 6: (4S,5R)-/er/-Butyl 4-((4-(5-(5-cyano-6-(isopropylamino)pyridin-3-yI)- l,3,4-thiadiazol-2-yl)-2,6-dimethylphenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (113). Intermediate 113 can be prepared by one skilled in the art from intermediate 112 using standard procedures. Typically, intermediate 112 is dissolved in an appropriate solvent such as DMF and heated in the presence of a palladium catalyst and an appropriate cyanide donor such as KCN.
[0294] Step 7: 5-(5-(4-((2S,3R)-2-Amino-3-hydroxybutoxy)-3,5-dimethylphenyl)- l,3,4-thiadiazol-2-yl)-2-(isopropylamino)nicotinonitrile ditrifluoroacetate salt. The title compound was synthesized from intermediate 113 using the same or an analogous synthetic procedure to that of (2R,3R)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol
dihydrochloride salt in Example 3. Ή NMR (400 MHz, DMSO-t¼) δ 8.85 (s, 1H), 8.37 (s, 1H), 7.72 (s, 2H), 4.45 (m, 1H), 4.15 (m, 2H), 4.05 (m, 1H), 3.62 (m, 1H), 2.40 (s, 6H), 1.28 (m, 9H); MS (EI) for C23H28N602S, found 453.20 (MH+).
[0295] Using the same or analogous synthetic techniques as in Example 14 and substituting with appropriate reagents, the following compound was prepared.
[0296] 5-(5-(4-((2S,3R)-2-Amino-3-hydroxybutoxy)-3,5-dichlorophenyl)-l,3,4- thiadiazol-2-yl)-2-(isopropylamino)nicotinonitrile ditrifluoroacetate salt.. Ή NMR (400 MHz, DMSO-4 δ 8.87 (s, 1H), 8.39 (s, 1H), 8.10 (s, 2H), 4.50 (m, 1H), 4.40 (m, 2H), 4.19 (m, 1H), 3.70 (m, 1H), 1.30 (m, 9H); MS (EI) for C2iH22Cl2N602S, found 493.10 (MH+). Example 15
(S)-2-Amino-3-(2,6-dichloro-4-(5-(2-(isopropylamino)-6-methylpyridin-4-yI)-l,3>4- thiadiazol-2- l)phenoxy)propyl dihydrogen phosphate
Figure imgf000114_0001
Figure imgf000115_0001
[0297] Step 1: (R)- r/-Butyl l-(2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)-3-hydroxypropan-2-ylcarbamate (114). (2R)-2-Amino-3-{[2,6-dichloro-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyridin-4- yl}-l ,3,4-thiadiazol-2-yl)phenyl]oxy}propan-l-ol (345 mg, 0.736 mmol), prepared as described in Example 2, was dissolved in dioxane (7.5 mL) to which was added triethylamine (0.3 mL, 2.22 mmol) and boc anhydride (325 mg, 1.49 mmol). The resulting reaction mixture was stirred at RT for 3 h, after which time it was diluted with water and extracted with EtOAc (3x). The combined organic extractions were washed with sat'd NaCl (lx), dried (Na2S04), concentrated in vacuo and dried under high vacuum overnight. The resulting oil was dissolved in a minimal amount of EtOAc to which was added hexanes with vigorous stirring. The resulting solids were filtered, washed with hexanes and air-dried to give 114 (306 mg, 73%). Ή NMR (400 MHz, DMSO-i¾) δ 8.15 (s, 2H), 6.83 (d, 2H), 6.79 (d, 1H), 6.74 (d, 1H), 4.81 (t, 1H), 4.20 (m, 1H). 4.05 (m, 2H), 3.83 (m, 1H), 3.52 (m, 2H), 2.35 (s, 3H), 1.47 (s, 9H), 1.16 (d, 6H); MS (EI) for C25H3iCl2N504S, found 568.2 (MH+).
[0298] Step 2: (S)-tert-Butyl l-(di-te/-/-butoxyphosphoryloxy)-3-(2,6-dichloro-4-(5-(2- (isopropylamino)-6-methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)propan-2- ylcarbamate (115). Compound 114 (306 mg, 0.54 mmol) was dissolved in a 3% solution of tetrazole in acetonitrile (10.5 mL). Di-/ert-butyl diethylphosphoramidite (602 mg, 2.41 mmol) was added and the resulting mixture was stirred at RT overnight. The reaction mixture was then cooled in an ice bath and w-chloroperbenzoic acid (461 mg, 2.67 mmol) was added with continued stirring in the ice bath for 2 h. The reaction mixture was then diluted with EtOAc and washed with sat'd NaHC03 (3x), sat'd NaCl (lx), dried (Na2S04) and
concentrated in vacuo. The crude residue was purified by column chromatography (25% EtOAc in hexanes followed by 25% EtOAc, 2% TEA in hexanes) to give 115 (189 mg, 46%). Ή NMR (400 MHz, OMSO-d6) δ 8.13 (s, 2H), 7.05 (d, 1H), 6.80 (d, 2H), 6.73 (d, 1H), 4.15- 3.95 (comp m, 6H), 2.33 (s, 3H), 1.40 (s, 18H), 1.37 (s, 9H), 1.14 (d, 6H); MS (EI) for C33H48C12N507PS, found 760.2 (MH+).
[0299] Step 3: (S)-2-Amino-3-(2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,3,4-thiadiazol-2-yl)phenoxy)propyl dihydrogen phosphate.
Compound 115 (189 mg, 0.25 mmol) was dissolved in a minimal amount of DCM to which was added 4 M HC1 in dioxane (5.0 mL). The resulting mixture was stirred at RT until the reaction was deemed complete by LC-MS. The reaction mixture was diluted with a mixture of acetonitrile and diethyl ether and vigorously stirred and sonicated for several minutes. The resulting solids were filtered, washed with diethyl ether and dried under high vacuum for several hours. The dried solids were dissolved in a mixture of water and acetonitrile and lyophilized to give the title compound (109 mg, 69%). Ή NMR (400 MHz, CF3C02D) δ 8.15 (br s, 2H), 7.60 (br s, 1H), 7.23 (br s, 1H), 4.90-4.45 (comp m, 5H), 4.15 (m, 1H), 2.72 (s, 3H), 1.48 (d, 6H); MS (EI) for C2oH24Cl2N505PS, found 548.0 (MH+).
[0300] Using the same or analogous synthetic techniques as in Example 15 and substituting with appropriate reagents, the following compounds were prepared.
[0301] (lR,2S)-2-Amino-3-{[5-chloro-2-fluoro-4-(5-{6-methyl-5-[(l- methylethyl)amino]pyridin-2-yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}-l-methylpropyl dihydrogen phosphate dihydrochloride salt. Ή NMR (400 MHz, DMSO-i¾) 6 8.5 (bs, 2H, -NH2), 8.1 (d, 1H), 8.0 (d, 1H), 7.7 (d, 1H), 7.1 (d, 1H), 4.6 (m, 1H), 4.5 (m, 1H), 4.4 (m, 1H), 3.7 (m, 2H), 2.4 (s, 3H), 1.4 (d, 3H), 1.2 (d, 6H); MS (EI) for C2iH26ClFN505PS, found 546 (MH+).
[0302] (lS,2S)-2-Amino-3-{[5-chloro-2-fluoro-4-(5-{6-methyl-5-[(l- methylethyl)amino]pyridin-2-yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}-l-methylpropyI dihydrogen phosphate dihydrochloride salt. Ή NMR (400 MHz, DMSO-i¾) δ 8.4 (bs, 2H, -NH2), 8.1 (d, 1H), 8.0 (d, 1H), 7.6 (d, 1H), 7.0 (d, 1H), 4.6 (m, 1H), 4.5-4.3 (m, 2H), 3.7 (m, 2H), 2.4 (s, 3H), 1.4 (d, 3H), 1.2 (d, 6H); MS (EI) for C2,H26C1FN505PS, found 546 (MH+).
[0303] (lR,2S)-2-Amino-3-{[2,6-dichloro-4-(5-{2-methyl-6-[(l- methylethyI)amino]pyridin-4-yl}-l,3,4-thiadiazoI-2-yl)phenyl]oxy}-l-methylpropyl dihydrogen phosphate dihydrochloride salt. Ή NMR (400 MHz, CF3C02D) δ 8.0 (m, 2H), 7.5 (m, 1H), 7.2 (m, 1H), 5.2 (m, 1H, -NH), 4.7-4.5 (m, 2H), 4.3 (m, 1H), 4.1 (m, 1H), 3.3 (m, 1H), 2.6 (s, 3H), 1.6 (m, 3H), 1.4 (m, 6H); MS (EI) for C21H26C12N505PS, found 562 (MH+).
[0304] (2S)-2-Amino-3-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,3,4-thiadiazol-2-yl)phenyl]oxy}propyl dihydrogen phosphate dihydrochloride salt. Ή NMR (400 MHz, CF3C02D) 6 8.1 (m, 1H), 7.5 (m, 1H), 7.4 (m, 1H), 7.2 (m, 1H), 4.8-4.6 (m, 3H), 4.4 (m, 1H), 4.1 (m, 1H), 3.3 (m, 1H), 2.6 (s, 3H), 1.4 (m, 6H); MS (EI) for C20H24ClFN5O5PS, found 532 (MH+).
[0305] (lR,2S)-2-Amino-3-{[2-chloro-6-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,3i4-thiadiazol-2-yl)phenyl]oxy}-l-methylpropyI dihydrogen phosphate dihydrochloride salt. Ή NMR (400 MHz, CF3C02D) δ 8.0 (m, 1H), 7.9 (m, 1H), 7.6 (m, 1H), 7.2 (m, 1H), 5.3 (m, 1H), 4.9 (m, 1H), 4.7 (m, 1H), 4.3 (m, 1H), 4.2 (m, 1H), 2.7 (m, 3H), 1.7 (m, 3H), 1.5 (m, 6H); MS (EI) for C2,H26C1FN505PS, found 546 (MH+).
[0306] (lR,2S)-2-Amino-3-{[5-chloro-2-fluoro-4-(5-{6-methyl-4-[(l- methylethyl)amino]pyridin-2-yl}-l,3j4-thiadiazol-2-yl)phenyl]oxy}-l-methylpropyl dihydrogen phosphate trifluoroacetate salt. Ή NMR (400 MHz, CD3OD) δ 8.3 (d, 1H), 7.6 (d, 1H), 7.3 (s, 1H), 6.8 (s, 1H), 4.6 (m, 2H), 4.4 (m, 1H), 4.0 (m, 1H), 3.8 (m, 1H), 2.6 (s, 3H), 1.5 (d, 3H), 1.3 (m, 6H); MS (EI) for C2iH26ClFN505PS, found 546 (MH+).
Example 16
5-{5-[4-((R)-2-Amino-3-hydroxy-propoxy)-5-fluoro-2-methyl-phenyl]-[l,3i4]thiadiazol-
2-yl}-2-isopropoxy-benzonitrile
Figure imgf000117_0001
[0307] To a stirred soln of 3-cyano-4-isopropoxy-benzoic acid (100 mg, 0.487 mmol) in DMF (4 mL) was added EDCI (89 mg, 0.47 mmol) and HOBt (63 mg, 0.47 mmol) at RT. After 2 h compound 1-49 (194 mg, 0.487 mmol) was added. After 17 h, the rxn was quenched with sat'd NaHC03 aq soln (15 mL). The mixture was extracted with EtOAc (2x15 mL). The combined organics were washed with brine, dried over sodium sulfate and concentrated. The resulting residue was purified via Combiflash on silica gel (30- 50% ethyl acetate in heptanes) to afford compound 1-51.
[0308] A soln of compound 1-51 (200 mg, 0.342 mmol) and Lawesson's reagent (162 mg, 0.400 mmol) in 2-methyltetrahydrofuran (4 mL) was warmed to 90°C in a sealed tube. After 18 h, the solvent was removed under reduced pressure. The residue was purified via Combiflash column (10-30% EtOAc in heptane) to afford compound 1-52.
[0309] A soln of compound 1-52 (70 mg, 0.12 mmol) in HC1 (4N in 1 ,4-dioxane) was stirred at RT for 17 h. The volatiles were removed under reduced pressure. The residue was basifed with sat'd NaHC03 soln (10 mL) and the mixture was extracted with EtOAc (3x10 mL). The combined organics were washed with brine, dried over sodium sulfate and concentrated. The resulting solid was triturated with ether to afford the title compound.
[0310] The following compounds were synthesized in a similar fashion from the appropriate intermediates:
Figure imgf000118_0001
Figure imgf000119_0001
Example 17
l-{5-Chloro-4-[5-(3-chloro-4-isopropoxy-phenyl)-[l,3,4]thiadiazol-2-yl]-2-fluoro- phenoxymethyl}-2,2,2-trifluoro-ethylamine
Figure imgf000120_0001
[0311] To compound 1-13 (204 mg, 0.290 mmol) in DMF (8 mL) was added thiophenol (75 uL, 0.73 mmol) and K2C03 (977 mg, 708 mmol). After 18 h, additional thiophenol (225 uL, 2.19 mmol) was added and the mixture was warmed to 65 °C. After 3 h, the rxn was cooled and diluted with sat'd sodium bicarbonate aq soln (30 mL) and extracted with EtOAc (3x15 mL). The combined organics were washed with sat'd sodium bicarbonate aq soln (3x10 mL), brine (10 mL), dried over magnesium sulfate, filtered and concentrated. The resulting crude material was purified on silica gel using a gradient of 0-1% methanol in DCM. Material from the column was triturated with ether and then recrystallized from DCM-hexanes to afford the title compound.
Example 18
(R)-2- Amino-4- {5-chloro-4- [5-(3-chIoro-4-isopropoxy-phenyl)- [1 ,3,4] thiadiazol-2-yI] -2- -phenoxy}-butan-l-ol
Figure imgf000121_0001
[0312] Compound 1-89 (86.0 mg, 0.206 mmol) and compound 1-38 (60.1 mg, 0.227 mmol) were dissolved in DMF (2.0 mL). TEA (100 μΐ,, 0.721 mmol) was added followed by TBTU (82.1 mg, 0.247 mmol). The rxn soln was stirred at RT overnight. The mixture was diluted with EtOAc (30 mL) and washed in turn with water (3x10 mL) and sat'd NaCl aq soln (5 mL). The organics were dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash chromatography using 10-100% EtO Ac/heptane as the gradient to afford compound 1-90.
[0313] To a dry microwave rxn vial was added Lawesson's reagent (43.6 mg, 0.105 mmol). Under nitrogen, compound 1-90 (98 mg, 0.156 mmol) dissolved in 2-methyltetrahydrofuran (2 mL) was added. The rxn soln was heated at 90°C for 16 h. The volatiles were evaporated under reduced presuure. The resulting crude solid was dissolved in EtOAc (40 mL). Sat'd NH4C1 aq soln (5 mL) was added and the NH4C1 solid precipitated. Water (5 mL) was added and the solid dissolved in the aq phase. The layers were separated. The organic layer was further washed with sat'd NaHC03 aq soln (5 mL) and sat'd NaCl aq soln (3 mL). The organics were dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash column chromatography using 5-50% EtOAc in heptane as the gradient to afford compound 1-92. [0314] Compound 1-92 (49 mg, 78 μπιοΐ) was dissolved in DCM (0.5 mL) in a 50-mL rbf. While stirring, 4 HC1 in 1 ,4-dioxane (0.50 mL, 2.0 mmol) was added. The rxn mixture was stirred at RT for 4 h. A few drops of MeOH was added and continued to stir at RT overnight. Diethyl ether anhydrous (30 mL) was added and stirred at RT overnight. The rxn mixture was filtered through a Buchner funnel. The solid was further washed with ether (5x10 mL). The pdt was dissolved in MeOH (2.8 mL) and water (0.2 mL) and filtered, then purified by reverse phase HPLC (5-95% ACN in water containing 0.1% TFA as the gradient, 20 min run). The pdt fractions were collected and basified with IA K2CO3 aq soln (0.8 mL) to pH = 8.5. The soln was then extracted with EtOAc (100 mL). The organic layer was washed with sat'd NaCl aq soln (10 mL). The organics were concentrated to afford the title compound.
Example 19
5-{5-[4-((R)-3-Amino-4-hydroxy-butoxy)-2-chloro-5-fluoro-phenyl]-[l,3»4]thiadiazol-2- -2-isopropoxy-benzonitrile
Figure imgf000122_0001
1-103 1-104
Figure imgf000122_0002
1-105
[0315] 3-Cyano-4-isopropoxy-benzoic acid (44.5 mg, 0.213 mmol) and compound 1-103 (1 10 mg, 0.255 mmol) were dissolved in DMF (2.0 mL). TEA (74 μί, 0.53 mmol) was added followed by TBTU (84.6 mg, 0.255 mmol). The rxn mixture was stirred at RT overnight. The mixture was diluted with EtOAc (30 mL) and the layers were separated. The organic layer was washed with water (3x10 mL) and sat'd NaCl aq soln (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash chromatography using 10-100% EtOAc in heptane as the gradient to afford compound 1-104. [0316] To a dry microwave rxn vial was added Lawesson's reagent (34.6 mg, 0.083 mmol). Under nitrogen, compound 1-104 (77.0 mg, 0.124 mmol) dissolved in 2- methyltetrahydrofuran (2 mL) was added. The rxn soln was heated at 90°C in a heating block for 15 h. The rxn mixture was diluted in EtOAc (40 mL). Sat'd NH4C1 aq soln (5 mL) was added and NH4C1 solid precipitates. Water (5 mL) was added and the solid dissolves in the aq phase. The layers were separated. The organic layer was further washed in turn with sat'd NaHC03 aq soln (5 mL) and sat'd NaCl aq soln (3 mL). The organics were dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash column chromatography using 10-100% EtOAc in heptane as the gradient to afford compound 1-105.
[0317] HC1 (4.0 in 1,4-dioxane, 1.0 mL, 4.0 mmol) was added to the rbf containing compound 1-105 (57 mg, 0.092 mmol). The rxn mixture was stirred at RT for 2 h. The volatiles were evaporated under reduced pressure. The crude pdt was diluted with EtOAc (50 mL) and washed with sat'd NaHC03 aq soln (3x10 mL). The aq phase was extracted with DCM (5x35 mL). The combined organics were concentrated. The resulting crude pdt was dissolved in DMSO and purified by reverse phase HPLC using 5-95% ACN in water as the gradient. The pdt fractions were collected and basified with 1 K2C03 aq soln (0.45 mL) to pH = 8.5. The soln was then extracted with EtOAc (2x50 mL). The combined organics were washed with sat'd NaCl aq soln (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to afford the title compound.
Example 20
5- {5- [4-((R)-2- Amino-3-hydroxy-propoxy)-2-chloro-5-fluoro-phenyl] -[1 ,3,4] thiadiazol-2- yl}-2-isopropoxy-nicotinonitrile
Figure imgf000124_0001
[0318] To a stirred soln of compound 1-98 (55.7 mg, 0.270 mmol) in DMF (2.0 mL) were added EDCI (51.8 mg, 0.270 mmol) and HOBt (36.5 mg, 0.270 mmol) at RT. After 2 h, compound 1-50 (1 13 mg, 0.270 mmol) was added. After 3 h, the rxn was quenched with sat'd NaHC03 aq soln (15 mL). The mixture was extracted with EtOAc (2x35 mL). The combined organics were washed with brine, dried over sodium sulfate and concentrated. The resulting residue was purified via Combi-flash on silica gel (0- 50% ethyl acetate in heptanes) to afford compound 1-99.
[0319] A soln of compound 1-99 (120 mg, 0.200 mmol) and Lawesson's reagent (51 mg, 0.12 mmol) in 2-methyltetrahydrofuran (2 mL) was heated at 90°C in a sealed tube for 16 h. The solvent was removed under reduced pressure. The residue was dissolved in DCM and filtered through a plug of silica gel and rinsed with 30% EtOAc in hexanes. The fitrate was concentrated and the resulting residue was purified by flash chromatography using a solvent gradient of heptane to 35% EtAOc in heptane to provide coumpound I- 100.
[0320] Compound 1-100 (76 mg, 0.13 mmol) was added 4 HC1 in 1,4-dioxane (1.6 mL, 6.3 mmol) and stirred for 1 h. Solvent was removed under reduced pressure. The residue was purified by reverse phase HPLC using a gradient of 10-90% ACN in water containing 0.1% FA. The fractions were combined, basified by sat'd NaHC03 to pH = 8, and concentrated to remove acetonitrile. A white solid was formed. The solid was filtered, rinsed with water, and dried under high vacuum to afford the title compound.
[0321] The following compounds were synthesized in a similar fashion from the appropriate intermediates:
Figure imgf000125_0001
Example 21
(R)-2-Amino-4-{5-chloro-4-[5-(8-chloro-6-trifluoromethyl-imidazo[l,2-a]pyridin-2-yl)- 1 ,3.4] thiadiazol-2-yl] -2-fluoro-phenoxy }-butan-l -ol
Figure imgf000126_0001
[0322] Compound 1-109 (200 mg, 0.443 mmol) and compound 1-30 (120 mg, 0.488 mmol) were suspended in dry THF (16 mL) in a 50-mL rbf under N2. The flask was cooled in an ice/water bath. KHMDS (0.5 in toluene, 0.98 mL, 0.49 mmol) was added dropwise in a period of 10 min. The resulting rxn soln was stirred at 0°C for 2 h. At 0°C, additional compound 1-30 (120 mg, 0.488) was added followed by slow addition of additional KHMDS soln (0.98 mL, 0.49 mmol). After 2 h at 0°, the rxn was quenched with slow addition of sat'd NH C1 aq soln (12 mL). The mixture was then diluted with EtOAc (60 mL) and water (6 mL). The layers were separated and the organic layer was further washed with water (6 mL) and sat'd NaCl aq soln (6 mL). The organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash chromatography using 10-50% EtOAc in heptane as the gradient to afford compound 1-110.
[0323] To compound 1-110 (120 mg, 0.177 mmol) was added 4.0 HC1 in 1 ,4-dioxane (5.0 mL, 20 mmol). The mixture was stirred at RT for 5 h. Diethyl ether anhydrous (50 mL) was added and stirred at RT for 6 h. The rxn mixture was filtered through a Buchner funnel and washed with diethyl ether (2x10 mL). The solid was then evaporated to afford the pdt as a HCI salt. The pdt salt was dissolved in DMSO and purified by reverse phase HPLC (5-95% ACN/water containing 0.1% TFA as the gradient). The pdt fractions were combined and lyophilized overnigh to afford the title compound as a TFA salt. Example 22
(R)-2-Amino-3-{5-chloro-2-fluoro-4-[5-(2-isobutyl-6-methyl-pyridin-4-yl)- [l,3,4]thiadiazol-2-yl]-phenoxy}-propan-l-ol
Figure imgf000127_0001
[0324] To a soln of compound 1-77 (50.0 mg, 0.259 mmol) in DMF (5 mL) were added EDCI (50 mg, 0.26 mmol) and HOBt (35 mg, 0.26 mmol). After stirring for 2 h, compound I- 50 (105 mg, 0.251 mmol) was added. After stirring for 18 h the mixture was diluted with sat'd aq ammonium chloride (20 mL) and extracted with EtOAc (4x15 mL). The extract was dried over sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by combiflash chromatography using a 0-60% (5: 1 EtOAc:MeOH soln) in heptane gradient to afford compound 1-78.
[0325] To a soln of compound 1-78 (95 mg, 0.16 mmol) in 2-methyltetrahydrofuran (5 mL) was added Lawesson's reagent (0.80 g, 0.20 mmol). The mixture was heated to 90°C overnight. After 16 h the mixture was concentrated under a stream of nitrogen then diluted in 60% EtOAc in heptanes and passed through a pad of silica. The filtrate was concentrated. The resulting crude pdt was purified by flash chromatography using a 0-40% (5:1 EtOAc:MeOH) in heptanes gradient to afford compound 1-79. [0326] To a soln of compound 1-79 in MeOH (2 mL) was added 4.0 HC1 in 1,4-dioxane (10 mL, 40 mmol). After stirring for 2 h the mixture was concentrated to dryness under a stream of nitrogen. A 2M NaOH soln (2 mL) was added and the mixture was extracted with EtOAc (4x10 mL). The extracts were washed with brine (2x10 mL), dried over sodium sulfate and concentrated. The crude material was taken up in diethyl ether and filtered to afford the title compound.
[0327] The following compounds were synthesized in a similar fashion from the appropriate intermediates:
Figure imgf000128_0001
Example 23
Preapration of (R)-2-Amino-4-{5-chloro-2-fluoro-4-[5-(2-isopropylamino-6-methyl- ridin-4-yl)-[l,3,4]thiadiazol-2-yl]-phenoxy}-butan-l-ol
Figure imgf000129_0001
Figure imgf000129_0002
[0328] Compound 1-150 (200 mg, 0.525 mmol) and compound 1-30 (161 mg, 0.656 mmol) were dissolved in dry THF (10 mL) in a 100 mL rbf under N2. The flask was cooled in an ice/water bath. KHMDS (0.5 in toluene, 1.58 mL, 0.788 mmol) was added dropwise over a period of 20 min. The resulting rxn soln was stirred at 0°C for 1 hr then slowly warmed to RT overnight. The rxn mixture was cooled to 0°C again and quenched with slow addition of sat'd NH4C1 aq soln (1 1 mL). Water (5.5 mL) was added and the mixture becomes homogeneous again. Sat'd NaHC03 aq soln (22 mL) was added. The mixture was extracted with EtOAc (50 mL). The organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash chromatography using 0-10% MeOH in DCM as the gradient to afford compound 1-31.
[0329] To a soln of compound 1-31 (315 mg, 0.520 mmol) in MeOH (2 mL) was added HCI (4.0 in 1 ,4-dioxane, 1.3 mL, 5.2 mmol). The rxn mixture was stirred at RT for 1 h. Diethyl ether (50 mL) was added and the mixture was stirred over the weekend. The mixture was filtered. The solid was further washed with ether (25 mL). The solid was then evaporated to afford the title compound as a 2HC1 salt. The pdt salt was dissolved in MeOH (5 mL) and filtered through a PL-HC03MP SPE cartridge (StratoSpheres™ SPE, Part No.: PL3540- C603), washing with MeOH (3x5 mL). The filtrate was concentrated to afford the title compound (free base). [0330] The following compound was synthesized in a similar fashion from the appropriate intermediate:
Figure imgf000130_0002
Example 24
(2R,3S)-3-Amino-4-{2,6-dichloro-4-[5-(3,4-diethoxy-phenyl)-[l,3,4]thiadiazol-2-yl]- henoxy}-butan-2-ol
Figure imgf000130_0001
[0331] To a soln of 3,4-diethoxy-benzoic acid (100 mg, 0.476 mmol) in DMF (4.0 mL) were added EDCI (1 15 mg, 0.600 mmol) and HOBt (81 mg, 0.60 mmol). After 2 h, compound I- 112 (200 mg, 0.446 mmol) was added. After 17 h, the rxn was quenched with sat'd NaHC03 aq soln (15 mL). The mixture was extracted with ethyl acetate (2x20 mL). The combined organics were washed with brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified via Combi-flash column on silica gel (0-40% ethyl acetate in heptanes) to afford compound 1-113.
[0332] A soln of compound 1-113 (180 mg, 0.281 mmol) and Lawesson's reagent (227 mg, 0.560 mmol) in 2-methyltetrahydrofuran (6 mL) was heated to 90°C in a sealed tube. After 5 h, the solvent was removed under reduced pressure. Water (15 mL) was added to the residue and the mixture was extracted with ethyl acetate (2x15 mL). The combined organics were further washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified via Combi-flash column on silica gel (0-15%> ethyl acetate in heptanes) to afford compound 1-114.
[0333] The mixture of compound 1-114 (80.0 mg, 0.125 mmol) in HC1 (4N in 1,4-dioxane, 1.0 mL, 4.0 mmol) was stirred at RT for 17 h. The volatiles were removed under reduced pressure. The residue was basified with 2 NaOH aq soln and extracted with EtOAc (3x10 mL). The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was triturated with ether to afford the title compound.
Example 25
(R)-3- {5-Chloro-4-[5-(3-chloro-4-isopropoxy-phenyl)- [ 1 ,3,4] thiadiazol-2-y l]-2-fluoro- phenoxy}-2-dimethylamino-propan-l-ol
Figure imgf000131_0001
1-133
[0334] Compound 1-131 (25 mg, 43 μηιοΐ) and sodium cyanoborohydride (7.0 mg, 0.21 mmol) were dissolved in MeOH (5 mL). To this soln was added formaldehyde soln (37 wt.% aq, 17 μί, 0.23 mmol) and the rxn was stirred for 18 h. The rxn mixture was concentrated. The resulting crude pdt was diluted with CH2C12 and poured into H20. The aq phase was separated and extracted twice with CH2C12. The organics were combined, dried (Na2S04), decanted and concentrated to afford compound 1-133.
[0335] Compound 1-133 (56 mg, 91 μηιοΐ) was dissolved in THF (2 mL). To this soln was added TBAF (1.0 in THF, 0.14 mL, 0.14 mmol). The rxn was stirred for 2 h then diluted with EtOAc and poured into sat'd NH4C1 aq soln. The aq phase was separated and extracted twice with EtOAc. The combined organics were dried (Na2S04), decanted and concentrated. The crude residue was purified via flash chromatography (Si02, 0-10% MeOH-CH2Cl2) to afford the title compound.
Example 26
(2R S)-3-Amino-4-{5-chloro-2-fluoro-4-[5-(2-isopropylamino-6-methyl-pyridin-4-yl)- -2-yl]-phenoxy}-butan-2-ol
Figure imgf000132_0001
[0336] Compound 1-150 TFA salt (83.0 mg, 0.168 mmol) and compound 1-22 (124 mg, 0.504 mmol) were dissolved in dry THF (4 mL) in a 100 mL rbf under N2. The flask was cooled in an ice/water bath. KHMDS soln (0.5 in toluene, 1.3 mL, 0.67 mmol) was added dropwise. The rxn soln was stirred at 0°C for 1 h then slowly warmed to RT in 1 h. The water batch was removed and the rxn mixture was stirred at RT for 2 h. The rxn mixture was cooled to 0°C again and quenched with slow addition of sat'd NH4C1 aq soln (6 mL). The mixture was diluted with EtOAc (40 mL) and sat'd NaHC03 aq soln (5 mL). The layers were separated and the organic phase was further washed with water (2x10 mL). The organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash chromatography using 0-10% MeOH/DCM as the gradient to afford compound 1-23. [0337] To a soln of compound 1-23 (122 mg, 0.201 mmol) in MeOH (2.0 mL) was added HCl soln (4.0 in 1 ,4-dioxane, 0.90 mL, 3.6 mmol). The mixture was stirred at RT overnight. The volatiles were evaporated under reduced pressure to afford the crude pdt. EtOAc (15 mL) and sat'd NaHC03 aq soln (10 mL) were added. The layers were separated and the aq layer was extracted with EtOAc (3x25 mL). The combined organics were washed with water (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to afford the title compound.
Example 27
(R)-2-Amino-3-{5-chloro-2-fluoro-4-[5-(2-isopropylamino-6-methyl-pyridin-4-yl)- l,354]thiadiazol-2-yl]-phenoxy}-propan-l-ol
Figure imgf000133_0001
[0338) Compound 1-150 TFA salt (60.0 mg, 0.121 mmol) and compound 1-17 (84.0 mg, 0.363 mmol) were dissolved in dry THF (3.0 mL) in a 100 mL rbf under N2. The flask was cooled in an ice/water bath for 5 min. KHMDS (0.5 in toluene, 0.97 mL, 0.49 mmol) was added dropwise. The resulting rxn soln was stirred at 0°C for 2 h then slowly warmed to RT in 1.5 h and continued to stir at RT for 1 h. The mixture was cooled to 0°C again and quenched with slow addition of sat'd NH4C1 aq soln (4.5 mL). The mixture was diluted with EtOAc (50 mL) and sat'd NaHC03 aq soln (5 mL). The layers were separated and the organic layer was further washed with water (1 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt was purified by normal phase flash chromatography using 0-10% MeOH/DCM as the gradient to afford compound 1-18. [0339] Compound 1-18 (84.0 mg, 0.142 mmol) was dissolved in 4 HC1 in 1,4-dioxane (10 mL, 40 mmol) and stirred at RT. MeOH (2 mL) was added to dissolve the formed ppt. After stirring for 2 h the mixture was concentrated to dryness under a stream of nitrogen. A sat'd sodium bicarbonate soln (5 mL) was added and the mixture was extracted with EtOAc (4x10 mL). The extracts were washed with brine (2x10 mL), dried over sodium sulfate and concentrated. The pure material was crashed out of EtOAc by addition of heptanes to provide the title compound.
Example 28
N-((R)-2-{5-Chloro-4-[5-(3-chloro-4-isopropoxy-phenyl)-[l,3,4]thiadiazol-2-yl]-2-fluoro- phenoxy}-l-hydroxymethyl-ethyl)-acetamide
Figure imgf000134_0001
[0340] Compound EX00075518 (50 mg, 0.106 mmol) is suspended in DCM (10 mL). TEA (0.037 mL, 0.265 mmol) is then added. Acetic anhydride dissolved in DCM (1 mL) is then added dropwise. DMF (0.5 mL) is then added. The rxn mixture is stirred at RT for 60 h. Additional acetic anhydride (one drop) is added and the rxn mixture changes from clowdy to a clear soln. LCMS indicates that the rxn is complete. The mixtue is diluted with DCM (50 mL) and washed with water (3x10 mL), then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting crude pdt is purified by normal phase column chromatography using 1-10% MeOH in DCM as the gradient to afford the title compound.
[0341] The following compound is synthesized in a similar fashion from the appropriate intermediates: HPLC-MS
Compound structure
[M + H]+ Retention time (min)
N -N CI
507.3 0.79
0
Example 29
N-((R)-2- {5-Chloro-4-[5-(3-chloro-4-isopropoxy-pheny 1)- [ 1 ,3,4] thiadiazol-2-yl] -2-fluoro- -l-hydroxymethyl-ethyl)-formamide
Figure imgf000135_0001
1-132 BI00677640
[0342] Compound 1-132 (100 mg, 0.163 mmol) is dissolved in THF. To this soln is added TBAF (1.0 in THF, 0.24 mL, 0.24 mmol) and the rxn is stirred for 2 h. The rxn mixture is diluted with EtOAc and poured into sat'd NH4C1 aq soln. The aq phase is separated and extracted twice with EtOAc. The organic layers are combined, dried (Na2S04), decanted and concentrated. The crude residue is purified via flash chromatography (Si02, 0- 10% MeOH-CH2Cl2) to afford the title compound.
Example 30
N-((R)-3-{5-Chloro-2-fluoro-4-[5-(2-isopropylamino-6-methyl-pyridin-4-yl)- [l,3,4]thiadiazol-2-yl]-phenoxy}-l-hydroxymethyl-propyl)-acetamide
Figure imgf000135_0002
[0343] Compound BI00677638 (60 mg, 0.1 1 mmol) is suspended in DCM (9 mL). TEA (70 μί, 0.50 mmol) is then added. Acetic anhydride (1 1 iL, 0.12 mmol) dissolved in DCM (1 mL) is then added dropwise. The rxn mixture is stirred at RT for 1 h. The mixture is diluted with EtOAc (50 mL) and washed with water (3x10 mL). The organics are dried over anhydrous sodium sulfate, filtered and concentrated. The resulting crude pdt is purified by normal phase column chromatography using 0-7% MeOH in DCM as the gradient to afford the title compound.
[0344] The following compound is synthesized in a similar fashion from the appropriate intermediate:
Figure imgf000136_0002
Example 31
((R)-3-{5-Chloro-2-fluoro-4-[5-(2-isopropylamino-6-methyI-pyridin-4-yl)- l,354]thiadiazol-2-yl]-phenoxy}-l-hydroxymethyI-propyl)-carbamic acid methyl ester
Figure imgf000136_0001
[0345] Compound BI00677638 (46.4 mg, 99.6 μιηοΐ) and K2CO3 (17.4 mg, 0.126 mmol) are dissolved in THF (1 mL) and water (1 mL). This mixture is cooled to 0°C. Methyl chloroformate (8.4 μί, 0.11 mmol) is added dropwise. The ice/water bath is removed and the rxn mixture is stirred at RT overnight. The rxn mixture is diluted with water (50 mL) and extracted with EtOAc (3x25 mL). The combined organics are dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude pdt is purified by normal phase flash column chromatography using 0-10% MeOH in DCM as the gradient. The resulting pdt is still not pure and purified again by reverse phase prepartive HPLC using 5-90% ACN/water containing 0.1% TFA as the gradient (20 min run). The pdt fractions are combined and lyophilized overnight to afford a solid. This solid is dissolved in MeOH (5 mL) and filtered through a PL-HCO3MP SPE cartriage (StratoSpheres™ SPE, Part No.: PL3540-C603) and washed with MeOH (3x5 mL). The filtrate is concentrated to afford the title compound. Biological Examples
[0346] Suitable in vitro assays for measuring SI PI and S 1P5 agonist activity are known in the art. All Compounds in Table 1 were tested in one or more of the following biological assays and were found to be agonists of S1P1 and/or S1P5. As such compounds of Formula I are useful for treating diseases, particularly autoimmune disease in which SI PI and/or S1P5 activity contributes to the pathology and/or symptomatology of the disease, for example, multiple sclerosis and graft-versus host disease. Suitable in vivo models for autoimmune diseases are known to those of ordinary skill in the art and are also described below, e.g. models for autoimmune-mediated inflammation, multiple sclerosis, graft-versus host disease, and osteoporosis. Following the examples disclosed herein, as well as that disclosed in the art, a person of ordinary skill in the art can determine the SI PI and S1P5 agonist activity of a compound of this invention and its usefulness for treating a disease.
Biological Example 1
CNG cAMP Assay
[0347] Frozen HEK293 cells expressing the CNG channel and SI Pi (BD Biosciences, San Jose, CA) are thawed and plated into the wells of a black, clear bottom, 384- well CellBind plate (Corning, Corning, NY) at 14,000 cells per well. HEK293 cells expressing the CNG channel and CB1 (BD Biosciences) are cultured and plated under the same conditions. The cells are incubated for 16 h at 37 °C in complete DMEM medium (Invitrogen Carlsbad, CA) containing 10% FBS (HyClone Logan, UT), 250 μg/mL geneticin (Invitrogen), and 1 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO). A membrane potential dye (BD Biosciences) is added and the plates are incubated for 2-2.5 h at room temperature.
[0348] Test compounds are tested at maximum concentrations of 10 μΜ. Compounds are diluted in DMSO (10 concentration points, 3-fold each) and added to the assay plate at final DMSO concentrations of 1.8%. For each compound, there are duplicate assay plates and each assay plate have duplicate wells per concentration point. Test compounds are added to the cells in a DPBS solution containing 25 μΜ Ro 20-1724 (Sigma-Aldrich), 500 nM of the A2b receptor agonist NEC A (Sigma-Aldrich) and 10 nM (EC95) of SIP (Avanti Alabaster, AL) and incubated for 90 min. The assay plate is read before compound addition (T0) and after the 90 min incubation (T90) using an EnVision plate reader (PerkinElmer, Waltham, MA) at an excitation wavelength of 350 nm and an emission wavelength of 590 nm. The T90/T0 ratio is determined for each concentration of the test compounds. The percent agonist activity is determined as [(test compound - DMSO alone control) / (NECA alone control- DMSO alone control) * 100]. The percent activities are plotted against compound concentration to determine EC50 using XLFit (IDBS, Alameda, CA). The control used for calculating rEC50 in the SI Pi CNG agonist assay is DMSO.
Biological Example 2
S1P1 β-arrestin Recruitment Assay
[0349] For the Tango™ β-arrestin recruitment assay, the cytoplasmic C-terminus of SI Pi was tethered to the tTA transcriptional activator with a linker that contains a cleavage site for the Nla protease from tobacco etch virus (TEV protease). The C-terminus of the human P-arrestin2 protein was fused to TEV protease. Binding of an agonist recruits the β-arrestin- TEV fusion protein to the receptor resulting in cleavage of the linker and released of tTA to enter the nucleus and subsequently activated a tTA-dependent luciferase reporter gene.
[0350] Assay 2a: Frozen HEK293 cells transiently transfected with receptor cDNAs for SI Pi (Invitrogen) are thawed and suspended in 10 mL of Pro293a-CDM culture medium (Invitrogen) supplemented with 4 mM L-Glutamine (Invitrogen), IX Pen/Strep (100 units/mL penicillin and 100 g/mL streptomycin, Invitrogen) and 0.1% fatty acid free BSA (Sigma- Aldrich). Cells are added to the wells of a 384- well white opaque bottom assay plate (PerkinElmer) at 3,000-6,000 cells per well and the plate is incubated for approximately 4 h in a 37 °C incubator. Test compounds are tested at maximum concentrations of 10 μΜ for the agonist assays. Compounds are diluted in DMSO (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration. For each compound, there are duplicate assay plates and each assay plate has duplicate wells per concentration point. The plate is incubated at 37 °C for 30 min. The efficacy control is 5 μΜ SIP (Avanti). Following agonist addition, the assay plates are incubated in a 37 °C incubator for 16-18 h. Luciferase assay reagent is added and luminescence measured in an EnVision plate reader (PerkinElmer). To determine agonist activity, percent activity is calculated as [(test compound - background) / (positive control- background) * 100], where background is the luminescence of the DMSO alone control and the positive control is the luminescence from cells incubated with the efficacy control 5 μΜ SIP. The percent activities are plotted against compound concentration to determine EC50 using XLFit (IDBS).
[0351] Assay 2b: Alternatively, U20S cells expressing the reporter gene and SlPi (Invitrogen) were added to the wells of a 384-well white opaque bottom assay plate (PerkinElmer) at 0.3125 X 106 cells per well. The cells were serum starved for 48 h in Freestyle medium (Invitrogen). Test compounds were tested at maximum concentrations of 1 μΜ for the agonist assay. Compounds were diluted in DMSO (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration. The efficacy control was 1 μΜ SIP (Avanti). For each compound, there were duplicate assay plates and each assay plate had duplicate wells per concentration point. The plate was incubated overnight at 37 °C. The GeneBLAzer β-lactamase assay reagent (Invitrogen) was added and the plates were incubated for an additional 2 h at room temperature. Fluorescence was measured using an EnVision plate reader (PerkinElmer, Waltham, MA) at an excitation wavelength of 409 nm and emission wavelengths of 460 nm and 530 nm. The emission intensity at each wavelength was background subtracted against wells containing medium only and the F460nm F530nm ratio determined for each concentration of the test compounds. Percent activity was calculated as [(test compound ratio - DMSO ratio) / (positive control ratio - DMSO ratio) * 100], where the positive control and DMSO ratios are from cells incubated with the efficacy control 1 μΜ SIP and 1% DMSO, respectively. The percent activities were plotted against compound concentration to determine EC50 using XLFit (IDBS).
Biological Example 3
hSIPIR GTPyS and GTP-Eu Binding Assays
[0352] Assay 3 a: The hSlPU? GTPyS binding assay was carried out at room temperature in 96 well non-binding surface assay plates. The reaction in each well contained 4 μg hSIPIR (hEdgl) membrane protein (Lonza), 30μΜ GDP, 0.1 nM [35S]GTPyS, 0.25% fatty acid free BSA, and serially diluted hSIPIR agonist compound in 200 μΐ assay buffer (25mM Tris- HC1 PH 7.9, 100 mM NaCl, 3 mM MgC12, and 0.2 mM EGTA). After one hour of incubation, 0.9 mg of WGA (Wheat Germ Agglutinin) SPA beads in 50 of assay buffer was added to each well. The SPA beads were spun down after an additional one hour incubation. The radioactivity of the bound GTPyS was counted by reading the assay plate using a MicroBeta.
[0100] Assay 3b: The DELFIA GTP-Eu binding assay (PerkinElmer) is a time-resolved fluorometric assay based on GDP-GTP exchange. CHO cell membranes (Lonza) expressing human SI Pi are incubated in 96-well filter plates (Pall, East Hills, NY) in a final volume of 100 μϋνκΙΙ buffer containing 40 μg/mL membrane, 50 mM HEPES, 2 μΜ GDP, 10 mM MgCl2, 100 mM NaCl, 500 μ /ηιί Saponin and test compound. Test compounds are tested at maximum concentrations of 10 μΜ. Compounds are diluted (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration. For each compound, there are duplicate assay plates and each assay plate has duplicate wells per concentration point. The plates are incubated for 30 min at room temperature on a plate shaker at low speed. GTP-Eu is added to each well (10 μί,, 10 nM final concentration) and the plate is incubated for an additional 30 min with slow shaking. The wells are washed with ice cold GTP washing buffer (3 X 150 μΙ_.) using a vacuum manifold and the assay plates read in an EnVision plate reader (PerkinElmer) at an excitation wavelength of 340 nm and an emission wavelength of 615 nm. To determine agonist activity, percent activity is calculated as [(test compound - background)/ (positive control- background) * 100], where background is the fluorescence in absence of compound and the positive control is the fluorescence from membranes incubated with 1 μΜ SIP (Avanti). The percent activities are plotted against compound concentration to determine IC50 or EC50 using XLFit (IDBS).
Biological Example 4
hSlP5R GTPyS Binding Assay
[0353] The hSlPi?5 GTPyS binding assay was carried out at room temperature in 96-well non-binding surface assay plates. The reaction in each well contained 5 μg hSlP/?5 (hEdg8) membrane protein from CHO cells expressing hSlPR5, 30 μΜ GDP, 0.1 nM [35S]GTPyS, 0.25% fatty acid free BSA, and serially-diluted Compound of the Invention in 200 μΐ, assay buffer (25 mM Tris-HCl PH 7.9, 100 mM NaCl, 3 mM MgC12, and 0.2 mM EGTA). After one hour of incubation, 0.9 mg of WGA (Wheat Germ Agglutinin) SPA beads in 50 μΐ, of assay buffer was added to each well. The SPA beads were spun down after an additional one hour incubation. The radioactivity of the bound GTPyS was counted by reading the assay plate using a MicroBeta.
Biological Example 5
S1P3 β-arrestin Recruitment Assay
[0354] This assay was conducted using the procedures described in Assay 2a, replacing S1P1 with SlP3.
Biological Example 6
S1P2 β-arrestin Recruitment Assay
[0355] This assay was conducted using the procedures described in Assay 2a, replacing S1P1 with SlP2.
Table 3: Results
[0356] Table 3 gives EC50 data (unless otherwise indicated) for the compounds in Table 1 and Table 2 and are in nM units. Assay 2b is the Tango™ β-arrestin Recruitment Assay in U20S cells, as described in Biological Example 2. Assay 3a is the hS IP 17? GTPyS Binding Assay as described in Biological Example 3. Assay 4 is the hSlP5R GTPyS Binding Assay as described in Biological Example 4. Assay 5 is the Tango™ β-arrestin Recruitment Assay in HEK293 cells, as described in Biological Example 5.
[0357] EC50's were measured unless otherwise noted. An "*" indicates that rEC50 (relative EC50) was measured rather than EC50.
[0358] For assay 5, F means the compound has an EC50 or relative EC50 of less than or equal to 250 nM and G means the compound has an EC50 or relative EC5o of greater than 250 nM.
[0359] For assays 3a, 2b and 4, "A" means the compound has an EC50 or relative EC50 of less than or equal to 10 nM. "B" means the compound has an EC50 or relative EC50 greater than 10 nM but less than or equal to 50 nM. "C" means the compound has an EC50 or relative EC50 greater than 50 nM but less than or equal to 250 nM. "D" means the compound has an EC50 or relative EC50 greater than 250 nM but less than or equal to 2800 nM. "E" means the compound has an EC50 or relative EC50 greater than 2800 nM but less than 10000 nM. In the table, "nt" means the Compound was not tested and "na" means the compound was tested but had no measurable activity under the assay conditions employed.
Figure imgf000141_0001
Table 3
Entry Assay
Name
No. 3a 2b 4 5
4-[5-(4-{[(2S,3S)-2-amino-3-hydroxybutyl]oxy}-2-
6 cyano-5-fluorophenyl)-l ,3,4-thiadiazol-2-yl]-6-[(l - A B nt na methylethyl)amino]pyridine-2-carbonitrile
(2^,3S)-3-amino-4-{[2,6-dichloro-4-(5-{2-methyl-6-
7 [( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4- A A nt na thiadiazol-2-yl)phenyl]oxy } butan-2-ol
(l^,2S)-2-amino-3-{[2,6-dichloro-4-(5-{2-methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4-
8 A A nt G thiadiazol-2-yl)phenyl]oxy }- 1 -methylpropyl
dihydrogen phosphate
4-[5-(4-{[(25,3i?)-2-amino-3-hydroxybutyl]oxy}-2-
9 chloro-5 -fluorophenyl)- 1 ,3 ,4-thiadiazol-2-yl]-6- [( 1 - A A nt na methylethyl)amino]pyridine-2-carbonitrile
(2i?,3S)-3 -amino-4- { [5 -chloro-2-fluoro-4-(5- { 6-
10 methyl-4- [( 1 -methylethyl)amino]pyridin-2-yl } - 1 ,3 ,4- B C nt na thiadiazol-2-yl)phenyl]oxy } butan-2-ol
4-[5-(4-{[(2S,3i?)-2-amino-3-hydroxybutyl]oxy}-2-
1 1 cyano-5-fluorophenyl)-l,3,4-thiadiazol-2-yl]-6-[(l - A B nt na methylethyl)amino]pyridine-2-carbonitrile
(2S)-2-amino-3-{[5-chloro-2-fluoro-4-(5-{2-methyl- 6-[( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4-
12 A A nt 45 thiadiazol-2-yl)phenyl]oxy}propyl dihydrogen
phosphate
(2i?)-2-amino-3 - { [5 -chloro-2-fluoro-4-(5 - { 2-methyl-
13 6- [( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4- A B nt na thiadiazol-2-yl)phenyl]oxy }propan- 1 -ol
(2tf,3S)-3-amino-4-{[2-chloro-6-fluoro-4-(5-{2-
14 methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4- A A nt na thiadiazol-2-yl)phenyl]oxy}butan-2-ol
( 1 fl,2S 2-amino-3- { [2-chloro-6-fluoro-4-(5- {2- methyl-6- [( 1 -methy lethyl)amino]pyridin-4-yl } - 1 ,3 ,4-
15 A A nt F thiadiazol-2-yl)phenyl]oxy } - 1 -methylpropyl
dihydrogen phosphate
( 1 tf,2S)-2-amino-3 - { [5 -chloro-2-fluoro-4-(5- { 6- methyl-4-[(l -methylethyl)amino]pyridin-2-yl}-l ,3,4-
16 A A nt G thiadiazol-2-yl)phenyl]oxy}-l-methylpropyl
dihydrogen phosphate Table 3
Entry Assay
Name
No. 3a 2b 4 5
(R)-2-amino-3-(2,6-dichloro-4-(5-(2-
17 (isopropylamino)-6-methylpyridin-4-yl)- 1,3,4- A A nt na thiadiazol-2-yl)phenoxy)propan- 1 -ol
(2R,3 S)-3 -amino-4-(4-(5 -(2-(isopropylamino)-6-
18 methylpyridin-4-yl)- 1 ,3,4-thiadiazol-2-yl)-2,6- A A nt na dimethylphenoxy)butan-2-ol
(2R,3S)-3-amino-4-(2-fluoro-4-(5-(2-
19 (isopropylamino)-6-methylpyridin-4-yl)- 1,3,4- B nt nt na thiadiazol-2-yl)-5-methylphenoxy)butan-2-ol
5-(5-(4-((2S,3R)-2-amino-3-hydroxybutoxy)-3,5-
20 dimethylphenyl)- 1 ,3 ,4-thiadiazol-2-yl)-2- A C nt na
(isopropylamino)nicotinonitrile
5-(5-(4-((2S,3R)-2-amino-3-hydroxybutoxy)-3,5-
21 dichloropheny 1)- 1 ,3 ,4-thiadiazol-2-yl)-2- B B nt na
(isopropylamino)nicotinonitrile
3-(5-chloro-2-fluoro-4-(5-(2-(isopropylamino)-6-
22 methylpyridin-4-yl)- 1 ,3 ,4-thiadiazol-2- B D nt na yl)phenyl)azetidin-3-ol
(R)-2-amino-3-(2-chloro-4-(5-(2-(isopropylamino)-6-
23 methylpyridin-4-yl)- 1 ,3 ,4-thiadiazol-2-yl)-6- A A nt na methylphenoxy)propan- 1 -ol
(S)-2-amino-3-(2,6-dichloro-4-(5-(2- (isopropylamino)-6-methylpyridin-4-yl)- 1 ,3,4-
24 A A nt na thiadiazol-2-yl)phenoxy)propyl dihydrogen
phosphate
3 -(5-chloro-4-(5 -(3 -chloro-4-isopropoxyphenyl)-
25 1 ,3 ,4-thiadiazol-2-yl)-2-fluorophenoxy)- 1,1,1- nt C nt nt trifluoropropan-2-amine
(R)-5-(5-(4-(2-amino-3-hydroxypropoxy)-5-fluoro-2-
26 methylphenyl)- 1 ,3 ,4-thiadiazol-2-yl)-2- nt A nt nt isopropoxybenzonitrile
(R)-2-amino-3 -(4-(5 -(3 -chloro-4-isopropoxyphenyl)-
27 l,3,4-thiadiazol-2-yl)-2-fluoro-5- nt C nt nt methylphenoxy)propan- 1 -ol Table 3
Entry Assay
Name
No. 3a 2b 4 5
(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(5-
28 isopropoxy-6-methy lpyridin-2-yl)- 1 ,3 ,4-thiadiazol-2- nt C nt nt yl)phenoxy)propan- 1 -ol
(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(2-
29 isopropoxy-6-methylpyridin-4-yl)- 1 ,3,4-thiadiazol-2- nt C nt nt yl)phenoxy)propan- 1 -ol
(R)-2-amino-4-(5-chloro-4-(5-(3-chloro-4-
30 isopropoxyphenyl)-l,3,4-thiadiazol-2-yl)-2- nt A nt nt fluorophenoxy)butan- 1 -ol
(R)-2-amino-3-(5-chloro-4-(5-(5-chloro-6-
31 ethoxypyridin-3-yl)-l ,3,4-thiadiazol-2-yl)-2- nt D nt nt fluorophenoxy)propan- 1 -ol
(R)-2-amino-3-(5-chloro-4-(5-(5-chloro-6-
32 isopropoxypyridin-3 -yl)-l,3 ,4-thiadiazol-2-yl)-2- nt C nt nt fluorophenoxy)propan- 1 -ol
(R)-5-(5-(4-(3-amino-4-hydroxybutoxy)-2-chloro-5-
33 fluorophenyl)-l,3,4-thiadiazol-2-yl)-2- nt A nt nt isopropoxybenzonitrile
N-(l-(5-chloro-4-(5-(3-chloro-4-isopropoxyphenyl)-
34 1 ,3 ,4-thiadiazol-2-yl)-2-fluorophenoxy)-3 - nt C nt nt hydroxypropan-2-yl)acetamide
(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(6-
35 isopropoxy-5 -methy lpyridin-3 -yl)- 1 ,3 ,4-thiadiazol-2- nt C nt nt yl)phenoxy)propan- 1 -ol
(R)-2-amino-3-(2-fluoro-4-(5-(6-isopropoxy-5-
36 methylpyridin-3 -yl)- 1 ,3 ,4-thiadiazol-2-yl)-5 - nt D nt nt methylphenoxy)propan- 1 -ol
(R)-5-(5-(4-(2-amino-3-hydroxypropoxy)-2-chloro-5-
37 fluorophenyl)- 1 ,3 ,4-thiadiazol-2-yl)-2- nt B nt nt isopropoxynicotinonitrile
(R)-2-amino-3-(5-chloro-4-(5-(3,4-diethoxyphenyl)-
38 nt A nt nt 1 ,3,4-thiadiazol-2-yl)-2-fluorophenoxy)propan-l -ol Table 3
Entry Assay
Name
No. 3a 2b 4 5
(R)-2-amino-3 -(5-chloro-4-(5 -(3 ,5 -diethoxyphenyl)-
39 nt B nt nt 1 ,3,4-thiadiazol-2-yl)-2-fluorophenoxy)propan-l -ol
(R)-2-amino-3-(5-chloro-4-(5-(2,4-diethoxyphenyl)-
40 nt B nt nt 1 ,3,4-thiadiazol-2-yl)-2-fluorophenoxy)propan-l -ol
(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(2-isobutyl-6-
41 methoxypyridin-4-yl)-l,3,4-thiadiazol-2- nt A nt nt yl)phenoxy)propan- 1 -ol
(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(2-isobutyl-6-
42 methylpyridin-4-yl)- 1 ,3 ,4-thiadiazol-2- nt A nt nt yl)phenoxy)propan- 1 -ol
(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(2-
43 (isopropyl(methyl)amino)-6-methylpyridin-4-yl)- nt C nt nt
1 ,3,4-thiadiazol-2-yl)phenoxy)propan-l -ol
(R)-2-amino-3-(5-chloro-4-(5-(4-ethoxy-3-
44 methoxyphenyl)- 1 ,3 ,4-thiadiazol-2-yl)-2- nt B nt nt fluorophenoxy)propan- 1 -ol
(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(2-isobutyl-6-
45 methylpyrimidin-4-yl)- 1 ,3 ,4-thiadiazol-2- nt D nt nt yl)phenoxy)propan- 1 -ol
(3S)-3-amino-4-(5-chloro-4-(5-(3,4-diethoxyphenyl)-
46 nt B nt nt l,3,4-thiadiazol-2-yl)-2-fluorophenoxy)butan-2-ol
(R)-2-amino-4-(5-chloro-2-fluoro-4-(5-(2-
47 (isopropylamino)-6-methylpyridin-4-yl)- 1,3,4- nt nt nt nt thiadiazol-2-yl)phenoxy)butan- 1 -ol
(R)-N-(l-(5-chloro-4-(5-(3-chloro-4-
48 isopropoxyphenyl)- 1 ,3 ,4-thiadiazol-2-yl)-2- nt nt nt nt fluorophenoxy)-3-hydroxypropan-2-yl)formamide
(S)-2-amino-4-(5-chloro-2-fluoro-4-(5-(2-
49 (isopropylamino)-6-methylpyridin-4-yl)- 1,3,4- nt nt nt nt thiadiazol-2-yl)phenoxy)butan- 1 -ol Table 3
Entry Assay
Name
No. 3a 2b 4 5
(2R,3S)-3-amino-4-(5-chloro-2-fluoro-4-(5-(2-
50 isobutyl-6-methylpyridin-4-yl)-l,3,4-thiadiazol-2- nt nt nt nt yl)phenoxy)butan-2-ol
(R)-N-(4-(5-chloro-2-fluoro-4-(5-(2- (isopropylamino)-6-methylpyridin-4-yl)- 1 ,3 ,4-
51 nt nt nt nt thiadiazol-2-yl)phenoxy)- 1 -hydroxybutan-2- yl)acetamide
(S)-N-(4-(5-chloro-2-fluoro-4-(5-(2- (isopropylamino)-6-methylpyridin-4-yl)- 1 ,3,4-
52 nt nt nt nt thiadiazol-2-yl)phenoxy)- 1 -hydroxybutan-2- yl)acetamide
(2R,3S)-3-amino-4-(2,6-dichloro-4-(5-(3,4-
53 diethoxyphenyl)- 1 ,3,4-thiadiazol-2- nt nt nt nt yl)phenoxy)butan-2-ol
(R)-3-(5-chloro-4-(5-(3-chloro-4-isopropoxyphenyl)-
54 l,3,4-thiadiazol-2-yl)-2-fluorophenoxy)-2- nt nt nt nt
(dimethylamino)propan- 1 -ol
(2R,3S)-3-amino-4-(2,6-dichloro-4-(5-(5-isobutyl-4-
55 methylpyridin-2-yl)- 1 ,3 ,4-thiadiazol-2- nt nt nt nt y l)phenoxy)butan-2 -ol
(2R,3S)-3-amino-4-(5-chloro-2-fluoro-4-(5-(5-
56 isobutyl-4-methylpyridin-2-yl)-l,3,4-thiadiazol-2- nt nt nt nt yl)phenoxy)butan-2-ol
(R)-methyl 4-(5-chloro-2-fluoro-4-(5-(2- (isopropylamino)-6-methylpyridin-4-yl)- 1,3,4-
57 nt nt nt nt thiadiazol-2-yl)phenoxy)- 1 -hydroxybutan-2- ylcarbamate
N-((2S,3R)-l-(5-chloro-2-fluoro-4-(5-(2-isobutyl-6-
58 methylpyridin-4-yl)- 1 ,3 ,4-thiadiazol-2-yl)phenoxy)- nt nt nt nt
3 -hydroxybutan-2-y l)acetamide
Biological Example 7
Lymphocyte PD Assay in Mice
[0360] Suppression of circulating lymphocytes is assessed as a pharmacodynamic (PD) endpoint in 6-10 week old C57B1/6 male mice (Taconic Farms, Germantown, NY). Upon arrival, mice are acclimated to the vivarium (12 h light cycle, 12 h dark cycle) for a minimum of 3 days prior to the initiation of a study. During the study, animals are provided food and water ad libitum and housed in a room conditioned at 70-75 °F. All animals are examined daily for health assessment.
[0361] Compounds of the Invention are suspended or dissolved in vehicle for administration of 0.1 mL/animal based on mean body weight of group. Compounds are administered using a disposable gavage needle (20G, Braintree Scientific, Braintree, MA). Blood is collected into EDTA-coated tubes (Microvette 100 with EDTA, Sarstedt, Newton, NC) from the retro-orbital sinus of isoflurane-anesthetized animals 24 or 32 h post-dose. Mice are then euthanized by cervical dislocation. For some experiments, samples of tissues are collected for measurement of compound levels. Samples are frozen immediately on dry ice and stored at -18 °C until assayed. After collection, blood samples are placed on a rock 'n roller mixer (Drew Scientific, Inc., Waterbury, CT) for at least 10 min and then a complete blood count (CBC) analysis is performed using the Hemavet 1700 Flexible Veterinary Multi- Species Hematology System (Drew Scientific). Samples are then placed on ice and, within 4 h, centrifuged to obtain plasma which is then stored frozen at 20 °C until analysis for compound levels. CBC readouts include white blood cells; total and % of total for the following: neutrophils, lymphocytes, monocytes, eosinophils, basophils, and nucleated red blood cells (RBC); RBC; hemoglobin; hematocrit; mean corpuscular volume, hemoglobin (HGB) total and concentration; RBC distribution width; platelets; and mean platelet volume. All mouse PD data consists of group sizes of 8 and are expressed as means ± SEM. Statistical analysis of each experimental endpoint is conducted with GraphPad Prism. All compound exposure data are based on n = 4/group and are expressed as means ± SD.
Biological Example 8
Lymphocyte PD Assay in Rats
[0362] Suppression of circulating lymphocytes is assessed as a PD endpoint in jugular vein canulated Sprague Dawely (SD) rats 6-8 weeks of age and weighing approximately 200 g (Taconic Farms, Germantown, NY). Prior initiating a study, rats are acclimated to the vivarium facility (12 h light cycle, 12 h dark cycle) for a minimum of 2 days. During a study, animals are provided food and water ad libitum and housed in a room conditioned at 70-75 °F and 60% relative humidity. All animals are examined daily for health assessment.
[0363] The Compound of the Invention are formulated and animals is dosed at a volume of 4 mL/kg. At indicated time points, whole blood is collected via jugular vein canulas into EDTA-coated tubes and hematology analysis is performed on an Abbott Cell-Dyn 3700 hematology analyzer. Readouts include white blood cells (total, differential, and % of total), neutrophils, lymphocytes, monocytes, eosinophils, basophils, RBC, HGB, hematocrit, mean corpuscle volume, mean corpuscle HGB concentration, RBC distribution width, platelets, and mean platelet volume.
Biological Examples 9-12
In vivo models
Biological Example 9: Delay ed-type Hypersensitivity (DTH) Model
[0364] Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. Signaling of SIP through S1P1 has been shown to exclusively modulate egress of lymphocyte including 70% of activated T cells from lymph nodes. Delayed-type hypersensitivity (DTH) is an immune response mediated by a variety of inflammatory cells, including neutrophils, macrophages and T cells (Kobayashi et al. 2001, Black 1999). DTH develops in two phases, a sensitization phase, in which T cells are sensitized and memory T cells are formed, and an elicitation phase, in which T cell recall responses are induced upon secondary challenge with antigen. This second phase results in recruitment of inflammatory cells such as neutrophils and macrophages to the injection site of an intradermally applied antigen in a previously sensitized host, which causes swelling 24 h to 48 h post antigen challenge. The DTH assay (primarily done in mice) is an in vivo manifestation of a cell-mediated immunity reaction, and the response to antigen representation modulated by immunosuppressive treatment can be measured.
[0365] C57B1/6 male mice (10 mice per group) are immunized on day zero by subcutaneous injection at the base of the tail with 100 μί of 2 mg/mL methylated BSA emulsified with Complete Freunds Adjuvans (CFA, Sigma). Once-daily for twice-daily administration of a Compound of the Invention occurs for 10 days. On day 10 after immunization, mice receive a second booster injection at the base of tail of an emulsified mixture of 2 mg/mL methylated BSA in Incomplete Freund's Adjuvans. On day 13 animals are challenged subcutaneously in the left hind footpad with 20 μΐ, of 10 mg/mL methylated BSA in sterile water (water for injection). Animals are injected with an equal volume of sterile water into the right hind footpad as a control. Twenty four hours later (dose day 14) the right and left hind foot paws are transected at the medial and lateral malleolus, weighed, and the weight difference induced by injected antigen determined and compared to weight differences of vehicle treated non-sensitized and sensitized control groups. The increase in paw weights comparing left and right hind paw for each treatment group are analyzed for differences of treatment with a Compound of the Invention compared to vehicle control group using the Mann- Whitney non-parametric test statistic with minimal significance level set at p<0.05.
Biological Example 10: Allograft Model
[0366] The rodent allograft model is an in vivo assay for assessing tissue rejection (ie, from transplantation) in response to chronic and/or sub-chronic immunosuppressive treatment (Chiba et al, 2005). Rejection is caused by T lymphocytes of the recipient responding to the foreign major histocompatibility complex of the donor graft. The transplanted organ (eg, skin) represents a continuous source of HLA alloantigens capable of inducing a rejection response at any time post transplantation. Because it cannot be eliminated, the allograft continuously activates the immune system, resulting in lifelong overproduction of cytokines, constant cytotoxic activity, and sustained alteration in the graft vasculature. Therefore, lifelong immunosuppression is required to ensure allograft survival. In this model skin from donor rats (male Lewis; histocompatibility i?T-l') is surgically engrafted onto a dorsal region of recipient rats (male F344; histocompatibility ?T-llvl). Administration of compound occurs immediately after surgery for a predetermined duration. Skin allografts are monitored daily for rejection.
[0367] On the day of surgery male Lewis donor rats are anesthetized with Isoflurane and skin aseptically harvested from the tail. Male F344 acceptor rats (8 per group) previously shaved (1-2 days prior to surgery) in the designated engraftment area are anesthetized with Isoflurane and a full thickness skin graft bed on the medial flank removed and discarded. The skin graft bed removed is equivalent in size to the donor skin to be engrafted. The prepared donor skin is then secured on the prepared graft bed with spot tissue glue or by 4 to 8 non-silk sutures, and covered with sterile Vaseline gauze and wrapped with a bandage. All surgery takes place on heated pads with sterile surgical equipment. Animals are monitored and turned every 20 minutes until ambulatory before returning to cages, water and food. Initiation of administration of a Compound of the Invention (once-daily or twice-daily) occurs when the animals fully recovered from anesthesia for a period of 14 days. On day 5 post-surgery, surgical bandaging is removed and the grafts assessed daily for rejection (necrosis of the graft tissue following by scabbing and sloughing from the graft bed site). An allograft is scored as "rejected" when it sloughed from the graft bed site. A positive effect in this model is delayed rejection of the allograft in response to treatment with a Compound of the Invention when compared to vehicle-treated control animals.
Biological Example 11: Experimental autoimmune encephalomyelitis (EAE) Model
[0368] Multiple sclerosis is a demyelinating disease of the CNS. The main features of the disease are focal areas of demyelination and inflammation mediated by macrophages and t-lymphocytes. These cells develop in the peripheral lymphoid organs and travel to the CNS causing an autoimmune response. The development of T cells is controlled largely by the expression of various cytokines as well as cellular adhesion molecules. The EAE model today is the most thoroughly studied animal model for human autoimmune diseases. Mice are immunized with myelin-derived peptide PLP and clinical parameters of disease (bodyweight loss and paralysis) are monitored daily. The endpoint is the analysis of the extent of inflammation in brain and spinal cord.
[0369] C57B1/6 mice develop chronic paralysis after immunization with MOG35.55 peptide. Mice develop EAE 8-14 days after immunization and stay chronically paralyzed for 30-40 days after onset of disease. Female C57B1/6 mice are subcutaneously injected with MOG35-55 peptide emulsified in Complete Freund's Adjuvant at two sites on the back, injecting 0.1 mL at each site. Within 2 h of injection, pertussis toxin (aids in brain penetration of the MOG peptide) is administered intraperitoneally. A second injection of pertussis toxin is administered 22-26 h after the MOG35-55 peptide injection. Onset of EAE is typically 7 days after immunization. EAE is scored on a scale of 0-5 with 0 being no obvious changes in motor functions, while 5 indicates complete paralysis. Mice are administered a Compound of the Invention (once-daily or twice-daily) on the day of MOG35-55 peptide injection and monitored for paralysis and compared to vehicle-treated control animals. A positive effect in this model is delayed onset/severity of EAE.
Biological Example 12: Osteoporosis model
[0370] Methods described in Nature 2009, 458(7237), 524-528, which is herein incorporated by reference, can be used to determine whether a Compound of the Invention is able to prevent bone density loss.
[0371] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding. The invention has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. It will be obvious to one of skill in the art that changes and modifications may be practiced within the scope of the appended claims. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled. All patents, patent applications and publications cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application or publication were so individually denoted.

Claims

What is claimed is:
1. A compound of the formula:
Figure imgf000152_0001
or a stereoisomer or mixture of isomers thereof and optionally as a pharmaceutically
acceptable salt thereof, where
R is heterocycloalkyl optionally substituted with one or two groups which groups are
hydroxyl, tert-butyl-dimethylsilyloxy, or alkoxycarbonyl; or R is according to
formula
Figure imgf000152_0002
m is 1 or 2;
R1 is hydrogen or -P(0)(OR6)2;
R2 and R2a are independently hydrogen, cyano, halo, alkyl, haloalkyl, alkoxy, or haloalkoxy;
Ring A is phenyl, pyrimidinyl, or pyridinyl;
R3 is alkylamino, alkoxy, alkyl, or heterocycloalkyloxy;
R4 is halo, alkyl, alkoxy, alkoxycarbonayl, cyano, or hydroxy;
provided that when R3 is alkylamino, alkoxy, or alkyl, then R4 is halo, alkyl, alkoxy,
alkoxycarbonyl, or cyano; and
when R3 is heterocycloalkyloxy, then R4 is hydroxy;
R5 is hydrogen or alkyl;
R5a is hydrogen, alkyl, formyl, alkylcarbonyl, or alkoxycarbonyl;
each R6 is independently hydrogen or alkyl;
each R7 and R7a is independently hydrogen or alkyl;
R7b and R7c are independently hydrogen or alkyl; and
R7d is -OR1 or halo;
R8 is hydrogen, alkyl, or hydroxyalkyl;
provided that the compound of formula I is neither one of the following compounds nor a pharmaceutically acceptable salt of one of the following compounds, (2i?)-2-amino-3 - { [5-chloro-4-(5- { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluoropheny l]oxy } propan- 1 -ol ;
(2 j?)-2-amino-3- { [4-(5- { 3-bromo-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluorophenyl]oxy } propan- 1 -ol;
2-amino-3-{[5-chloro-4-(5-{5-chloro-6-[(l-methylethyl)oxy]pyridin-3-yl}-l,3,4-thiadiazol-2- yl)-2-fluoropheny l]oxy } propan- 1 -ol ;
(2S)-2-amino-3 - { [5-chloro-4-(5 - { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy } propan- 1 -ol ;
(2S)-2-amino-3- { [4-(5- { 3-bromo-4-[( 1 -methylethyl)oxy]phenyl} - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluorophenyl]oxy } propan- 1 -ol;
(2/?)-2-amino-3 - { [4-(5- { 3 -bromo-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluorophenyl]oxy} propyl dihydrogen phosphate;
(2S)-2-amino-3-{ [5-chloro-4-(5-{3-chloro-4-[(l -methylethyl)oxy]phenyl}-l ,3,4-thiadiazol-2- yl)-2-fluorophenyl]oxy} propyl dihydrogen phosphate;
2-amino-3-{ [5-chloro-4-(5-{6-chloro-5-[(l -methylethyl)oxy]pyridin-2-yl}- 1 ,3,4-thiadiazol-2- yl)-2-fluorophenyl]oxy } propan- 1 -ol;
(2i?)-2-amino-3- { [5-chloro-4-(5- {3-chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy} propyl dihydrogen phosphate;
(2i?)-2-amino-3 - [(5-chloro-4- { 5- [3 -chloro-4-(propyloxy)pheny 1]- 1 ,3 ,4-thiadiazol-2-yl } -2- fluorophenyl)oxy]propan- 1 -ol;
(27?)-2-amino-3- { [5 -chloro-4-(5 - { 5-chloro-6-[( 1 -methylethyl)oxy]pyridin-3 -yl } - 1 ,3 ,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}propan- 1 -ol;
(2i?)-2-amino-3- { [5 -chloro-4-(5 - { 6-chloro-5-[( 1 -methylethyl)oxy]pyridin-2-yl } - 1 ,3 ,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}propan-l-ol;
(2i?)-2-amino-3-[(5-chloro-4-{5-[3-chloro-4-(ethyloxy)phenyl]-l,3,4-thiadiazol-2-yl}-2- fluorophenyl)oxy]propan- 1 -ol;
(2i?)-2-amino-3-{[5-chloro-4-(5-{3,5-dichloro-4-[(l-methylethyl)oxy]phenyl}-l,3,4-thiadiazol-
2-yl)-2-fluorophenyl]oxy }propan- 1 -ol;
(2i?)-2-amino-3 - { [4-(5- { 3 -bromo-5-[( 1 -methylethyl)oxy ]phenyl } - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluorophenyl]oxy}propan-l-ol; (2S)-2-amino-3 - { [4-(5- { 3-bromo-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2-yl)-5- chloro-2-fluorophenyl]oxy} propyl dihydrogen phosphate;
(2S)-2-amino-3 - { [5 -chloro-4-(5 - { 5 -chloro-6- [( 1 -methylethyl)oxy]pyridin-3 -yl } - 1 ,3 ,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}propan- 1 -ol;
(2S)-2-amino-3- { [5-chloro-4-(5- {6-chloro-5-[( 1 -methylethyl)oxy]pyridin-2-yl } - 1 ,3,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}propan-l-ol;
(27?)-2-amino-3-{[3-chloro-4-(5-{3-chloro-4-[(l-methylethyl)oxy]phenyl}-l,3,4-thiadiazol-2- yl)phenyl]oxy}propan-l -ol;
5-[5-(4-{ [(27?)-2-amino-3-hydroxypropyl]oxy}-2-chloro-5-fluorophenyl)- 1 ,3,4-thiadiazol-2-yl]- 2-[(l-methylethyl)oxy]benzonitrile;
(2J?)-2-amino-3 - { [2,6-dichloro-4-(5- { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)phenyl]oxy } propan- 1 -ol ;
(2i?)-2-amino-3- { [2-chloro-4-(5- {3-chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-6-fluorophenyl]oxy } propan- 1 -ol;
(2i?)-2-amino-3 - { [5 -chloro-2-fluoro-4-(5- { 3 -fluoro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4- thiadiazol-2-y l)pheny 1] oxy } propan- 1 -ol ;
(2i?)-2-amino-3 - { [5 -chloro-4-(5 - { 3 -chloro-4- [( 1 -methylethyl)amino]phenyl } - 1 ,3 ,4-thiadiazol-
2-yl)-2-fluorophenyl]oxy } propan- 1 -ol ;
(25)-2-amino-3 - { [5 -chloro-4-(5 - { 3 -chloro-4- [( 1 -methylethyl)amino]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy} propan- l-ol;
(2R,3S)-3 -amino-4- { [5-chloro-4-(5- { 5 -chloro-6-[( 1 -methylethyl)amino]pyridin-3 -yl } - 1 ,3 ,4- thiadiazol-2-yl)-2-fluorophenyl]oxy}butan-2-ol;
( 1 i?,2S)-2-amino-3- { [5-chloro-4-(5- { 3 -cyano-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-
2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
( 1 i?,2S)-2-amino-3- { [5 -chloro-4-(5- { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
( 1 S,2S)-2-amino-3 - { [5-chloro-4-(5- { 3 -cyano-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
5.[5-(4-{[(2/?,35)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2- yl]-2-[(l-methylethyl)oxy]benzonitrile; (27?,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{3-methyl-4-[(l-methylethyl)oxy]phenyl}-l,3,4- thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2S,35)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{3-methyl-4-[(l-methylethyl)oxy]phenyl}-l,3,4- thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2/?,3<S)-3 -amino-4- { [5 -chloro-2-fluoro-4-(5- {2-methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol ;
(2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyridin-4-yl}- 1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol ;
( 1 S,2i?)-2-amino-3- { [5-chloro-4-(5- { 3-chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
(2R,3S)-3 -amino-4- { [5-chloro-2-fluoro-4-(5- { 5 -methyl-6- [( 1 -methylethyl)amino]pyridin-3-yl } - l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{5-methyl-6-[(l-methylethyl)amino]pyridin-3-yl}- l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2i?,3 ?)-3-amino-4- { [5 -chloro-2-fluoro-4-(5- { 5 -methyl-6-[( 1 -methylethyl)amino]pyridin-3 -yl } - 1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol ;
(2i?,3i?)-3-amino-4- { [5-chloro-2-fluoro-4-(5- {6-[( 1 -methylethyl)amino]pyridin-3-yl} - 1 ,3 ,4- thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
( 1 S,25)-2-amino-3- { [5 -chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
( 1 i?,27?)-2-amino-3 - { [5 -chloro-4-(5- { 3 -chloro-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}-l-methylpropyl dihydrogen phosphate;
(2S,3i?)-3-amino-4- { [5-chloro-4-(5- { 3 -chloro-4- [( 1 -methy lethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy } butan-2-ol ;
(2S,3S)-3 -amino-4- { [5-chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol- 2-yl)-2-fluorophenyl]oxy}butan-2-ol;
(2i?,35)-3 -amino-4- { [5-chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-
2-yl)-2-fluorophenyl]oxy } butan-2-ol ;
(2i?,3i?)-3 -amino-4- { [5-chloro-4-(5- { 3 -chloro-4-[( 1 -methylethyl)oxy]pheny 1 } - 1 ,3 ,4-thiadiazol-
2-yl)-2-fluorophenyl]oxy}butan-2-ol; 5-[5-(4-{[(2i?)-2-amino-3-hydroxypropyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2-yl]- 2-(ethyloxy)benzonitrile;
(2S)-2-amino-3- { [5-chloro-4-(5- { 3 -cyano-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4-thiadiazol-2- yl)-2-fluorophenyl]oxy} propyl dihydrogen phosphate;
(2/?)-2-amino-3- { [5-chloro-4-(5 - { 5 -chloro-6- [( 1 -methylethyl)amino]pyridin-3 -yl } - 1 ,3 ,4- thiadiazol-2-yl)-2-fluorophenyl]oxy } propan- 1 -ol;
5-[5-(4-{[(2S,3/?)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2- yl]-2-[(l-methylethyl)oxy]benzonitrile;
5-[5-(4-{[(2S,3S)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2- yl]-2-[(l -methylethyl)oxy]benzonitrile;
5-[5-(4-{[(2^,3 ?)-2-amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,3,4-thiadiazol-2- yl]-2-[(l-methylethyl)oxy]benzonitrile;
( 1 i?,2S)-2-amino-3 - { [5-chloro-2-fluoro-4-(5 - { 2-methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl } - 1 ,3,4-thiadiazol-2-yl)phenyl]oxy}-l -methylpropyl dihydrogen phosphate;
(lS,2S)-2-amino-3-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyridin-4-yl}- 1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy }- 1 -methylpropyl dihydrogen phosphate;
(2S,3i?)-3-amino-4- { [5-chloro-2-fluoro-4-(5- { 5-methyl-6-[( 1 -methylethyl)amino]pyridin-3-yl}- l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol;
(2i?,3i?)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l -methylethyl)amino]pyridin-4-yl}-
1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol ;
(2S,3i?)-3 -amino-4- { [5 -chloro-2-fluoro-4-(5- { 2-methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } -
1 ,3 ,4-thiadiazol-2-yl)phenyl]oxy } butan-2-ol ;
(l/?,2S)-2-amino-3-{[5-chloro-2-fluoro-4-(5-{3-methyl-4-[(l-methylethyl)oxy]phenyl}-l,3,4- thiadiazol-2-yl)phenyl]oxy}-l -methylpropyl dihydrogen phosphate;
( 1 i?,2i?)-2-amino-3- { [5-chloro-2-fluoro-4-(5- {2-methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl} -
1 ,3,4-thiadiazol-2-yl)phenyl]oxy }-l -methylpropyl dihydrogen phosphate;
( 1 S,2£)-2-amino-3 - { [5 -chloro-2-fluoro-4-(5- { 2-methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } -
1 ,3,4-thiadiazol-2-yl)phenyl]oxy }-l -methylpropyl dihydrogen phosphate;
(2R,3S)-3 -amino-4- { [5-chloro-2-fluoro-4-(5- { 6-methyl-5 - [( 1 -methylethyl)amino]pyridin-2-yl } - l,3,4-thiadiazol-2-yl)phenyl]oxy}butan-2-ol; ( 1 S,25)-2-amino-3 - { [5 -chloro-2-fluoro-4-(5- { 3 -methyl-4- [( 1 -methylethyl)oxy]phenyl } - 1 ,3 ,4- thiadiazol-2-yl)phenyl]oxy}-l-methylpropyl dihydrogen phosphate;
(2S,3S)-3-amino-4-(5-chloro-2-fluoro-4-{5-^
thiadiazol-2-yl } phenoxy)butan-2-ol; and
(25',35 -3-amino-4-(5-chloro-2-fluoro-4-{5-[6-methyl-4-(propan-2-ylamino)pyridin-2-yl]-l ,3,4- thiadiazol-2-yl } phenoxy)butan-2-ol.
2. The com ound of claim 1 according to one of the formulae,
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000158_0002
Figure imgf000158_0003
157 or
Figure imgf000159_0001
Figure imgf000159_0002
Figure imgf000159_0003
Figure imgf000160_0001
The com ound of claim 1 according to one of the formulae
Figure imgf000160_0002
8. The compound of any one of claims 1-7, wherein R1 is hydrogen.
9. The compound of any one of claims 1-7, wherein R1 is -P(0)(OR6)2.
10. The compound of any one of claims 1-9, wherein R2 and R2a are independently cyano, halo, or alkyl.
1 1. The compound of any one of claims 1 -9, wherein R2 and R2a are independently cyano, chloro, fluoro, or methyl.
12. The compound of any one of claims 1-1 1 , wherein R3 is alkylamino, alkoxy, or alkyl and R4 is halo, alkyl, alkoxy, alkoxycarbonyl, or cyano. The compound of any one of claims 1-11, wherein R is alkylamino, alkoxy, or alkyl and R4 is methyl, methoxy, ethoxy, ethoxycarbonyl, cyano, or chloro.
The compound of any one of claims 1-1 1, wherein R3 is ethoxy, isopropoxy, isopropylamino, or isobutyl and R4 is methyl, methoxy, ethoxy, ethoxycarbonyl, cyano, or chloro.
15. The compound of any one of claims 1-1 1, wherein R3 is heterocycloalkyloxy and R4 is hydroxy.
16. The compound of any one of claims 1-1 1 , wherein R3 is alkylamino and R4 is alkyl, alkoxycarbonyl, or cyano, or R3 is heterocycloalkyloxy and R4 is hydroxy.
17. The compound of any one of claims 1-16, wherein R5 is hydrogen and R5a is
hydrogen, alkyl, alkylcarbonyl, or alkoxycarbonyl.
18. The compound of any one of claims 1-16, wherein R3 is hydrogen; R a is hydrogen
The compound of any one of claims 1-18, wherein R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl- dimethylsilyloxy, or alkoxycarbonyl or R is according to one of the formulae,
Figure imgf000161_0001
20. The compound of claim 19, wherein each R7 and R7a is hydrogen.
21. The compound of claim 19 or 20, wherein R is hydrogen.
The compound of any one of claims 19-21, wherein R is hydrogen or alkyl.
23. The compound of claim 22, wherein R is hydrogen.
The compound of any one of claims 19-21, wherein R is hydrogen or alkyl.
The compound of claim 24, wherein R 0 is hydrogen or methyl.
26. The compound of claim 24, wherein R c is ethyl.
27. The compound of claim 24, wherein R c is hydrogen
The compound of any one of claims 1-18, wherein R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl- dimeth lsilylox , or alkoxycarbonyl or R is according to one of the formulae,
Figure imgf000162_0001
29. The compound of claim 28, wherein each R7 and R7a is hydrogen.
30. The compound of claim 28 or 29, wherein R7c is hydrogen or alkyl
31. The compound of claim 30, wherein R7c is hydrogen or methyl.
32. The compound of claim 30, wherein R is methyl.
33. The compound of claim 30, wherein R7c is hydrogen.
34. The compound of any one of claims 1-18, wherein R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl- dimethylsilyloxy or alkoxycarbonyl; or R is according to formula
Figure imgf000163_0001
wherein R7, R7a, and R7c are hydrogen; R7b is hydi alkyl; and R is hydi
The compound of any one of claims 1-18, wherein R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl- dimeth lsilyloxy, or alkoxycarbonyl; or R is according to the formula
Figure imgf000163_0002
, wherein R7, R7a, and R7c are hydrogen; R7b is hydrogen or alkyl; and
R is hydrogen.
36. The compound of any one of claims 1-18, wherein R is heterocycloalkyl optionally substituted with one or two groups which groups are hydroxyl, tert-butyl- dimethylsilyloxy, or alkoxycarbonyl.
37. The Compound of claim 1 optionally as a pharmaceutically acceptable salt thereof, selected from the list of compounds in Tables 1 and 2.
38. A pharmaceutical composition which comprises a compound of any of claims 1-37 or a single stereoisomer or a mixture of isomers thereof, additionally optionally as a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.
39. A method for treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound, or a single stereoisomer or a mixture of isomers thereof, of any of claims 1-37 optionally as a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 38.
40. The method of claim 39 where the disease is an autoimmune disease.
41. The method of claim 40 where the autoimmune disease is multiple sclerosis, psoriasis, inflammatory bowel disease, graft-versus-host disease, autoimmune-induced inflammation, or rheumatoid arthritis.
42. The method of claim 39 where the disease is osteoporosis.
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