WO2017004609A1 - Modulateurs thiadiazole de s1p et procédés de fabrication et d'utilisation - Google Patents

Modulateurs thiadiazole de s1p et procédés de fabrication et d'utilisation 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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English (en)
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Wei Xu
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Exelixis, Inc.
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Publication of WO2017004609A1 publication Critical patent/WO2017004609A1/fr

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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

L'invention concerne des composés de la formule : dans laquelle chacune des variables sont telles que définis dans la description, ainsi que des procédés de fabrication et d'utilisation des composés en tant qu'agonistes de S1P1 et/ou de S1P5 pour le traitement, par exemple, d'une maladie auto-immune.
PCT/US2016/040895 2015-07-02 2016-07-02 Modulateurs thiadiazole de s1p et procédés de fabrication et d'utilisation WO2017004609A1 (fr)

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WO2018211324A1 (fr) * 2017-05-17 2018-11-22 Oppilan Pharma Ltd, Promédicaments pour traiter une maladie
CN113527281A (zh) * 2020-04-20 2021-10-22 昆山彭济凯丰生物科技有限公司 杂环化合物及其制备方法和应用

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Publication number Priority date Publication date Assignee Title
WO2018211324A1 (fr) * 2017-05-17 2018-11-22 Oppilan Pharma Ltd, Promédicaments pour traiter une maladie
CN113527281A (zh) * 2020-04-20 2021-10-22 昆山彭济凯丰生物科技有限公司 杂环化合物及其制备方法和应用
CN113527281B (zh) * 2020-04-20 2023-12-22 昆山彭济凯丰生物科技有限公司 杂环化合物及其制备方法和应用

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