WO2017004608A1 - Oxadiazole modulators of s1p methods of making and using - Google Patents

Abstract

The invention is directed to Compounds of Formula (I): wherein each variable is defined herein, as well as methods of making and using the compounds as agonists of S1P1 and/or S1P5 for instance for treating graft versus host disease and autoimmune diseases.

Description

OXADIAZOLE MODULATORS OF SIP AND METHODS OF MAKING AND

USING PRIORITY CLAIM

[0001] This application claims priority to United States Provisional Application Serial No. 62/188,293, filed July 2, 2015. The entire contents of the aforementioned application are Incorporated herein by reference.

BACKGROUND OF THE INVENTION

Statement Regarding Joint Research Agreement

[0002] One or more inventions contained in this application were developed under a joint research agreement as defined in the Cooperative Research and Technology Enhancement Act of 2001 between Exelixis, Inc. and Boehringer Ingelheim International,GmbH.

Field of the Invention

[0003] This invention relates to the field of agonists of Sphingosine 1 -Phosphate Type 1 Receptor (S1P1R or SI PI) and/or Type 5 Receptor (S1P5R or S1P5), and methods of their use.

Summary of the Related Art

[0004] Sphingosine 1 -phosphate (S 1 P) 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 S1P1 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 SI PI receptor are thought to induce long-term down-regulation of S1P1 on lymphocytes, thereby inhibiting the migration of lymphocytes toward SIP. The consequential decrease in trafficking and infiltration of antigen-specific T cells provides a means of immunomodulating activity that can be useful in the treatment of various immune- related conditions such as graft versus host disease and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosis. Therefore, agonists of S1P1R are potentially useful immunosuppressants for the treatment of a variety of autoimmune conditions.

[0005] The myelin sheath that surrounds neural cell axons is required to insulate neural axons and allow rapid movement of electrical impulses through the myelinated nerve fiber. Demyelination, or loss of the integrity of the myelin sheath is the hallmark of autoimmune neurodegenerative diseases, including multiple sclerosis. The myelin sheath in the central nervous system is produced by oligodendrocytes. Mature, myelin-producing

oligodendrocytes express SIP receptor transcripts in relative abundance of

S1P5>S1P3>S1P1, with undetectable levels of S1P4. Fingolimod (FTY720), a sphingosine- 1 -phosphate (SIP) analogue that has been used successfully in clinical trials as a systemic immunomodulatory therapy for multiple sclerosis, readily accesses the central nervous system. In vitro studies using either mature adult human oligodendrocytes or oligodendrocyte precursor cells demonstrate that fmgolomid or SIP has significant effects on oligodendrocyte process elongation and retraction as well as on cell survival and apoptosis. These studies further demonstrated that the effects seen were, at least in part, due to S1P5. These results indicate that S1P5 may play an important role in the beneficial effect observed with fmgolomid treatment of multiple sclerosis.

[0006] Sphingosine-1 -phosphate (SIP) has been shown to regulate the migration of osteoclast precursors, demonstrating a role in bone mineral homeostasis and suggesting a role in treating bone-destroying disorders such as rheumatoid arthritis and osteoporosis {Nature 2009, 458(7237), 524-528).

[0007] S1P3 has been associated with acute toxicty 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 S 1P1 and/or S1P5, without being active for S1P3, are desirable.

SUMMARY OF THE INVENTION

[0008] The following only summarizes certain aspects of the invention and is not intended to be limiting in nature. These aspects and other aspects and embodiments are described more fully below. All references cited in this specification are hereby incorporated by reference in their entirety. In the event of a discrepancy between the express disclosure of this specification and the references incorporated by reference, the express disclosure of this specification shall control.

[0009] The invention provides compounds that are agonists of SI PI and/or S1P5 and that are useful in the treatment of graft versus host disease and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and systemic lupus erythematosis, in mammals. This invention also provides methods of making the compound, methods of using such compounds in the treatment of graft versus host disease and autoimmune diseases, especially in humans, and to pharmaceutical compositions containing such compounds.

[0010] A first aspect of the invention provides compounds of embodiment (1) of Formula I:

I

or a stereoisomer or mixture of isomers thereof and optionally as a pharmaceutically

acceptable salt thereof, where

R¹ is hydrogen or -P(0)(OR⁶)₂;

R² and R^2a are independently hydrogen, cyano, halo, alkyl, haloalkyl, alkoxy, or haloalkoxy; Ring A is phenyl or 6-membered heteroaryl;

each R³ is independently cyano, halo, alkyl, haloalkyl, hydroxy, alkoxy, haloalkoxy,

alkoxyalkyloxy, cycloalkyloxy, heterocycloalkyloxy, nitro, phenyl, amino, alkylamino, dialkylamino, aminocarbonyl, alkylcarbonylamino, or alkoxycarbonylamino;

n is 0, 1, or 2;

R⁴ is hydrogen, alkyl, cyano, halo, haloalkyl, or hydroxyalkyl;

R⁵ is hydrogen or alkyl;

R^5a is hydrogen, alkyl, alkylcarbonyl, or alkoxycarbonyl;

each R⁶ is independently hydrogen or alkyl;

R⁷, R^7a, R^7b, and R^7c are independently hydrogen or alkyl; and

R is hydrogen, alkyl, or hydroxyalkyl.

[0011] In a second aspect, the invention is directed to a pharmaceutical composition which comprises 1) a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt thereof and 2) a pharmaceutically acceptable carrier, excipient, or diluent.

[0012] In a third aspect, the invention provides a method for treating a disease, disorder, or syndrome which method comprises administering to a patient a therapeutically effective amount of a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I or a single stereoisomer or mixture of isomers thereof, optionally as a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0013] In a fourth aspect, the Invention is directed to a method of making a Compound of the Invention which method comprises:

reacting an intermediate of formula 116, or a salt thereof:

116

where PG is a nitrogen-protecting group, and R², R^2a, and R^7c are as defined in the Summary of the Invention for a Compound of Formula I; with an intermediate of formula 117:

117

where n, Ring A, R³, and R⁴ are as defined in the Summary of the Invention for a Compound of Formula I to yield an intermediate of formula 118:

118

and followed by deprotection and ring opening to yield a Compound of the Invention the formula:

wherein R², R^2a, R³, R⁴, R^7c, ring A, and n are as defined for Formula I, and optionally separating individual isomers; and optionally modifying any of the R², R^2a, R³, R⁴, and R^7c groups; and optionally forming a pharmaceutically acceptable salt, hydrate, solvate or combination thereof. DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

The following abbreviations and terms have the indicated meanings throughout:

Abbreviation Meanin

[0015] The symbol "-" means a single bond, "=" means a double bond, "≡" means a triple bond, " " means a single or double bond. The symbol 'ΊΛΛ /" refers to a group on a double-bond as occupying either position on the terminus of a double bond to which the symbol is attached; that is, the geometry, E- or Z-, of the double bond is ambiguous. When a group is depicted removed from its parent formula, the "~" symbol will be used at the end of the bond which was theoretically cleaved in order to separate the group from its parent structural formula.

[0016] When chemical structures are depicted or described, unless explicitly stated otherwise, all carbons are assumed to have hydrogen substitution to conform to a valence of four. For example, in the structure on the left-hand side of the schematic below there are nine hydrogens implied. The nine hydrogens are depicted in the right-hand structure. Sometimes a particular atom in a structure is described in textual formula as having a hydrogen or hydrogens as substitution (expressly defined hydrogen), for example, -CH₂CH₂-. It is understood by one of ordinary skill in the art that the aforementioned descriptive techniques are common in the chemical arts to provide brevity and simplicity to description of otherwise complex structures.

[0017] If a group "R" is depicted as "floating" on a ring system, as for example in the formula:

then, unless otherwise defined, a substituent "R" may reside on any atom of the ring system, assuming replacement of a depicted, implied, or expressly defined hydrogen from one of the ring atoms, so long as a stable structure is formed.

[0018] If a group "R" is depicted as floating on a fused ring system, as for example in the formulae:

then, unless otherwise defined, a substituent "R" may reside on any atom of the fused ring system, assuming replacement of a depicted hydrogen (for example the -NH- in the formula above), implied hydrogen (for example as in the formula above, where the hydrogens are not shown but understood to be present), or expressly defined hydrogen (for example where in the formula above, "Z" equals =CH-) from one of the ring atoms, so long as a stable structure is formed. In the example depicted, the "R" group may reside on either the 5-membered or the 6-membered ring of the fused ring system.

[0019] When a group "R" is depicted as existing on a ring system containing saturated carbons, as for example in the formula:

where, in this example, "y" can be more than one, assuming each replaces a currently depicted, implied, or expressly defined hydrogen on the ring; then, unless otherwise defined, where the resulting structure is stable, two "R's" may reside on the same carbon. A simple example is when R is a methyl group; there can exist a geminal dimethyl on a carbon of the depicted ring (an "annular" carbon). In another example, two R's on the same carbon, including that carbon, may form a ring, thus creating a spirocyclic ring (a "spirocyclyl" group) structure with the depicted ring as for example in the formula:

[0020] Although all moieties are generally referred to as their monovalent form (e.g., alkyl, aryl), those skilled in the art will understand from the context and standard valence rules when di-, tri-, etc., valent radicals are intended. So, for example, alkyl can refer to a monovalent alkyl radical or a divalent radical (i.e., alkylene).

[0021] "Administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., surgery, radiation, and chemotherapy, etc.), "administration" and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.

[0022] "Alkyl" means a linear saturated hydrocarbon radical of one to six carbon atoms or a branched saturated hydrocarbon radical of three to six carbon atoms, e.g., methyl, ethyl, propyl, 2-propyl, butyl (including all isomeric forms), or pentyl (including all isomeric forms), and the like.

[0023] "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.

[0024] "Alkoxy" means an -OR group where R is alkyl group as defined herein.

Examples include methoxy, ethoxy, propoxy, isopropoxy, and the like.

[0025] "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.

[0026] "Alkoxyalkyloxy" means an -OR group where R is alkoxyalkyl as defined herein.

[0027] "Alkoxycarbonyl" means a -C(0)R group where R is alkoxy, as defined herein.

[0028] "Alkoxycarbonylamino" means an -NHR group where R is alkoxycarbonyl as defined herein.

[0029] "Alkylamino" means an -NHR group where R is alkyl, as defined herein.

[0030] "Alkylaminoalkyl" means an alkyl group substituted with one or two alkylamino groups, as defined herein. [0031] "Alkylcarbonyl" means a -C(0)R group where R is alkyl, as defined herein.

[0032] "Alkylcarbonylamino" means an -NHR group where R is alkylcarbonyl, as defined herein.

[0033] "Alkylsulfonyl" means an -S(0)₂R group where R is alkyl, as defined herein, e.g. methylsulfonyl, isopropylsulfonyl.

[0034] "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.

[0035] "Amino" means -NH₂.

[0036] "Aminocarbonyl" means a -C(0)NH₂ group.

[0037] "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.

[0038] "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.

[0039] "Carboxy" means a -C(0)OH group.

[0040] "Cycloalkyl" means a monocyclic or fused bicyclic, saturated or partially unsaturated (but not aromatic), hydrocarbon radical of three to ten carbon ring atoms. Fused bicyclic hydrocarbon radical includes bridged ring systems. Unless stated otherwise, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. One or two ring carbon atoms may be replaced by a -C(O)-, -C(S)-, or -C(=NH)- group. More specifically, the term cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl, or cyclohex-3-enyl, and the like.

[0041] "Cycloalkyloxy" means an -OR group where R is cycloalkyl as defined herein.

[0042] "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.N-methylethylamino, and the like.

[0043] "Fused ring" means a polycyclic ring that contains bridged or fused rings; that is, where two rings have more than one shared atom in their ring structures. In this application, fused ring systems are not necessarily all aromatic ring systems. Typically, but not necessarily, fused rings share a vicinal set of atoms, for example naphthalene or 1,2,3,4- tetrahydro-naphthalene. A spiro ring system is not a fused ring system by this definition, but fused ring systems of the invention may themselves have spiro rings attached thereto via a single ring atom of the fused ring system. In some examples, as appreciated by one of ordinary skill in the art, two adjacent groups on an aromatic system may be fused together to form a ring structure. The fused ring structure may contain heteroatoms and may be optionally substituted with one or more groups. It should additionally be noted that saturated carbons of such fused groups (i.e. saturated ring structures) can contain two substitution groups.

[0044] "Halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.

[0045] "Haloalkoxy" means an -OR' group where R' is haloalkyl as defined herein, e.g., trifluoromethoxy or 2,2,2-trifluoroethoxy, and the like.

[0046] "Haloalkyl" mean an alkyl group substituted with one or more halogens, specifically one to five halo atoms, e.g., trifluoromethyl, 2-chloroethyl, and 2,2-difluoroethyl, and the like.

[0047] "Heteroaryl" means a monocyclic, fused bicyclic, or fused tricyclic, radical of 5 to 14 ring atoms containing one or more, specifically one, two, three, or four ring heteroatoms independently selected from -0-, -S(0)_n- (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. One or two ring carbon atoms of any nonaromatic rings comprising a bicyclic or tricyclic radical may be replaced by a -C(O)-, -C(S)-, or -C(=NH)- group. R is hydrogen, alkyl, hydroxy, alkoxy, acyl, or alkylsulfonyl. 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. More specifically, the term heteroaryl includes, but is not limited to, 1 ,2,4-triazolyl, 1,3,5-triazolyl, phthalimidyl, pyridinyl, pyrrolyl, imidazolyl, thienyl, furanyl, indolyl, 2,3-dihydro-lH-indolyl (including, for example, 2,3-dihydro-lH-indol-2-yl or 2,3-dihydro- lH-indol-5-yI, and the like), isoindolyl, indolinyl, isoindolinyl, benzimidazolyl, benzodioxol- 4-yl, benzofuranyl, cinnolinyl, indolizinyl, naphthyridin-3-yl, phthalazin-3-yl, phthalazin-4- yl, pteridinyl, purinyl, quinazolinyl, quinoxalinyl, tetrazoyl, pyrazolyl, pyrazinyl,

pyrimidinyl, pyridazinyl, oxazolyl, isooxazolyl, oxadiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl (including, for example, tetrahydroisoquinolin-4-yl or tetrahydroisoquinolin-6-yl, and the like), pyrrolo[3,2-c]pyridinyl (including, for example, pyrrolo[3,2-c]pyridin-2-yl or pyrrolo[3,2-c]pyridin-7-yl, and the like), benzopyranyl, thiazolyl, isothiazolyl, thiadiazolyl, benzothiazolyl, benzothienyl, and the derivatives thereof, or N-oxide or a protected derivative thereof.

[0048] "Heteroatom" refers to O, S, N, and P.

[0049] "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. One or two ring carbon atoms may be replaced by a -C(O)-, -C(S)-, or -C(=NH)- group. Fused bicyclic radical includes bridged ring systems. Unless otherwise stated, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. When the point of valency is located on a nitrogen atom, R^y is absent. More specifically the term heterocycloalkyl includes, but is not limited to, azetidinyl, pyrrolidinyl, 2-oxopyrrolidinyl, 2,5-dihydro-lH-pyrrolyl, piperidinyl, 4-piperidony , morpholinyl, piperazinyl, 2-oxopiperazinyl, tetrahydropyranyl, 2-oxopiperidinyl,

thiomorpholinyl, thiamorpholinyl, perhydroazepinyl, pyrazolidinyl, imidazolinyl,

imidazolidinyl, dihydropyridinyl, tetrahydropyridinyl, oxazolinyl, oxazolidinyl,

isoxazolidinyl, thiazolinyl, thiazolidinyl, quinuclidinyl, isothiazolidinyl, octahydroindolyl, octahydroisoindolyl, decahydroisoquinolyl, tetrahydrofuryl, and tetrahydropyranyl, and the derivatives thereof and N-oxide or a protected derivative thereof.

[0050] "Heterocycloalkyloxy" means an -OR group where R is hetero cycloalkyl as defined herein.

[0051] "Hydroxyalkyl" means an alkyl group substituted with at least one, in another example with one, two, or three, hydroxy groups.

[0052] "Spirocyclyl" or "spirocyclic ring" refers to a ring originating from a particular annular carbon of another ring. For example, as depicted below, a ring atom of a saturated bridged ring system (rings B and B'), but not a bridgehead atom, can be a shared atom between the saturated bridged ring system and a spirocyclyl (ring A) attached thereto. A spirocyclyl can be carbocyclic or heteroalicyclic.

[0053] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. One of ordinary skill in the art would understand that with respect to any molecule described as containing one or more optional substituents, only sterically practical and/or synthetically feasible compounds are meant to be included. "Optionally substituted" refers to all subsequent modifiers in a term. So, for example, in the term "optionally substituted arylC].g alkyl," optional substitution may occur on both the "Ci_-8 alkyl" portion and the "aryl" portion of the molecule may or may not be substituted.

[0054] "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.

[0055] 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 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.

[0056] Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,

2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid,

2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid, and salicylic acid and the like. [0057] Examples of a pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Specific salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,

2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine,

theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine,

N-methylglucamine, polyamine resins, and the like. Exemplary organic bases are

isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine."Platin(s)," and "platin-containing agent(s)" include, for example, cisplatin, carboplatin, and oxaliplatin.

[0058] "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.

[0059] "Stereoisomer" means any of two or more isomers containing the same atoms bonded to each other in an identical manner but differing from each other in the spatial arrangement of the atoms or groups of atoms. "Stereoisomer" includes, for example, an enantiomer, a geometric isomer, a diastereomer, a rotamer, cis-isomer, trans-isomer, and conformational isomer. The names and illustration used in this application to describe compounds of the invention, unless indicated otherwise, are meant to encompass all possible stereoisomers and any mixture, racemic or otherwise, thereof.

[0060] "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.

[0061] "Treating" or "treatment" of a disease, disorder, or syndrome, as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a human, i.e. causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by one of ordinary skill in the art.

[0062] "Yield" for each of the reactions described herein is expressed as a percentage of the theoretical yield.

Embodiments of the Invention

[0063] The following paragraphs present a number of embodiments of compounds of the invention. In each instance the embodiment includes the recited compounds, as well as a single stereoisomer or mixture of stereoisomers thereof, as well as a pharmaceutically acceptable salt thereof.

[0064] The invention further comprises subgenera of embodiment (1) in which the substituents are selected as any and all combinations of structural formula (I), R¹, R², R^2a, R³, R⁴, R⁵, R^5a, R⁷, R^7a, R^7b, R^7c, R⁸ and n as defined herein, including without limitation, the following: [°°65] Structural Formula I is one of formulae (I-a) - (i-b). (Il-a - fll-d). (Hl-a) -

(IV-a) (IV-b)

(IV-c) (IV-d)

IV-g) (IV-h)

(V-e) (V-f)

(VI-e) (Vl-f)

[0066] R¹ is selected from one of the following groups (la) - (lg):

(la) hydrogen.

(lb) -P(0)(OR⁶)₂.

(lc) -P(0)(OR⁶)₂, wherein each R⁶ is independently alkyl.

(Id) -P(0)(OR⁶)₂, wherein each R⁶ is independently hydrogen or t-butyl.

(le) -P(0)(OR⁶)₂, wherein each R⁶ is independently t-butyl.

(If) hydrogen or -P(0)(OH)₂.

(ig) -P(0)(OH)₂.

[0067] R² and R^2a are selected from one of the following groups (2a) - (2m):

(2a) R² and R^2a are independently cyano, halo, or alkyl.

(2b) R² and R^2a are independently halo or alkyl.

(2c) R² and R^2a are each independently halo.

(2d) R² and R^2a are each chloro.

(2e) R² is chloro or fluoro and R^2a is chloro or fluoro.

(2f) R² is chloro and R^2a is fluoro.

(2g) R² and R^2a are each independently alkyl.

(2h) R² and R^2a are each methyl.

(2i) R² is alkyl and R^2a is halo. (2j) R^z is methyl and R is halo.

(2k) R^ is methyl and R ^a is chloro or fluoro

(21) R is methyl and R is fluoro.

(2m) R is methyl and R is chloro.

[0068] each R is independently selected from one of the following groups (3a) -

(3D):

(3a) each R³ is independently alkyl, haloalkyl, alkoxy, haloalkoxy, alkoxyalkyloxy, cycloalkyloxy, heterocycloalkyloxy, alkylamino, or dialkylamino.

(3b) each R³ is independently alkyl, haloalkyl, alkoxy, haloalkoxy, alkylamino, or dialkylamino.

(3c) each R³ is independently alkyl, alkoxy, or alkylamino.

(3d) each R³ is independently alkyl or alkoxy.

(3e) each R is independently alkyl or alkylamino.

(3f) each R³ is independently alkoxy or alkylamino.

(3g) each R³ is independently alkyl.

(3h) each R³ is independently alkoxy.

(3i) each R³ is independently alkylamino.

(3j) each R³ is isopropoxy, isopropylamino, or isobutyl.

(3k) each R³ is isopropoxy or isopropylamino.

(31) each R³ is isopropoxy or isobutyl.

(3m) each R³ is isopropylamino or isobutyl.

(3n) each R³ is isopropoxy.

(3o) each R is isopropylamino.

(¾ ) each R is isobutyl.

[0069] R⁴ is selected from one of the following groups (4a) - (4m);

(4a) R⁴ is alkyl, cyano, halo, haloalkyl, or hydroxyalkyl.

(4b) R⁴ is methyl, cyano, chloro, or hydroxymethyl.

(4c) R⁴ is alkyl, cyano, halo, or hydroxyalkyl.

(4d) R⁴ is alkyl, cyano, halo;.

(4e) R⁴ is methyl, cyano, halo.

(4f) R⁴ is methyl, cyano, chloro.

(4g) R⁴ is alkyl.

(4h) R⁴ is methyl.

(4i) R⁴ is cyano. (4j) R⁴ is halo.

(4k) R⁴ is chloro.

(41) R⁴ is hydroxyalkyl.

(4m) R⁴ is hydroxymethyl.

[0070] R⁵ and R^5a are selected from one of the following groups (5a) - f5<T);

(5a) R⁵ is hydrogen and R^5a is hydrogen, alkyl, alkylcarbonyl, or alkoxycarbonyl.

(5b) R⁵ is hydrogen and R^5a is alkylcarbonyl or alkoxycarbonyl.

(5c) R⁵ is hydrogen and R^5a is hydrogen or alkyl.

(5d) R⁵ is hydrogen; R^5a is hydrogen.

[0071] n, when present, is selected from one of the following groups (6a) - (6f):

(6a) n is 0, 1, or 2.

(6b) n is O or l .

(6c) n is 1 or 2.

(6d) n is 0.

(6e) n is 1.

(6f) n is 2.

[0072] R⁷. R^7a. R^7b. R^7c and R⁸ are selected from one of the following groups (7a) - (7g):

(7a) R⁷ and R^7a are each hydrogen; R^7b and R^7c are independently hydrogen or alkyl; and R⁸ is hydrogen, alkyl, or hydroxyalkyl.

(7b) R⁷, R^7a, and R^7b are each hydrogen; R^7c is hydrogen or alkyl; and R⁸ is hydrogen, alkyl, or hydroxyalkyl.

(7c) R⁷, R^7a, R^7b and R⁸ are each hydrogen; and R^7c is hydrogen or alkyl.

(7d) R⁷, R^7a, R^7b and R⁸ are each hydrogen; and R^7c is hydrogen or methyl.

(7e) R⁷, R⁷ , R^7b and R⁸ are each hydrogen; and R^7c is alkyl.

(7f) R⁷, R^7a, R^7b and R⁸ are each hydrogen; and R^7c is methyl.

(7g) R⁷, R^7a, R^7b , R^7c and R⁸ are hydrogen.articular embodiments of this aspect of the invention include compounds of any one of the formulae (I), (I-a) - (I-b), (Il-a) - (II-d), (III- a) - (III-c), (IV-a) - (IV-i), (V-a) - (V-f), and (Vl-a) - (Vl-f), each as defined in each of the following rows, wherein each entry is a group number as defined above (e.g., (6e) refers to n is 1), and a dash "-" indicates that the variable is as defined for formula (I) or defined according to any one of the applicable variable definitions l(a)-7(g) [e.g., when R¹ is a dash, it can be either as defined for Formula (I) or any one of definitions (la)-(lg)]: E_bidtmomen ∞

¾

« w

Ό ·- a ¾ ¾ e r-~ Ό

e se 06 a ¾ a e

« a

¾

- - 3b 4a - 6e - (l)-26 - - 3c 4a - 6e 7a

(l)-2 - - 3b 4b - 6e - (l)-27 - - 3c 4b - 6e 7a

(l)-3 - - 3b 4d - 6e - (l)-28 - - 3c 4d - 6e 7a

(l)-4 - - 3b 4f - 6e - (l)-29 - - 3c 4f - 6e 7a

(l)-5 - - 3b 4h - 6e - (l)-30 - - 3c 4h - 6e 7a

(l)-6 - - 3c 4a - 6e - (1)-31 - - 3f 4a - 6e 7a

(l)-7 - - 3c 4b - 6e - (l)- E^bdit3momen2 - - 3f 4b - 6e 7a

(l)-8 - - 3c 4d - 6e - (l)-33 - - 3f 4d - 6e 7a

(l)-9 - - 3c 4f - 6e - (l)-34 - - 3f 4f - 6e 7a

(l)-lO - - 3c 4h - 6e - (l)-35 - - 3f 4h - 6e 7a

(l)-ll - - 3f 4a - 6e - (l)-36 - - 3j 4a - 6e 7a

(D-12 - - 3f 4b - 6e - (l)-37 - - 3j 4b - 6e 7a

(D-13 - - 3f 4d - 6e - (l)-38 - - 3j 4d - 6e 7a

(D-14 - - 3f 4f - 6e - (l)-39 - - 3j 4f - 6e 7a

(D-15 - - 3f 4h - 6e - (l)-40 - - 3j 4h - 6e 7a

(D-16 - - 3j 4a - 6e - (1)-41 - - 3b 4a - 6e 7c

(D-17 - - 3j 4b - 6e - (l)-42 - - 3b 4b - 6e 7c

(D-18 - - 3j 4d - 6e - (l)-43 - - 3b 4d - 6e 7c

(D-19 - - 3j 4f - 6e - (l)-44 - - 3b 4f - 6e 7c

(D-20 - - 3j 4h - 6e - (l)-45 - - 3b 4h - 6e 7c

(1)-21 - - 3b 4a - 6e 7a (l)-46 - - 3c 4a - 6e 7c

(D-22 - - 3b 4b - 6e 7a (D-47 - - 3c 4b - 6e 7c

(l)-23 - - 3b 4d - 6e 7a (l)-48 - - 3c 4d - 6e 7c

(l)-24 - - 3b 4f - 6e 7a (l)-49 - - 3c 4f - 6e 7c

(l)-25 - - 3b 4h - 6e 7a (l)-50 - - 3c 4h - 6e 7c

E_bditmomen ∞ ∞

Pi Pi es « ea

¾ <¾ Ρί

¾ i Pi a Ό e Ό Ό -S

«8 Ρί 8 Pi 8 8

5 Pi

¾ ¾ a

Pi ¾ ¾ Pi Pi Pi

[1)-101 If 2b 3f 4d 5e 6e 7c [1)-126 la 2e 3j 4d 5e 6e 7c

[1)-102 la 2b 3f 4d 5e 6e 7c ;i)-127 lc 2e 3j 4d 5e 6e 7c

(1)-103 lc 2b 3f 4d 5e 6e 7c (1)-128 If 2b 3f 4f 5e 6e 7c

(1)-104 If 2a 3f 4d 5e 6e 7c (1)-129 la 2b 3f 4f 5e 6e 7c1)-105 la 2a 3f 4d 5e 6e 7c [1)-130 lc 2b 3f 4f 5e 6e 7c

[1)-106 lc 2a 3f 4d 5e 6e 7c [1)-131 If 2a 3f 4f 5e 6e 7c

[1)-107 If 2e 3f 4d 5e 6c 7c [1)- E^bdi1^tmom^en32 la 2a 3f 4f 5e 6e 7c

:i)-108 la 2e 3f 4(1 5e 6e 7c [1)-133 lc 2a 3f 4f 5e 6e 7c

:i)-109 lc 2e 3f 4(1 5e 6e 7c [1)-134 If 2e 3f 4f 5e 6e 7ci)-iio If 2b 3c 4(1 5e 6e 7c [1)-135 la 2e 3f 4f 5e 6c 7c

[1)-111 la 2b 3c 4d 5e 6e 7c [1)-136 lc 2e 3f 4f 5e 6c 7c

[1)-112 lc 2b 3c 4d 5e 6e 7c [1)-137 If 2b 3c 4f 5e 6c 7c

[1)-113 If 2a 3c 4d 5e 6e 7c [1)-138 la 2b 3c 4f 5e 6c 7ci)-114 la 2a 3c 4d 5e 6e 7c [1)-139 lc 2b 3c 4f 5e 6c 7c

(1)-115 lc 2a 3c 4(1 5e 6e 7c (1)-140 If 2a 3c 4f 5e 6e 7c

[1)-116 If 2e 3c 4d 5e 6e 7c [1)-141 la 2a 3c 4f 5e 6c 7c

[1)-117 la 2e 3c 4d 5e 6e 7c [1)-142 lc 2a 3c 4f 5e 6c 7c1)-118 lc 2e 3c 4d 5e 6e 7c (1)-143 If 2e 3c 4f 5e 6e 7c

[1)-119 If 2b 3j 4d 5e 6e 7c (1)-144 la 2e 3c 4f 5e 6c 7c

[1)-120 la 2b 3j 4d 5e 6e 7c (1)-145 lc 2e 3c 4f 5e 6c 7c

[l m lc 2b 3j 4d 5e 6e 7c (1)-146 If 2b 3j 4f 5e 6c 7c

:i)-122 If 2a 3j 4(1 5e 6e 7c (1)-147 la 2b 3j 4f 5e 6e 7c

[1)-123 la 2a 3j 4d 5e 6e 7c (1)-148 lc 2b 3j 4f 5e 6c 7c

[1)-124 lc 2a 3j 4d 5e 6e 7c [1)-149 If 2a 3j 4f 5e 6c 7c

[1)-125 If 2e 3j 4d 5e 6e 7c (1)-150 la 2a 3j 4f 5e 6e 7c

E_bditmomen ∞ 00

PS OS

eg « u

¾ OS PS

OS S

¾

OS Ό

«a os «a a PS a

« OS OS c β

S

m 1/1 a

OS OS PS OS OS PS PS os 1)-201 la 2b 3j 4f 5a 6e 7c [l)-226 lc 2e 3c 4d 5e 6e 7a

[l)-202 lc 2b 3j 4f 5a 6e 7c [l)-227 If 2b 3j 4d 5e 6e 7a

(l)-203 If 2a 3j 4f 5a 6e 7c [l)-228 la 2b 3j 4d 5e 6e 7a

[l)-204 la 2a 3j 4f 5a 6e 7c [l)-229 lc 2b 3j 4d 5e 6e 7al)-205 lc 2a 3j 4f 5a 6e 7c (l)-230 If 2a 3j 4d 5e 6e 7a

[l)-206 If 2e 3j 4f 5a 6e 7c [1)-231 la 2a 3j 4d 5e 6e 7al)-207 la 2e 3j 4f 5a 6e 7c [l)-2 E^bditmmo^en32 lc 2a 3j 4d 5e 6e 7a l)-208 lc 2e 3j 4f 5a 6e 7c [l)-233 If 2e 3j 4d 5e 6e 7a

[l)-209 If 2b 3f 4d 5e 6e 7a (l)-234 la 2e 3j 4d 5e 6e 7a

[1)-210 la 2b 3f 4d 5e 6e 7a [l)-235 lc 2e 3j 4d 5e 6c 7a

[1)-211 lc 2b 3f 4d 5e 6e 7a [l)-236 If 2b 3f 4f 5e 6c 7a

[1)-212 If 2a 3f 4d 5e 6e 7a (l)-237 la 2b 3f 4f 5e 6e 7a

[1)-213 la 2a 3f 4d 5e 6e 7a [l)-238 lc 2b 3f 4f 5e 6e 7a

[1)-214 lc 2a 3f 4d 5e 6e 7a (l)-239 If 2a 3f 4f 5e 6c 7a

[1)-215 If 2e 3f 4d 5e 6e 7a [l)-240 la 2a 3f 4f 5e 6e 7a

[1)-216 la 2e 3f 4d 5e 6e 7a [1)-241 lc 2a 3f 4f 5e 6c 7a

[1)-217 lc 2e 3f 4d 5e 6e 7a [l)-242 If 2e 3f 4f 5e 6e 7a )-218 If 2b 3c 4d 5e 6e 7a ;i)-243 la 2e 3f 4f 5e 6e 7a i)-219 la 2b 3c 4d 5e 6e 7a l)-244 lc 2e 3f 4f 5e 6c 7al)-220 lc 2b 3c 4d 5e 6e 7a [l)-245 If 2b 3c 4f 5e 6e 7a

[1)-221 If 2a 3c 4d 5e 6e 7a [l)-246 la 2b 3c 4f 5e 6e 7a

[l)-222 la 2a 3c 4d 5e 6e 7a [l)-247 lc 2b 3c 4f 5e 6e 7a

[l)-223 lc 2a 3c 4d 5e 6e 7a [l)-248 If 2a 3c 4f 5e 6e 7a

[l)-224 If 2e 3c 4d 5e 6e 7a (l)-249 la 2a 3c 4f 5e 6c 7a l)-225 la 2e 3c 4d 5e 6e 7a l)-250 lc 2a 3c 4f 5e 6c 7a

E_bditmomen oe

PS &S

« « r-

PS PS PS PS Ό Ό

S e c Ό

e

ee ee PS S a

«s ¾ PS a c PS m « M 1/1

S PS PS S PS

«- *~

PS S

(1)-301 lc 2b 3c 4f 5a 6e 7a [1)-310 lc 2b 3j 4f 5a 6e 7a

(l)-302 If 2a 3c 4f 5a 6e 7a [D-311 If 2a 3j 4f 5a 6e 7a

[l)-303 la 2a 3c 4f 5a 6e 7a (1)-312 la 2a 3j 4f 5a 6e 7al)-304 lc 2a 3c 4f 5a 6e 7a (1)-313 lc 2a 3j 4f 5a 6e 7a

[l)-305 If 2e 3c 4f 5a 6e 7a D-314 If 2e 3j 4f 5a 6e 7ai)-306 la 2e 3c 4f 5a 6e 7a (1)-315 la 2e 3j 4f 5a 6e 7a

:i)-307 lc 2e 3c 4f 5a 6e 7a 1)- E3^bditmomen16 lc 2e 3j 4f 5a 6e 7ai)-308 If 2b 3j 4f 5a 6e 7a

[l)-309 la 2b 3j 4f 5a 6e 7a

[0074] Another aspect of the Invention provides a pharmaceutical composition which comprises a compound of any one of Formulae I, (I-a) - (I-b), (Il-a) - (Il-d), (Ill-a) - (III-c), (IV- a) - (IV-i), (V-a) - (V-f), and (Vl-a) - (Vl-f), 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.

[0075] 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 Formulae I, (I-a) - (I-b), (Il-a) - (Il-d), (Ill-a) - (III-c), (IV-a) - (IV-i), (V-a) - (V-f), and (Vl-a) - (Vl-f), 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.

[0076] Another aspect of the invention is directed to a method of treating an autoimmune disease, disorder, or syndrome comprising administering to a patient a therapeutically effective amount of a compound of Formulae I, (I-a) - (I-b), (Il-a) - (Il-d), (Ill-a) - (III-c), (IV-a) - (IV-i), (V-a) - (V-f), and (Vl-a) - (Vl-f), or a compound selected from Table 1 or Table 2 (below), or selected from any of the above embodiments, optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof. In one embodiment the autoimmune disease is multiple sclerosis. In another embodiment, the disease is psoriasis. In another embodiment, the disease is inflammatory bowel disease. In another embodiment the autoimmune disease is graft-versus-host disease. In another embodiment, the disease is inflammation caused by an autoimmune disease.

[0077] Another aspect of the invention comprises compositions for treating an autoimmune disease, disorder, or syndrome which composition comprises a pharmaceutically acceptable carrier, excipient, and/or diluent together with a therapeutically effective amount of a compound of Formulae I, (I-a) - (I-b), (Il-a) - (Il-d), (Ill-a) - (III-c), (IV-a) - (IV-i), (V-a) - (V-f), and (VI- a) - (Vl-f), or a compound selected from Table 1 or Table 2 (below), or selected from any of the above embodiments, optionally as a pharmaceutically acceptable salt or pharmaceutical composition thereof. In another embodiment, the disease is an autoimmune disease. In another embodiment the autoimmune disease is multiple sclerosis. In another embodiment, the disease is psoriasis. In another embodiment, the disease is inflammatory bowel disease. In another embodiment the autoimmune disease is graft-versus-host disease. In another embodiment, the disease is inflammation caused by an autoimmune disease.

[0078] Another aspect of the invention comprises use of a compound of Formulae I, (I-a) - (I-b), (Il-a) - (Il-d), (Ill-a) - (III-c), (IV-a) - (IV-i), (V-a) - (V-f), and (Vl-a) - (Vl-f), 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 the treatment of an autoimmune disease, disorder, or syndrome. In another embodiment, the disease is an autoimmune disease. In another embodiment the autoimmune disease is multiple sclerosis. In another embodiment, the disease is psoriasis. In another embodiment, the disease is inflammatory bowel disease. In another embodiment the autoimmune disease is graft-versus-host disease. In another embodiment, the disease is inflammation caused by an autoimmune disease.

Representative Compounds

[0079] Representative compounds of Formula I are depicted below. The examples are merely illustrative and do not limit the scope of the invention in any way. Compounds of the invention are named according to systematic application of the nomenclature rules agreed upon by the International Union of Pure and Applied Chemistry (IUPAC), International Union of Biochemistry and Molecular Biology (IUBMB), and the Chemical Abstracts Service (CAS). Names were generated using ACD/Labs naming software or ChemDraw Ultra 10.0.

Table 1

methylethyl)amino]pyridine-3-carbonitrile

CH₃ dihydrogen phosphate

Table 2

General Administration

[0080] In one aspect, the invention provides pharmaceutical compositions comprising an inhibitor of SI PI and/or S1P5 according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent. In certain other specific embodiments, administration is by the oral route. Administration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration or agents for serving similar utilities. Thus, administration can be, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, specifically in unit dosage forms suitable for simple administration of precise dosages.

[0081] The compositions will include a conventional pharmaceutical carrier or excipient and a compound of the invention as the/an active agent, and, in addition, may include carriers and adjuvants, etc.

[0082] 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.

[0083] If desired, a pharmaceutical composition of the invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.

[0084] The choice of formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills or capsules) and the bioavailability of the drug substance. Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4,107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1 ,000 nm in which the active material is supported on a crosslinked matrix of macromolecules. U.S. Pat. No. 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability. [0085] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0086] 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.

[0087] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, cellulose derivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, magnesium stearate and the like (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

[0088] 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. [0089] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.

[0090] 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.

[0091} 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.

[0092] 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.

[0093] 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.

[0094] Generally, depending on the intended mode of administration, the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a suitable pharmaceutical excipient. In one example, the composition will be between about 5% and about 75% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.

[0095] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease-state in accordance with the teachings of this invention.

[0096] The compounds of the invention, or their pharmaceutically acceptable salts or solvates, are administered in a therapeutically effective amount which will vary depending upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease-states, and the host undergoing therapy. The compounds of the present invention can be administered to a patient at dosage levels in the range of about 0.1 to about 1,000 mg per day. For a normal human adult having a body weight of about 70 kilograms, a dosage in the range of about 0.01 to about 100 mg per kilogram of body weight per day is an example. The specific dosage used, however, can vary. For example, the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to one of ordinary skill in the art.

[0097] If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described above and the other pharmaceutically active agent(s) within its approved dosage range. Compounds of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a combination formulation is inappropriate.

General Synthesis

[0098] 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); Rodd'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). These schemes are merely illustrative of some methods by which the compounds of this invention can be synthesized, and various modifications to these schemes can be made and will be suggested to one skilled in the art having referred to this disclosure. The starting materials and the intermediates of the reaction may be isolated and purified if desired using conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography and the like. Such materials may be characterized using conventional means, including physical constants and spectral data.

[0099] Unless specified to the contrary, the reactions described herein take place at atmospheric pressure and over a temperature range from about -78 °C to about 150 °C, more specifically from about 0 °C to about 125 °C and more specifically at about room (or ambient) temperature, e.g., about 20 °C. Unless otherwise stated (as in the case of a hydrogenation), all reactions are performed under an atmosphere of nitrogen.

[00100] 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. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference for all purposes.

[0100] The compounds of the invention, or their pharmaceutically acceptable salts, may have asymmetric carbon atoms or quaternized nitrogen atoms in their structure. Compounds of the Invention that may be prepared through the syntheses described herein may exist as single stereoisomers, racemates, and as mixtures of enantiomers and diastereomers. The compounds may also exist as geometric isomers. All such single stereoisomers, racemates and mixtures thereof, and geometric isomers are intended to be within the scope of this invention. Some of the compounds of the invention may exist as tautomers. For example, where a ketone or aldehyde is present, the molecule may exist in the enol form; where an amide is present, the molecule may exist as the imidic acid; and where an enamine is present, the molecule may exist as an imine. All such tautomers are within the scope of the invention.

[0101] The present invention also includes isotopically-labeled compounds, which are identical to those recited in Formula I, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C, ^l4C, ¹⁵N, ¹⁸0, ^,70, ³¹P, ³²P, ³⁵S, ^l8F, and ³⁶C1, respectively. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e., ^I4C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically-labelled 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-labelled reagent for a non-isotopically-labelled reagent.

[0102] The present invention also includes N-oxide derivatives and protected derivatives of compounds of the Invention. For example, when compounds of the Invention contain an oxidizable nitrogen atom, the nitrogen atom can be converted to an N-oxide by methods well known in the art. When compounds of the Invention contain groups such as hydroxy, carboxy, thiol or any group containing a nitrogen atom(s), these groups can be protected with a suitable "protecting group" or "protective group". A comprehensive list of suitable protective groups can be found in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc. 1991, the disclosure of which is incorporated herein by reference in its entirety. For example nitrogen protecting groups include, but are not limited to Boc, Fmoc, benzyl, trityl, and the like. The protected derivatives of compounds of the Invention can be prepared by methods well known in the art.

[0103] Methods for the preparation and/or separation and isolation of single stereoisomers from racemic mixtures or non-racemic mixtures of stereoisomers are well known in the art. For example, optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. Enantiomers (R- and S-isomers) may be resolved by methods known to one of ordinary skill in the art, for example by: formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallization; via formation of diastereoisomeric derivatives which may be separated, for example, by crystallization, selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where a desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step may be required to liberate the desired enantiomeric form. Alternatively, specific enantiomer may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents or by converting on enantiomer to the other by asymmetric transformation. For a mixture of enantiomers, enriched in a particular enantiomer, the major component enantiomer may be further enriched (with concomitant loss in yield) by recrystallization.

[0104] In addition, the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

[0105] The chemistry for the preparation of the compounds of this invention is known to those skilled in the art. In fact, there may be more than one process to prepare the compounds of the invention. The following examples illustrate but do not limit the invention. All references cited herein are incorporated by reference in their entirety. [0106] The compounds of formula I may be made using the general synthetic methods described below, which also constitute part of the invention.

GENERAL SYNTHETIC METHODS

[0107] The invention additionally provides methods for making compounds of formula I. The compounds of the invention may be prepared by the general methods and examples presented below, and methods known to those of ordinary skill in the art and reported in the chemical literature. Unless otherwise specified, solvents, temperatures, pressures, and other reaction conditions may be readily selected by one of ordinary skill in the art. Specific procedures are provided in the Synthetic Examples section. Intermediate substituted benzoic acids and their precursors (benzoic acids esters, benzamides or benzonitriles) are commercially available or are prepared by methods known to those skilled in the art (see WO201011316). 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 N-hydroxyamidines of formula III and IV are prepared from the corresponding substituted nitrile by methods known to those skilled in the art.

[0108] The methods described below and in the Synthetic Examples section may be used to prepare the compounds of formula I. In the Schemes below, R², R^2a, R³, R⁴, 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 R², R^2a, R³, R⁴, and R^7c groups; and optionally forming a pharmaceutically acceptable salt, hydrate, solvate or combination thereof.

Scheme I

[0109] As illustrated above, a carboxylic acid of formula II is reacted with a N- hydroxyamidine of formula HI under coupling conditions well known in the art such as by treatment with SOCl₂ or, N-(3-dimethylaminopropyl)-A -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, 0-(benzotriazol-l-yl- N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence of a base such as Et₃N or N,N-diisopropylethylamine (DIPEA) in a suitable solvent such as DMF or dimethylacetamide to provide an intermediate acyl N-hydroxyamidine which can be isolated or cyclized in situ upon warming to 80-100°C to afford intermediate oxadiazole IV. Reaction of IV (where PG is a BOC group) with a suitable acid such as HCl in a suitable solvent such as dioxane, THF or methanol provides the desired compound of formula I.

[0110] An alternate approach that may be used to obtain compounds of formula I is illustrated in Scheme II. 16 040892

Scheme II

IV

[0111] As illustrated above, a carboxylic acid of formula II is reacted with and N- hydroxyamidine of formula V, where X is a halogen such as fluorine, under coupling conditions well known in the art such as by treatment with SOCl₂ or, N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride in the presence of 1-hydroxylbenzotriazole (HOBt) or, benzotriazol-l-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP) or, O-(7- azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or, O- (benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence or absence of a base such as Et₃N or N,N-diisopropylethylamine (DIPEA) in a suitable solvent such as DMF or dimethylacetamide to provide an intermediate acyl hydroxyamidine which can be isolated or cyclized in situ upon warming to 80-100°C to afford intermediate oxadiazole VI. Reaction of VI with an alcohol VII (where PG is a BOC group) in the presence of a base such as sodium hydride in a suitable solvent such as DMF or THF provides a compound of formula IV. Treatment of intermediate IV as shown in Scheme I with a suitable acid such as HCl in a suitable solvent such as dioxane, THF or methanol provides the desired compound of formula I.

[0112] Another approach that may be used to obtain compounds of formula I is illustrated in Scheme III. Scheme III

IV

[0113] Reaction of VI, where X is a halogen such as fluorine, with a base such as tetrabutylammonium hydroxide in a suitable solvent such as dioxane or aqueous NaOH or aqueous KOH in a suitable solvent such as acetonitrile provides a compound of formula VIII. Alkylation of intermediate VIII with a suitable electrophile, for this example compound Vila (X is I, Br, or CI) where PG is an amine protecting group such as BOC in the presence of a base such as NaH, LDA or KHMDS in a suitable solvent such as DMF, DMSO or THF provides a compound of formula IV. Alternately, reacting intermediate VIII and an alcohol Vila (X is OH) under Mitsunobu conditions in the presence of PPh₃ and di-tert-butyl azodicarboxylate in a suitable solvent such as THF provides intermediate IV. Reacting intermediate IV as shown in Scheme I with a suitable acid such as HCl in a suitable solvent such as dioxane, THF or methanol provides the desired compound of formula I.

[0114] Yet another method of preparing compounds of formula I with various R⁴'s is shown in Scheme IV.

Scheme

[0115] Reaction of nitrile IX (intermediate IV where R⁴ is cyano) with a reducing agent such as diisobutyl aluminum hydride in a suitable solvent such as dichloromethane followed by a quench with an acid such as tartaric acid in water provides an aldehyde of formula X. Reduction of aldehyde X with a suitable reducing agent such as lithium aluminum hydride in a suitable solvent such as THF provides an alcohol of formula XI. Reacting intermediate XI (intermediate IV where R⁴ is -CH₂OH) as shown in Scheme I with a suitable acid such as HC1 in a suitable solvent such as dioxane, THF or methanol provides the desired compound of formula I.

[0116] General Methods: All reactions were run at room temperature unless note otherwise. All compounds were characterized by one or all of the following methods: Ή NMR, HPLC, HPLC-MS, and melting point.

[0117] Retention times (RT) are reported in Table 3 using one of the following methods:

HPLC Time Mobile Phase Flow Column

0 70 30 2.5

1.7 5 95 2.5

Agilent Zorbax CI 8 SB 3.5um

2 2 5 95 2.5

4.6x30mm cartridge

2.1 70 30 2.5

2.3 70 30 2.5

0 99 1 2.5

1.7 50 50 2.5

Agilent Zorbax CI 8 SB 3.5um

3 2 5 95 2.5

4.6x30mm cartridge

2.1 5 95 2.5

2.3 99 1 2.5

0 95 5 1.5

7 5 95 1.5

Agilent Zorbax Eclipse XDB-C8

4 9 5 95 1.5

5um 4.6x150mm

9.3 95 5 1.5

10 95 5 1.5

0 99 1 2.5

1.6 80 20 2.5

1.7 5 95 2.5 Agilent Zorbax CI 8 SB 3.5um

5

2 5 95 2.5 4.6x30mm cartridge

2.1 99 1 2.5

2.3 99 1 2.5

0 99 1 1.5

2 80 20 1.5

7 5 95 1.5 Agilent Zorbax Eclipse XDB-C8

6

9 5 95 1.5 5um 4.6x150mm column

9.3 99 1 1.5

10 99 1 1.5

0 88 12 1.5

0.25 70 30 1.5 Agilent SB-C18 1.8um 3x50mm

7 0.3 60 40 1.5 column

1.19 5 95 1.5

1.75 0 100 1.5

0 60 40 1.5

Agilent Eclipse C8 1.8um

8 1.19 15 85 1.5

3x50mm column

1.75 0 100 1.5

0 95 5 1.5

0.25 50 50 1.5

Agilent SB-AQ 1.8um 3x50mm

9 0.3 70 30 1.5

column

1.3 10 90 1.5

1.7 0 100 1.5

0 95 5 1.5 Agilent SB-C18 1.8um 3x50mm

10 3.8 10 90 1.5 column

4.5 0 100 1.5

Synthetic Examples

Intermediate 2

-Butyl 4-(hydroxymethyl)-2,2-dimethyloxazolidine-3-carboxylate

1

[0118] (iS)-ter/-Butyl 4-(hydroxymethyl)-2,2-dimethyloxazolidine-3-carboxylate (2). To an ice cold solution of commercially available 1 (35 g, 135 mmol) in THF MeOH (500 mL (95:5)) was added LiBH₄ (5.9 g, 271 mmol) portionwise and the suspension was stirred for 2 h at RT. The reaction mixture was cooled and quenched with ice. Solvent was removed under reduced pressure, and water was added. The aqueous layer was extracted with EtOAc, concentrated and chromatographed (EtOAc:hexanes, 3:7) to yield 2 (23 g, 74 % yield). Intermediate 7

-tert-Butyl 4-(hydroxymethyl)-2,2,5-trimethyloxazoIidine-3-carboxylate

6 7

[0119] Step 1: (2R,3R)-Methyl 2-amino-3-hydroxybutanoate (4). To MeOH (120 niL) cooled in an ice bath was added acetyl chloride (20 mL, 282 mmol) dropwise with stirring. The mixture was stirred in the ice bath for 15 min after which time D-allothreonine 3 (1 1.31 g, 94.9 mmol) was added. The reaction mixture was removed from the ice bath and heated to reflux for several hours. After cooling to RT, the reaction mixture was concentrated in vacuo. The crude product was used as is in the next reaction.

(0120] Step 2: (2R,3R)-Methyl 2-(tert-butoxycarbonylamino)-3-hydroxybutanoate (5).

Crude 4 (94.9 mmol), THF (300 mL) and triethylamine (21.6 g, 213 mmol) were combined and cooled in an ice bath. Boc anhydride (31.9 g, 146 mmol) was added and the reaction mixture was allowed to warm to RT and stirred for several days. The reaction mixture was concentrated in vacuo and the resulting residue was partitioned between sat'd NaHC0₃ and diethyl ether. The phases were separated and the aqueous phase was extracted with diethyl ether (2x). The combined diethyl ether extractions were washed with sat'd NaHC0₃ and sat'd NaCl, dried (Na₂S0₄), concentrated and dried under high vacuum overnight. The resulting residue was sonicated in hexanes followed by stirring in hexanes for several hours. Stirring was stopped and the mixture allowed to settle. The hexanes were decanted off the resulting oil. The oil was dissolved in DCM and reconcentrated in vacuo followed by drying under high vacuum to give compound 5 (12.68 g, 57% over 2 steps from compound 3). ^lH NMR (400 MHz, DMSO-i¾) δ 7.02 (d, 1H), 4.92 (m, 1H), 3.88 (m, 1H), 3.80 (m, 1H), 3.60 (s, 3H), 1.35 (s, 9H), 1.06 (d, 3H).

[0121] Step 3: (4R,5R)-3-tert-Butyl 4-methyl 2,2,5-trimethyloxazolidine-3,4- dicarboxylate (6). Compound 5 (12.68 g, 54.4 mmol), toluene (150 mL), dimethoxypropane (75 mL, 610 mmol) and p-toluenesulfonic acid monohydrate (1.4 g, 7.4 mmol) were combined and stirred at 80 °C for 6 h. After cooling to RT, the reaction mixture was concentrated in vacuo and the resulting oil was partitioned between sat'd NaHC0₃ and diethyl ether. Phases were separated and the aqueous phase was extracted with diethyl ether (2x). The combined diethyl ether extractions were washed with sat'd NaCl, dried (Na₂S0₄), concentrated, and dried under high vacuum overnight to give compound 6 (1 1.7 g, 79%).). Ή NMR (400 MHz, DMSO-fi ) δ 4.40

(m, 1H), 4.28 (m, 1H), 3.65 (s, 3H), 1.56 (d, 3H), 1.39 (s, 3H), 1.31 (s, 9H), 1.07 (d, 3H).

[0122] Step 4: (4S,5R)-tert-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 the addition was complete, the reaction mixture was removed from the ice bath and stirred at RT overnight. The reaction was followed by LC-MS and NMR and additional lithium borohydride was added in 500 mg aliquots as needed until the reaction was complete. Once reaction was complete, the reaction mixture was cooled in an ice bath then slowly added to a cold solution of 7.5% aqueous citric acid solution. The mixture was stirred for 30 min then extracted with EtOAc (3x). The combined

EtOAc extractions were washed with sat'd NaCl, dried (Na₂S0₄) and concentrated in vacuo. The resulting crude material was purified by flash chromatography (20% EtOAc in hexanes) to give compound 7 as a colorless oil (6.9 g, 66%). Ή NMR (400 MHz, DMSO-<&) δ 4.65 (m, 1H), 4.20

(m, 1H), 3.70-3.60 (m, 1H), 3.50-3.35 (m, 2H), 1.41 (s, 9H), 1.40 (s, 6H), 1.22 (d, 3H).

Intermediate 10

2-(Isopropylamino)-6-methylisonicotinic acid

[0123] Step 1: Ethyl 2-(isopropylamino)-6-methylisonicotinate (9). Ethyl 2-chloro-6- methylpyridine-4-carboxylate 8 (550 mg, 2.75 mmol), Pd(OAc)₂ (28 mg, 0.13 mmol), BINAP (156 mg, 0.25 mmol), Cs₂C0₃ (2.4 g, 7.5 mmol) and 1,4-dioxane (25 mL) were combined in a sealed tube. N₂ was bubbled into the mixture for a few minutes and isopropylamine (885 mg, 15 mmol) was added. The sealed mixture was heated to 85 °C for 20 h. The mixture was filtered through Celite and washed with EtOAc. The solvents were removed in vacuo. Purification by flash column chromatography gave 9 (530 mg, 86%).

[0124] Step 2: 2-(IsopropyIamino)-6-methyIisonicotinic 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 .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%).

Example 1

(2R S)-3-Amino-4-(2,6-dichIoro-4-{5-[2-(isopropylamino)-6-methylpyridin-4-yl]-l,2,^

-3-yl}phenoxy)butan-2-oI

[0125] Step 1: (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-cyanophenoxy)methyl)-2,2,5- trimethyloxazolidine-3-carboxyIate (12). To a solution of 3,5-dichloro-4-fluorobenzonitrile 11 (836 mg, 4.40 mmol) and intermediate 7 (1.19 g, 4.84 mmol) in 10 mL of anhydrous THF, cooled at 0 °C with an ice bath, was added NaH (194 mg, 60% in mineral oil) in portions with stirring over 2 min. The reaction mixture was warmed to RT and stirred at RT for 4 h. The reaction was quenched by addition of ice water. The mixture was extracted with EtOAc (3x). The organic phase was washed with water, brine, dried with MgS0₄, and concentrated in vacuo to give an off-white solid. The solid was triturated with hexanes and filtered to afford compound 12 as a white solid (1.48 g, 81% yield), which was used as is for subsequent reactions. [0126] Step 2: (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-(N- hydroxycarbamimidoyl)phenoxy)-methyl)-2,2,5-trimethyloxazolidine-3-carboxylate (13).

Compound 12 (1.31 g, 3.15 mmol), hydroxylamine hydrochloride (1.33 g, 19 mmol), triethylamine (2.23 g, 22 mmol) and EtOH (15 mL) were combined in a sealed tube and stirred at RT for 20 min, then heated with stirring at 80 °C for 6 h. After removal of solvent in vacuo, the mixture was dissolved in EtOAc, which was then washed with water (3x), brine, dried with MgS0₄, and concentrated in vacuo to give compound 13 as a foamy white solid in quantitative yield (1.42 g). Ή NMR (400 MHz, DMSO-< ₆) δ 9.89 (s, 1H), 7.77 (s, 1H), 7.75 (s, 1H), 5.98 (s, 2H), 4.37 (m, 1H), 4.20 (m, 1H), 4.07 (m, 1H), 3.87 (m, 1H), 1.51-1.42 (m, 12H), 1.27 (s, 6H).

[0127] Step 3: (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,2,4-oxadiazol-3-yl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (14). A mixture of intermediate 10 (383 mg, 1.97 mmol), EDCI HC1 (378 mg, 1.97 mmol), and HOBT (272 mg, 1.97 mmol) in DMA (4 mL) as a white slurry was stirred at RT for 5 min. To the mixture was added a solution of hydroxyamidine 13 (735 mg, 1.64 mmol) in DMA (4 mL). The resulting white slurry was stirred at RT for 10 min, and then heated at 80 °C with stirring. Upon heating for 5 min, the reaction mixture turned into a clear solution. After heating at 80 °C for 16 h, the reaction mixture was cooled to RT, diluted with water, and extracted with EtOAc (3x). The organic phase was washed with water and brine, dried (MgS0₄), and concentrated in vacuo. The crude product was chromatographed on silica gel column (EtOAc:hexane = 1 :4) to afford compound 14 (550 mg, 55.3% yield). Ή NMR (400 MHz, DMSO-i¾ 6 8.12 (s, 1H), 8.10 (s, 1H), 7.00 (s, 2H), 6.85 (d, 1H), 4.40 (m, 1H), 4.30 (m, 1H), 4.1 1-3.91 (m, 3H), 2.38 (s, 3H), 1.52-1.43 (m, I2H), 1.29 (s, 6H), 1.17 (d, 6H).

[0128] Step 4: (2R,3S)-3-Amino-4-(2,6-dichloro-4-{5-[2-(isopropylamino)-6- methylpyridin-4-yl]-l,2,4-oxadiazol-3-yl}phenoxy)butan-2-ol. Compound 14 (545 mg, 0.90 mmol) was dissolved in 10 mL of 4M HCl in dioxane with stirring at RT followed by addition of 5 mL of MeOH. After stirring at RT for 30 min, the resulting yellow solution was concentrated in vacuo. The residue was dissolved in water, basified with 1 M NaOH to pH>13, and extracted with EtOAc (3x). The organic phase was washed with water, brine, dried with MgS0₄, and concentrated in vacuo to give the title compound as a yellow solid (380 mg, 91% yield). Ή NMR (400 MHz, OMSO-d₆) δ 8.09 (s, 2H), 6.99 (s, 2H), 6.85 (d, 1H), 4.61 (d, 1H), 4.21 (dd, U 2016/040892

IH), 4.06 (m, IH), 3.95 (t, IH), 3.61 (m, IH), 2.99 (m, IH), 2.38 (s, 3H), 1.66 (br s, 2H), 1.16 (d, 6H), 1.13 (d, 3H); MS (EI) for C₂,H₂₅C1₂N₅0₃, found 466.2 (MH+).

Using the same or analogous synthetic techniques as in Example 1 and substituting with appropriate reagents, the following compounds were prepared.

[0129] (2R,3S)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol. 1H NMR (400 MHz, CD₃OD) δ 7.90 (d, J=11.5, IH), 7.63 (s, IH), 7.52 (d, J=7.5, IH), 7.32 (s, IH), 4.53 (dd, J=10.4, 3.6, 1H), 4.42 (dd, J=10.1, 8.1, IH), 4.19 (dd, J=11.3, 4.9, 2H), 3.74 -3.58 (m, 3H), 2.66 (m, IH), 2.65 (s, 3H), 1.36 (dd, J=l 1.6, 6.4, 6H); MS (EI) for C₂iH₂₅ClFN₅0₃, found 450 (MH⁺).

[0130] (2R,3S)-3-Amino-4-{[2-nuoro-5-methyl-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol. Ή-NMR (400 MHz, DMSO-i¾)D D δ 7.79 (d, IH), 7.26 (d, IH), 6.97 (br s, 2H), 6.84 (d, IH), 4.67 (br s, IH), 4.20 (m, IH), 4.02 (m, 2H), 3.54 (m, 3H), 2.93 (m, IH), 2.59 (s, 3H), 2.38 (s, 3H), 1.15 (d, 6H); MS (EI) for C₂₂H₂₈FN₅0₃, found 430.0 (MH+).

[0131] (2R,3S)-3-Amino-4-(2-chloro-6-fluoro-4-{5-[2-(isopropylamino)-6- methylpyridin-4-yl]-l,2,4-oxadiazol-3-yl}phenoxy)butan-2-ol. 1H NMR (400 MHz, CDC1₃) 5 8.02 (s, IH), 7.85 (d, IH), 7.14 (s, IH), 6.89 (m, IH), 4.68 (d, IH), 4.36 (m, IH), 4.16 (t, IH), 3.95 - 3.90 (m, 2H), 3.22 (m, IH), 2.47 (s, 3H), 1.30 - 1.26 (dd, 6H); MS (EI) for C₂,H₂₅C1FN₅0₃, found 450.2 (MH⁺)

[0132] (2R,3S)-3-Amino-4-{[2,6-dimethyl-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol. Ή NMR (400 MHz, DMSO-<¾) δ 8.38 (br s, 2H), 7.80 (s, 2H), 7.45 (s, IH), 7.20 (s, IH), 4.25 (m, IH), 4.08 (m, IH), 4.00 (m, 2H), 3.48 (m, IH), 2.55 (s, 3H), 2.38 (s, 6H), 1.22 (d, 6H), 1.20 (d, 3H); MS (EI) for C₂₃H₃,N₅0₃, found 4.26.30 (MH+).

Example 2

(2R)-2-Amino-3<2-fluoro-4-{5-[2-(isopropyIamm

-yl}-5-methyIphenoxy)propan-l-ol

[0133] Step 1: l-Bromo-4,5-difluoro-2-methylbenzene (16). To a ice cooled flask containing 3,4-difluorotoluene 15 (27 g, 210 mmol) and iron powder (600 mg, 10 mmol) was added dropwise Br₂ (40 g, 250 mmol). The reaction was stirred for 12 h while it was slowly warmed to RT. Water was added and the resulting mixture was extracted with diethyl ether. The ether layer was washed with sodium thiosulfate solution, brine and dried over Na₂S0₄. Removal of ether gave the desired bromotoluene 16 (36 g, 83%). Ή-NMR (400MHz, DMSO-d6):D D 8 7.78 (t, 1H), 7.52 (t, 1H), 2.29 (s, 3H).

[0134] Step 2: 4,5-Difluoro-2-methylbenzonitriIe (17). 1 -Bromo-4,5-difluoro-2- methylbenzene 16 (2 g, 9.6 mmol), CuCN (1.0 g, 1 1 mmol) and NMP (5 mL) were combined in a sealed tube and heated to 160 °C with stirring for 12 h. Due to the volatility of the resulting benzonitrile 13, the crude reaction mixture was used as is in the next reaction.

[0135] Step 3: (S)-tert-Butyl 4-((4-cyano-2-fluoro-5-methylphenoxy)methyl)-2,2- dimethyloxazolidine-3-carboxylate (18). The crude reaction mixture of compound 17 was cooled to 0 °C. The alcohol 2 (2.0 g, 8.7 mmol) was added followed by the addition of KO^lBu (2.2 g, 20 mmol). The mixture was stirred for 3 h at RT. Water was added to quench the reaction. The crude product was extracted with EtOAc. Purification by flash column chromatography gave compound 18 (1.36 g, 43%).

[0136] Step 4: (S)-tert-Butyl 4-((2-fluoro-4-(N'-hydroxycarbamimidoyl)-5- methylphenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate (19). Intermediate 19 was synthesized from intermediate 18 in good yield (>90%) using the same or an analogous synthetic procedure to that of intermediate 13 in Example 1.

[0137] Step 5: (S)-tert-Butyl 4-((2-fluoro-4-(5-(2-(isopropylamino)-6-methylpyridin-4- yl)-l,2,4-oxadiazol-3-yl)-5-methylphenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate (20). To a mixture of carboxylic acid 10 (582 mg, 3 mmol), HOBT (445 mg, 3.3 mmol) and DCM (30 mL) was added N,N'-diisopropylcarbodiimide (415 mg, 3.3 mmol). The mixture was stirred at RT for 12 h followed by the addition of amidoxime 19 (1.2 g, 3 mmol). The stirring was continued for 3 h. The diisopropylurea was filtered off and the filtrate was concentrated. The residue was dissolved in BuOH (20 mL) and was heated to 110 °C for 3 h. Removal of BuOH and purification by flash column chromatography gave the desired compound 20 (837 mg, 50%). 1H-NMR (400 MHz, CDC1₃):D D δ 7.88 (t, 1H), 7.15 (s, 1H), 7.01 (dd, 1 H), 6.90 (s, 1H), 4.68 (d, 1H), 4.40-3.90 (m, 6H), 2.66 (s, 3H), 2.46 (s, 1H), 1.60-1.48 (m, 15H).

[0138] Step 6: (2R)-2-Amino-3-(2-fluoro-4-{5-[2-(isopropylamino)-6-methyIpyridin-4- yI]-l,2,4-oxadiazol-3-yl}-5-methylphenoxy)propan-l-ol. To a solution of compound 20 (837 mg, 1.5 mmol) in MeOH (10 mL) was added 4 N HC1 in dioxane (5 mL). The mixture was stirred for 4 h. Volatiles were removed in vacuo. The residue was diluted with water (25 mL) and was washed with diethyl ether (2 x 15 mL). The aqueous phase was basified with NaOH (pH~12) and extracted with EtOAc. The organic phase was washed with brine and dried over Na₂S0₄. Removal of the EtOAc gave pure title compound (443 mg, 70%). Ή NMR (400 MHz, DMSO-i¾) δ 8.20 (br s, 1H), 7.82 (d, 1H), 7.32 (d, 1H), 7.08 (br s, 1H), 4.35 (m, 2H), 4.15 (m, 1H), 3.52 (m, 2H), 3.08 (m, 1H), 2.60 (s, 3H), 2.45 (s, 3H), 1.20 (d, 6H); MS (EI) for C2iH₂₆FN₅03, found 416.0 (MH+).

[0139] Using the same or analogous synthetic techniques as in Example 2 and substituting with appropriate reagents, the following compounds were prepared.

[0140] (2R)-2-Amino-3-{[2,6-dichloro-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyridin- 4-yI}-l,2,4-oxadiazol-3-yl)phenyI]oxy}propan-l-ol. Ή NMR (400 MHz,

δ 8.09 (s, 2H), 6.99 (s, 2H), 6.85 (d, 1H), 4.67 (t, 1H), 4.08 (m, 2H), 3.95 (m, 1H), 3.54-3.34 (m, 2H), 3.14 (m, 1H), 2.38 (s, 3H), 1.72 (br s, 2H), 1.17 (d, 6H); MS (EI) for C₂₀H2₃Cl₂N₅O3, found 452.2 (MH+).

[0141] (2R)-2-Amino-3-{[2,6-dimethyI-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyrid 4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}propan-l-ol. Ή NMR (400 MHz, CD₃OD) δ 7.88 (s, 2H), 7.61 (s, 1H), 7.35 (s, 1H), 4.20 (m, 1H), 4.08 (m, 2H), 3.93 (m, 2H), 3.73 (m, 1H), 2.65 (s, 3H), 2.40 (s, 6H), 1.40 (d, 6H); MS (EI) for C₂2H₂₉N₅03, found 412.25 (MH+).

Example 3

(2R)-2-Amino-3-{[2-chloro-6-methyl-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyri^

-oxadiazol-3-yl)phenyl]oxy}propan-l-ol

[0142] Step 1: 2-ChIoro-4-bromo-6-methylphenol (22). To a solution of 2-chloro-6- methylphenol 21 (5 g, 35 mmol) in AcOH (70 mL) was added NBS (6.2 g, 35 mmol). The solution was stirred at RT for 12 h. AcOH was removed under reduced pressure. The residue was diluted with EtOAc, washed with saturated Na₂C0₃ solution and dried over Na₂S0₄. Removal of the solvents gave desired compound 22 (4 g, 52%) which was used in the next step without further purification.

[0143] Step 2: (S)-tert-Butyl 4-((4-bromo-2-chIoro-6-methylphenoxy)methyl)-2,2- dimethyIoxazolidine-3-carboxylate (23). To a solution of compound 22 (4 g, 18 mmol), intermediate 2 (4.8 g, 21.7 mmol) and triphenylphosphine (7.0 g, 27 mmol) in DCM (80 mL) was added diisopropylazodicarboxylate (5.4 g, 27 mmol). The reaction mixture was stirred at RT for 3 h. Water was added and the product was extracted with DCM. Purification by flash column chromatography gave the desired product 23 (5.8 g, 73%).

[0144] Step 3: (S)-tert-Butyl 4-((2-chloro-4-cyano-6-methylphenoxy)methyl)-2,2- dimethyloxazoIidine-3-carboxylate (24). Compound 23 (5.68 g, 13 mmol), Zn(CN)₂ (920 mg, 7.8 mmol), Pd(dppf)Cl₂, DCM (475 mg, 0.65 mmol) and polymethylhydrosiloxane (390 mg) were dissolved in DMA/H₂0 (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 the desired compound 24 (4.5 g, 91%).

[0145] Step 4: (S)-tert-Butyl 4-((2-chloro-4-(N'-hydroxycarbamimidoyl)-6- methylphenoxy)methyl)-2,2-dimethyIoxazolidine-3-carboxylate (25). Intermediate 25 was synthesized from intermediate 24 using the same or an analogous synthetic procedure to that of intermediate 13 in Example 1.

[0146] Step 5: (S)-tert-Butyl 4-((2-chloro-4-(5-(2-(isopropylamino)-6-methylpyridin-4- yl)-l,2,4-oxadiazol-3-yl)-6-methylphenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate (26). Intermediate 26 was synthesized from intermediates 10 and 25 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

[0147] Step 6: (2R)-2-Amino-3-{[2-chloro-6-methyl-4-(5-{2-methyl-6-[(l- methylethyI)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}propan-l-ol. The title compound was synthesized from intermediate 26 using the same or an analogous synthetic procedure to that of (2R)-2-amino-3-(2-fluoro-4-{5-[2-(isopropylamino)-6-methylpyridin-4-yl]- l,2,4-oxadiazol-3-yl}-5-methylphenoxy)propan-l -ol in Example 2. 1H NMR (400 MHz, DMSO- d₆) δ 7.90 (m, 2H), 6.97 (m, 2H), 6.84 (d, 1H), 4.66 (t, 1H), 4.05 (m, 1H), 3.93 (m, 1H), 3.80 (m, 1H), 3.50 (m, 1H), 3.45 (m, 1H), 3.09 (m, 1H), 2.39 (s, 3H), 2.38 (s, 3H), 1.63 (br s, 2H), 1.16 (d, 6H); MS (EI) for C₂iH₂₆ClN₅0₃, found 432.2 (MH+). Example 4

(2S,3S)-3-Amino-4- { [5-chloro-2-fluoro-4-(5- {2-methyl-6- [(1-methy lethyl )aniino] pyridin-4- -l,2,4-oxadiazoI-3-yI)phenyI]oxy}butan-2-oI dihydrochloride salt

[0148] Step 1: (4S,5S)-tert-Butyl 4-((5-chIoro-4-cyano-2-fluorophenoxy)methyl)-2,2,5- trimethyloxazolidine-3-carboxylate (29). Intermediate 28 was made in an analogous manner to intermediate 7 using the appropriate enantiomerically pure starting material in place of compound 3. Intermediate 29 was synthesized from commercially available 27 using the same or an analogous synthetic procedure to that of intermediate 12 in Example 1.

[0149] Step 2: (4S,5S)-tert-Butyl 4-((5-chIoro-2-fluoro-4-(N'- hydroxycarbamimidoyl)phenoxy)-methyl)-2,2,5-trimethyIoxazolidine-3-carboxylate (30). Intermediate 30 was synthesized from intermediate 29 using the same or an analogous synthetic procedure to that of intermediate 13 in Example 1.

[0150] Step 3: (4S,5S)-tert-Butyl 4-((5-chloro-2-fluoro-4-(5-(2-(isopropylamino)-6- methylpyridin-4-yl)-l,2,4-oxadiazol-3-yl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (31). Intermediate 31 was synthesized from intermediates 10 and 30 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

[0151] Step 4: (2S,3S)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol

dihydrochloride salt. To stirred solution of 25 (0.15 g, 0.254 mmol) in EtOH (0.5 mL) was added ethanolic HCl (1.5 mL) at 0 °C and reaction was stirred at RT for 1 h. Solvent was removed and the obtained solid residue was washed with diethyl ether and acetonitrile. The resulting crude compound was purified by preparative HPLC. The obtained TFA salt was dissolved in EtOH to which was added ethanolic HC1 (1 mL) at 0 °C. The solution was stirred at RT for 45 min. Solvent was removed and the solid residue washed with diethyl ether and hexane to afford the title compound (140 mg, 63 %) as a yellow solid. 1H NMR (400 MHz, CD₃OD) δ 7.9 (d, 1H), 7.6 (s, 1H), 7.5 (d, 1H), 7.3 (s, 1H), 4.5-4.3 (m, 2H), 4.2-4.0 (m, 2H), 3.5 (m, 1H), 2.6 (s, 3H), 1.4 (m, 9H); MS (EI) for C₂₁H2₅C1FN₅0₃, found 450 (MH+).

Example 5

(2R,3S)-3-Amino-4-{[5-chloro-4-(5-{3-chloro-4-[(l-methylethyl)oxy]phenyl}

-3-yl)-2-fluorophenyl]oxy}butan-2-ol hydrochloride salt

[0152] Step 1: 3-Chloro-4-isopropoxybenzonitrile (33). A stirring suspension of 32 (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 such that the internal reaction temperature did not exceed 10 °C. After 2 h, the reaction mixture was cooled and quenched with ice. THF was removed under reduced pressure, water was added to the residue and the resulting mixture was extracted with EtOAc. The organic phases were dried and concentrated in vacuo to afford 33 (24 g, 95.6%) which was used as such for the next step.

[0153] Step 2: 3-Chloro-4-isopropoxybenzoic acid (34). To a stirring solution of 33 (57 g, 291 mmol) in EtOH (120 mL) was added 12% aqueous KOH solution (300 mL ) and the reaction mixture was heated to 100 °C for 3 h. The reaction mixture was then cooled and acidified with HC1 to pH 5. A yellow solid precipitated out which was filtered, washed with water and dried. The resulting solid was stirred with hexane for 5 min and the hexane decanted. The resulting solid was resuspended in hexane, filtered, and washed with hexane to afford 34 as a white solid (45 g, 72%).

[0154] Step 3: (4S,5R)-tert-Butyl 4-((5-chloro-4-(5-(3-chloro-4-isopropoxyphenyl)-l,2,4- oxadiazol-3-yI)-2-fluorophenoxy)methyl)-2,2,5-trimethyloxazolidine-3-carboxylate (36).

Intermediate 35 was synthesized using the same or an analogous synthetic procedure to that of intermediate 30 in Example 4, substituting intermediate 7 for intermediate 28. Intermediate 36 was synthesized from intermediates 34 and 35 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

[0155] Step 4: (2R,3S)-3-Amino-4-{[5-chloro-4-(5-{3-chloro-4-[(l- methylethyl)oxy]phenyl}-l,2,4-oxadiazol-3-yl)-2-fluorophenyl]oxy}butan-2-ol

hydrochloride salt. The title compound was synthesized from intermediate 36 using the same or an analogous synthetic procedure to that of (2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2- methyl-6-[(l-methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol dihydrochloride salt in Example 4. Ή NMR (400 MHz, DMSO^) 6 8.4 (bs, 2H), 8.2 (s, 1H), 8.1 (d, 1H), 8.0 (d, 1H), 7.6 (d, 1H), 7.4 (d, 1H), 5.5 (m, 1H), 4.9 (m, 1H), 4.5 (m, 1H), 4.4 (m, 1H), 4.0 (m, 1H), 3.5 (m, 1H), 1.4 (d, 6H), 1.2 (d, 3H); MS (EI) for C21H22CI2FN3O4, found 469.95 (MH+).

Example 6

5-[3-(4-{[(2S,3R)-2-Amino-3-hydroxybutyl]oxy}-2-chIoro-5-fluorophenyl)-l,2,4-oxadiazol- 5-yI]-2-[(l-methylethyl)oxy]benzonitrile hydrochloride salt

[0156] Step 1: Methyl 4-hydroxy-3-iodobenzoate (38). A stirred solution of methyl-4- hydroxybenzoate 37 (20 g, 132 mmol) in AcOH (100 mL) was heated to 65 °C. A solution of IC1 (21.3g, 7.0 mL, 132 mmol) in AcOH (25 mL) was added dropwise over 40 min. Stirring at 65 °C was continued for 8 h followed by stirring at RT for 16 h. The precipitated product was isolated via filtration, washed with water and dried under vacuum to give 38 (20 g, 55%) as a white solid.

[0157] Step 2: Methyl 3-cyano-4-hydroxybenzoate (39). To a stirred solution of 38 (12 g, 44 mmol) in DMF (60 mL) was added CuCN (4.4 g, 48 mmol) and NaCN (0.24g, 4.8 mmol) followed by heating to 105 °C for 18 h. The reaction mixture was allowed to cool to RT, and any precipitates were removed via filteration and washed with ethyl acetate. The filtrate was diluted with water (200 mL), extracted with EtOAc, dried over Na₂S0₄ and concentrated in vacuo to afford 39 (6.2 g, 80%) as a light yellow solid.

[0158] Step 3: Methyl 3-cyano-4-isopropoxybenzoate (40). To as stirred solution of compound 39 (6.2 g, 34 mmol) in DMF (25 mL) was added 2-bromopropane (6.34 g, 52 mmol) and K₂C0₃ (14 g, 103 mmol) and the resulting mixture was heated to 90 °C for 14 h. After cooling to RT, the reaction mixture was diluted with water (200 mL) and extracted with DCM. The combined organic layers were dried over Na₂S0₄, concentrated, and purified by column chromatography to give 40 (7.0 g, 91%) as a thick oil.

[0159] Step 4: 3-Cyano-4-isopropoxybenzoic acid (41). To a stirred solution of 40 (7.0 g, 315 mmol) in a mixture of EtOH (30 mL) and THF (30 mL) was added 2M NaOH (20 mL, 41 mmol) and the resulting mixture was stirred at RT for 4 h. The solvent was removed in vacuo and water (100 mL) was added to the resulting residue. The resulting aqueous mixture was acidified with 2N HC1 and extracted with EtOAc. The EtOAc phase was washed with water and brine and dried over Na₂S0₄. The crude compound was stirred in 10% diethyl ether in hexane, filtered and dried to afford 41 (6 g, 92 %) as an off-white solid. [0160] Step 5: (4S,5R)-tert-Butyl 4-((5-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-l,2,4- oxadiazol-3-yl)-2-fluorophenoxy)methyl)-2,2,5-trimethyIoxazolidine-3-carboxylate (42).

Intermediate 42 was synthesized from intermediates 41 and 35 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

[0161] Step 6: 5-[3-(4-{[(2S,3R)-2-Amino-3-hydroxybutyl]oxy}-2-chloro-5- fluorophenyl)-l,2,4-oxadiazol-5-yl]-2-[(l-methylethyl)oxy]benzonitrile hydrochloride salt.

The title compound was synthesized from intermediate 42 using the same or an analogous synthetic procedure to that of (2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l - methylethyl)amino]pyridin-4-yl}-l ,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol dihydrochloride salt in Example 4. Ή NMR (400 MHz, DMSO-<¾) δ 8.5 (s, IH), 8.4 (m, 3H), 7.9 (d, IH), 7.65 (d, IH), 7.55 (d, IH), 5.4 (m, IH), 5.0 (m, IH), 4.5 (m, IH), 4.3 (m, IH), 4.0 (m, IH), 3.4 (m, IH), 1.4 (d, 6H), 1.2 (d, 3H); MS (EI) for C₂₂H₂₂C1FN₄0₄, found 461 (MH+).

[0162] Using the same or analogous synthetic techniques as in Example 5 and substituting intermediate 35 with intermediate 30, the following compound was prepared.

[0163] 5-[3-(4-{[(2S,3S)-2-Amino-3-hydroxybutyl]oxy}-2-chloro-5-fluorophenyl)-l,2,4- oxadiazol-5-yl]-2-[(l-methylethyl)oxy]benzonitrile hydrochloride salt. Ή NMR (400 MHz, DMSO^) δ 8.5 (s, IH), 8.4 (m, IH), 8.2 (bs, 2H, -NH₂), 7.9 (d, IH), 7.62 (d, IH), 7.58 (d, IH), 5.6 (m, IH), 5.0 (m, IH), 4.5-4.3 (m, IH), 3.9 (m, IH), 3.4 (m, IH), 1.4 (d, 6H), 1.2 (d, 3H); MS (EI) for C₂₂H₂₂C1FN₄0₄, found 461 (MH+).

Example 7

(2R S)-3-amino-4-(2,6-dichloro-4-(5-(5-(isopropylamino)-6-methylpyridin-2-yl)-l,2,4- oxadiazol-3-yl)phenoxy)butan-2-ol dihydrochloride salt

46 ₄₇

[0164] Step 1: 6-Chloro-2-methylpyridin-3-amine (44). To a stirred solution of 43 (5 g, 29 mmol) in EtOH (20 mL) and cone. HCI (20 mL) was added Fe powder (16.2 g, 289 mmole) in small portions at RT over 30 min. Stirring was continued at RT for another 30 min. The solvent was distilled off under reduced pressure. Water was added and the resulting mixture was neutralized with NaHC0₃. EtOAc was added and the biphasic mixture was filtered through Celite and washed with EtOAc. The phases of the filtrate were separated and the organic layer was washed with water, brine, dried over Na₂S0₄, and concentrated to afford 44 (4.1 g, 99 %) as a yellow solid.

[0165] Step 2: 6-Chloro-N-isopropyl-2-methylpyridin-3-amine (45). To a stirred solution of 44 (4.81 g, 33.75 mmol) and acetone (2.74 g, 47.2 mmol) in dichloroethane (60 mL) was added NaBH(OAc)₃ (10.713 g, 50.53 mmol) and AcOH (3.44 g, 57.2 mmol) at RT. The reaction was stirred for 16 h followed by dilution with IN NaOH. The aqueous solution was extracted with DCM and the organic layer was washed with water, brine, dried over Na₂S0₄ and concentrated to afford 45 (6.16 g, 98%).

[0166] Step 3: 5-(Isopropylamino)-6-methylpicolinonitrile (46). To a stirred solution of 45 (6.2 g, 33.5 mmol) in DMF (50 mL) was added Zn(CN)₂ (5.5 g, 46.84 mmol) and tetrakis(triphenylphosphine)palladium (0) (5.8 g, 5.01 mmol) at 25 °C. The reaction was stirred at 130 °C for 16 h. After completion, the reaction mixture was absorbed on silica gel and chromatographed to obtain 46 (5 g, 85 %) as thick liquid.

[0167] Step 4: 5-(Isopropylamino)-6-methylpicolinic acid (47). To a stirred solution of 46 (5.0 g, 28.4 mmol) in EtOH (40 mL) was added 20 % aqueous KOH (40 mL) and the reaction mixture was refluxed for 12 h. Solvent was removed in vacuo. The resulting aqueous mixture was neutralized to pH 5 with citric acid solution and extracted with EtOAc. Organic layer was dried, concentrated and washed with pentane to afford 47 (2.8 g, 50%) as a white solid.

[0168] Step 5: (4S,5R)-tert-Butyl 4-((2,6-dichIoro-4-(5-(5-(isopropylamino)-6- methylpyridin-2-yl)-l,2,4-oxadiazol -yl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3- carboxylate (48). Intermediate 48 was synthesized from intermediates 47 and 13 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

[0169] Step 6: (2R,3S)-3-amino-4-(2,6-dichloro-4-(5-(5-(isopropylamino)-6- methylpyridin-2-yl)-l^,4-oxadiazol-3-yl)phenoxy)butan-2-ol dihydrochloride salt. The title compound was synthesized from intermediate 48 using the same or an analogous synthetic procedure to that of (2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-oI dihydrochloride salt in Example 4. Ή NMR (400 MHz, CD₃OD) δ 8.38 (d, 1H), 8.22 (s, 2H), 7.60 (d, 1H), 4.40 (m, 2H), 4.11 (m, 1H), 3.98 (m, 1H), 3.71 (m, 1H), 2.75 (s, 3H), 1.37 (m, 9H); MS (EI) for C₂iH₂₅Cl₂N₅0₃, found 466.10 (MH+).

Using the same or analogous synthetic techniques as in Example 7 and substituting with appropriate reagents, the following compounds were prepared.

[0170] (2R)-2-Amino-3-{[2,6-dimethyl-4-(5-{6-me

2-yl}-l,2,4-oxadiazoI-3-yl)phenyl]oxy}propan-l-ol. Ή NMR (400 MHz, CD₃OD) δ 8.25 (d, 1H), 7.90 (s, 2H), 7.45 (d, 1H), 4.08 (m, 2H), 3.92 (m, 3H), 3.75 (m, 2H), 2.65 (s, 3H), 2.40 (s, 6H), 1.35 (d, 6H); MS (EI) for C₂₂H₂₉N₅0₃, found 412.25 (MH+).

[0171] (2R,3S)-3-Amino-4-{[2-fluoro-5-methyl-4-(5-{6-methyl-5-[(l- methylethyl)amino]pyridin-2-yl}-l,2,4-oxadiazoI-3-yl)phenyl]oxy}butan-2-ol. Ή NMR (400 MHz, CD₃OD) 6 8.35 (d, 1H), 7.95 (d, 1H), 7.55 (d, 1H), 7.22 (d, 1H), 4.45 (m, 2H), 4.20 (m, 1H), 3.93 (m, 1H), 3.62 (m, 1H), 2.70 (s, 3H), 2.68 (s, 3H), 1.38 (d, 6H), 1.36 (d, 3H); MS (EI) for C₂₂H₂₈FN₅0₃, found 430 (MH+).

Example 8

(2R,3S)-3-amino-4^2,6-dichloro-4-(5-(6-(isopropylam

oxadiazoI-3-yl)phenoxy)butan-2-ol dihydrochloride salt

[0172] Step 1: Methyl 6-fluoro-5-methylnicotinate (50). A mixture of 5-methyl-6-fluoro- nicotinic acid 49 (7.0 g, 45 mmol), K₂C0₃ (13.7 g, 99 mmol), methyl iodide (9.58 g, 67 mmol) and DMF (200 mL) was stirred for 16 h at RT. After dilution with water (50 mL), the reaction mixture was extracted with EtOAc (50 mL). The organic phase was washed successively with saturated aqueous NaHC0₃ (20 mL), brine (2 x 20 mL) and dried (Na₂S0₄). Filtration and evaporation of the solvent gave 50 (6.5 g, 85%).

[0173] Step 2: Methyl 6-(isopropylamino)-5-methylnicotinate (51). Isopropylamine (50 mL) and 50 (6.5 g, 38 mmol) were combined in a sealed tube and heated at 90 °C for 12 h. After cooling to RT, volatiles were removed in vacuo. The resulting residue was diluted with water and extracted with EtOAc. The combined organic layers were dried over Na₂S0₄ and concentrated to afford 51 (7.9 g, 98.4%).

[0174] Step 3: 6-(Isopropylamino)-5-methylnicotinic acid (52). A stirred mixture of 51 (4.5 g, 21 mmol) in cone. HCI (20 mL) was heated to 80 °C for 12 h. After cooling to RT, the reaction mixture was concentrated in vacuo. The resulting residue was dried under high vacuum, washed with pentane and diethyl ether and azeotroped with toluene to afford 52 (4.0 g, 95%) as the hydrochloride salt.

[0175] Step 4: (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-(5-(6-(isopropylamino)-5- methylpyridin-3-yl)-l,2,4-oxad.azol-3-yl)phenoxy)methy^

carboxylate (53). Intermediate 53 was synthesized from intermediates 52 and 13 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

[0176] Step 5: (2R,3S)-3-amino-4-(2,6-dichIoro-4-(5-(6-(isopropylamino)-5- methylpyridin-3-yl)-l,2,4-oxadiazol-3-yl)phenoxy)butan-2-ol dihydrochloride saltThe title compound was synthesized from intermediate 53 using the same or an analogous synthetic procedure to that of (2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl } - 1 ,2,4-oxadiazol-3 -yl)phenyl]oxy } butan-2-ol dihydrochloride salt in Example 4. Ή NMR (400 MHz, CD₃OD) δ 8.6 (s, 1H), 8.4 (s, 1H), 8.2 (s, 2H), 4.4 (m, 2H), 4.2 (m, 2H), 3.7 (m, 1H), 2.4 (s, 3H), 1.45 (d, 6H), 1.3 (d, 3H); MS (EI) for C₂iH₂₅Cl₂N₅0₃, found 466 (MH+).

Example 9

(2R,3S)-3-amino-4-(2,6-dichloro-4-(5^4-(isopropylamino)-6-methylpyridin-2-yI)-l,2,4- -3-yl)phenoxy)butan-2-ol dihydrochloride salt

[0177] Step 1: 6-Methyl-4-nitropicolinonitriIe (55). A mixture of 4-nitro-2-picoline-N- oxide 54 (6 g, 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 80 mL of water, cooled to -10 °C and a solution of sodium cyanide (7.7 g, 152 mmol) in water (55 mL) added dropwise with stirring over 40 min under an atmosphere of N₂. The mixture was then stirred for 3 h at the same temperature, and then diluted with EtOAc (200 mL) and water (100 mL). After the mixture was stirred for about 1 h, it was allowed to stand overnight. The organic layer was separated, washed with water, dried over Na₂S0₄ and concentrated. The crude product was purified by column chromatography to afford 55 (7 g, 99%) as a yellow solid.

[0178] Step 2: 6-Methyl-4-nitropicolinic acid (56). A solution 55 (7 g, 42 mmol) in 90% H₂S0₄ (40 mL) was heated at 120 °C for 2 h and then allowed to cool to RT. A solution of sodium nitrite (7.63 g, 1 13 mmol, 2.65 equiv) in water (15 mL) was added dropwise over a period of 30 min while maintaining the temperature between 12-15 °C. The reaction was stirred for 30 min at RT then for 1 h at 80 °C. The solution was cooled to RT and poured onto crushed ice. After stirring the yellow solution for 10 min, 200 mL of water was added and a solid was precipitated out. The product was filtered, washed with a small amount of water and dried in vacuo to yield 56 as a light yellow crystalline solid (7 g, 76%).

[0179] Step 3: 4-Bromo-6-methylpicolinic acid (57). A solution of 56 (6 g, 32.7 mmol) in 48% hydrobromic acid (65 mL ) was heated at 100 °C overnight and was then cool to RT. The solution was then evaporated to dryness in vacuo to give crude 57 in near quantative yield which was directly used in the next step.

[0180] Step 4: 4~(Isopropylamino)-6-methylpicoIinic acid (58). To a stirred solution of crude 57 (32.7 mmol) in butanol (120 mL) was added isopropylamine (8.2 g, 139 mmol) and the mixture was heated to 1 17 °C for 2 days. The solvent was removed in vacuo and the resulting residue was purified by column chromatography to afford 58 (5.0 g, 78% over 2 steps from compound 56) as a brown solid.

[0181] Step 5: (4S,5R)-tert-ButyI 4 (2,6-dichloro-4<5-(4-(isopropylamino)-6- methylpyridin-2-yl)-l,2,4-oxadiazol-3-yl)phenoxy)m

carboxylate (59). Intermediate 59 was synthesized from intermediates 58 and 13 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

[0182] Step 6: (2R,3S)-3-amino-4-(2,6-dichloro-4-(5-(4-(isopropyIamino)-6- methylpyridin-2-yl)-l,2,4-oxadiazol-3-yl)phenoxy)butan-2-ol dihydrochloride saltThe title compound was synthesized from intermediate 59 using the same or an analogous synthetic procedure to that of (2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l ,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol dihydrochloride salt in Example 4. Ή NMR (400 MHz, CD₃OD) δ 8.28 (br s, 2H), 7.60 (br s, 1H), 6.98 (s, 1H), 4.40 (m, 2H), 4.19 (m, 1H), 4.00 (m, 1H), 3.72 (m, 1H), 2.66 (s, 3H), 1.35 (m, 9H); MS (EI) for C2iH25Cl₂N₅0₃, found 466.05 (MH+). Example 10

5-[3^4-{[(2S R)-2-Amino-3-hydroxybutyl]oxy}-3,5-dichlorophenyl)-l,2,4-oxadiazol-5-yl]- 2-[(l-methy]ethyl)amino]pyridine-3-carbonitrile di-trifluoroacetate salt

[0183] Step 1: Methyl 5-bromo-6-chloronicotinate (61). A mixture of 5-bromo-6- chloronicotinic acid 60 (0.2 g, 0.84 mmol), K₂C0₃ (0.3 g, 2.1 mmol) and methyl iodide (0.178 g, 1.2 mmol) in DMF (10 mL) was stirred for 16 h at RT. After completion, the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (2 x 20 mL). The combined organic extracts were washed with saturated aqueous NaHC0₃ solution (20 mL), brine (2 x 20 mL) and dried (Na₂S0₄) and concentrated to afford ester 61 (0.2 g, 94.7%).

[0184] Step 2: Methyl 5-bromo-6-(isopropyIamino)nicotinate (62). In a sealed tube, to a mixture of isopropylamine (30 mL) and 61 (10 g, 39.9 mmol) was added triethylamine (5 mL) and the resulting mixture heated to 90 °C for 12 h. After completion it was cooled to RT and volatiles were removed in vacuo. The obtained residue was diluted with water and extracted with EtOAc. The combined organic layers were dried over Na₂S0₄ and concentrated to afford 62 (10 g, 91.8%).

[0185] Step 3: Methyl 5-cyano-6-(isopropylamino)nicotinate (63). To a stirred solution of 62 (1.5 g, 5.4 mmol) in DMF (5 mL) was added Zn(CN) ₂ (1.59 g, 13.6 mmol) and tetrakis(tirphenylphosphine)palladium (0) (0.78 g, 0.68 mmol) at 25 °C. The reaction mixture was stirred at 90 °C for 4 h. After completion, the reaction mixture was absorbed onto silica gel and chromatographed to afford 63 (1.0 g, 83 %).

[0186] Step 4: 5-Cyano-6-(isopropylamino)nicotinic acid (64). To a stirred solution of 63 (4.8 g, 20.4 mmol) in MeOH (50 mL) was added LiOH (1.367 g, 32.4 mmol) in water (5 mL) and the stirring was continued for 2 h at RT. After completion, the reaction mixture was diluted with water (100 mL) and extracted with diethyl ether. The pH was adjusted to 2-3 with IN HC1 and extracted with EtOAc. The EtOAc phase was dried over Na₂S0₄ and concentrated under reduced pressure to obtain compound 64 (3.8 g, 84% yield) as a white solid.

[0187] Step 5: (4S,5R)-tert-Butyl 4-((2,6-dichloro-4-(5-(5-cyano-6-

(isopropylamino)pyridin-3-yl)-l,2,4-oxadiazoI-3-yI)phenoxy)methyl)-2,2,5- trimethyloxazolidine-3-carboxylate (65). Intermediate 65 was synthesized from intermediates 64 and 13 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

[0188] Step 6: 5-[3-(4-{[(2S,3R)-2-Amino-3-hydroxybutyl]oxy}-3,5-dichlorophenyl)- l,2,4-oxadiazol-5-yI]-2-[(l-methylethyl)amino]pyridine-3-carbonitrile di-trifluoroacetate salt. The title compound was synthesized from intermediate 65 using the same or an analogous synthetic procedure to that of (2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l ,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol dihydrochloride salt in Example 4, substituting TFA for HC1 and DCM for EtOH and without converting the resulting TFA salt from the preparative HPLC purification to the corresponding HC1 salt. Ή NMR (400 MHz, CD₃OD) δ 9.05 (s, IH), 8.52 (s, IH), 8.20 (s, 2H), 4.55 (m, IH), 4.38 (m, 2H), 4.18 (m, IH), 3.70 (m, IH), 1.30 (d, 9H); MS (EI) for C₂,H₂₂C1₂N₆0₃, found 477.00 (MH+). Using the same or analogous synthetic techniques as in Example 10 and substituting with appropriate reagents, the following compound was prepared.

[0189] 5-[3-(4-{[(2S,3R)-2-Amino-3-hydroxybutyl]oxy}-3,5-dimethylphenyl)-l,2,4- oxadia₂ol-5-yl]-2-[(l-methylethyl)amino]pyridine-3-carbonitrile di-trifluoroacetate salt. 1H

NMR (400 MHz, CD₃OD) 5 9.00 (s, IH), 8.48 (s, IH), 7.82 (s, 2H), 4.52 (m, IH), 4.15 (m, 2H), 4.02 (m, IH), 3.62 (m, IH), 2.40 (s, 6H), 1.28 (d, 9H); MS (EI) for C₂₃H₂₈N₆0₃, found 437.25 (MH+). Example 11

5-(3-(4-((2S,3R)-2-amino-3-hydroxybutoxy)-3,5-dichlorophenyl)-l,2,4-oxadiazol-5-yl)-2- isopropoxynicotinonitrile trifluoroacetic acid salt

[0190] Steps 1-4: 5-Cyano-6-isopropoxynicotinic acid (68). Intermediate 68 can be prepared by one skilled in the art from commercially available 60 in four steps using the standard procedures outlined in the scheme above.

[0191] Step 5: (4S,5R)-tert-ButyI 4-((2,6-dichloro-4-(5-(5-cyano-6-isopropoxypyridin-3- yI)-l,2,4-oxadiazol-3-yl)phenoxy)methyl)-2,2,5-trimethyloxazolidine-3-carboxylate (69).

Intermediate 69 was synthesized from intermediates 68 and 13 using the same or an analogous synthetic procedure to that of intermediate 14 in Example 1.

10192] Step 6: 5-(3-(4-((2S,3R)-2-amino-3-hydroxybutoxy)-3,5-dichlorophenyl)-l,2,4- oxadiazol-5-yl)-2-isopropoxynicotinonitrile trifluoroacetic acid saltThe title compound was synthesized from intermediate 69 using the same or an analogous synthetic procedure to that of (2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyridin-4-yl}- l,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol dihydrochloride salt in Example 4, substituting TFA for HCI and DCM for EtOH and without converting the resulting TFA salt from the preparative HPLC purifⁱcation to the corresponding HCl salt. Ή NMR (400 MHz, CD₃OD) δ 9.20 (s, 1H), 8.85 (s, 1H), 8.21 (s, 2H), 5.60 (m, 1H), 4.40 (m, 2H), 4.18 (m, 1H), 3.72 (m, 1H), 1.48 (d, 6H) 1.32 (d, 3H); MS (EI) for C₂iH₂₁Cl₂N₅0₄, found 478.05 (MH+).

Example 12

(lR,2S)-2-Amino-3-{[5-chloro-2-nuoro-4-^

yl}-l,2,4-oxadiazoI-3-yl)phenyl]oxy}-l-methylpropyl dihydrogen phosphate

dih drochloride salt

[0193] Step 1: tert-Butyl (2S,3R)-l-(5-chloro-2-fluoro-4-(5-(2-(isopropylamino)-6- methyIpyridin-4-yl)-l,2,4-oxadiazol-3-yl)phenoxy)-3-hydroxybutan-2-ylcarbamate (70).

(2R,3S)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l-methylethyl)amino]pyridin-4-yl}- l,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol was synthesized using the same or analogous synthetic techniques as in Example 1 and substituting with appropriate reagents. To a stirred solution of (2R,3S)-3-Amino-4-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l ,2,4-oxadiazol-3-yl)phenyl]oxy}butan-2-ol (0.6 g, 1.33 mmol) and triethylamine (0.4 mL, 2.88 mmol) in THF (20 mL) was added Boc anhydride (0.4 g, 1.99 mmol) at RT and the reaction mixture was stirred for 12 h. Solvent was removed and water was added to the reaction mixture. The resulting solid was filtered and washed with diethyl ether to obtain 70 (0.7 g, 94 %) which was used as such in the next step. [0194] Step 2: tert-Butyl (2S,3 )-l-(5-chIoro-2-fluoro-4-(5-(2-(isopropylamino)-6- methyIpyridin-4-yl)-l,2,4-oxadiazol-3-yl)phenoxy)-3-(di-tert-butoxyphosphoryloxy)butan- 2-yIcarbamate (71). To a ice cooled solution of 70 (0.7 g, 1.27 mmol) in DCM (10 mL) was added di-tert-butyl diethylphosphoramidite (1.26 mL, 5.09 mmol) followed by tetrazole (15 mL, 0.45M solution in CH₃CN) and the reaction mixture was stirred at RT for 3 h. Hydrogen peroxide (15 mL, 30 %) was then added to the reaction mixture at 0 °C and stirring was continued for 30 min at 0 °C. A saturated solution of sodium thiosulphate (40 mL) was then added dropwise and the reaction mixture was stirred at the same temperature for 2 h. The reaction mixture was then extracted with DCM (3x) and the combined DCM layers were washed with brine, dried and concentrated to give 71 (0.6 g) as oil which was used as such in the next step.

[0195] Step 3: (lR,2S)-2-Amino-3-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazoI-3-yI)phenyl]oxy}-l-methylpropyl dihydrogen phosphate dihydrochloride salt. To an ice cold solution of 71 (0.6 g, 0.8 mmol) in EtOH (10 mL) was added ethanolic HCl (20 mL), and the reaction mixture was stirred at RT for 1 h. Solvent was then removed and the solid obtained was washed with diethyl ether and acetonitrile. The crude compound was purified by Prep HPLC and lyophilized. The obtained TFA salt was dissolved in ethanol, cooled to 0 °C and ethanolic HCl added. The resulting mixture was stirred at RT for 45 min then concentrated in vacuo. The resulting solid was washed with ether and hexane to afford (lR,2S)-2-amino-3-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}-l-methylpropyl dihydrogen phosphate dihydrochloride salt (70 mg, 12 %) as a pale yellow solid. 1H NMR (400 MHz, DMSO-4 δ 8.4 (m, 2H), 7.9 (d, 1H), 7.7 (d, 1H), 4.7-4.5 (m, 2H), 4.4 (m, 1H), 4.2 (m, 1H), 3.8 (m, 1H), 2.5 (m, 3H), 1.4 (d, 3H), 1.2 (d, 6H); MS (EI) for C₂,H₂₆C1FN₅0₆P, found 530 (MH+). Using the same or analogous synthetic techniques as in Example 12 and substituting with appropriate reagents, the following compounds were prepared.

[0196] (lS,2S)-2-Amino-3-{[5-chloro-2-fluoro-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yl}-l,2,4-oxadiazol-3-yl)phenyl]oxy}-l-methyIpropyl dihydrogen phosphate dihydrochloride salt. Ή NMR (400 MHz, CD₃OD) δ 7.9 (d, 1H), 7.6 (s, 1H), 7.5 (d, 1H), 7.3 (s, 1H), 4.8 (m, 1H, -NH), 4.6-4.4 (m, 2H), 4.2 (m, 1H), 4.1 (m, 1H), 3.8 (m, 1H), 2.6 (s, 3H), 1.6 (m, 3H), 1.4 (m, 6H); MS (EI) for C₂iH₂₆ClFN₅0₆P, found 530 (MH+). [0197] (lR,2S)-2-amino-3-{[2-fluoro-5-methyl-4-(5-{2-methyl-6-[(l- methylethyl)amino]pyridin-4-yI}-l,2,4-oxadiazol-3-yl)phenyI]oxy}-l-methylpropyl dihydrogen phosphate. ^lH NMR (400 MHz, DMSO-i¾ δ 7.81 (d, IH), 7.32 (d, IH), 6.96 (m, 2H), 6.84 (d, IH), 4.53 (m, IH), 4.45 (m, IH), 4.24 (m, IH), 4.06 (m, IH), 3.61 (m, IH), 2.61 (s, 3H), 2.38 (s, 3H), 1.29 (d, 3H), 1.16 (d, 6H); MS (EI) for

found 510.0 (MH+).

Example 13

5-{3-[4-((R)-2-Amino-3-hydroxy-propoxy)-2-chloro-5-fluoro-phenyl]-[l,2,4]oxadiazol-5-yl}-

2-isopropoxy-nicotinonitriIe (29, Table 2)

[0198] A-1 (2.0 g, 8.5 mmol) was heated in SOCl₂ (8 mL, 110 mmol) at 80°C for 1 hour. Excess SOCl₂ was removed under reduced pressure to afford A-2. [0199] A-2 (2.2 g, 8.5 mmol) was heated in /-PrOH (10 mL) at 80°C for 1 hour. Excess i- PrOH was removed under reduced pressure. The residue was purified by flash chromatography (using a solvent gradient of 0-20% EtOAc in heptane) to afford A-3.

[0200] A-3 (2.5 g, 8.8 mmol) and z-PrOH (2.7 mL, 35.2 mmol) were dissolved in dry THF (25 mL) under N₂. The mixture was cooled to 0°C and a 1M solution of KOtBu in THF (10.6 mL, 10.56 mmol) was added. The reaction was stirred for 1 hour at 0°C. To the reaction was added saturated aqueous NH₄C1 solution (30 mL) and EtOAc (50 mL). The layers were separated and the organic phase was extracted with EtOAc (50 mL). The combined organic layer was washed with brine (10 mL), dried over Na₂S0₄ and concentrated. The residue was purified by flash chromatography (using a solvent gradient of 0-20% EtOAc in heptane) to afford A-4 (2.5 g) as colorless oil.

[0201] A-4 (1 g, 3.31 mmol), Zn(CN)₂ (805 mg, 6.86 mmol), and Pd[P(Ph)₃]₄ (1 15 mg, 0.1 mmol) in DMF (10 mL) was heated at 120°C for 3 hours. The mixture was cooled to room temperature, saturated aqueous NaHC0₃ (15 mL) was added and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na₂S0₄, filtered and concentrated. The residue was purified by flash chromatography (using a solvent gradient of 0-30% EtOAc in heptane) to afford A-5.

[0202] A solution of A-5 (690 mg, 2.78 mmol), LiOH-H₂0 (233 mg, 5.56 mmol) in dioxane (1 mL) and H₂0 (5 mL) was stirred for 4 hours. The solvent was removed under reduced pressure. Water and DCM were added. The mixture was acidified to pH=2 with IN HC1 aqueous solution. The organic layer was separated. The inorganic layer was extracted with DCM. The organic layers were combined, washed with brine, dried over Na₂S0₄, filtered and concentrated to afford A-6.

[0203] A-6 (31 mg, 0.15 mmol) in DMF (1 mL) was added EDC-HC1 (29 mg, 0.15 mmol) and HOBt (24 mg, 0.18 mmol). The mixture was stirred for 50 minutes, and compound B-l (63 mg, 0.15 mmol) was added. After 50 minutes, the mixture was warmed at 95°C for 6 hours. The reaction was diluted with saturated aqueous NaHC0₃ and extracted with EtOAc. The organic phase was washed with water, brine, dried over Na₂S0₄, filtered and concentrated. The residue was purified by flash chromatography (using a solvent gradient of 0-25% EtOAc in heptane) to afford compound A-7. [0204] To compound A-7 (62 mg, 0.1 1 mmol) was added 4M HCI in dioxane (1.3 mL, 5.3 mmol). After 1 hour, the solvent was removed under reduced pressure. The residue was purified by reverse phase HPLC (using a solvent gradient of 10 to 90% of acetonitrile in water, with 0.1% formic acid). The material from the column was made basic with saturated NaHC0₃ solution (pH=8), partially concentrated to remove acetonitrile. The resulting solid was filtered, rinsed with water and dried to afford compound the title compound.

[0205] The following compounds were synthesized in similar fashion from the appropriate intermediates: Compound 25, table 3; Compound 26, table 3; Compound 27, table 3; Compound 28, table 3; Compound 32, table 3.

Example 14

(R)-2-Amino-3-{5-chloro-2-fluoro-4-[5-(5-hydroxymethyl-6-isopropoxy-pyridin-3-yl)- [l,2,4]oxadiazol-3-yl]-phenoxy}-propan-l-ol (33, Table 2)

[0206] To a chilled (-78°C) solution of compound A-7 (25 mg, 0.04 mmol) in DCM (0.5 mL) was added a 1M solution of DIB AH in DCM (0.2 mL, 0.2 mmol). After 1 hour, the reaction was quenched with 1M aqueous tartaric acid (0.17 mL), stirred for 5 minutes, extracted with DCM, washed with brine, dried over Na₂S0₄, filtered, and concentrated. The residue was purified by flash chromatography (using a solvent gradient of 0-30% EtOAc in heptane) to afford A-8.

[0207] To a chilled (0°C) solution of compound A-8 (14 mg, 0.02 mmol) in THF (0.5 mL) was added LiAlH₄ (1.8 mg, 0.05 mmol). After 2 hours, the mixture was quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried, and concentrated to afford A-9.

[0208] To compound A-9 (12 mg, 0.02 mmol) was added a solution of 4M of HC1 in dioxane (0.3 mL, 1.2 mmol). After 1 hour, the solvent was removed under reduced pressure. The residue was purified by reverse phase HPLC (using a solvent gradient of 10 to 90% of acetonitrile in water, with 0.1% FA). Desired fractions were made basic with saturated aqueous NaHC0₃ (pH=8), and concentrated to remove acetonitrile. The organic was extracted by EtOAc, dried over Na₂S0₄, filtered and concentrated to afford.

[0209] The following compound was synthesized in similar fashion from the appropriate intermediates: Compound 34, table 3.

Example 15

(R)-2-Amino-3-{5-chloro-2-fluoro-4-[5-(2-isopropoxy-6-methyl-pyridiii-4-yl)- [l,2,4]oxadiazoI-3-yl]-phenoxy}-propan-l-oI (30, Table 2).

[0210] To a solution of compound 4-1 (7 g, 64.1 mmol) in DMF (50 mL) was added sodium hydride (2.6 g, 65 mmol) at room temperature under a stream of nitrogen. After 20 minutes, 2- bromopropane (18 mL, 192 mmol) was added. After 2 hours, the reaction was quenched with saturated aqueous ammonium chloride and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated. The crude residue was diluted with heptanes and filtered. The filtrate was concentrated to afford compound 4-2.

[0211] To a solution of 4-2 (5.1 g, 33.73 mmol) in DCM (40 ml) was added m-CPBA (10.0 g, 44.62 mmol) at room temperature. After 16 hours, the reaction mixture was diluted with DCM and water, and the aqueous layer was extracted with DCM. The combined organic layers were washed with saturated aqueous NaHC0₃, brine, dried over sodium sulfate, filtered and concentrated. The crude was purified by silica gel chromatography using a gradient of 0-50% methanol in CH₂C1₂ to afford 4-3.

[0212] A solution of compound 4-3 (3.9 g, 23.3 mmol) in acetonitrile (50 mL) was added TMSCN (15.5 mL, 116.3 mmol) and DIPEA (21 mL, 117 mmol) was warmed at reflux. After 3 hours, the mixture was diluted with saturated aqueous ammonium chloride, concentrated and then extracted with EtOAc. The organic layers were washed with brine, dried over sodium sulfate and concentrated. The crude was filtered through a pad of silica gel using 20% EtO Ac/heptanes to afford compound 4-4.

[0213] To a solution of compound 4-4 (0.56 g, 3.2 mmol) in EtOH (20 mL) was added potassium hydroxide (1.8 g, 32 mmol) and water (5 mL). The mixture was warmed at reflux. After 18 hours, the mixture was concentrated, made acidic with IN HCl and extracted with EtO Ac. The organic layers were washed with brine, dried over sodium sulfate and concentrated to afford compound 4-5.

[0214] To a solution of compound 4-5 (0.033 g, 0.18 mmol) in DMF (3 mL) was added EDC-HC1 (0.035 g, 0.18 mmol), and HOBT (0.025 g, 0.18 mmol). After 30 minutes, compound 1-6 (0.070 g, 0.17 mmol) was added. After 30 minutes, the mixture was warmed at 95°C. After 5 hours, the mixture was diluted with saturated ammonium chloride, extracted with EtOAc, washed with brine, dried over Na₂S0 , filtered and concentrated. The residue was purified by silica gel chromatography using a 0-50% gradient of EtOAc in heptanes to afford compound 4-6.

[0215] To compound 1-7 in MeOH (2 mL) was added a solution of 4M HCl in dioxane (10 mL). After 2 hours, the mixture was concentrated to dryness, 2M aqueous NaOH (2 mL) was added and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated. The crude material was triturated with ether to afford the title compound.

[0216] The following compound was prepared in similar fashion from the appropriate intermediates: Compound 31, table 3

[0217] Preparation of Intermediate 5-5

5-5

[0218] A mixture of 2-chloro-4,5-difluorobenzonitrile (7.5 g, 43.2 mmol) and 50% aqueous hydroxylamine (9.0 mL, 146.8 mmol) in MeOH (50 mL) was warmed at 65°C. After 2 hours, the reaction was cooled and concentrated to remove the methanol resulting in a solid. The mixture was diluted with minimal water and the solid was collected by filtration and washed with cold water to afford compound 5-5.

16 040892

Example 16

(2R S)-3-Amino-4-{5-chloro-2-fluoro-4-[5-(2-isobutyI-6-methyl-pyridin-4-yl)- [l,2,4]oxadiazoI-3-yI]-phenoxy}-butan-2-ol (35, Table 2)

[0219] To a mixture of compound 5-1 (6.7 g, 36.1 mmol) in DMF (34 mL) was added 2- methyl- 1-propenylboronic acid pinacol ester (7.2 g, 39.5 mmol) and potassium carbonate (8.8 g, 63.6 mmol). The mixture was degassed for 5 minutes, [(Ph)₃P]₄Pd (713.0 mg, 0.6 mmol) was added and the mixture was warmed at 120°C. After 3 hours, the reaction was cooled and diluted with saturated aqueous ammonium chloride and extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium bicarbonate, brine, dried over magnesium sulfate, filtered and concentrated. The crude material was passed through a pad of silica gel eluting with 5% ethyl acetate-heptane to afford compound 5-2. Mixed fraction were purified a second time eluting with 15% dichloromethane in heptane to afford addition 5-2.

[0220] A mixture of compound 5-2 (7.4 g, 36.1 mmol), Pt0₂-hydrate (200 mg, 0.9 mmol) in methanol (20 mL) was hydrogenated under 1 atmosphere. After 3 days, the mixture was diluted with filter agent and filtered through filter agent and concentrated to afford compound 5-3.

[0221] To a solution of compound 5-3 (6.8 g, 33 mmol) in methanol (50 mL) was added a solution of sodium hydroxide (4 g, 0.1 mol) in 50 mL of water. After 24 hours, the mixture was concentrated diluted with water (25 mL) and the pH of the mixture was adjusted with 12N aqueous HC1 (8.3 mL, 100 mmol) in 25 mL of water. The aqueous was extracted with ethyl acetate and then concentrated. The residue was diluted with ethyl acetate and combined with the ethyl acetate extracts. The organic layers were dried over magnesium sulfate, filtered and concentrated. The crude solid was triturated with ether-heptane to afford in three crops compound 5-4.

[0222] To compound 5-4 (4.5 g, 23.3 mmol) in DMF (35 mL) was added EDC-HC1 (4.58 g, 23.9 mmol) followed by HOBt (3.25 g, 24.1 mmol). After 1 hour, compound 5-5 (4.9 g, 23.7 mmol) was added. After 10 minutes, the mixture was warmed at 85°C. After 7 hours, the reaction was diluted with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated. The crude material was purified on silica gel (using a gradient of 0-100% ethyl acetate in heptane) to afford compound 5-6.

[0223] To a mixture of compound 5-6 (165 mg, 0.450 mmol) and compound 5-7 (161 mg, 0.66 mmol) in DMF (3 mL) was added 60% sodium hydride in mineral oil (30 mg, 0.75 mmol). After 30 minutes, the reaction was diluted with saturated aqueous ammonium chloride and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated. The crude material was purified on silica gel (using a gradient of 0-100 ethyl acetate-heptane) to afford compound 5-8.

[0224] To a solution compound 5-8 (135 mg, 0.23 mmol) in methanol (10 mL) was added 4N HC1 in dioxane (1 mL, 4 mmol). After 18 hours, the mixture was concentrated and made basic with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The combined anic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated.

The solid was triturated with ether-heptane to afford the title compound.

See Synthetic Example Section of HPLC-MS methods.

Biological Examples

[0225] Suitable in vitro assays for measuring S1P1 and S1P5 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 S I PI and/or S1P5. As such compounds of Formula I are useful for treating diseases, particularly autoimmune disease in which SI PI and/or S1P5 activity contributes to the pathology and/or symptomatology of the disease, for example, multiple sclerosis and graft-versus host disease. Suitable in vivo models for autoimmune diseases are known to those of ordinary skill in the art and are also described below, e.g. models for autoimmune-mediated inflammation, multiple sclerosis, graft-versus host disease, and osteoporosis. Following the examples disclosed herein, as well as that disclosed in the art, a person of ordinary skill in the art can determine the S1P1 and S1P5 agonist activity of a compound of this invention and its usefulness for treating a disease.

Biological Example 1

CNG cAMP Assay

[0226] Frozen HEK293 cells expressing the CNG channel and SIP] (BD Biosciences, San Jose, CA) are thawed and plated into the wells of a black, clear bottom, 384-well CellBind plate (Corning, Corning, NY) at 14,000 cells per well. HEK293 cells expressing the CNG channel and CB1 (BD Biosciences) are cultured and plated under the same conditions. The cells are incubated for 16 h at 37 °C in complete DMEM medium (Invitrogen Carlsbad, CA) containing 10% FBS (HyClone Logan, UT), 250 μg/mL geneticin (Invitrogen), and 1 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO). A membrane potential dye (BD Biosciences) is added and the plates are incubated for 2-2.5 h at room temperature.

[0227] Test compounds are tested at maximum concentrations of 10 μΜ. Compounds are diluted in DMSO (10 concentration points, 3 -fold each) and added to the assay plate at final DMSO concentrations of 1.8%. For each compound, there are duplicate assay plates and each assay plate have duplicate wells per concentration point. Test compounds are added to the cells in a DPBS solution containing 25 μΜ Ro 20-1724 (Sigma-Aldrich), 500 nM of the A2b receptor agonist NEC A (Sigma-Aldrich) and 10 nM (EC₉₅) of SIP (Avanti Alabaster, AL) and incubated for 90 min. The assay plate is read before compound addition (To) and after the 90 min incubation (T₉₀) using an EnVision plate reader (PerkinElmer, Waltham, MA) at an excitation wavelength of 350 nm and an emission wavelength of 590 nm. The T₉o/T₀ ratio is determined for each concentration of the test compounds. The percent agonist activity is determined as [(test compound - DMSO alone control) / (NEC A alone control- DMSO alone control) * 100]. The percent activities are plotted against compound concentration to determine EC₅₀ using XLFit (IDBS, Alameda, CA). The control used for calculating rEC50 in the SIPi CNG agonist assay is DMSO. Biological Example 2

S1P1 β-arrestin Recruitment Assay

[0228] For the Tango™ β-arrestin recruitment assay, the cytoplasmic C-terminus of SI Pi was tethered to the tTA transcriptional activator with a linker that contains a cleavage site for the Nla protease from tobacco etch virus (TEV protease). The C-terminus of the human P-arrestin2 protein was fused to TEV protease. Binding of an agonist recruits the β-arrestin-TEV fusion protein to the receptor resulting in cleavage of the linker and released of tTA to enter the nucleus and subsequently activated a tTA-dependent luciferase reporter gene.

[0229] 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 μg/mL streptomycin, Invitrogen) and 0.1% fatty acid free BSA (Sigma- Aldrich). Cells are added to the wells of a 384-well white opaque bottom assay plate (PerkinElmer) at 3,000-6,000 cells per well and the plate is incubated for approximately 4 h in a 37 °C incubator. Test compounds are tested at maximum concentrations of 10 μΜ for the agonist assays. Compounds are diluted in DMSO (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration. For each compound, there are duplicate assay plates and each assay plate has duplicate wells per concentration point. The plate is incubated at 37 °C for 30 min. The efficacy control is 5 μΜ SIP (Avanti). Following agonist addition, the assay plates are incubated in a 37 °C incubator for 16-18 h. Luciferase assay reagent is added and luminescence measured in an En Vision plate reader (PerkinElmer). To determine agonist activity, percent activity is calculated as [(test compound - background) / (positive control- background) * 100], where background is the luminescence of the DMSO alone control and the positive control is the luminescence from cells incubated with the efficacy control 5 μΜ SIP. The percent activities are plotted against compound concentration to determine EC50 using XLFit (IDBS).

[0230] Assay 2b : Alternatively, U20S cells expressing the reporter gene and SI Pi (Invitrogen) were added to the wells of a 384-well white opaque bottom assay plate (PerkinElmer) at 0.3125 X 10⁶ cells per well. The cells were serum starved for 48 h in Freestyle medium (Invitrogen). Test compounds were tested at maximum concentrations of 1 μΜ for the agonist assay. Compounds were diluted in DMSO (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration. The efficacy control was 1 μΜ SIP 6 040892

(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 F4₆onm F₅30nm ratio determined for each concentration of the test compounds. Percent activity was calculated as [(test compound ratio - DMSO ratio) / (positive control ratio - DMSO ratio) * 100], where the positive control and DMSO ratios are from cells incubated with the efficacy control 1 μΜ SIP and 1% DMSO, respectively. The percent activities were plotted against compound concentration to determine EC₅₀ using XLFit (IDBS).

Biological Example 3

hSIPIR GTPyS and GTP-Eu Binding Assays

[0231] Assay 3a: The hSIPIR GTPyS binding assay was carried out at room temperature in 96 well non-binding surface assay plates. The reaction in each well contained 4 μg hSIPIR (hEdgl) membrane protein (Lonza), 30μΜ GDP, 0.1 nM [³⁵S]GTPyS, 0.25% fatty acid free BSA, and serially diluted hSIPIR agonist compound in 200 μΐ, assay buffer (25mM Tris-HCl PH 7.9, 100 mM NaCl, 3 mM MgC12, and 0.2 niM EGTA). After one hour of incubation, 0.9 mg of WGA (Wheat Germ Agglutinin) SPA beads in 50 μΐ, of assay buffer was added to each well. The SPA beads were spun down after an additional one hour incubation. The radioactivity of the bound GTPyS was counted by reading the assay plate using a MicroBeta.

[0232] 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 SIP] are incubated in 96-well filter plates (Pall, East Hills, NY) in a final volume of 100 μΙ νβΙΙ buffer containing 40 μg/mL membrane, 50 mM HEPES, 2 μΜ GDP, 10 mM MgCl₂, 100 mM NaCl, 500 μg/mL Saponin and test compound. Test compounds are tested at maximum concentrations of 10 μΜ. Compounds are diluted (10 concentration points, 3-fold each) and added to the assay plate at a 1% final DMSO concentration. For each compound, there are duplicate assay plates and each assay plate has duplicate wells per concentration point. The plates are incubated for 30 min at room temperature on a plate shaker at low speed. GTP-Eu is added to each well (10 μί, 10 nM final concentration) and the plate is incubated for an additional 30 min with slow shaking. The wells are washed with ice cold GTP washing buffer (3 X 150 μΐ,) 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 ran. To determine agonist activity, percent activity is calculated as [(test compound - background)/ (positive control- background) * 100], where background is the fluorescence in absence of compound and the positive control is the fluorescence from membranes incubated with 1 μΜ SIP (Avanti). The percent activities are plotted against compound concentration to determine IC₅₀ or EC₅₀ using XLFit (IDBS).

Biological Example 4

hSlP5R GTPyS Binding Assay

[0233] The hSlPR5 GTPyS binding assay was carried out at room temperature in 96-well non-binding surface assay plates. The reaction in each well contained 5 μg hSlPR5 (hEdg8) membrane protein from CHO cells expressing hSlPR5, 30 μΜ GDP, 0.1 nM [³⁵S]GTPyS, 0.25% fatty acid free BSA, and serially-diluted Compound of the Invention in 200 xL assay buffer (25 mM Tris-HCl PH 7.9, 100 mM NaCl, 3 mM MgC12, and 0.2 mM EGTA). After one hour of incubation, 0.9 mg of WGA (Wheat Germ Agglutinin) SPA beads in 50 μΐ, of assay buffer was added to each well. The SPA beads were spun down after an additional one hour incubation. The radioactivity of the bound GTPyS was counted by reading the assay plate using a MicroBeta.

Biological Example 5

S1P3 β-arrestin Recruitment Assay

[0234] This assay was conducted using the procedures described in Assay 2a, replacing S1P1 with SlP3.

Biological Example 6

S1P2 β-arrestin Recruitment Assay

[0235] This assay was conducted using the procedures described in Assay 2a, replacing SI PI with SI P2.

[0236] Table 4 gives EC₅₀ data (unless otherwise indicated) for the compounds in Tables 1 and 2 and are in nM units. Assay 2b is the Tango™ β-arrestin Recruitment Assay in U20S cells, as described in Biological Example 2. Assay 3a is the hSIPIR GTPyS Binding Assay as described in Biological Example 3. Assay 4 is the hSlP5R GTPyS Binding Assay as described in Biological Example 4. Assay 5 is the Tango™ β-arrestin Recruitment Assay in HEK293 cells, as described in Biological Example 5.

[0237] EC₅₀'s were measured unless otherwise noted. An "*" indicates that rECso (relative EC₅o) was measured rather than EC₅₀.

[0238] For assay 5, F means the compound has an EC₅₀ or relative EC₅₀ of less than or equal to 250 nM and G means the compound has an EC50 or relative EC50 of greater than 250 nM.

[0239] For assays 3a, 2b and 4, "A" means the compound has an EC50 or relative EC₅₀ of less than or equal to 10 nM. "B" means the compound has an EC₅₀ or relative EC₅₀ greater than 10 nM but less than or equal to 50 nM. "C" means the compound has an EC₅₀ or relative EC o greater than 50 nM but less than or equal to 250 nM. "D" means the compound has an EC₅₀ or relative EC₅₀ greater than 250 nM but less than or equal to 2800 nM. "E" means the compound has an EC₅₀ or relative EC₅₀ greater than 2800 nM but less than 10000 nM. In the table, "nt" means the Compound was not tested and "na" means the compound was tested but had no measurable activity under the assay conditions employed.

Table 4

Assay (nM)

Entry

Name

No. 3a 2b 4 5

(li?,25)-2-amino-3-{[5-chloro-2-fluoro-4-(5-{2- methyl-6- [( 1 -methylethyl)amino]pyridin-4-yl } -

5 A A nt F l,2,4-oxadiazol-3-yl)phenyl]oxy}-l- methylpropyl dihydrogen phosphate

(2S,3S)-3-amino-4-{[5-chloro-2-fluoro-4-(5-{2-

6 methyl-6-[(l-methylethyl)amino]pyridin-4-yl}- A B nt na

1 ,2 ,4-oxadiazol-3 -y l)phenyl] oxy } butan-2-ol

( 15,25)-2-amino-3- { [5-chloro-2-fluoro-4-(5- {2- methyl-6- [( 1 -methyl ethyl)amino]pyridin-4-yl } -

7 A A nt G

1 ,2,4-oxadiazol-3 -yl)phenyl]oxy } - 1 - methylpropyl dihydrogen phosphate

(2R)-2-Amino-3-(2-fluoro-4- { 5-[2-

8 (isopropylamino)-6-methylpyridin-4-yl]-l,2,4- A A nt na oxadiazol-3-yl}-5-methylphenoxy)propan-l-ol

(2R,3 S)-3-Amino-4-(2,6-dichloro-4- { 5-[2-

9 (isopropylamino)-6-methylpyridin-4-yl]- 1 ,2,4- A A nt G oxadiazol-3-yl}phenoxy)butan-2-ol

(2R,3S)-3-Amino-4-{[2-fluoro-5-methyl-4-(5-

10 {2-methyl-6-[( 1 -methylethyl)amino]pyridin-4- A B nt na yl } - 1 ,2,4-oxadiazol-3 -yl)phenyl]oxy }butan-2-ol

(2R,3 S)-3-Amino-4-(2-chloro-6-fluoro-4- { 5-[2-

11 (isopropylamino)-6-methylpyridin-4-yl]- 1 ,2,4- A A nt G oxadiazol-3 -y 1 } phenoxy)butan-2-ol

(2R)-2-Amino-3- { [2,6-dichloro-4-(5- {2-

12 methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl } - A A A G

1 ,2,4-oxadiazol-3 -yl)phenyl]oxy } propan- 1 -ol

(2R)-2-Amino-3-{[2,6-dimethyl-4-(5-{2-

13 methyl-6- [( 1 -methyl ethyl)amino]pyridin-4-yl } - A A nt na

1 ,2,4-oxadiazol-3 -y l)phenyl]oxy } propan- 1 -ol

(2R)-2-Amino-3-{[2,6-dimethyl-4-(5-{6-

14 methyl-5- [( 1 -methylethyl)amino]pyridin-2-yl } - B D nt na

1 ,2,4-oxadiazol-3-yl)phenyl]oxy }propan-l -ol Table 4

Entry Assay (nM)

Name

No. 3a 2b 4 5

(2R,3 S)-3-Amino-4- { [2,6-dimethyl-4-(5- {2-

15 methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl } - A A nt G

1 ,2,4-oxadiazol -3 -y l)phenyl] oxy } butan-2-ol

(2R,3S)-3-Amino-4-{[2-fluoro-5-methyl-4-(5-

16 { 6-methyl-5 - [( 1 -methylethyl)amino]pyridin-2- B C nt na y 1 } - 1 ,2,4-oxadiazol-3 -yl)pheny 1] oxy } butan-2-ol

5-[3-(4-{[(2S,3R)-2-Amino-3- hydroxybutyl]oxy}-3,5-dimethylphenyl)-l ,2,4-

17 A B nt na oxadiazol-5-yl]-2-[(l- methylethyl)amino]pyridine-3-carbonitrile

5-[3-(4-{[(2S,3R)-2-Amino-3- hydroxybuty 1] oxy } -3 ,5 -dichloropheny 1)- 1,2,4-

18 A A nt na oxadiazol-5-yl]-2-[(l - methylethyl)amino]pyridine-3-carbonitrile

(2R,3S)-3-amino-4-(2,6-dichloro-4-(5-(5-

19 (isopropylamino)-6-methylpyridin-2-yl)- 1 ,2,4- A B nt na oxadiazol-3-yl)phenoxy)butan-2-ol

5-(3-(4-((2S,3R)-2-amino-3-hydroxybutoxy)-

20 3,5-dichlorophenyl)-l,2,4-oxadiazol-5-yl)-2- A A nt na isopropoxynicotinonitrile

(2R,3S)-3-amino-4-(2,6-dichloro-4-(5-(6-

21 (isopropylamino)-5-methylpyridin-3-yl)- 1 ,2,4- A D nt na oxadiazol-3-yl)phenoxy)butan-2-ol

(2R,3S)-3-amino-4-(2,6-dichloro-4-(5-(4-

22 (isopropylamino)-6-methylpyridin-2-yl)- 1 ,2,4- B B nt na oxadiazol-3-yl)phenoxy)butan-2-ol

(2R)-2-Amino-3-{[2-chloro-6-methyl-4-(5-{2-

23 methyl-6-[( 1 -methylethyl)amino]pyridin-4-yl} - A A nt G

1 ,2,4-oxadiazol-3-yl)phenyl]oxy}propan-l -ol

(lR,2S)-2-amino-3-{[2-fluoro-5-methyl-4-(5- {2-methyl-6-[( 1 -methylethyl)amino]pyridin-4-

24 A A nt F yl } - 1 ,2,4-oxadiazol-3 -yl)phenyl]oxy } - 1 - methylpropyl dihydrogen phosphate Table 4

Entry Assay (nM)

Name

No. 3a 2b 4 5

(R)-5-(3-(4-(2-amino-3-hydroxypropoxy)-2-

25 chloro-5-fluorophenyl)- 1 ,2,4-oxadiazol-5-yl)-2- nt A nt nt isopropoxybenzonitrile

(R)-2-amino-3-(5-chloro-4-(5-(3-chloro-4-

26 isopropoxyphenyl)- 1 ,2,4-oxadiazol-3-yl)-2- nt A nt nt fluorophenoxy)propan-l -ol

(R)-2-amino-3-(5-chloro-4-(5-(5-chloro-6-

27 ethoxypyridin-3-yl)-l,2,4-oxadiazol-3-yl)-2- nt C nt nt fluorophenoxy)propan- 1 -ol

(R)-2-amino-3-(5-chloro-4-(5-(5-chloro-6-

28 isopropoxypyridin-3-yl)-l,2,4-oxadiazol-3-yl)- nt B nt nt

2-fluorophenoxy)propan- 1 -ol

(R)-5-(3-(4-(2-amino-3-hydroxypropoxy)-2-

29 chloro-5-fluorophenyl)-l ,2,4-oxadiazol-5-yl)-2- nt A nt nt isopropoxynicotinonitrile

(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(5-

30 isopropoxy-6-methylpyridin-2-yl)- 1 ,2,4- nt B nt nt oxadiazol-3-yl)phenoxy)propan- 1 -ol

(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(2-

31 isopropoxy-6-methylpyridin-4-yl)- 1 ,2,4- nt C nt nt oxadiazol-3 -yl)phenoxy)propan- 1 -ol

5-(3-(4-((2S)-2-amino-3-hydroxybutoxy)-2-

32 chloro-5-fluorophenyl)- 1 ,2,4-oxadiazol-5-yl)-2- nt A nt nt isopropoxynicotinonitrile

(R)-2-amino-3-(5-chloro-2-fluoro-4-(5-(5-

33 (hydroxymethyl)-6-isopropoxypyridin-3-yl)- nt B nt nt

1 ,2,4-oxadiazol-3-yl)phenoxy)propan- 1 -ol

(3S)-3-amino-4-(5-chloro-2-fluoro-4-(5-(5-

34 (hydroxymethyl)-6-isopropoxypyridin-3-yl)- nt B nt nt l,2,4-oxadiazol-3-yl)phenoxy)butan-2-ol

(2R,3S)-3-amino-4-(5-chloro-2-fluoro-4-(5-(2-

35 isobutyl-6-methylpyridin-4-y 1)- 1 ,2,4-oxadiazol- nt B nt nt

3 -yl)phenoxy)butan-2-ol Biological Example 7

Lymphocyte PD Assay in Mice

[0240] Suppression of circulating lymphocytes is assessed as a pharmacodynamic (PD) endpoint in 6-10 week old C57B1/6 male mice (Taconic Farms, Germantown, NY). Upon arrival, mice are acclimated to the vivarium (12 h light cycle, 12 h dark cycle) for a minimum of 3 days prior to the initiation of a study. During the study, animals are provided food and water ad libitum and housed in a room conditioned at 70-75 °F. All animals are examined daily for health assessment.

[0241] Compounds of the Invention are suspended or dissolved in vehicle for administration of 0.1 mL/animal based on mean body weight of group. Compounds are administered using a disposable gavage needle (20G, Braintree Scientific, Braintree, MA). Blood is collected into EDTA-coated tubes (Microvette 100 with EDTA, Sarstedt, Newton, NC) from the retro-orbital sinus of isoflurane-anesthetized animals 24 or 32 h post-dose. Mice are then euthanized by cervical dislocation. For some experiments, samples of tissues are collected for measurement of compound levels. Samples are frozen immediately on dry ice and stored at -18 °C until assayed. After collection, blood samples are placed on a rock 'n roller mixer (Drew Scientific, Inc., Waterbury, CT) for at least 10 min and then a complete blood count (CBC) analysis is performed using the Hemavet 1700 Flexible Veterinary Multi-Species Hematology System (Drew Scientific). Samples are then placed on ice and, within 4 h, centrifuged to obtain plasma which is then stored frozen at 20 °C until analysis for compound levels. CBC readouts include white blood cells; total and % of total for the following: neutrophils, lymphocytes, monocytes, eosinophils, basophils, and nucleated red blood cells (RBC); RBC; hemoglobin; hematocrit; mean corpuscular volume, hemoglobin (HGB) total and concentration; RBC distribution width; platelets; and mean platelet volume. All mouse PD data consists of group sizes of 8 and are expressed as means ± SEM. Statistical analysis of each experimental endpoint is conducted with GraphPad Prism. All compound exposure data are based on n = 4/group and are expressed as means ± SD.

Biological Example 8

Lymphocyte PD Assay in Rats

[0242] Suppression of circulating lymphocytes is assessed as a PD endpoint in jugular vein canulated Sprague Dawely (SD) rats 6-8 weeks of age and weighing approximately 200 g (Taconic Farms, Germantown, NY). Prior initiating a study, rats are acclimated to the vivarium facility (12 h light cycle, 12 h dark cycle) for a minimum of 2 days. During a study, animals are provided food and water ad libitum and housed in a room conditioned at 70-75 °F and 60% relative humidity. All animals are examined daily for health assessment.

[0243] The compounds of the invention are formulated and animals are dosed at a volume of 4 mL/kg. At indicated timepoints, whole blood is collected via jugular vein canulas into EDTA-coated tubes and hematology analysis is performed on an Abbott Cell-Dyn 3700 hematology analyzer. Readouts include white blood cells (total, differential, and % of total), neutrophils, lymphocytes, monocytes, eosinophils, basophils, RBC, HGB, hematocrit, mean corpuscule volume, mean corpuscule HGB concentration, RBC distribution width, platelets, and mean platelet volume.

Biological Examples 9-12

In vivo models

Biological Example 9: Delayed-type Hypersensitivity (DTH) Model

[0244] Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. Signaling of SIP through SlPl has been shown to exclusively modulate egress of lymphocyte including 70% of activated T cells from lymph nodes. Delayed-type hypersensitivity (DTH) is an immune response mediated by a variety of inflammatory cells, including neutrophils, macrophages and T cells (Kobayashi et al. 2001, Black 1999). DTH develops in two phases, a sensitization phase, in which T cells are sensitized and memory T cells are formed, and an elicitation phase, in which T cell recall responses are induced upon secondary challenge with antigen. This second phase results in recruitment of inflammatory cells such as neutrophils and macrophages to the injection site of an intradermally applied antigen in a previously sensitized host, which causes swelling 24 h to 48 h post antigen challenge. The DTH assay (primarily done in mice) is an in vivo manifestation of a cell-mediated immunity reaction, and the response to antigen representation modulated by immunosuppressive treatment can be measured.

[0245] C57B1/6 male mice (10 mice per group) are immunized on day zero by subcutaneous injection at the base of the tail with 100 iL of 2 mg/mL methylated BSA emulsified with Complete Freunds Adjuvans (CFA, Sigma). Once-daily for twice-daily administration of a Compound of the Invention occurrs for 10 days. On day 10 after immunization, mice receive a second booster injection at the base of tail of an emulsified mixture of 2 mg/mL methylated BSA in Incomplete Freund's Adjuvans. On day 13 animals are challenged subcutaneously in the left hind footpad with 20 of 10 mg/mL methylated BSA in sterile water (water for injection). Animals are injected with an equal volume of sterile water into the right hind footpad as a control. Twenty four hours later (dose day 14) the right and left hind foot paws are transected at the medial and lateral malleolus, weighed, and the weight difference induced by injected antigen determined and compared to weight differences of vehicle treated non-sensitized and sensitized control groups. The increase in paw weights comparing left and right hind paw for each treatment group are analyzed for differences of treatment with a Compound of the Invention compared to vehicle control group using the Mann- Whitney non-parametric test statistic with minimal significance level set at p<0.05.

Biological Example 10: Allograft Model

[0246] The rodent allograft model is an in vivo assay for assessing tissue rejection (ie, from transplantation) in response to chronic and/or sub-chronic immunosuppressive treatment (Chiba et al, 2005). Rejection is caused by T lymphocytes of the recipient responding to the foreign major histocompatibility complex of the donor graft. The transplanted organ (eg, skin) represents a continuous source of HLA alloantigens capable of inducing a rejection response at any time post transplantation. Because it cannot be eliminated, the allograft continuously activates the immune system, resulting in lifelong overproduction of cytokines, constant cytotoxic activity, and sustained alteration in the graft vasculature. Therefore, lifelong immunosuppression is required to ensure allograft survival. In this model skin from donor rats (male Lewis; histocompatibility RT-1¹) is surgically engrafted onto a dorsal region of recipient rats (male F344; histocompatibility RT-l^lvl). Administration of compound occurs immediately after surgery for a predetermined duration. Skin allografts are monitored daily for rejection.

[0247] On the day of surgery male Lewis donor rats are anesthetized with Isoflurane and skin aseptically harvested from the tail. Male F344 acceptor rats (8 per group) previously shaved (1-2 days prior to surgery) in the designated engraftment area are anesthetized with Isoflurane and a full thickness skin graft bed on the medial flank removed and discarded. The skin graft bed removed is equivalent in size to the donor skin to be engrafted. The prepared donor skin is then secured on the prepared graft bed with spot tissue glue or by 4 to 8 nonsilk sutures, and covered with sterile Vaseline gauze and wrapped with a bandage. All surgery takes place on heated pads with sterile surgical equipment. Animals are monitored and turned every 20 minutes until ambulatory before returning to cages, water and food. Initiation of administration of a Compound of the Invention (once-daily or twice-daily) occurrs when the animals fully recovered from anesthesia for a period of 14 days. On day 5 post-surgery, surgical bandaging is removed and the grafts assessed daily for rejection (necrosis of the graft tissue following by scabbing and sloughing from the graft bed site). An allograft is scored as "rejected" when it sloughed from the graft bed site. A positive effect in this model is delayed rejection of the allograft in response to treatment with a Compound of the Invention when compared to vehicle-treated control animals.

Biological Example 11: Experimental autoimmune encephalomyelitis (EAE) Model

[0248] Multiple sclerosis is a demyelinating disease of the CNS. The main features of the disease are focal areas of demyelination and inflammation mediated by macrophages and t-lymphocytes. These cells develop in the peripheral lymphoid organs and travel to the CNS causing an autoimmune response. The development of T cells is controlled largely by the expression of various cytokines as well as cellular adhesion molecules. The EAE model today is the most thoroughly studied animal model for human autoimmune diseases. Mice are immunized with myelin-derived peptide PLP and clinical parameters of disease (bodyweight loss and paralysis) are monitored daily. The endpoint is the analysis of the extent of inflammation in brain and spinal cord.

[0249] C57B1/6 mice develop chronic paralysis after immunization with MOG₃₅-₅₅ peptide. Mice develop EAE 8-14 days after immunization and stay chronically paralyzed for 30-40 days after onset of disease. Female C57B1/6 mice are subcutaneously injected with MOG35-55 peptide emulsified in Complete Freund's Adjuvant at two sites on the back, injecting 0.1 mL at each site. Within 2 h of injection, pertussis toxin (aids in brain penetration of the MOG peptide) is administered intraperitoneally. A second injection of pertussis toxin is administered 22-26 h after the MOG₃5-5₅ peptide injection. Onset of EAE is typically 7 days after immunization. EAE is scored on a scale of 0-5 with 0 being no obvious changes in motor functions, while 5 indicates complete paralysis. Mice are administered a Compound of the Invention (once-daily or twice- daily) on the day of MOG_35-55 peptide injection and monitored for paralysis and compared to vehicle-treated control animals. A positive effect in this model is delayed onset/severity of EAE. Biological Example 12: Osteoporosis model

[0250] Methods described in Nature 2009, 458(7237), 524-528, which is herein incorporated by reference, can be used to determine whether a Compound of the Invention is able to prevent bone density loss.

[0251] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding. The invention has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. It will be obvious to one of skill in the art that changes and modifications may be practiced within the scope of the appended claims. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled. All patents, patent applications and publications cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application or publication were so individually denoted.

Claims

What is claimed is:

1. A compound of the formula,

or a stereoisomer or mixture of isomers thereof and optionally as a pharmaceutically acceptable salt thereof, where

R¹ is hydrogen or -P(0)(OR⁶)₂;

R² and R^2a are independently hydrogen, cyano, halo, alkyl, haloalkyl, alkoxy, or haloalkoxy;

Ring A is phenyl or 6-membered heteroaryl;

each R³ is independently cyano, halo, alkyl, haloalkyl, hydroxy, alkoxy, haloalkoxy, alkoxyalkyloxy, cycloalkyloxy, heterocycloalkyloxy, nitro, phenyl, amino, alkylamino, dialkylamino, aminocarbonyl, alkylcarbonylamino, or alkoxy carbony lamino ;

n is 0, 1, or 2;

R⁴ is hydrogen, alkyl, cyano, halo, haloalkyl, or hydroxyalkyl;

R⁵ is hydrogen or alkyl;

R^5a is hydrogen, alkyl, alkylcarbonyl, or alkoxycarbonyl;

each R⁶ is independently hydrogen or alkyl;

R⁷, R^7a, R^7b, and R^7c are independently hydrogen or alkyl; and

R is hydrogen, alkyl, or hydroxyalkyl.

2. The compound of claim 1 according to one of the formulae,

or

3. The compound of claim 1 according to one of the formulae,

The compound of claim 1 according to one of the formulae,

6. The compound of claim 1 according to one of the formulae,

7. The compound of claim 1 according to one of the formulae,

8. The compound of any one of claims 1 7, wherein R¹ is hydrogen. 9. The compound of any one of claims 1 7, wherein R¹ is -P(0)(OR⁶)₂, wherein each R⁶ is independently alkyl.

10. The compound of any one of claims 1 7, wherein R¹ is hydrogen or -P(0)(OH)₂. 1 1. The compound of any one of claims 1 10, wherein R² and R^2a are independently cyano, halo, or alkyl.

12. The compound of any one of claims 1 10, wherein R² and R^2a are independently halo or alkyl.

13. The compound of any one of claims 1 ■10, wherein R² is chloro or fluoro and R^2a is chloro or fluoro.

14. The compound of any one of claims 1-13, wherein each R³ is independently alkyl, alkoxy, or alkylamino.

15. The compound of any one of claims 1-13, wherein each R³ is independently alkoxy or alkylamino.

16. The compound of any one of claims 1-13, wherein each R³ is isopropoxy,

isopropylamino, or isobutyl.

17. The compound of any one of claims 1-16, wherein R⁴ is alkyl, cyano, halo.

18. The compound of any one of claims 1-16, wherein R⁴ is methyl, cyano, chloro.

19. The compound of any one of claims 1-18, wherein R⁵ is hydrogen and R^5a is hydrogen, alkyl, alkylcarbonyl, or alkoxycarbonyl.

20. The compound of any one of claims 1-8, wherein R⁵ is hydrogen; R^5a is hydrogen;

21. The compound of any one of claims 1 -20, wherein n, when present, is 1.

22. The compound of any one of claims 1 -21 , wherein R⁷ and R^7a are each hydrogen; R^7b and R^7c are independently hydrogen or alkyl; and R⁸ is hydrogen, alkyl, or hydroxyalkyl.

23. The compound of any one of claims 1-21, wherein R⁷, R^7a, R^7b and R⁸ are each hydrogen; and R^7c is hydrogen or alkyl.

24. The compound of claim 1 optionally as a pharmaceutically acceptable salt thereof,

selected from compounds numbers 1-35, as numbered in Tables 1 and 2.

25. A pharmaceutical composition which comprises a compound of any of claims 1-24 or a single stereoisomer or a mixture of isomers thereof, additionally optionally as a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

26. A method for treating a disease, disorder, or syndrome which method comprises

administering to a patient a therapeutically effective amount of a compound, or a single stereoisomer or a mixture of isomers thereof, of any of claims 1-24 optionally as a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 25.

27. The method of claim 26 where the disease is an autoimmune disease.

28. The method of claim 27 where the autoimmune disease is multiple sclerosis, psoriasis, inflammatory bowel disease, graft-versus-host disease, autoimmune-induced inflammation, or rheumatoid arthritis.

29. The method of claim 26 where the disease is osteoporosis.