WO2008091967A1

WO2008091967A1 - Chemical compounds

Info

Publication number: WO2008091967A1
Application number: PCT/US2008/051832
Authority: WO
Inventors: Hongfeng Deng; Ghotas Evindar; Gang Yao
Original assignee: SmithKline Beecham Corp
Current assignee: SmithKline Beecham Corp
Priority date: 2007-01-26
Filing date: 2008-01-24
Publication date: 2008-07-31
Anticipated expiration: 2009-07-26

Abstract

The invention provides compounds Formula (I), their preparation, and their use as pharmaceutically active immunosuppressive agents for the treatment of autoimmune disorders, organ transplant rejection, disorders associated with an activated immune system, as well as other disorders modulated by lymphopenia or SlP receptors.

Description

Attorney Docket No. PPI-304-1

CHEMICAL COMPOUNDS

BACKGROUND OF THE INVENTION

The sphingosine-1 -phosphate (SlP) receptors 1-5 constitute a family of seven transmembrane G-protein coupled receptors. These receptors, referred to as SlPl to S1P5, are activated via binding by sphingosine-1 -phosphate, which is produced by the sphingosine kinase-catalyzed phosphorylation of sphingosine. SlP receptors are cell surface receptors involved in a variety of cellular processes, including cell proliferation and differentiation, cell survival, cell invasion, lymphocyte trafficking, and cell migration. Sphingosine-1 -phosphate is found in plasma and a variety of other tissues, and exerts autocrine and paracrine effects, including regulating the secretion of growth factors.

Administration of SlP to an animal results in sequestration of lymphocytes into the lymph nodes and Peyers patches without causing lymphocyte depletion. This activity, which is of potential utility in treating diseases or conditions associated with inappropriate immune response, including transplant rejection, autoimmune diseases, as well as other disorders modulated by lymphocyte trafficking, is believed to proceed via activation of the SlPl receptor. Administration of SlP in vivo has been shown to cause hypotension and bradycardia, which are believed to be due to signaling through one or more of the other SlP receptors, i.e. S1P2 to S1P5. Accordingly, there is a need for compounds which are potent and selective agonists of the SlP-I receptor.

SUMMARY OF THE INVENTION

These and other needs are met by the present invention which is directed to a compound of formula I

or a pharmaceutically acceptable salt thereof, wherein: Attorney Docket No. PPI-304-1

Ri is alkyl, alkoxy, oxaalkyl, thiaalkyl, azaalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl group, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted cycloalkylalkyl, or substituted or unsubstituted heterocycloalkylalkyl;

is an optionally substituted aryl or heteroaryl group, wherein

".^/vw " indicate points of attachment and X1-X5 are each independently C, C-R' N, N-R", S, or O, wherein R' is hydrogen, halo, or alkyl and R" is hydrogen or alkyl;

R2 is hydrogen, halogen, cyano, straight chain or branched Ci-Cβ-alkyl, straight chain or branched Ci-Cβ-alkoxy, straight chain or branched halo-Ci-C₆-alkyl (e.g., trifluoromethyl), straight chain or branched halo-Ci-Cβ-alkoxy, Ci-C₆-alkoxy-Ci-C6-alkyl, hydroxyl-Ci-Cβ- alkyl, carboxy-Ci-Ce-alkyl, d-C₆-alkyl-SO₂ Or N(R)R', where R and R' are each independently hydrogen, straight chain or branched Ci-C₆-alkyl, straight chain or branched Ci-Cβ-alkoxy, straight chain or branched halo-Ci-Cβ-alkyl, straight chain or branched halo- Ci-Cβ-alkoxy, Ci-C₆-alkoxy-Ci-C₆-alkyl, hydroxyl-Ci-Ce-alkyl, carboxy-Ci-Ce-alkyl or Ci- C₆-alkyl-SO2, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; and z is an integer from 1 to 3;

Q is -CH₂NR-, -CH₂NR(CO)-, -NH(CO)-, -(CO)NH-, -(CO)-, -0-, -S-, -SO-, -SO₂-, -NRSO₂-, -SO₂-NR-, aryl or heteroaryl, wherein R is hydrogen or straight chain or branched Ci-C_δ-alkyl;

R₃ is -OH, -CO₂R⁹, -CH₂=CH(CO)OR⁹, -OPO₂R¹⁰R¹¹, -OPO₃R¹⁰R¹¹, - CH₂PO₃R¹⁰R¹¹, -OPO₂(S)R¹⁰R¹¹ Or -C(Y)(X)PO₃R¹⁰R¹¹, wherein X is hydroxyl or halide; Y is H or halide; R⁹ is H, straight chain or branched Ci-Cβ-alkyl, or a substituted or unsubstituted aryl group; and

R¹⁰ and R¹¹ are each independently H, straight chain or branched Ci-Cβ-alkyl, a substituted or unsubstituted aryl group or selected from the group consisting of: Attorney Docket No. PPI-304-1

R₄ is H, Ci-Ce-alkyl, hydroxy-Ci-C₆-alkyl, aryl, or heteroaryl;

R_5a and R_Jb each independently selected from H, straight chain or branched Ci-Cβ- alkyl, or C3-C6-cycloalkyl; taken together Rs₃ and Rs_b may form a substituted or unsubstituted C2-C8 fused carbocyclic ring or substituted or unsubstituted C2-Q0 fused heterocyclic rings, which may contain one or more heteroatoms and may be saturated or unsaturated; and m and n are each independently 0, 1, 2, or 3.

The invention also provides a compound which is:

Attorney Docket No. PPI-304-1

Attorney Docket No. PPI-304-1

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as well as pharmaceutically acceptable salts, phosphate derivatives, phosphate mimics, or phosphate precursor analogs thereof.

The invention is also directed to a method of treating an autoimmune disorder, comprising, administering to a subject in need thereof a pharmaceutically acceptable amount of a compound of the invention.

The invention is also directed to a method treating transplant rejection comprising, administering to a subject in need thereof a pharmaceutically acceptable amount of a compound of the invention. Attorney Docket No. PPI-304-1

The invention is also directed to a pharmaceutical composition, comprising a compound of the invention admixed with a pharmaceutically acceptable carrier.

The invention is also directed to a process for making a compound of the invention as provided herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions The following definitions are used, unless otherwise described.

"Halogen" or "halo" means fluoro (F), chloro (Cl), bromo (Br), or iodo (I).

The term "hydrocarbon" used alone or as a suffix or prefix, refers to any structure comprising only carbon and hydrogen atoms up to 14 carbon atoms.

The term "hydrocarbon radical" or "hydrocarbyl" used alone or as a suffix or prefix, refers to any structure as a result of removing one or more hydrogens from a hydrocarbon.

The term "alkyP used alone or as a suffix or prefix, refers to monovalent straight or branched chain hydrocarbon radicals comprising 1 to about 12 carbon atoms.

The term "alkylene" used alone or as suffix or prefix, refers to divalent straight or branched chain hydrocarbon radicals comprising 1 to about 12 carbon atoms, which serves to links two structures together.

The term "cycloalkyl" used alone or as suffix or prefix, refers to a saturated or partially unsaturated monovalent ring-containing hydrocarbon radical comprising at least 3 up to about 12 carbon atoms.

The term "aryl" used alone or as suffix or prefix, refers to a monovalent hydrocarbon radical having one or more polyunsaturated carbon rings having aromatic character, and comprising 5 up to about 14 carbon atoms.

The term "heterocycle" used alone or as a suffix or prefix, refers to a ring-containing structure or molecule having one or more multivalent heteroatoms, independently selected from N, O and S, as a part of the ring structure and including at least 3 and up to about 20 atoms in the ring(s). Heterocycle may be saturated or unsaturated, containing one or more double bonds, and heterocycle may contain more than one ring. When a heterocycle contains more than one ring, the rings may be fused or unfused. Fused rings generally refer to at least two rings share two atoms therebetween. Heterocycle may have aromatic character or may not have aromatic character. Attorney Docket No. PPI-304-1

The terms "heterocyclic group", "heterocyclic moiety", "heterocyclic", or "heterocyclo" used alone or as a suffix or prefix, refers to a radical derived from a heterocycle by removing one or more hydrogens therefrom.

The term "heterocyclyl" used alone or as a suffix or prefix, refers a monovalent radical derived from a heterocycle by removing one hydrogen therefrom.

The term "heteroaryl" used alone or as a suffix or prefix, refers to a heterocyclyl having aromatic character.

Heterocycle includes, for example, monocyclic heterocycles such as: aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane 2,3-dihydrofuran, 2,5-dihydrofuran tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydro-pyridine, piperazine, morpholine, thiomorpholine, pyran, thiopyran, 2,3-dihydropyran, tetrahydropyran, 1 ,4-dihydropyridine, 1 ,4-dioxane, 1 ,3-dioxane, dioxane, homopiperidine, 2,3,4,7-tetrahydro-lH-azepine homopiperazine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxeρin, and hexamethylene oxide. In addition, heterocycle includes aromatic heterocycles (heteroaryl groups), for example, pyridine, pyrazine, pyrimidine, pyridazine, thiophene, furan, furazan, pyrrole, imidazole, thiazole, oxazole, pyrazole, isothiazole, isoxazole, 1 ,2,3-triazole, tetrazole, 1,2,3- thiadiazole, 1,2,3-oxadiazole, 1 ,2,4-triazole, 1,2,4-thiadiazole, 1 ,2,4-oxadiazole, 1 ,3,4- triazole, 1,3,4-thiadiazole, and 1 ,3,4-oxadiazole. Additionally, heterocycle encompass polycyclic heterocycles, for example, indole, indoline, isoindoline, quinoline, tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline, 1,4-benzodioxan, coumarin, dihydrocoumarin, benzo furan, 2,3-dihydrobenzo furan, isobenzo furan, chromene, chroman, isochroman, xanthene, phenoxathiin, thianthrene, indolizine, isoindole, indazole, purine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, phenanthridine, perimidine, phenanthroline, phenazine, phenothiazine, phenoxazine, 1,2-benzisoxazole, benzothiophene, benzoxazole, benzthiazole, benzimidazole, benztriazole, thioxanthine, carbazole, carboline, acridine, pyrolizidine, and quinolizidine.

In addition to the polycyclic heterocycles described above, heterocycle includes polycyclic heterocycles wherein the ring fusion between two or more rings includes more than one bond common to both rings and more than two atoms common to both rings.

Examples of such bridged heterocycles include quinuclidine, diazabicyclo[2.2.1]heptane and 7-oxabicyclo[2.2.1 Jheptane.

Heterocyclyl includes, for example, monocyclic heterocyclyls, such as: aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, Attorney Docket No. PPI-304-1

pyrazolidinyl, pyrazolinyl, dioxolanyl, sulfolanyl, 2,3-dihydrofuranyl, 2,5-dihydrofiiranyl, tetrahydrofliranyl, thiophanyl, piperidinyl, 1 ,2,3,6-tetrahydro-pyridinyl, piperazinyl, moφholinyl, thiomorpholinyl, pyranyl, thiopyranyl, 2,3-dihydropyranyl, tetrahydropyranyl, 1,4-dihydropyridinyl, 1 ,4-dioxanyl, 1 ,3-dioxanyl, dioxanyl, homopiperidinyl, 2,3,4,7- tetrahydro-lH-azepinyl, homopiperazinyl, 1 ,3-dfoxepanyl, 4,7-dihydro-l,3-dioxepinyl, and hexamethylene oxidyl.

In addition, heterocyclyl includes aromatic heterocyclyls or heteroaryl, for example, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl, furyl, furazanyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1 ,2,3-triazolyl, tetrazolyl, 1 ,2,3- thiadiazolyl, 1,2,3-oxadiazolyl, 1 ,2,4-triazolyl, 1,2,4-thiadiazolyl, 1 ,2,4-oxadiazolyl, 1,3,4- triazolyl, 1 ,3,4-thiadiazolyl, and 1 ,3,4 oxadiazolyl.

Additionally, heterocyclyl encompasses polycyclic heterocyclyls (including both aromatic or non-aromatic), for example, indolyl, indolinyl, isoindolinyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, 1 ,4-benzodioxanyl, coumarinyl, dihydrocoumarinyl, benzofuranyl, 2,3-dihydrobenzofuranyl, isobenzofuranyl, chromenyl, chromanyl, isochromanyl, xanthenyl, phenoxathiinyl, thianthrenyl, indolizinyl, isoindolyl, indazolyl, purinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, phenanthridinyl, perimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, 1 ,2-benzisoxazolyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrolizidinyl, and quinolizidinyl.

In addition to the polycyclic heterocyclyls described above, heterocyclyl includes polycyclic heterocyclyls wherein the ring fusion between two or more rings includes more than one bond common to both rings and more than two atoms common to both rings. Examples of such bridged heterocycles include quinuclidinyl, diazabicyclo[2.2.1]heptyl; and 7-oxabicyclo[2.2.1]heptyl.

The term "six-membered" used as prefix refers to a group having a ring that contains six ring atoms.

The term "five-membered" used as prefix refers to a group having a ring that contains five ring atoms.

A five-membered heteroaryl ring is a heteroaryl with a ring having five ring atoms wherein 1 , 2 or 3 ring atoms are independently selected from N, O and S. Exemplary five- membered ring heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1 ,2,3-triazolyl, tetrazolyl, 1 ,2,3-thiadiazolyl, 1 ,2,3- Attorney Docket No. PPI-304-1

oxadiazolyl, 1 ,2,4-triazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,3,4-triazolyl, 1,3,4- thiadiazolyl, and 1,3,4- oxadiazolyl.

A six-membered ring heteroaryl is a heteroaryl with a ring having six ring atoms wherein 1 , 2 or 3 ring atoms are independently selected from N, O and S. Exemplary six- membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.

The term "cycloalkylalkyl" refers to an alkyl group substituted with a cycloalkyl group.

The term "heterocycloalkylalkyl" refers to an alkyl group substituted with an heterocycloalkyl group. The term "aralkyl" refers to an alkyl group substituted with an aryl group.

The term "heteroaralkyl" refers to an alkyl group substituted with an heteroaryl group.

Unless otherwise specified, the term "substituted", when used as a prefix, refers to a structure, molecule or group, wherein one or more hydrogens are replaced with one or more alkyl groups, or one or more chemical groups containing one or more heteroatoms selected from N, O, S, F, Cl, Br, I, and P. Exemplary chemical groups containing one or more heteroatoms include heterocyclyl, -NO₂, -O-alkyl, halo, -CF₃, -CO₂H, -CO₂R, -NH₂, -SH, - NHR, -NR₂, -SR, -SO₃H, -SO₂R, -S(O)R, -CN, -OH, -C(O)NR₂, -NRC(O)R, oxo (=O), imino (=NR), thio (=S), and oximino (=N-OR), wherein each "R" is alkyl as defined above. For example, substituted phenyl may refer to nitrophenyl, pyridylphenyl, methoxyphenyl, chlorophenyl, aminophenyl, an so on, wherein the nitro, pyridyl, methoxy, chloro, and amino groups may replace any suitable hvdrogen on the phenyl ring.

The term "alkoxy" used alone or as a suffix or prefix, refers to radicals of the general -O-alkyl, Exemplary alkoxy groups includes methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, isobutoxy, cyclopropylnethoxy, allyloxy, and propargyloxy. The term "amine" or "amino" used alone or as a suffix or prefix, refers -NH₂.

The term "alkylamino" used alone or as a suffix or prefix, refers -NH(alkyl). The term "dialkylamino" used alone or as a suffix or prefix, refers -NH(alkyl)₂.

"Acyl" used alone, as a prefix or suffix, means -C(O)-R, wherein R hydrogen, hydroxyl, amino, alkylamino, dialkylamino, or alkoxy, any of which may be substituted as provided by the defiontion of "substituted" given above. Acyl groups include, for example, acetyl, propionyl, benzoyl, phenyl acetyl, carboethoxy, and dimethylcarbamoyl.

Some of the compounds in the present invention may exist as stereoisomers, including enantiomers, diastereomers, and geometric isomers. All of these forms, including (R), (S), Attorney Docket No. PPI-304-1

epimers, diastereomers, cis, trans, syn, anti, solvates (including hydrates), tautomers, and mixtures thereof, are contemplated in the compounds of the present invention.

The invention also relates to salts of the compounds of the invention and, in particular, to pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. The salts can be, for example, salts with a suitable acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, benzoic acid, pamoic acid, alginic acid, methanesulfonic acid, naphthalenesulfonic acid, and the like. Also included are salts of cations such as ammonium, sodium, potassium, lithium, zinc, copper, barium, bismuth, calcium, and the like; or organic cations such as tetralkylammonium and trialkylammonium cations. Combinations of the above salts are also useful. Salts of other acids and/or cations are also included, such as salts with trifluoroacetic acid, chloroacetic acid, and trichloroacetic acid.

The invention also includes different crystal forms, hydrates, and solvates of the compounds of the invention.

Invention Compounds

We turn now to a compound of formula I.

A specific value for Ri is hydrogen. Another specific value for Ri is heptyl. Another specifc value for Ri is hexyl. Another specific value for Ri is pentyl. Another specific value

for Ri is-"' ~^^ ^"1^ . Another specific value for Ri is phenyl. Another specific value for Ri is cyclopentyl. Another specific value for Ri is cyclohexyl. Another specific value for Ri is a cyclopentyl or cyclohexyl group, bearing an alkyl substitutent, such as a propyl group, Attorney Docket No. PPI-304-1 although other alkyl substituents may be employed. Another specific value for Ri is

. Another specific value for Ri is . Another specific value for

Ri is benzyl. Another specific value for R] is

A specific value for

. Another

specific value for

A specific value for

. Another specific value for

is . Another specific value for ² is

Another specific value for

² is . Another specific value for

is . Another specific value for ² is ^Me .

Another specific value for

R₂ : IS Attorney Docket No. PPI-304-1

A specific value for Q is

^s . Another specific value for Q is

. Another specific value for Q is -NH-(C=O)- in either bonding orientation. Another specific value for Q is -NMe-(C=O)- in either bonding orientation.

A specific value for R₃ is -OH. Another specific value for R3 is OPO2H2. Another specific value for R3 is OPO2Me₂. Another specific value for R₃ is OPO₂Et₂. Another specific value for R₃ is -CO₂H. Another specific value for R₃ is -CO₂Me or -CO₂Et.

A specific value for R₄ is hydrogen. Another specific value for R₄ is methyl. Another specific value for R₄ is hydroxymethyl.

Specific values for R_5a and Rs_b independently include hydrogen or alkyl.

The compounds of the present invention include R₂ which is a selectivity enhancing moiety. The term "selectivity enhancing moiety (SEM)" is defined in United States Application Ser. No. 11/349069 filed on February 6, 2006 which is assigned to the assignee of the present application, the contents of which are incorporated herein by reference, refers to one or more moieties that provide an enhancement in the selectivity of the compound to which they are attached for the SlPl receptor, as compared to the compound not containing the moiety or moieties. The SEM confers selectivity to the compound to which it is attached for the SlPl receptor as compared to, for example, the S 1P2 to S1P5 receptors. The enhancement conferred to a compound by the SEM may be measured by, for example, determining the binding specificity of a compound for the S lPl receptor and one or more of the other S 1 P receptors wherein enhancement conferred to a compound by the SEM may be in the form of increased potency. The SEM of the present application is defined in one embodiment as for R₂.

More specifically, the SEM is a halo-substituted alkyl group such as CF₃, CF₂CF₃, CF₂CF₂CF₃, CFHCF₃, CH₂CF₃, CH₂CH₂CF₃, CHCl₂, or CH₂Cl.

In certain embodiments, the SEM may possess a selectivity enhancing orientation (SEO). The term "selectivity enhancing orientation" or "SEO," is defined in United States Application Ser. No. 11/349069 filed on February 6, 2006 which is assigned to the assignee of the present application, the contents of which are incorporated herein by reference and as Attorney Docket No. PPI-304-1

used herein refers to the relative selectivity enhancement of a compound based on the orientation of the SEM as well as the additional substitutents on the ring, either alone or in combination with each other. In particular, the SEO may result from the orientation of the SEM on the ring to which it is attached, in relation to any other ring and/or moiety attached to

the same ring. In one embodiment, the SEM on ^"^^ is in the ortho position relative to X3 in Formula I. In another specific embodiment, the SEM is in the meta position relative to X₃.

Thus, a specific value for R2 is trifluoromethyl. Another specific value for R2 is fluoro. Another specific value for R₂ is chloro. Another specific value for R2 is bromo. Another specific value for R₂ is cyano. Another specific value for R₂ is methyl.

A specific group of compounds of the invention are compounds of formula I which are of formula IA.

Another specific group of compounds of the invention are compounds of formula I which are compounds of formula IB.

Another specific group of compounds of the invention are compounds of formula I which are compounds of formula IC. Attorney Docket No. PPI-304-1

Biological Activity of Invention Compounds

Lymphopenia Assay

Several of the compounds described herein were evaluated for the ability to induce lymphopenia in mice. Male C57B1/6 mice were divided into groups of three. A control group received the 3% BSA vehicle only. The other groups received a single dose of either a specified dose of test compound in vehicle administered orally (PO) and intravenously (IV). After 6 hours, the mice were anesthesized with isoflurane and approximately 250 μL of blood was removed from the retroorbital sinus and collected in an EDTA microtainer, mixed with an anticoagulant and placed on a tilt table until complete blood count (CBC) analysis. Oral administration (10 mg/K) of these compounds induced increased lymphopenia versus the vehicle.

Binding to SlPl or S1P3 Receptors

In certain embodiments, the compounds of the invention selective for the SlPl receptor as compared to one or more of the other SlP receptors. For example, one set of compounds includes compounds which are selective for the SlPl receptor relative to the S1P3 receptor. Compounds selective for the SlPl receptor can be agonists of the SlPl receptor, significantly weaker agonists of one or more other receptors and/or antagonists of one or more other receptors. A compound is "selective" for the S lPl receptor relative to a second receptor, if the EC50 of the compound for the second receptor is at least two-fold greater than the EC50 for the SlPl receptor. The EC50 of a compound is determined using the ³⁵S-GTPyS binding assay, as described in WO 03/061567, the entire contents of which are incorporated herein by reference. Additionally or alternatively, a compound is "selective" for the Sl Pl receptor relative to a second receptor, if the IC₅₀ of the compound for the second receptor is at least two-fold greater than the ICs₀ for the SlPl receptor. The IC₅₀ of a Attorney Docket No. PPI-304-1

compound is determined using the [³³P]sphingosine 1 -phosphate binding assay, as described in Davis, M.D. et ai, Sphingosine 1 -Phosphate Analogs as Receptor Antagonists. J. Biol. Chem. (2005) 280:9833-9841 , the entire contents of which are incorporated herein by this reference. The terms "agonist" or "SlPl receptor agonist" as used herein include the compounds described herein which bind to and/or agonize the SlPl receptor. In one embodiment, the SlP receptor agonists have an IC50 for the SlPl receptor of about 100 nM - 0.25 nM, about 50 nM - 0.25 nM, about 25 nM - 0.5 nM, about 100 nM or less, about 75 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 1 nM or less, about 0.5 nM or less, or about 0.25 nM or less. The compounds' IC₅₀ for the SlPl receptor can be measured using the binding assays described in Example 13 or those described in WO 03/061567. Compounds of the invention generally had an IC50 in the range of 100 pM (picomolar) to 100 M.

Ranges intermediate to the above recited values are also intended to be part of this invention. For example, ranges using a combination of any of the above recited values as upper and/or lower limits are intended to be included.

In a further embodiment, the SlP receptor agonist has an IC₅0 value for the S1P3 receptor of about 10 nM - 10,000 nM, about 100 nM - 5000 nM, about 100 nM - 3000 nM, about 10 nM or greater, about 20 nM or greater, about 40 nM or greater, about 50 nM or greater, about 75 nM or greater, or about 100 nM or greater. In another embodiment, the Sl P compound of the invention binds the S1P3 receptor with an IC₅₀ of 1000 nM or greater, 2000 nM or greater, 3000 nM or greater, 5000 nM or greater, 10,000 nM or greater. The IC50 for of S1P3 receptor can be measured using the binding assays described herein or those described in WO 03/061567, the entire contents of which are incorporated herein by reference.

In addition, it should be understood that the ranges intermediate to the above recited values are also intended to be part of this invention. For example, ranges using a combination of any of the above recited values as upper and/or lower limits are intended to be included. In yet another embodiment, the S lP receptor agonists described herein have an IC₅₀ value for the SlPl receptor that is about 5-fold lower, about 10-fold lower, about 20-fold lower, about 50-fold lower, about 100-fold lower, about 200-fold lower, about 500-fold lower or about 1000-fold lower than their IC₅₀ value for the S1 P3 receptor. Attorney Docket No. PPI-304-1

The ability of several of the compounds described herein to bind to the SlPl or S1P3 receptor was also tested as follows.

For the membrane preparation, plasmid DNA was transfected into HEK 293 T cells using the FuGENE 6 transfection protocol (publicly available from Roche). Briefly, subconfluent monolayers of HEK 293 T cells were transfected with the DNA mixture containing FuGENE 6 (using a 1 :3 ratio). The dishes containing the cells were then placed in a tissue culture incubator (5% CO₂, 37°C). The cells were harvested 48 hours after addition of the DNA by scraping in HME buffer (in mM: 20 HEPES, 5 MgCl₂, 1 EDTA, pH 7.4, 1 itiM PMSF) containing 10% sucrose on ice, and disrupted using a Dounce homogenizer. After centrifugation at 800 g, the supernatant was diluted with HME without sucrose and centrifuged at 17,000 g for 1 hour. This crude membrane pellet was resuspended in HME with sucrose, aliquoted, and snap-frozen by immersion in liquid nitrogen. The membranes were stored at -70 ⁰C. Protein concentration was determined spectroscopically by Bradford protein assay.

For the binding assay, [³³P] sphingosine 1 -phosphate (obtained from American Radiolabeled Chemicals, Inc) was added to membranes in 200 μl in 96-well plates with assay concentrations of 2.5 pM [³³P]sphingosine 1 -phosphate, 4 mg/ml BSA, 50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, and 5 μg of protein. Binding was performed for 60 minutes at room temperature with gentle mixing and terminated by collecting the membranes onto GF/B filter plates. After drying the filter plates for 10 minutes, 50 μl of Microscint40 was added to each well, and filter-bound radionuclide was measured on a Packard Top Count. Nonspecific binding was defined as the amount of radioactivity remaining in the presence of excess of unlabeled S lP.

For example,

had an IC50 of value of SlPl = 1.9 nM; S1P3 = >10 000 nM; and Selectivity >5000

In one embodiment, the compounds of the invention do not include the compounds described in WO 06/020951 A2, WO 05/041899A2, WO 04/010949A2, WO 04/024673 Al Attorney Docket No. PPI-304-1

and WO 02/064616, and USSN 11/349069, filed February 2, 2006; the entire contents of each of which are hereby incorporated herein by reference.

Methods of Using Invention Compounds In one embodiment, the invention relates to a method for treating a subject suffering from a sphingosine 1 -phosphate associated disorder, comprising administering to a subject an effective amount of a compound of the invention; that is, a compound of formula I or compounds otherwise described herein, such that the subject is treated for a sphingosine 1- phosphate associated disorder. The term "sphingosine 1 -phosphate associated disorder" includes disorders, diseases or conditions which are associated with or caused by a misregulation in SlP receptor function and/or signaling or SlP receptor ligand function. The term also includes diseases, disorders or conditions which can be treated by administering to a subject an effective amount of a sphingosine 1 -phosphate receptor agonist. Such disorders include disorders that are associated with an inappropriate immune response and conditions associated with an overactive immune response, e.g., autoimmune diseases.

"Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent such as a compound of formula I to a subject who has a shingosine 1 -phosphate associated disorder as described herein, with the purpose to cure, heal, alleviate, delay, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term "treatment" or "treating" is also used herein in the context of administering agents prophylactically.

In some embodiments, the efficacy of the compounds of the present invention can be measured by comparing a value, level, feature, characteristic, property, etc. to a "suitable control". A "suitable control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" is a value, level, feature, characteristic, property, etc. determined prior to administering a composition of the present invention. For example, the immune response, etc. can be determined prior to introducing a compound of the invention into a cell or subject. In another embodiment, a "suitable control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" is a predefined value, level, feature, characteristic, property, etc. For example a "suitable control" can be a pre-defined level of binding to a specified Sl P receptor. Attorney Docket No. PPI-304-1

An additional embodiment of the invention pertains to a method for treating a subject suffering from a sphingosine 1 -phosphate associated disorder, comprising administering to a subject a compound, such that the subject is treated for a sphingosine 1 -phosphate associated disorder by a compound of the invention; that is, a compound of formulae I or compounds otherwise described herein.

The present invention is also directed to a method of selectively treating a sphingosine 1 -phosphate associated disorder, comprising administering to a subject an effective amount of a compound of the invention, e.g., compounds of any of Formulae I or compounds otherwise described herein, such that the subject is selectively treated for a sphingosine 1 -phosphate associated disorder. In certain embodiments, the sphingosine 1 -phosphate associated disorder is a sphingosine l-phosphate-(l) associated disorder. In a particular embodiment, the sphingosine l-ρhosρhate-(l) associated disorder is selectively treated as compared with a sphingosine l-phosphate-(3) associated disorder.

In another embodiment, the present invention provides a method of treating a condition associated with an activated immune system. Such diseases or disorders include rejection of transplanted organs, tissue or cells; graft-versus-host diseases brought about by transplantation; autoimmune syndromes including rheumatoid arthritis; systemic lupus erythematosus; antiphospholipid syndrome; Hashimoto's thyroiditis; lymphocytic thyroiditis; multiple sclerosis; myasthenia gravis; type I diabetes; uveitis; episcleritis; scleritis; Kawasaki's disease, uveo-retinitis; posterior uveitis; uveitis associated with Behcet's disease; uveomeningitis syndrome; allergic encephalomyelitis; chronic allograft vasculopathy; postinfectious autoimmune diseases including rheumatic fever and post-infectious glomerulonephritis; inflammatory and hyperproliferative skin diseases; psoriasis; psoriatic arthritis; atopic dermatitis; myopathy; myositis; osteomyelitis; contact dermatitis; eczematous dermatitis; seborrhoeic dermatitis; lichen plan us; pemph igus; bullous pemphigoid; epidermolysis bullosa; urticaria; angioedema; vasculitis; erythema; cutaneous eosinophilia; acne; scleroderma; alopecia areata; keratoconjunctivitis; vernal conjunctivitis; keratitis; herpetic keratitis; dystrophia epithelialis corneas; corneal leukoma; ocular pemphigus; Mooren's ulcer; ulcerative keratitis; scleritis; Graves' ophthalmopathy; Vogt-Koyanagi- Harada syndrome; sarcoidosis; pollen allergies; reversible obstructive airway disease; bronchial asthma; allergic asthma; intrinsic asthma; extrinsic asthma; dust asthma; chronic or inveterate asthma; late asthma and airway hyper-responsiveness; bronchiolitis; bronchitis; endometriosis; orchitis; gastric ulcers; ischemic bowel diseases; inflammatory bowel diseases; necrotizing enterocolitis; intestinal lesions associated with thermal burns; coeiiac Attorney Docket No. PPI-304-1

disease; proctitis; eosinophilic gastroenteritis; mastocytosis; Crohn's disease; ulcerative colitis; vascular damage caused by ischemic diseases and thrombosis; atherosclerosis; fatty heart; myocarditis; cardiac infarction; aortitis syndrome; cachexia due to viral disease; vascular thrombosis; migraine; rhinitis; eczema; interstitial nephritis; IgA-induced nephropathy; Goodpasture's syndrome; hemolytic- uremic syndrome; diabetic nephropathy; glomerulosclerosis; glomerulonephritis; tubulointerstitial nephritis; interstitial cystitis; multiple myositis; Guillain-Barre syndrome; Meniere's disease; polyneuritis; multiple neuritis; myelitis; mononeuritis; radiculopathy; hyperthyroidism; Basedow's disease; thyrotoxicosis; pure red cell aplasia; aplastic anemia; hypoplastic anemia; idiopathic thrombocytopenic purpura; autoimmune hemolytic anemia; autoimmune thrombocytopenia; agranulocytosis; pernicious anemia; megaloblastic anemia; anerythroplasia; osteoporosis; fibroid lung; idiopathic interstitial pneumonia; dermatomyositis; leukoderma vulgaris; ichthyosis vulgaris; photoallergic sensitivity; cutaneous T cell lymphoma; polyarteritis nodosa; Huntington's chorea; Sydenham's chorea; myocardosis; myocarditis; scleroderma; Wegener's granuloma; Sjogren's syndrome; adiposis; eosinophilic fascitis; lesions of gingiva, periodontium, alveolar bone, substantia ossea dentis; male pattern alopecia or alopecia senilis; muscular dystrophy; pyoderma; Sezary's syndrome; hypophysitis; chronic adrenal insufficiency; Addison's disease; ischemia-reperfusion injury of organs which occurs upon preservation; endotoxin shock; pseudomembranous colitis; colitis caused by drug or radiation; ischemic acute renal insufficiency; chronic renal insufficiency; lung solid cancer; malignancy of lymphoid origin; acute or chronic lymphocytic leukemias; lymphoma; psoriasis; pulmonary emphysema; cataracts; siderosis; retinitis pigmentosa; senile macular degeneration; vitreal scarring; corneal alkali burn; dermatitis erythema; ballous dermatitis; cement dermatitis; gingivitis; periodontitis; sepsis; pancreatitis; peripheral artery disease; carcinogenesis; solid cancer tumors; metastasis of carcinoma; hypobaropathy; autoimmune hepatitis; primary biliary cirrhosis; sclerosing cholangitis; partial liver resection; acute liver necrosis; cirrhosis; alcoholic cirrhosis; hepatic failure; fulminant hepatic failure; late-onset hepatic failure; "acute-on-chronic" liver failure.

As used herein, the term "subject" includes warm-blooded animals, e.g. , mammals, including humans, cats, dogs, horses, bears, lions, tigers, ferrets, rabbits, mice, cows, sheep, pigs, etc. In a particular embodiment, the subject is a primate. In a specific embodiment, the primate is a human.

As used herein, the term "administering" to a subject includes dispensing, delivering or applying a compound of the invention in a pharmaceutical formulation (as described Attorney Docket No. PPI-304-1

herein), to a subject by any suitable route for delivery of the compound to the desired location in the subject, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, topical delivery, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.

As used herein, the term "effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to treat the condition in a subject. An effective amount of a compound of the invention, as defined herein, may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the compound are outweighed by the therapeutically beneficial effects.

A therapeutically effective amount of a compound of the invention (i.e., an effective dosage) may range from about O.001 to 30 mg/kg body weight, for example, about O.Ol to 25 mg/kg body weight, for example, about 0.1 to 20 mg/kg body weight. It is to be understood that all values and ranges between those listed are intended to be encompassed by the present invention. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound of the invention can include a single treatment or, for example, can include a series of treatments. It will also be appreciated that the effective dosage of the compound used for treatment may increase or decrease over the course of a particular treatment. The methods of the invention further include administering to a subject a therapeutically effective amount of a compound of the invention in combination with another pharmaceutically active compound known to treat the disease or condition, e.g., an immunomodulatory agent or an anti- inflammatory agent. Pharmaceutically active compounds that may be used depend upon the condition to be treated, but include as examples cyclosporin, rapamycin, FK506, methotrexate, etanercept, infliximab, adalimumab, non-steroidal anti-inflammatory agents, eye looxygenase-2 -inhibitors, such as celecoxib and rofecoxib, and corticosteroids. Other suitable compounds can be found in Harrison's Principles of Internal Medicine, Thirteenth Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y., N. Y.; and the Physicians Desk Reference 50th Edition 1997, Oradell New Jersey, Attorney Docket No. PPI-304-1

Medical Economics Co., the complete contents of which are expressly incorporated herein by reference. The compound of the invention and the additional pharmaceutically active compound may be administered to the subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times).

Pharmaceutical Compositions Comprising Invention Compounds

The present invention also provides pharmaceutically acceptable formulations and compositions comprising one or more compounds of the invention; that is, compounds of formula I or compounds otherwise described herein. In certain embodiments, the compound of the invention is present in the formulation in a therapeutically effective amount; that is, an amount effective to treat a sphingosine 1 -phosphate associated disorder.

Accordingly, in one embodiment, the invention pertains to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention; that is, compounds of formula I or compounds otherwise described herein, and a pharmaceutically acceptable carrier.

In another embodiment, the invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention; that is, compounds of formula I or compounds otherwise described herein; and instructions for using the compound to treat a sphingosine 1 -phosphate associated disorder in a subject.

The term "container" includes any receptacle for holding the pharmaceutical composition. For example, in one embodiment, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions can contain information pertaining to the compound's ability to perform its intended function, e.g. , treating, preventing, or reducing a sphingosine 1 -phosphate associated disorder in a subject. Attorney Docket No. PPI-304-1

Another embodiment of the invention relates to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention; that is, a compound of formula I or compounds otherwise described herein, and instructions for using the compound to selectively treat a sphingosine 1 - phosphate associated disorder in a subject.

Such pharmaceutically acceptable formulations typically include one or more compounds of the invention as well as one or more pharmaceutically acceptable carriers and/or excipients. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compounds of the invention, use thereof in the pharmaceutical compositions is contemplated.

Supplementary pharmaceutically active compounds known to treat transplant or autoimmune disease, i.e., immunomodulatory agents and anti- inflammatory agents, as described above, can also be incorporated into the compositions of the invention. Suitable pharmaceutically active compounds that may be used can be found in Harrison's Principles of Internal Medicine.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions, or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor El™ (BASF, Parsippany, NJ.) Attorney Docket No. PPI-304-1

or phosphate buffered saline (PBS). In all cases, the pharmaceutical composition must be sterile and should be fluid to the extent that easy syringability exists. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The 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 dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the compound of the invention in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the compound plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compound of the invention can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also include an enteric coating. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a Attorney Docket No. PPI-304-1

disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds of the invention are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g. , a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the compounds of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. The present pharmaceutical compositions can also be prepared in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1, U.S. Pat. No. 5,455,044 and U.S. Pat. No. 5,576,018, and U.S. Pat. No. 4,883,666, the contents of all of which are incorporated herein by reference.

The compounds of the invention can also be incorporated into pharmaceutical compositions which allow for the sustained delivery of the compounds to a subject for a period of at least several weeks to a month or more. Such formulations are described in published PCT application no. WO 02/74247, incorporated herein by reference.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used Attorney Docket No. PPI-304-1

herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of a compound of the invention calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are dictated by and directly dependent on the unique characteristics of the compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such compounds for the treatment of individuals.

This invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents, patent applications cited throughout this application are incorporated herein by reference. It should be understood that the use of any of the compounds described herein is within the scope of the present invention and is intended to be encompassed by the present invention and is expressly incorporated herein for all purposes.

EXAMPLES General Approach to Synthesis of Biaryl-Thiadiazoles

Synthesis of biaryl-thiazoles is described in Schemes IA and IB. Reaction of allyl alcohol with substituted 4-fluorobenzoic acid 1 afforded the substituted ether-benzoic acid 2 (R₂ = O-allyl). Substituted benzoic acid 2 (R₂ = O-allyl, Cl, Br, I) were coupled with hydrazine to afford benzohydrazide 3. Reaction of benzohydrazide 3 with orthogonally- protected amino acid 4 in the presence of HATU, followed by Lawesson's reagent-mediated cyclization, provided thiadizole 6 in good yield. Removal of the allyl protecting group in 6 (R2 = O-allyl) using Pd(PPh3)4 generated the desired phenol intermediate 7. Reaction of phenol intermediate 7 with triflic anhydride to generated triflate 8. Palladium catalized cross coupling of 8 or 6a (R₂ = Cl, Br, or I) with Zn(CN)₂ afforded nitrile intermediate 9. Nitrile intermediate 9 was then condensed with hydroxylamine to give JV-hydroxybenzimidamide, which upon thermal-mediated coupling with the desired carboxylic acid afforded oxadiazole 10. Removal of the protecting groups provided compound 11. Alternatively, Suzuki cross-coupling of the intermediate triflate 8a or 6a (R₂ = Cl, Br, or I) with various boronic acids or esters, followed by deprotection afforded compound 12. Reaction of the alcohol 11 or 12 with diethyl chlorophosphate followed by deprotection with TMSBr gave the corresponding phosphate. Attorney Docket No. PPI-304-1

Scheme IA

allyl alcohol hydrazine, HATU

KOΕu, THF DIPEA, DCIWDMF

5a 6a

R₂ = O-allyl, Cl, Br, or I

^

R₂ = O-allyl Et₂NH, THF Et₃N, DCM

7a 6a R₂ =CI, Br, or I 8a R₂ = Oπ

Attorney Docket No. PPI-304-1

Scheme IB

protected Lawes son's amino acid reagent

HATU, DIPEA DCM

5b 6b

HCI

Pd(PPh₃)₄

2) Acid, HATU E^{t2NH' THF}

General Approach to the Synthesis of Biaryl-Amides

Synthesis of biaryl-amides is described in Scheme 2. Condensation of the substituted nitrile 1 with hydroxylamine gave the desired N-hydroxybenzimidamide 2, which upon thermal-mediated coupling with the desired carboxylic acid and heat afforded oxadiazole 4. Hydro genation of the nitro group in oxadiazole 4 gave aniline 5. Coupling of aniline 5 with various protected amino acids afforded intermediate 6, which upon deprotection yielded biaryl-amide 7. The desired alcohols 7 were reacted with diethyl chlorophosphate followed by deprotection with TMSBr to generate the corresponding phosphates.

Scheme 2

Attorney Docket No. PPI-304-1

deprotection

General Protocol for the Synthesis of Substituted 4-(Allyloxy)benzoic acid (2)

To a solution of allyl alcohol (1.05 equivalents) in anhydrous THF was added potassium ^-butyloxide (2.05 equivalents). The mixture was heated at 65 ⁰C for 10 minutes then substituted 4-fluorobenzoic acid (1) (1.00 equivalent) in THF was added. The resultant solution was heated at 65 ⁰C for 1 -3 hours. After cooling down to room temperature, the reaction was diluted with ethyl acetate and washed with 10% KHSO4 (Ix), and saturated NaCl (1 time). The organic layer was dried over MgSθ4, filtered, and the solvent was removed in vacuo to afford intermediate 2.

Attorney Docket No. PPI-304-1

4-(AHyloxy)-3-(trifluoromethyl)benzoic acid (2a)

The title compound was prepared from 4-fluoro-3-(trifluoromethyl)benzoic acid (Ia) in >99% (5.65 g) yield. HPLC retention time on a C8(2) column (30 x 3.00 mm, 3 μ) was 2.53 minutes with gradient 20-98% acetonitrile-H₂O (0.1 % trifluoro acetic acid (TFA)) in 4.0 minutes as mobile phase. ¹H NMR (400 MHz, DMSO-d₆) δ 13.10 (br s, IH), 8.15 (dd, IH, J = 8.8 Hz, J= 2.4 Hz ), 8.08 (d, IH, J= 2.4 Hz), 7.35 (d, IH, J= 8.8 Hz), 5.95-6.80 (m, IH), 5.38-5.45 (m, IH), 5.26-5.32 (m, I H), 4.77-4.82 (m, 2H).

General Protocol for the Synthesis of Substituted Benzohydrazide (3)

Benzoic acid 2 (1.0 equivalent) was stirred with HATU (1.1 equivalents) and Diisopropylethyl amine (DIEA (3.0 equivalents) in dichloromethane (DCM)-dimethyl formamide (DMF) (2:1) for 20 minutes. The solution was then added to a solution of hydrazine mono-hydrate (5.0) in DCM-DMF (2:1). The reaction mixture was stirred at room temperature for 1 hour, then diluted with ethyl acetate and washed with 10% NH₄CI (2x) and saturated NaCl (1 time). The organic layer was dried over MgSC^, filtered, and the solvent was removed in vacuo to afford benzohydrazide 3.

4-(Allyloxy)-3-(trifluoromethyl)benzohydrazide (3a)

The title compound was prepared from 4-(allyloxy)-3-(trifluoromethyl)benzoic acid (Ia) in >99% (6.00 g) yield. HPLC retention time on a C8(2) column (30 x 3.00 mm, 3 μ) Attorney Docket No. PPI-304-1

was 1 .79 min with gradient 20-98% acetonitrile-H₂O (0.1% TFA) in 4.0 minutes as mobile phase. MS (ESI, M+H⁺) = 261.09

General Protocol for the Synthesis of Acyl-benzohydrazide (5a)

(Λ)-3-(terf-Butoxycarbonyl)-2,2,4-trimethyloxazolidine-4-carboxylic acid 4 (1.0 equivalents) was stirred with HATU (1.1 equivalents) and DIEA (3.0 equivalents) in DCM- DMF (2:1) for 10 minutes followed by addition of substituted benzohydrazide 3 (1.0 equivalents). The reaction mixture was stirred at room temperature for 1 hour, then diluted with ethyl acetate and washed with 10% NH₄Cl (2 times) and saturated NaCl (1 time). The organic layer was dried over MgSO₄, filtered, and the solvent was removed in vacuo to afford acyl-benzohydrazide 5a.

(R)-tert-Buty\ 4-(2-(4-(aIlyloxy)-3-(trifluoromethyl)benzoyl)hydrazinecarbonyl)-2,2,4- trimcthyloxazolidine-3-carboxylate (5aa)

The title compound was prepared from 4-(allyloxy)-3- (trifluoromethyl)benzohydrazide 3a in >99% (11.51 g) yield. HPLC retention time on a C8(2) column (30 x 3.00 mm, 3 μ) was 2.92 minutes with gradient 20-98% acetonitrile-H₂O (0.1% TFA) in 4.0 minutes as mobile phase. MS (ESI, M+Na⁺) = 524.12 Attorney Docket No. PPI-304-1

General protocol for synthesis of phenyl-thiadiazole (6a)

A solution of acyl-benzohydrazide 5a (1.0 equivalent) in DCM was treated with Lawesson's reagent (1.0 equivalent) at 50 ⁰C overnight. The reaction was cooled down to room temperature and the supernatant was chromatographed on a silica gel column eluting with ethyl acetate in hexanes (0-40%, v/v) to afford phenyl-thiadiazole 6a.

(Λ)-tert-butyl 4-(5-(4-(allyloxy)-3-(trifluoromethyl)phenyl)-l^,4-thiadiazol-2-yl)-2,2,4- trimethyIoxazolidine-3-carboxylate (6a a)

The title compound was prepared from acyl-benzohydrazide 5aa in 88% (8.35 g) yield. HPLC retention time on a C8(2) column (30 x 3.00 mm, 3 μ) was 2.84 minutes with gradient 50-98% acetonitrile-H₂O (0.1% TFA) in 4.0 minutes as mobile phase. MS (ESI, M+H⁺) = 500.03; ¹H NMR (400 MHz, DMSO-d₆) δ 8.13-8.24 (m, 2H), 7.42 (d, IH, J= 8.4 Hz), 5.98-6.10 (m, IH), 5.40-5.50 (m, IH), 5.25-5.34 (m, I H), 4.80-4.84 (m, 2H), 4.10-4.40 (m, 2H), 1.88 (s, 3H), 1.66 (s, 3H), 1.56 (s, 3H), 1.41 (s, 3H), 1.18 (s, 6H).

General protocol for synthesis of phenol (7a)

Attorney Docket No. PPI-304-1

A solution of phenyl-thiadiazole 6a (1.0 equivalent) and Et2NH (1.5 equivalents) in THF was treated with Pd(PPh3)4 (0.02 to 0.05 equivalents) at room temperaturet for 1 -3 hours. The solvent removed in vacuo and the product was purified by silica gel column chromatography using the Combi-Flash system eluting with hexane-ethyl acetate (Hex:EtOAc).

(ΛHert-ButyU-CS-C^hydroxy-S-^rifluoiOmethyOphenyO-l^^-thiadiazoH-yl)-!^^- trimethyloxazolidine-3-carboxylate (7aa)

The title compound was prepared from phenyl-thiadiazole 6aa in 64% (4.86 g) yield. HPLC retention time on a C8(2) column (30 x 3.00 mm, 3 μ) was 2.06 minutes with gradient 50-98% acetonitrile-H₂O (0.1% TFA) in 4.0 minutes as mobile phase. MS (ESI, M+H⁺) = 460.04; ¹H NMR (400 MHz, DMSO-d₆) δ 11.40 (s, IH), 8.00-8.10 (m, 2H), 7.18 (d, IH, J= 8.8 Hz), 4.07-4.21 (m, 2H), 1.88 (s, 3H), 1.67 (s, 3H), 1.57 (s, 3H), 1.42 (s, 3H), 1.19 (s, 6H).

(Λ)-ferf-Butyl 2,2,4-triiiiethyl-4-(5-(3-(trifluoroinethyl)-4-

(trifluoromethylsulfonyloxy)phenyl)-l,3,4-thiadiazol-2-yl)oxazolidine-3-carboxylate

(8aa)

A solution of {R)-tert-bx\\y\ 4-(5-(4-hydroxy-3-(trifluoromethyi)phenyl)-l,3,4- thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3-carboxylate (7aa) (382 mg, 0.826 mmol) in DCM (10 mL) was cooled to 0 ⁰C and treated successively with triethyl amine (TEA) (0.1 15 mL, 0.826 mmol) and triflic anhydride (0.14 mL, 0.826 mmol). The reaction mixture was stirred at 0 ⁰C for 30 minutes, then aqueous NaHCO₃ solution was added and the mixture was Attorney Docket No. PPI-304-1

extracted with DCM. The organic layer was washed with brine, dried over anhydrous Na2SO₄, and evaporated under reduced pressure to give a residue, which was purified by SiCh column chromatograph (Hex:EtOAc 85 :15) to give (R)-tert-buty\ 2,2,4-trimethyl-4-(5-(3- (trifluoromethyl)-4-(trifluoromethylsulfonyloxy)phenyl)-l,3,4-thiadiazol-2-yl)oxazolidine-3- carboxylate in 74% (360 mg) yield. HPLC retention time on a C18 column (30 x 4.6 mm, 3.5 μ) was 2.76 minutes with gradient 50-95% acetonitrile-H₂O (0.1% TFA) in 3.5 minutes as mobile phase. MS (ESI, M+U⁺) = 592.4; ¹H NMR (400 MHz, CDCl₃) δ 8.35 (br s, IH), 8.23 (dd, IH, J= 8.4 Hz, J= 2 Hz), 7.65 (d, I H, J= 8.4 Hz), 4.24-4.17 (m, IH), 4.10 (br s, IH), 2.02 (s, 3H), 1.79-1.66 (m, 6H), 1.51 (br s, 4H), 1.30 (br s, 5H).

(K)-tert-Buty\ 4-(5-(4-cyano-3-(trifluoromethyl)phenyI)-l,3,4-thiadiazol-2-yl)-2,2,4- trimethyloxazolidine-3-carboxylate from corresponding triflate (9aa)

To a solution of the triflate 8ab (360 mg, 0.61 mmol) and Zn(CN)₂ (50 mg, 0.426 mmol) in DMF (2 mL) was added Pd(PPh₃)₄ (35 mg, 0.03 mmol). The reaction mixture was evacuated under high vacuum and refilled with nitrogen and stirred at 100 ⁰C for 3 hours. The reaction mixture was cooled to room temperature, and then was quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na₂SO4, and evaporated under reduced pressure to give an oil, which was purified by SiO₂ column chromatograph (Hex:EtOAc 4:1) to give (R)-tert-butyl 4-(5-(4-cyano-3- (trifluoromethyl)phenyl)- 1 ,3,4-thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3 -carboxylate in 95% (272 mg) yield. HPLC retention time on a Cl 8 column (30 x 4.6 mm, 3.5 μ) was 1.95 minutes with gradient 50-95% acetonitrile-H₂0 (0.1% TFA) in 3.5 minutes as mobile phase. MS (ESI, M+H⁴) = 469.4; ¹H NMR (400 MHz, CDCl₃) δ 8.40 (br s, IH), 8.25 (dd, IH, J = 7.2 Hz, J= 1.6 Hz), 7.97 (d, IH, J= 7.2 Hz), 4.42-4.18 (m, IH), 4.1 1 (br s, IH), 2.03 (s, 3H), 1.79-1.66 (m, 6H), 1.51 (br s, 4H), 1.30 (br s, 5H).

(Λ)-ferf-ButyΙ 4-(2-(4-bromobenzoyl)hydrazinecarbonyl)-2,2,4-trimethyloxazolidine-3- ca rboxyla te (5a b) Attorney Docket No. PPI-304-1

To a solution of 4-bromobenzoic acid (5.15 g, 35 mmol) and HATU (13.3 g, 35 mmol) in DMF (100 mL) was added DIPEA (18.3 mL, 105 mmol). The solution was stirred at rt for 5 min, cooled to 0 ⁰C and hydrazine hrdrate (5.1 mL, 105 mmol) was added in one portion. After being stirred for an sdditional 1 h, the reaction mixture was quenched with 1 N NaOH, extracted with EtOAc, dried over Na₂SO₄, concentrated to afforded the crude 4- bromobenzohydrazide as an colorless oil. HPLC retention time on a Cl 8 column (30 x 4.6 mm, 3.5 μ) was 0.96 min with gradient 10-95% acetonitrile-H₂O (0.1% TFA) in 3.5 min as mobile phase. MS (ESI, NH-H⁴) = 215.1.

To a solution of (i?)-3-(tert-butoxycarbonyl)-2,2,4-trimethyloxazolidine-4-carboxylic acid (9.0 g, 35 mmol) and HATU (13.3 g, 35 mmol) in DMF (70 mL) was added DIPEA (18.5 mL, 105 mmol). The resulting solution was left stirred for 5 min, treated with a solution of crude 4-bromobenzohydrazide (5a b) prepared above in THF (200 mL). The the reaction mixture was stirred over night at rt, concentrated in vacuo, quenched with saturated NaHCO₃, extracted with EtOAc, dried over Na₂SO₄, concentrated to afforded an oil which was purified by SiO₂ column chromatograph (n-hexane/EtOAc = 7 : 3) to leave (/?)-tert-butyl 4-(2-(4- bromobenzoyl)hydrazinecarbonyl)-2,2,4-trimethyloxazolidine-3-carboxylate as a white foam 93% (14.8 g) yield over two steps. HPLC retention time on a C18 column (30 x 4.6 mm, 3.5 μ) was 2.42 min with gradient 10-95% acetonitrile-H₂O (0.1 % TFA) in 3.5 min as mobile phase. MS (ESI, M+H⁺) = 456.3. ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, 2H, J= 8 Hz), 7.54 (d, 2H, J= 8 Hz), 4.50 (br s, IH), 3.75 (br s, IH), 1.68-1.66 (m, 6H), 1.57 (br s, 3H), 1.51 (br s, 9H).

(/?)-/ert-Butyl 4-(5-(4-bromophenyl)-l,3,4-thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3- carboxylate (6a b)

Attorney Docket No. PPI-304-1

To a solution of (R)-tert-butyl 4-(2-(4-bromobenzoyl)hydrazinecarbonyl)-2,2,4- trimethyloxazolidine-3-carboxylate (14.8 g, 32.4 mmol) in DCM (350 mL) was added Lawesson's reagent (13g, 32.4 mmol). The reaction mixture was allowed to stirred at 50 ⁰C overnight, concentrated to 100 mL and purified directly by SiC>₂ column chromatograph (n- hexane/EtOAc =85:15) to leave (R)-tert-butyl 4-(5-(4-bromophenyl)-l ,3,4-thiadiazol-2-yl)- 2,2,4-trimethyloxazolidine-3-carboxylate as a white solid. HPLC retention time on a Cl 8 column (30 x 4.6 mm, 3.5 μ) was 2.17 min with gradient 50-95% acetonitrile-H₂O (0.1% TFA) in 3.5 min as mobile phase. MS (ESI, M+H⁺) = 454.2. ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, 2H, J= 7.2 Hz), 7.61 (d, 2H, J= 7.2 Hz), 4.40-4.20 (m, IH), 4.09-4.07 (m, IH), 2.00 (s, 3H), 1.77-1.60 (m, 7H), 1.50 (s, 3H), 1.28 (s, 5H).

(R)-tert-Buty\ 4-(5-(4-cyanophenyl)-l,3,4-thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3- carboxylate from corresponding bromide (9a b)

To a solution of (R)-tert-Buty\ 4-(5-(4-bromophenyl)-l ,3,4-thiadiazol-2-yl)-2,2,4- trimethyloxazolidine-3-carboxylate (6ab) (97 mg, 0.21 mmol) and Zn(CN)₂ (18 mg, 0.15 mmol) in DMF (1 mL) was added Pd(PPh₃)₄ (25 mg, 0.0214 mmol). The reaction mixture was evacuated under high vacuum and refilled with nitrogen and stirred at 120 ⁰C for 6 h. After being cooled to rt, the reaction mixture was quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na₂SC^, and evaporated under reduced pressure to give an oil, which was purified by SiO₂ column chromatograph (n-hexane/EtOAc =4:1) to leave (R)-tert-butyl 4-(5-(4-cyanophenyl)- 1,3,4- thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3-carboxylate in 82% (70 mg) yield. HPLC retention time on a Cl 8 column (30 x 4.6 mm, 3.5 μ) was 1.45 min with gradient 50-95% acetonitrile-H₂O (0.1% TFA) in 3.5 min as mobile phase. MS (ESI, M+H+) = 401.5; ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, 2H, J= 6.8 Hz), 7.77 (d, 2H, J= 6.8 Hz), 4.41-4.13 (m, IH), 4.11-4.09 (m, IH), 2.01 (s, 3H), 1.78-1.60 (m, 6H), 1.50 (s, 4H), 1.29 (s, 5H). Attorney Docket No. PPI-304-1

(5)-2-amino-2-(5-(4-(5-heptyM,2,4-oxadiazol-3-yl)-3-(trif]uoroinethyl)phenyl)-l ,3,4- thiadiazol-2-yl)propan-l-ol (1 laa)

A mixture of (R)-tert-buty\ 4-(5-(4-cyano-3-(trifluoromethyl)phenyl)-l ,3,4-thiadiazol-2- yl)-2,2,4-trimethyloxazolidine-3-carboxylate (73 mg, 0.155 mmol), hydroxylamine hydrochloride (54 mg, 0.775 mmol) and TEA (0.11 mL, 0.775 mmol) in methanol was stirred at 50 ⁰C for 18 hours before it was poured on to 1 N aqueous NaOH. The solution was extracted with EtOAc and dried over anhydrous Na2SO4, the solvent was removed and the residue dried under high vacuum to leave (R)-tert-bvΛy\ 4-(5-(4-(N-hydroxycarbamimidoyl)- 3-(trifluoromethyl)phenyl)-l,3,4-thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3-carboxylate as a solid. HPLC retention time on a C 18 column (30 x 4.6 mm, 3.5 μ) was 1.81 minutes with gradient 10-95% acetonitrile-H₂O (0.1% TFA) in 3.5 minutes as mobile phase. MS (ESI, M+H⁴) = 502.3

To a solution of octanoic acid (0.025 mL, 0.155 mmol) in DMF (0.5 mL) was added

HATU (58 mg, 0.155 mmol) and DIPEA (0.26 mL, 1.5 mmol). After stirring for 10 min, the crude (R)-tert-buty\ 4-(5-(4-(N-hydroxycarbamimidoyl)-3-(trifluoromethyl)phenyl)- 1 ,3 ,4- thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3-carboxylate in DMF (0.5 mL) was added. The reaction mixture was stirred at room temperature for 45 minutes, then at 120 ⁰C for 1 hour. The reaction mixture was quenched with saturated aqueous NaHCθ₃ and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, and evaporated under reduced pressure to give an oil, which was purified by Siθ2 column chromatograph (Hex:EtOAc 3:1) to yield (R)-tert-butyl 4-(5-(4-(5-heptyl-l,2,4-oxadiazol-3- yl)-3-(trifluoromethyl)phenyl)-l ,3,4-thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3- carboxylate (7 mg). HPLC retention time on a C18 column (30 x 4.6 mm, 3.5 μ) was 3.75 minutes with gradient 50-95% acetonitrile-H2θ (0.1% TFA) in 3.5 minutes as mobile phase. MS (ESI, M+^ = 610.5

(Λ)-tert-butyl 4-(5-(4-(5-heptyl-l,2,4-oxadiazol-3-yl)-3-(trifluoromethyl)phenyl)-l ,3,4- thiadiazol-2-yl)-2,2,4-trimethyloxazolidine-3-carboxylate was treated with 1 mL of a solution Attorney Docket No. PPI-304-1

of 10% TFA in DCM +1% triisopropyl silane (TIPS). The reaction mixture was stirred at room temperature for 3 hours, dried under vacuum and was subjected directly to preparative HPLC purification (C8(2) column, Phenomenex LunalOO x 21.10 mm, 5μ, gradient with acetonitrile/H₂θ containing 0.1% TFA as mobile phase and gradient 25-65% in 20 minutes) to give (S)-2-amino-2-(5-(4-(5-heptyl- 1 ,2,4-oxadiazol-3-yl)-3 -(trifluoromethyl)phenyl)- 1 ,3,4- thiadiazol-2-yl)propan-l-ol. HPLC retention time on a C18 column (30 x 4.6 mm, 3.5 μ) was 2.19 minutes with gradient 10-95% acetonitrile-H₂O (0.1% TFA) in 3.5 minutes as mobile phase. MS (ESI, M+H⁺) = 470.3 ; ¹H NMR (400 MHz, CD₃OD) δ 8.54 (s, IH), 8.39 (dd, IH, J = 8.4 Hz, J = 1.6 Hz), 8.04 (d, IH, J= 8.4 Hz), 4.01 (AB, IH, J=X 1.6 Hz ), 3.94 (AB, IH, J =11.6 Hz ), 3.04 (t, 2H, J= 7.6 Hz), 1.91-1.86 (m, 5H), 1.45-1.32 (m, 8H), 0.92 (t, 3H, J = 6.4 Hz).

(5)-2-amino-2-(5-(4-(5-hexyl-l^,4-oxadiazol-3-yl)-3-(trifluoromethyl)phenyl)-l^,4- thiadiazol-2-yl)propan-l -ol (1 lab)

The title product was prepared analogously to compound 1 laa from intermediate 9aa (Scheme Ia) and was isolated as a brown solid. HPLC retention time on a C18 column (30 x 4.6 mm, 3.5 μ) was 2.06 minutes with gradient 10-95% acetonitrile-H₂O (0.1% TFA) in 3.5 minutes as mobile phase. MS (ESI, M+H*) = 456.6. ¹H NMR (400 MHz, CD₃OD) δ 8.54 (s, 1 H), 8.38 (dd, IH, J= 8.2 Hz, J= 1.6 Hz), 8.03 (d, IH, J= 7.6 Hz), 4.00 (AB, IH, J=I 1.2 Hz ), 3.93 (AB, 1 H, J=I 1.6 Hz ), 3.04 (t, 2H, J= 7.2 Hz), 1.91 -1.86 (m, 5H), 1.47-1.34 (m, 6H), 0.93 (t, 3H, J= 6.8 Hz).

(5)-2-amino-2-(5-(4-(5-hexyl-l,2,4-oxadiazol-3-yl)phenyl)-l ,3,4-thiadiazol-2-yl)propaii- l-ol (llac) Attorney Docket No. PPI-304-1

The title product was prepared analogously to compound l laa from intermediate 9ab (R₂ = Br ) (Scheme Ia) and was isolated as a brown solid. HPLC retention time on a Cl 8 column (30 x 4.6 mm, 3.5 μ) was 1.93 minutes with gradient 10-95% acetonitrile-H₂O (0.1% TFA) in 3.5 minutes as mobile phase. MS (ESI, M+H⁺) = 388.5; ¹H NMR (400 MHz, CD₃OD) δ 8.13 (d, IH, J= 8.4 Hz), δ 7.94 (d, IH, J = 8.8 Hz), δ 7.08 (d, IH, J= 8.4 Hz), δ 6.72 (d, IH, J= 8.8 Hz), δ 4.14 (q, 2H, J= 12 Hz), δ 2.95 (t, 2H, J= 7.2 Hz), δ 1.91-1.84 (m, 3H), δ 1.62 (s, 2H), δ 1.49-1.25 (m, 6H), δ 0.91 (t, 3H, J= 6.8 Hz).

(Λ)-tert-Butyl 4-(5-(4-(l-isopentyl-lH-pyrazoI-4-yl)-3-(trifluoromethyl)phenyl)-l,3,4- thiadiazol-2-yl)-2_r2,4-trimethyloxazolidine-3-carboxylate

A suspension oftriflate 8aa [(R)-tert-Buty\ 2,2,4-trimethyl-4-(5-(3-(trifluoromethyl)-

4-(trifluoromethylsulfonyloxy)phenyl)-l,3,4-thiadiazol-2-yl)oxazolidine-3-carboxylate] (60 mg, 0.1 mmol) and l-(3-methylbutyl)-4-(4,4,5,5-tetramethyl-l ,3,2-dioxaboralan-2-yl)-l/7- pyrazole (63 mg, 0.24 mmol), Pd(PPh₃)₄ (12 mg, 0.01 mmol), and barium hydroxide octahydrate (48 mg, 0.15 mmol) in dimethoxyethane (DME) was heated for 1 hour at 80 ⁰C using a microwave reactor. The reaction mixture was purified on Isco system with 0-30% ethyl acetate-hexane as eluent system to provide the title compound (45 mg, 77% yield). MS (ESI, M+H⁴) = 580.1 ; ¹H NMR (400 MHz, CDCl₃) δ 8.30 (br, IH), 8.10 (dd, IH, J= 2.0 Hz, J= 8.4 Hz), 7.66 (s, IH), 7.59 (s, IH), 7.56 (d, 1 H, J= 7.6 Hz), 4.43, 4.10 (AB, 2H, J= 7.6 Hz), 4.20 (t, 3H, J= 7.2 Hz), 2.02 (s, 3H), 1.82 (m, 2H), 1.61 (m, IH), 1.58 (s, 3H), 1.29 (s, 6H), 0.97 (d, 6H, J= 6.4 Hz) Attorney Docket No. PPI-304-1

(5)-2-Amino-2-(5-(4-(l-isopentyl-l//-pyrazol-4-yl)-3-(trifluoromethyl)phenyl)-l,3,4- thiadiazol-2-yl)propan-l-oI (12a)

Deprotection of 12a using 20% TFA-DCM afforded the title compound. MS (ESI,

M+H⁴) = 440.0; ¹H NMR (400 MHz, CD₃OD) δ 8.40 (d, IH, J= 2 Hz), 8.21 (dd, IH, J = 2.0 Hz, J = 8.4 Hz), 7.91 (s, IH), 7.12 (d, IH, J= 8.4 Hz), 7.68 (s, IH), 4.25 (t, 2H, J= 6.8 Hz), 4.00, 3.93 (AB^H₅ J= I LO HZ)₅ LSS (S₅ SH)₅ 1.81 (m, 2H)₅ 1.56 (m, IH), 0.97 (d, 6H, J= 6.4 Hz).

General Method for Phosphate Synthesis

R₃ = H or protecting group

To a solution of unprotected amino alcohol (1.0 equivalent) in dry CH₂CI₂ at room temperature was added excess diethyl chlorophosphate (10.0 equivalents) and triethylamine (20.0 equivalents) and the reaction was stirred for 12-18 hours. The reaction was monitored by LC-MS. The crude reaction mixture was then evaporated to dryness in vacuo. The resulting phospho-diester intermediate was reacted with excess bromotrimethylsilane (10.0- 20.0 equivalents) in dry DCM at room temperature over a period of 6-10 hours to afford the final phosphate which was purified by reverse-phase preparative HPLC after evaporation of the solvent and excess reagent.

(5)-2-Amino-2-(5-(4-(5-heptyl-l,2,4-oxadiazol-3-yl)-3-(trifluoromethyl)phenyl)-lβ,4- thiadiazoI-2-yl)propyl dihydrogen phosphate Attorney Docket No. PPI-304-1

The title product was obtained as a white solid in 1 1% (0.7 mg) yield from the alcohol precursor. HPLC retention time on a C18 column (30 x 4.6 mm, 3.5 μ) was 2.22 minutes with gradient 10-95% acetonitrile-H₂O (0.1% TFA) in 3.5 minutes as mobile phase. MS (ESI, M+H⁴) = 550.4.

Claims

Attorney Docket No. PPI-304-1CLAIMSWhat is claimed is:

1. A compound of formula I:

1 or a pharmaceutically acceptable salt thereof, wherein:

is an optionally substituted aryl or heteroaryl group, wherein

"^^/wv ^» indicate points of attachment and X1-X5 are C, C-R' N, N-R", S, or O, wherein R' is hydrogen, halo, or alkyl and R" is hydrogen, alkyl;

R₂ is hydrogen, halogen, cyano, straight chain or branched Ci-Cβ-alkyl, straight chain or branched Ci-Cβ-alkoxy, straight chain or branched halo-Ci-C₆-alkyl (e.g., trifluoromethyl), straight chain or branched halo-Ci-C₆-alkoxy, Ci-C₆-alkoxy-Ci-C₆-alkyl, hydroxyl-Ci-Cβ- alkyl, carboxy-Ci-C₆-alkyl, Ci-C₆-alkyl-SO₂ or N(R)R', where R and R' are each independently hydrogen, straight chain or branched Ci-Cβ-alkyl, straight chain or branched Ci-Cβ-alkoxy, straight chain or branched halo-Ci-C₆-alkyl, straight chain or branched halo- Ci-Cβ-alkoxy, Ci-Cβ-alkoxy-Ci-Cβ-alkyl, hydroxyl-Ci-Cβ-alkyl, carboxy-Ci-Cβ-alkyl or Q- C₆-alkyl-SO₂, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; and Attorney Docket No. PPI-304-1

z is an integer from 1 to 3 ;

Q is -CH₂NR-, -CH₂NR(CO)-, -NH(CO)-, -(CO)NH-, -(CO)-, -O-, -S-, -SO-, -SO₂-, -NRSO₂-, -SO₂-NR-, aryl or heteroaryl, wherein R is hydrogen or straight chain or branched Q-Q-alkyl; R₃ is -OH, -CO₂R⁹, -CH₂=CH(CO)OR⁹, -OPO₂R¹⁰R¹¹, -OPO₃R¹⁰R¹¹, -

CH₂PO₃R¹⁰R¹¹, -OPO₂(S)R¹⁰R¹¹ or -C(Y)(X)PO₃R¹⁰R¹¹, where X is hydroxy! or halide and Y is H or halide; or analogues of other carboxylate, phosphate or phosphonate isosteres not limited to those shown below; R⁹ is H, straight chain or branched Ci-Cβ-alkyl, or a substituted or unsubstituted aryl group; R¹⁰ and R¹¹ are each independently H, straight chain or branched Ci-C₆-alkyl, a substituted or unsubstituted aryl group or selected from, but not limited to, the prodrugs listed below:

R₄ is H, Ci-C₆-alkyl, hydroxy-Ci-Ce-alkyl, aryl, or heteroaryl; R_5a and R_5b each independently selected from H, straight chain or branched Ci-Cδ- alkyl, or C₃-C6-cycloalkyl; taken together R_5a and Rsb may form a substituted or unsubstituted C₂-Ce fused carbocyclic ring or substituted or unsubstituted C₂-CiO fused heterocyclic rings, which may contain one or more heteroatoms and may be saturated or unsaturated; and m and n are each, independently, an integer from O to 3.