Sphingosine 1-phosphate receptor modulators

Abstract

Structural formula (I): Provided is a compound having the formula: TIFF2023518967000013.tif38150, or a pharmaceutically acceptable salt, homolog, hydrate, or solvate thereof, wherein R is as defined herein. Such compounds serve as modulators of the sphingosine-1-phosphate receptor and have utility in treating conditions where activation of this receptor is medically indicated.

Description

[Technical Field]

[0001] Modulators of the sphingosine-1-phosphate receptor are provided for treating conditions in which activation of the receptor is medically indicated. [Background technology]

[0002] The S1P1 / EDG1 receptor is a G protein-coupled receptor (GPCR) and a member of the endothelial differentiation gene (EDG) receptor family. Endogenous ligands for the EDG receptor include lysophospholipids such as sphingosine-1-phosphate (S1P). Like all GPCRs, ligation of the receptor propagates second messenger signals via the activation of G proteins (alpha, beta, and gamma). The development of small molecule S1P1 agonists and antagonists has provided insight into several physiological roles of the S1P1 / S1P receptor signaling pathway. For this reason, S1P receptors have been divided into five subtypes (i.e., S1P1, S1P2, S1P3, S1P4, and S1P5), which are expressed in a wide variety of tissues and exhibit different cellular properties. Agonism of the S1P1 receptor inhibits lymphocyte trafficking and sequesters them in lymph nodes and other secondary lymphoid tissues, resulting in rapid and reversible lymphopenia, presumably due to receptor ligation on both lymphatic endothelial cells and the lymphocytes themselves (Rosen et al., Immunol. Rev., 195:160-177, 2003). Summary of the Invention

[0003] Briefly, modulators of sphingosine-1-phosphate receptors are provided for treating conditions in which activation of the receptor is medically indicated. In one embodiment, a compound of structural formula (I): [ka] wherein R is as follows: or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, is provided. DETAILED DESCRIPTION OF THE INVENTION

[0004] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Furthermore, the words "comprise," "include," and "have," when used herein, are open-ended terms and do not exclude the presence of additional elements or components.

[0005] The present invention relates to compounds that modulate S1P receptors, as well as related products and methods for making and using these compounds. S1P receptors are divided into five subtypes (i.e., S1P1, S1P2, S1P3, S1P4, and S1P5), which are expressed in a wide variety of tissues and exhibit different cellular specificities. The compounds disclosed herein modulate one or more of these subtypes. In one embodiment, the compound is an "S1P1" modulator, modulating subtype 1 of the sphingosine-1-phosphate receptor. In another embodiment, the compound modulates subtype 1 and another subtype, such as subtype 5. As used herein, "S1P1 modulator" is understood to include compounds that modulate only the S1P1 subtype or that modulate the S1P1 subtype as well as one or more other subtypes. In one embodiment, the S1P1 modulator modulates both the S1P1 and S1P5 subtypes.

[0006] As used herein, a "modulator" of the S1P1 receptor is a compound that, when administered to a subject, provides the desired integration with the target receptor, either by directly acting on the receptor itself or by a metabolite of the compound acting on the receptor. When administered to a subject, the compounds of the present invention modulate the S1P1 receptor by activating the receptor for signal transduction. Such compounds are also referred to herein as "agonists" or "S1P1 agonists." Such S1P1 agonists may be selective in their action on S1P1. For example, a compound selective in its action on S1P1 acts on S1P1 at a lower concentration than on other subtypes of the S1P receptor family.

[0007] Receptor agonists can be classified as either orthosteric or allosteric, and the S1P1 agonist of the present invention encompasses both classifications, either the compound itself acts on the receptor or the metabolite of the compound acts on the receptor.In certain embodiments, the compound of the present invention is an orthosteric agonist.Orthosteric agonists bind to the site on receptor that significantly overlaps with the binding of natural ligands, and reproduce the key interaction between natural ligands and receptors.Orthosteric agonists activate receptors through the molecular mechanism similar to that of natural ligands, and will compete with natural ligands and will competitively antagonize pharmacological agents that are competitive antagonists for natural ligands.

[0008] In certain other embodiments, the compounds of the present invention are allosteric agonists. Allosteric agonists bind to receptor sites that do not overlap, either partially or completely, with natural ligands, resulting in some degree of significant interaction. Allosteric agonists are true agonists, not allosteric potentiators. As a result, allosteric agonists only activate receptor signaling and do not require the submaximal concentration of the natural ligand. Allosteric agonists can be identified when antagonists known to compete with orthosteric ligands exhibit non-competitive antagonism. Allosteric agonist sites can also be mapped by receptor mutagenesis.

[0009] In one embodiment, a compound of structural formula (I): [ka] [In formula: R is alkyl. or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, is provided.

[0010] When used in formula (I), the following terms have the following meanings: "Alkyl" refers to a straight-chain, branched-chain, or cyclic alkyl group (cycloalkyl), saturated or unsaturated, having from 1 to about 20 carbon atoms (C 1-20 Alkyl typically refers to an alkyl group having 1 to 12 carbon atoms (C 1-12 alkyl), or in certain embodiments, 1 to 8 carbon atoms (C 1-8 alkyl), or in certain embodiments, 1 to 4 carbon atoms (C 1-4 alkyl), or in some embodiments, 1 to 3 carbon atoms (C 1-3Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Examples of unsaturated alkyl groups include alkenyl and alkynyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, cycloalkyl groups have 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl and the like.

[0011] In one embodiment, the compounds of structural formula (I), or pharmaceutically acceptable salts, homologs, hydrates, or solvates thereof, wherein alkyl is a straight or branched saturated alkyl having 1 to 8 carbon atoms (C 1-8 alkyl), or in some embodiments, 1 to 4 carbon atoms (C 1-4 alkyl), or in some embodiments, 1 to 3 carbon atoms (C 1-3 In a more specific embodiment, the alkyl is methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, sec-butyl, or t-butyl, or a pharmaceutically acceptable salt, homolog, hydrate, or solvate thereof.

[0012] In one embodiment, provided is a compound represented by Structural Formula (I), or a pharmaceutically acceptable salt, homolog, hydrate, or solvate thereof, wherein alkyl is cycloalkyl having 3 to 8 ring members, or in certain embodiments, cycloalkyl having 3 to 7, 3 to 6, or 3 to 5 ring members. In a more specific embodiment, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0013] Representative compounds of formula (I) are listed in Table 1. Table 1 [Table 1]

[0014] As noted above, the compounds of structural formula (I) also include pharmaceutically acceptable salts, homologs, hydrates or solvates thereof.

[0015] As is well known in the art, a "salt" includes organic compounds, such as carboxylic acids, sulfonic acids, or amines, in ionic form combined with a counterion. For example, an acid in its anionic form can form a salt with a metal cation, such as sodium, potassium, etc., e.g., NH +Or they may form salts with various amine cations, such as ammonium salts (including tetraalkylammonium salts, such as tetramethylammonium, and alkylammonium salts, such as tromethanamine salts), or other cations, such as trimethylsulfonium. A "pharmaceutically acceptable" or "pharmacologically acceptable" salt is a salt formed from an ion that is approved for human consumption and is generally nontoxic, such as a chloride salt or a sodium salt. A "zwitterion" is an internal salt that can be formed in a molecule having at least two ionizable groups, one forming an anion and the other forming a cation, which balance each other. For example, amino acids such as glycine can exist in the form of a zwitterion. "Zwitterions" are salts within the scope of the present invention. The compounds of the present disclosure may take the form of salts. The term "salt" includes addition salts of free acids or free bases of the compounds of the present disclosure. The salt may be a "pharmaceutically acceptable salt." The term "pharmaceutically acceptable salt" refers to a salt that has a toxicity profile within a range that makes it useful in pharmaceutical applications. Salts that are not pharmaceutically acceptable may nevertheless have properties, such as high crystallinity, that make them useful in practicing the present disclosure, e.g., useful in processes for synthesizing, purifying, or formulating the compounds of the present disclosure.

[0016] Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Suitable organic acids may be selected from the aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, or sulfonic classes of organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.

[0017] Suitable pharmaceutically acceptable base addition salts of the compounds of the present disclosure include, for example, metal salts, including alkali metal, alkaline earth metal, and transition metal salts, such as calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts prepared from basic amines, such as N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. Although pharmaceutically unacceptable salts are generally not useful as pharmaceuticals, such salts may be useful, for example, as intermediates in synthesizing compounds or in purifying them, for example, by recrystallization. All of these salts can be prepared by conventional means from the corresponding compounds, for example, by reacting the appropriate acid or base with the compound. The term "pharmaceutically acceptable salt" refers to a non-toxic inorganic or organic acid and / or base addition salt. See, for example, Gould et al., Salt Selection for Basic Drugs (1986), Int J. Pharm., 33, 201-217 (incorporated herein by reference).

[0018] Non-limiting examples of potential salts of the present disclosure include hydrochloride, citrate, glycolate, fumarate, malate, tartrate, mesylate, ethanesulfonate, cinnamate, isethionate, sulfate, phosphate, diphosphate, nitrate, hydrobromide, hydroiodide, succinate, formate, acetate, dichloroacetate, lactate, p-toluenesulfonate, palmitate, pidorate, pamoate, salicylate, 4-aminosalicylate, benzoate, 4-acetamidobenzoate, glutamate, aspartate, glycolate, adipate, alginate , ascorbate, besylate, camphorate, camphorsulfonate, camsylate, caprate, caproate, cyclamate, lauryl sulfate, edisylate, gentisate, galactarate, gluceptate, gluconate, glucuronate, oxoglutarate, hippurate, lactobionate, malonate, maleate, mandelate, napsylate, napadisylate, oxalate, oleate, sebacate, stearate, succinate, thiocyanate, undecylenate, and xinafoate.

[0019] The compounds of the present disclosure Isotopic compounds " is a compound in which one or more atoms of said compound are replaced by an isotope of that atom. For example, Isotopic compounds includes compounds in which deuterium replaces one or more hydrogen atoms of the compound, such as compounds of the present disclosure in which the methyl groups of the isopropoxy moiety of formulas IR and IS are fully or partially deuterated (e.g., (D3C)2CHO-). Isotopic compounds Isotopic substitutions that can be made to form include non-radioactive (stable) atoms such as deuterium and carbon-13, and radioactive (unstable) atoms such as tritium, carbon-14, iodine-123, iodine-125, and the like.

[0020] A "hydrate" is a compound that exists with water molecules in a composition. The composition can contain a stoichiometric amount of water, such as a monohydrate or dihydrate, or can contain a random amount of water. As used herein, "hydrate" refers to a solid form; that is, a compound in an aqueous solution may be hydrated, but the term is not a hydrate as used herein.

[0021] A "solvate" is a similar composition except that water is replaced with a solvent other than water. For example, methanol or ethanol can form an "alcoholate," which again can be stoichiometric or non-stoichiometric. As the term is used herein, "solvate" refers to a solid form; i.e., a solution of a compound in a solvent, although it may be solvated, is not a solvate as the term is used herein. The compounds disclosed herein can be prepared by techniques known to those skilled in the art, as well as by the procedures disclosed in the examples below.

[0022] Example General Method of Synthesis 1 H NMR (400 MHz) and 13 C NMR (100 MHz) was obtained in deuterated chloroform (CDCl), deuterated methanol (CDOD), or dimethyl sulfoxide-D (DMSO). NMR spectra were processed using Mestrec 5.3.0 and 6.0.1. 13C NMR peaks enclosed in brackets represent two rotamers of the same carbon. Mass spectrometry (LCMS) was obtained using an Agilent 1100 / 6110 HPLC system equipped with a Thompson ODS-A, 100A, 5µ (50 x 4.6 mm) column, using water + 0.1% formic acid as mobile phase A and acetonitrile + 0.1% formic acid as mobile phase B. The gradient was 20 to 100% mobile phase B over 2.5 min, then held at 100% for 2.5 min. The flow rate was 1 mL / min. For more hydrophobic compounds, the following gradient, designated Method 1, was used: 40 to 95% over 0.5 min, held at 95% for 8.5 min, then returned to 40% over 2 min, at a flow rate of 1 mL / min. The purity of the final compound was checked using Method 2 (5% for 1 minute, 5-95% over 9 minutes, then hold at 95% for 5 minutes, flow rate 1 mL / min). The enantiomeric excess was determined by integrating the separated peaks on a Chiralpak AD-H, 250 x 4.6 mm column, 5 μm particle size. The flow rate was 1 mL / min, and the mobile phase was isocratic. Unless otherwise noted, the chiral data provided uses this method. Alternatively, chiral separations, designated Chiral Method 1 and Chiral Method 2, were performed under the following conditions: Chiral Method 1 (Chiralpak AY-H, 250 x 4.6 mm column, 5 μm particle size; flow rate: 1 mL / min, and mobile phase: isocratic), and Chiral Method 2 (Chiralcel OZ-3, 250 x 4.6, 3 μm particle size, flow rate: 0.75 mL / min). Pyridine, dichloromethane (DCM), tetrahydrofuran (THF), and toluene used in the procedures were from Aldrich Sure-Seal bottles stored under nitrogen (N). All reactions were magnetically stirred and at the external reaction temperature. Chromatographic procedures were performed using a Combiflash RF flash purification system (Teledyne Isco) equipped with a Redisep (Teledyne Isco) silica gel (SiO) column.Preparative HPLC purification was performed on a Varian ProStar / PrepStar system using water containing 0.05% trifluoroacetic acid as mobile phase A and acetonitrile containing 0.05% trifluoroacetic acid as mobile phase B. The gradient was 10 to 80% mobile phase B over 12 minutes, then held at 80% for 2 minutes, then returned to 10% over 2 minutes, at a flow rate of 22 mL / min. Other methods similar to this procedure may also be utilized. Fractions were collected using a Varian Prostar fraction collector and evaporated using a Savant SpeedVac Plus vacuum pump. Microwave heating was performed using a Biotage Initiator microwave reactor equipped with a Biotage microwave vessel. The following abbreviations were used: ethanol (EtOH), carbonyldiimidazole (CDI), isopropanol (IPA), and 4-dimethylaminopyridine (DMAP).

[0023] Example 1 Synthesis of Compound No. 1 [ka]

[0024] Step 1: Synthesis of 3-ethoxy-1H-indene-7-carbonitrile (Intermediate 2): A stirred mixture of 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile (Intermediate 1) (20.0 g, 98 wt %, 18.6 g assay, 124.8 mmol) in absolute EtOH (20 mL), triethyl orthoformate (80 mL, 481 mmol), and methanesulfonic acid (0.88 mL, 12.5 mmol) in toluene (80 mL) was heated at 43-47 °C. After 1 hour, GC analysis indicated that the orthoformate had been consumed, leaving 12.8 area % of Intermediate 1. An additional charge of triethyl orthoformate (20 mL, 120.2 mmol) was made, and after 45 minutes, GC analysis indicated the presence of 1.5 area % of Intermediate 1. The batch was cooled to ambient temperature, then vigorously stirred and poured into 1 M aqueous KHPO (200 mL), maintaining a quench temperature of <15°C. The biphasic mixture was vigorously stirred for 10 minutes. The phases were separated, and the aqueous phase (pH 11) was back-extracted with toluene (100 mL). The organic phases were combined and distilled at atmospheric pressure, removing 340 mL of distillate. Toluene (500 mL) was added and distilled at atmospheric pressure, removing 500 mL of distillate. The distillation time was a total of 3 hours, with a temperature range of 80-120°C. At this point, the batch was stored at <5°C overnight. Excess orthoformate was removed by chasing under reduced pressure with ethyl acetate (100 mL) until distillation ceased. Another volume of ethyl acetate (100 mL) was added and then concentrated under reduced pressure until distillation ceased. A third volume of ethyl acetate (100 mL) was added and then concentrated under reduced pressure until distillation ceased, after which GC analysis confirmed that no orthoformate remained. The crude material was then stirred at 110° C. for 1 hour, converting the intermediate ketal to 3-ethoxy-1H-indene-7-carbonitrile (Intermediate 2). After cooling, the crude material (a flowing oil, 21.34 g) was purified by HPLC using mesitylene as the internal standard. 1 Intermediate 2 was assayed by H NMR. The oil was assayed to be 78.1 wt% product = 16.73 g assay, 90.0 mmol = 72.1% yield assay. The crude material was then purified by filtration through a silica gel plug, eluting with 15% EtOAc / hexane. Pure fractions were combined and used in the next step.1 H NMR (400MHz, d6-DMSO) δ 7.78(d,J=8.4,1H), 7.63(m,1H), 7.49(m,1H), 5.60(m,1H), 1.38(t,J=6,8Hz,1H), 1.19(t,J=6.8Hz,1H);LRMS:C 12 H 12 NO + Calculated for [M+H]: 186.2; Found: 186.2

[0025] Step 2: Synthesis of intermediate 3: A solution of 3-ethoxy-1H-indene-7-carbonitrile (Intermediate 2) in EtOAc / hexanes (650 mL) was concentrated under reduced pressure to approximately 17 mL, and isopropyl alcohol (IPA, 40 mL) was added. The solution was concentrated to approximately 17 mL, and a second volume of IPA (34 mL) was added. To this stirred solution was added aqueous hydroxylamine (50%, 30 mL, 455 mmol). The batch was then warmed to 35-40°C over 5 hours and then stirred at ambient temperature overnight. The batch was cooled to 0°C and seeded (50 mg), and the seed bed was stirred for 30 minutes to generate. Water (250 mL) was then added dropwise over approximately 1.5 hours. The batch was stirred at 0-20°C for 1 hour. The product was isolated by filtration, the cake washed with water (100 mL) and dried on the filter under vacuum and nitrogen atmosphere to give 3-ethoxy-N-hydroxy-1H-indene-7-carboximidamide (Intermediate 3) (20.8 g, 90% yield). 1 H NMR (400MHz, d6-DMSO) δ 9.61(s,1H), 7.43(m,1H), 7.32(m,2H), 5.77(s,1H), 5.41(s,1H), 4.08(q,J=6.8Hz,2H), 3.45(s,2H), 1.39(t,J=6.8Hz,3H);LRMS:C 12 H 15 N2O2 + Calculated for [M+H]: 219.2; Found: 219.1

[0026] Step 3: N-((3-cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-1H-indene-7-carboximidamide (Intermediate 4): A mixture of CDI (16.64 g, 102.6 mmol) and 3-cyano-4-isopropoxylbenzoic acid (21.06 g, 102.6 mmol) in DMF (83 mL) was stirred at 20 °C for 1 h. A solution of 3-ethoxy-N-hydroxy-1H-indene-7-carboximidamide (Intermediate 3) (20.8 g, 93.3 mmol) in DMF (40 mL) was added via addition funnel over approximately 5 min. After approximately 30 min, the batch became viscous, and an additional volume of DMF (40 mL) was added to aid stirring. At this point, HPLC assay indicated the reaction was complete. The resulting slurry was diluted with water (1.5 L), cooled to 0 °C, and isolated by filtration. The filter cake was washed with water (1.5 L) and the product was dried on the filter under a stream of nitrogen to give N-((3-cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-1H-indene-7-carboximidamide (Intermediate 4) as an off-white solid (34.8 g, 90% yield). 1 H NMR (400MHz, d6-DMSO) δ LRMS:C 23 H 24 N3O4 + Calculated for [M+H]: 406.4; Found: 406.2

[0027] Step 4: Synthesis of 5-(3-(3-ethoxy-1H-inden-7-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile (Intermediate 5) N-((3-cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-1H-indene-7-carboximidamide (Intermediate 4) (34.8 g, 83.97 mmol) was suspended in toluene (590 mL) and heated to reflux in a Dean-Stark apparatus for 18 hours. Approximately 2 mL was collected (theoretical 1.5 mL). The batch was cooled to ambient temperature, filtered through Celite, and concentrated in vacuo. The crude solid 5-(3-(3-ethoxy-1H-inden-7-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile (Intermediate 5) (30 g, 90% yield) was carried on directly to the next step. LRMS: C 23 H 22 N3O3 + Calculated [M+H]: 388.4; Found: 388.3

[0028] Step 5: Synthesis of 2-isopropoxy-5-(3-(1-oxo-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (Compound No. 1): Intermediate 5 (30 g, 75.57 mmol) is suspended in IPA / H2O (4:1) (300 mL). Catalytic H2SO4 (0.1 mL, 0.19 mmol) is added, and the resulting mixture is heated to reflux for 12 hours. The slurry is cooled to ambient temperature and stirred for 1 hour. The product is isolated by filtration and washed with IPA / H2O (4:1) (100 mL). After drying on the filter under vacuum for 1 hour, the wet cake is returned to the reactor and suspended in EtOAc (300 mL). The mixture is heated to reflux for 3 hours, then cooled to ambient temperature and stirred for 1 hour. The slurry is filtered, washed with EtOAc (100 mL), and dried on the filter under nitrogen to give 2-isopropoxy-5-(3-(1-oxo-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (compound no. 1) (22 g, 80% yield) as an off-white solid. 1H NMR (400MHz, d6-DMSO) δ 8.55(d,J=2.0Hz,1H), 8.44(m,2H), 7.88(d,J=7.6Hz,1H), 7.69(t,J=7.6Hz,1H), 7.57(d,J=9.2Hz,1H), 4.99( h,J=12.4Hz,1H), 3.46(dd,J1=5.6, J2=11.2Hz,2H), 2.76(dd,J1=5.6, J2=11.2Hz,2H), 1.45(d,J=12.4Hz,6H); 13 C NMR (100MHz, d6-DMSO) δ 205.9, 173.4, 167.4, 162.6, 154.2, 138.1, 134.7, 134.2, 133.9, 128.2, 1 25.9, 124.5, 115.8, 115.3, 114.9, 102.5, 72.6, 35.9, 27.3, 21.5;LRMS:C 21 H 18 N3O3 + Calculated [M+H]: 360.1; Found: 360.2; CHN analysis: Found: %C: 70.25, %H: 4.69; %N: 11.71; Theoretical: %C: 70.18; %H: 4.77; %N: 11.69

[0029] Example 2 compound 2 (5-(3-(1-hydroxy-1-methyl-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile) Synthesis of [ka] To a solution of 2-isopropoxy-5-(3-(1-oxo-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (100 mg, 0.28 mmol) in DCM (4 mL) was added ether (4 mL) followed by methylmagnesium bromide (0.158 mL, 0.4 mmol, 3 M in ether) at 20° C. The mixture was stirred at room temperature for 20 min, and 25% conversion was observed. An additional 0.15 mL of methylmagnesium bromide (3 M in ether) was added and stirred for another 30 min. The reaction mixture was then poured into ice water. 2 M aqueous HCl (5 mL) was added to the solution until the pH was approximately 1, and then extracted with EtOAc (20 mL). The organic layer was washed with brine, dried, concentrated, and then purified by ISCO to give the desired product: 5-(3-(1-hydroxy-1-methyl-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxy-benzonitrile (15 mg, 0.04 mmol, 15%). 1 H NMR (400 MHz, chloroform-d) δ ppm 1.48(d,J=8Hz,6H), 1.63(s,3H), 2.35(m,2H), 3.25(m,1H), 3.45(m,1H), 4.81(m,1H), 7.12(d,J=8Hz,1H ), 7.45(t,J=4Hz,1H), 7.50(d,J=8Hz,1H), 8.23(d,J=8Hz,1H), 8.35(d,J=8Hz,1H), 8.46(s,1H);ESIMS:C 22 H 21 As N3O3, measured value: m / z 376.0 (M+1)

[0030] Example 3 compound 3 (5-(3-(1-ethyl-1-hydroxy-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile) Synthesis of [ka] 5-(3-(1-Ethyl-1-hydroxy-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile was prepared in 23% yield according to the procedure described in Example 2, except that ethylmagnesium bromide was used instead of methylmagnesium bromide. 1 H NMR (400MHz, chloroform-d) δ ppm 1.0(m,3H), 1.48(d,J=8Hz,6H), 1.84(m,1H), 2.01(m,1H), 2.15(m,1H), 2.45(m,1H), 3.25(m,1H), 3.45(m,1H), 4.81(m,1H) , 7.12(d,J=8Hz,1H), 7.45(t,J=4Hz,1H), 7.50(d,J=8Hz,1H), 8.23(d,J=8Hz,1H), 8.35(d,J=8Hz,1H), 8.46(s,1H);ESIMS:C 23 H 23 As N3O3, measured value: m / z 390.0 (M+1)

[0031] Example 4 compound 4 (5-(3-(1-hydroxy-1-isopropyl-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile) Synthesis of [ka] 5-(3-(1-Hydroxy-1-isopropyl-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile (N39-034) was prepared in 20% yield according to the procedure described in Example 2, using isopropylmagnesium bromide instead of methylmagnesium bromide. 1H NMR (400 MHz, chloroform-d) δ ppm 0.80(d,J=8Hz,3H), 1.0(d,J=8Hz,3H), 1.48(d,J=8Hz,6H), 2.05(m,1H), 2.25(m,1H), 2.49(m,1H), 3.25(m,1H), 3.45(m,1H), 4.81 (m,1H), 7.12(d,J=8Hz,1H), 7.45(t,J=4Hz,1H), 7.50(d,J=8Hz,1H), 8.23(d,J=8Hz,1H), 8.35(d,J=8Hz,1H), 8.46(s,1H);ESIMS:C 24 H 25 As N3O3, measured value: m / z 404.7 (M+1)

[0032] Example 5 compound 5 (5-(3-(1-cyclopropyl-1-hydroxy-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile) Synthesis of [ka] To a solution of 2-isopropoxy-5-(3-(1-oxo-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (200 mg, 0.56 mmol) in DCM (4 ml) was added ether (8 ml), and then a solution of cyclopropylmagnesium bromide (0.189 ml, 0.94 mmol, 0.5 M in THF) was added to the above solution at 20° C. The mixture was stirred at room temperature for 20 minutes, and 25% conversion was observed. The reaction mixture was then poured into ice water. A 2 M aqueous solution of HCl (5 ml) was added to the solution until the pH was about 1; then it was extracted with EtOAc (20 ml). The organic layer was washed with brine, dried, and concentrated. The crude material was treated with excess NaBH4, and the product was reduced to the alcohol, which was then purified by ISCO to give the desired product: 5-(3-(1-cyclopropyl-1-hydroxy-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile (35 mg, 0.087 mmol, 16%). 1 H NMR (400MHz, chloroform-d) δ ppm 0.50(m,4H), 1.40(m,1H), 1.48(d,J=8Hz,6H), 2.15(m,1H), 2.35(m,1H), 3.25(m,1H), 3.45(m,1H), 4.81(m,1H), 7.12 (d,J=8Hz,1H), 7.45(t,J=4Hz,1H), 7.50(d,J=8Hz,1H), 8.23(d,J=8Hz,1H), 8.35(d,J=8Hz,1H), 8.46(s,1H);ESIMS:C 24 H 23 Measured value as N3O3: m / z 384.1 (M-18)

[0033] Example 6 In vitro biological assays GTPγS binding assay [ 35Binding assays for [S]-GTPγS were performed in a 96-well non-binding surface plate in a final volume of 200 μL. Test compounds were serially diluted in DMSO and added to the assay plate in a total volume of 0.4 μL using a Tecan D300E digital dispenser. Sphingosine-1-phosphate (S1P) control was prepared separately by preparing a 400 μM stock solution in 10 mM Na2CO3 containing 2% β-cyclodextrin from 100 nmoles of S1P pellet. Serial dilutions of S1P were made in complete assay buffer (20 mM HEPES, 10 mM MgCl2, 100 mM NaCl, 1 mM EDTA, 0.1% fatty acid-free bovine serum albumin [BSA], and 30 μg / mL saponin, pH 7.4) and transferred to wells previously containing 0.4 μL DMSO. All wells were then loaded with a total volume of 40 μL of complete assay buffer, except for the nonspecific binding (NSB) wells. To the NSB wells, 40 μL / well of 50 μM GTPγS (Sigma Aldrich, Catalog No. G8634, St. Louis, MO) was added to wells containing 0.4 μL of DMSO. The assay was initiated by adding 120 μL / well of CHO-S1P receptor membrane solution, containing 40 μg / mL membrane protein, 16.67 μM guanosine diphosphate (GDP; Sigma Aldrich, Catalog No. G7127, St. Louis, MO), and 2.5 mg / mL WGA PVT SPA beads in complete buffer. The assay plate was then sealed and incubated for 30 minutes at room temperature with gentle agitation. Next, 1 nM of [ 3540 μL / well of [S]-GTPγS (PerkinElmer, Cat. No. NEG030X250UC, Waltham, MA) was added to the assay plate to a final concentration of 200 pM, and the plate was further incubated for 40 minutes at room temperature with gentle agitation. The assay was terminated by centrifugation of the plate at 1000 rpm for 3 minutes in an Eppendorf 5810R centrifuge (Eppendorf, Hamburg, Germany), and G protein-bound radioactivity was quantified using a MicroBeta2 microplate scintillation counter (PerkinElmer, Waltham, MA).

[0034] Data for representative compounds assayed by the above techniques are shown in Table 2. Table 2 [Table 2]

[0035] Example 7 In vivo biological assays Determination of absolute oral bioavailability in rats Pharmacokinetic studies are performed in unfasted male Sprague-Dawley rats (Simonsen Laboratories or Harlan Laboratories). Rats are housed in an ALAAC-accredited facility, and the study is approved by the institutional Animal Care and Use Committee (IACUC). Animals are allowed to acclimate to the laboratory for at least 48 hours before the start of the experiment.

[0036] Compounds are formulated in 5% DMSO / 5% Thuen 20 and 90% purified water (intravenous infusion) or 5% DMSO / 5% Thuen 20 and 90% 0.1N HCl (oral gavage). The concentration of the dosing solution is confirmed by HPLC-UV. For intravenous administration, compounds are administered via an infusion pump into the jugular vein over 1 minute in manually restrained animals (n = 4 rats per compound). Oral administration is by gavage using a standard stainless steel gavage needle (n = 2-4 rats per compound). For both routes of administration, blood is collected at eight time points after dosing, with a final sample collected 24 hours after dosing. Aliquots of blood samples are transferred to 96-well polypropylene plates and frozen at -20°C until analysis.

[0037] After thawing the blood samples at room temperature, 5 μL of DMSO was added to each well. Proteins were precipitated by adding 150 μL of acetonitrile containing 200 nM of the internal standard (4-hydroxy-3-(alpha-iminobenzyl)-1-methyl-6-phenylpyrindin-2-(1H)-one) and 0.1% formic acid. The plate was mixed on a plate shaker for 1 minute to facilitate protein precipitation and then centrifuged at 3,000 rpm for 10 minutes to pellet the protein. The supernatant was transferred to a clean plate and centrifuged at 3,000 rpm for 10 minutes to pellet any remaining solids before being subjected to LC / MS / MS analysis. A reference calibration curve was prepared by spiking 5 μL of compound stock in DMSO into freshly collected EDTA rat blood. An eight-point standard curve ranging from 5 nM to 10,000 nM was included in each bioanalytical unit. The standards are treated similarly to the rat pharmacokinetic samples.

[0038] Concentrations in rat pharmacokinetic samples were measured using a standardized HPLC-LC / MS / MS method linked to an 8-point standard curve. The system consisted of an Agilent 1200 HPLC with a binary pump coupled to a Leap CTC Pal injector and an Applied Biosystems 3200 QTrap. Compounds were chromatographed on a 20x2mm 2µm Mercury cartridge on a Phenomenex Synergy Fusion RP with a Security Guard. A gradient method was used, with mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile, at flow rates varying between 0.7 and 0.8 mL / min. Ions were generated in positive ionization mode using an electrospray ionization (ESI) interface. A unique multiple reaction monitoring (MRM) method was developed for each compound. The heated nebulizer was set at 325°C with a nebulizer current of 4.8µA. Collision energies were used to generate daughter ions in the range of 29 and 39V. The specific mass transitions for each compound used for quantification are subjected to MRM to obtain peak area ratios. The limit of quantification for this method is typically 5 nM. Data is collected and analyzed using Analyst software version 1.4.2.

[0039] Blood concentration versus time data are analyzed using non-compartmental methods (WinNonlin version 5.2; Model 200 for oral administration and Model 202 for intravenous infusion). Absolute oral bioavailability (%) is calculated using the following formula: (Oral AUC x IV dose) / (IV AUC x Oral dose) x 100 It is calculated using

[0040] Lymphocytopenia With a mouse: Female C57BL6 mice (Simonsen Laboratories, Gilroy, CA) were housed in an ALAAC-accredited facility, and the study was approved by the institution's Institutional Animal Care and Use Committee (IACUC). Animals were allowed to acclimate to the laboratory for at least 5 days before the start of the experiment. Mice (n=3 / compound / time point) were administered compounds at 1–30 mg / kg via oral gavage in a vehicle consisting of 5% DMSO / 5% Thuen 20 and 90% 0.1 N HCl. Control mice were administered vehicle PO. Terminal whole blood samples were collected in EDTA from mice anesthetized with isoflurane via cardiac puncture. Whole blood was incubated with rat anti-mouse CD16 / CD32 (mouse BD Fc Block, #553141), PE-rat anti-mouse CD45R / B220 (BD #553089), APC-Cy7-rat anti-mouse CD8a (BD #557654), and Alexa Fluor 647-rat anti-mouse CD4 (BD #557681) on ice for 30 minutes. Red blood cells were lysed using BD Pharm Lyse Lysing Buffer (#555899), and leukocytes were analyzed by FACS. Lymphopenia was expressed as the percentage of leukocytes that were CD4 or CD8 positive T cells. The overall lymphopenic response over 24 hours was estimated by calculating the area under the effect curve (AUEC) using the linear trapezoidal method.

[0041] In rats: Male rats (Simonsen Laboratories, Gilroy, CA) were housed in an ALAAC-accredited facility, and the study was approved by the institution's Institutional Animal Care and Use Committee (IACUC). Animals were allowed to acclimate to the laboratory for at least 5 days before the start of the experiment. Rats (n=3 / compound / timepoint) were administered compounds at 1-30 mg / kg via oral gavage in a vehicle consisting of 5% DMSO / 5% Thuen 20 and 90% 0.1 N HCl. Control mice were administered vehicle PO. Whole blood was collected from rats anesthetized with isoflurane via the retro-orbital sinus, with a final sample collected in EDTA via cardiac puncture. Whole blood was incubated with mouse anti-rat CD32 (BD #550271), PE-mouse anti-rat CD45R / B220 (BD #554881), PECy5-mouse anti-rat CD4 (BD #554839), and APC-mouse anti-rat CD8a (eBioscience #17-0084) on ice for 30 minutes. Red blood cells were lysed using BD Pharm Lyse Lysing Buffer (#555899), and leukocytes were analyzed by BD FACSArray. Lymphopenia was expressed as the percentage of leukocytes that were CD4 or CD8 positive T cells. The overall lymphopenic response over 24 hours was estimated by calculating the area under the effect curve (AUEC) using the linear trapezoidal method.

[0042] Lymphocytopenia With a mouse: Female C57BL6 mice (Simonsen Laboratories, Gilroy, CA) were housed in an ALAAC-accredited facility, and the study was approved by the institution's Institutional Animal Care and Use Committee (IACUC). Animals were allowed to acclimate to the laboratory for at least 5 days before the start of the experiment. Mice (n=3 / compound / time point) were administered compounds at 1 mg / kg via oral gavage in a vehicle consisting of 5% DMSO / 5% Thuen 20 and 90% 0.1 N HCl. Control mice were administered vehicle PO. Terminal whole blood samples were collected in EDTA from mice anesthetized with isoflurane via cardiac puncture. Whole blood was incubated with rat anti-mouse CD16 / CD32 (mouse BD Fc Block, #553141), PE-rat anti-mouse CD45R / B220 (BD #553089), APC-Cy7-rat anti-mouse CD8a (BD #557654), and Alexa Fluor 647-rat anti-mouse CD4 (BD #557681) on ice for 30 minutes. Red blood cells were lysed using BD Pharm Lyse Lysing Buffer (#555899), and leukocytes were analyzed by FACS. Lymphopenia was expressed as the percentage of leukocytes that were CD4 or CD8 positive T cells. The overall lymphopenic response over 24 hours was estimated by calculating the area under the effect curve (AUEC) using the linear trapezoidal method.

[0043] In rats: Male rats (Simonsen Laboratories, Gilroy, CA) were housed in an ALAAC-accredited facility, and the study was approved by the institution's Institutional Animal Care and Use Committee (IACUC). Animals were allowed to acclimate to the laboratory for at least 5 days before the start of the experiment. Rats (n=3 / compound / time point) were administered compounds at 1 mg / kg via oral gavage in a vehicle consisting of 5% DMSO / 5% Thuen 20 and 90% 0.1N HCl. Control mice were administered vehicle PO. Whole blood was collected from rats anesthetized with isoflurane via the retro-orbital sinus, with a terminal sample collected in EDTA via cardiac puncture. Whole blood was incubated with mouse anti-rat CD32 (BD #550271), PE-mouse anti-rat CD45R / B220 (BD #554881), PECy5-mouse anti-rat CD4 (BD #554839), and APC-mouse anti-rat CD8a (eBioscience #17-0084) on ice for 30 minutes. Red blood cells were lysed using BD Pharm Lyse Lysing Buffer (#555899), and leukocytes were analyzed by BD FACSArray. Lymphopenia was expressed as the percentage of leukocytes that were CD4 or CD8 positive T cells. The overall lymphopenic response over 24 hours was estimated by calculating the area under the effect curve (AUEC) using the linear trapezoidal method.

[0044] The various embodiments described above may be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced herein and / or listed in the Application Data Sheet are hereby incorporated by reference in their entireties. Aspects of the embodiments can be modified, as necessary, to utilize concepts from various patents, applications, and publications to provide further embodiments. These and other variations can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. U.S. Provisional Application No. 63 / 001,085, filed March 27, 2020, and U.S. Provisional Application No. 63 / 018,333, filed April 30, 2020, are hereby incorporated by reference in their entireties.

Claims

1. Structural formula (I): 【Chemical 1】 wherein R is alkyl. or a pharmaceutically acceptable salt, isotope, hydrate or solvate thereof.

2. 2. The compound of claim 1, wherein the alkyl is a straight or branched saturated alkyl having 1 to 8 carbon atoms.

3. 3. The compound of claim 2, wherein the alkyl is a straight or branched saturated alkyl having 1 to 4 carbon atoms.

4. The compound of claim 3, wherein alkyl is methyl, ethyl, or isopropyl.

5. 2. The compound of claim 1, wherein R is cycloalkyl having 3 to 8 ring members.

6. The compound of claim 5, wherein the cycloalkyl has 3 to 6 ring members.

7. 7. The compound of claim 6, wherein R is cycloalkyl, wherein the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

8. 10. The compound of claim 1, wherein the compound has one of the following structures: or a pharmaceutically acceptable salt, isotopic compound, hydrate, or solvate thereof: 【Table 1】

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