BACKGROUND

Technical Field

Modulators of the sphingosine-1 -phosphate receptor are provided for treatment of a malcondition for which activation of the same is medically indicated.

Description of the Related Art

The SIPi/EDGi receptor is a G-protein coupled receptor (GPCR) and is a member of the endothelial cell differentiation gene (EDG) receptor family. Endogenous ligands for EDG receptors include lysophospholipids, such as sphingosine-1 -phosphate (SIP). Like all GPCRs, ligation of the receptor propagates second messenger signals via activation of G-proteins (alpha, beta and gamma). Development of small molecule SIPi agonists and antagonists has provided insight into some physiological roles of the SIPi/SIP-receptor signaling system. To this end, SIP receptors are divided into five subtypes (i.e., SIPi, SIP2, SIP3, SIP4 and SIP5), which subtypes are expressed in a wide variety of tissues and exhibit different cell specificity. Agonism of the SIPi receptor perturbs lymphocyte trafficking, sequestering them in lymph nodes and other secondary lymphoid tissue. This leads to rapid and reversible lymphopenia, and is probably due to receptor ligation on both lymphatic endothelial cells and lymphocytes themselves (Rosen et al, Immunol. Rev., 195:160-177, 2003). BRIEF SUMMARY

In brief, modulators of the sphingosine-1 -phosphate receptor are provided for treatment of a malcondition for which activation of the same is medically indicated.

In one embodiment, a compound is provided having the structure of

Formula (I): or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, wherein R is as defined below.

DETAILED DESCRIPTION OF THE DISCLOSURE As used in the specification and the appended claims, the singular forms

"a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Further, the words "comprising," "including" and “having" are open-ended terms as used herein, and do not preclude the existence of additional elements or components.

The present invention is directed to compounds which modulate an SIP receptor, as well as to related products and methods for their preparation and use. SIP receptors are divided into five subtypes (i.e., SIPi, SIP2, SIP3, SIP4 and SIP5), which subtypes are expressed in a wide variety of tissues and exhibit different cell specificity. The compounds disclosed herein modulate one or more of these subtypes. In one embodiment, the compounds are "SIPi" modulators as they modulate subtype 1 of a sphingosine-1 -phosphate receptor. In another embodiment, the compounds modulate subtype 1 and another subtype, such as subtype 5. As used herein, an "SIPi modulator” is understood to encompass compounds that modulate the SIPi subtype alone, or modulate the SIPi subtype as well as one or more other subtypes. In one embodiment, an SIPi modulator modulates both the SIPi subtype and the SIP5 subtype. As used herein, a “modulator” of the SIPi receptor is a compound which, when administered to a subject, provides the desired integration with the target receptor, either by way of the compound acting directly on the receptor itself, or by way of a metabolite of the compound acting on the receptor. Upon administration to a subject, the compounds of this invention modulate the SIPi receptor by activating on the receptor for signal transduction. Such compounds are also referred to herein as “agonists” or “SIPi agonists”. Such SIPi agonists can be selective for action on SIPi. For example, a compound selective for action on S lPi acts at a lower concentration on S lPi than on other subtypes of the SIP receptor family.

Receptor agonists may be classified as either orthosteric or allosteric, and SIPi agonists of this invention include both classifications, either by way of the compound or by way of a metabolite of the compound acting on the receptor. In certain embodiments, compounds of the invention are orthostatic agonists. An orthosteric agonist binds to a site in the receptor that significantly overlaps with the binding of the natural ligand and replicates the key interactions of the natural ligand with the receptor. An orthosteric agonist will activate the receptor by a molecular mechanism similar to that of the natural ligand, will be competitive for the natural ligand, and will be competitively antagonized by pharmacological agents that are competitive antagonists for the natural ligand.

In certain other embodiments, compounds of the invention are allosteric agonists. An allosteric agonist binds to a site in the receptor that makes some significant interactions that are partly or wholly non-overlapping with the natural ligand. Allosteric agonists are true agonists and not allosteric potentiators. Consequently, they activate receptor signaling alone and without a requirement for a sub-maximal concentration of the natural ligand. Allosteric agonists may be identified when an antagonist known to be competitive for the orthosteric ligand shows non-competitive antagonism. The allosteric agonist site can also be mapped by receptor mutagenesis.

In one embodiment, a compound is provided having the structure of

Formula (I): or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, wherein:

R is alkyl.

As used in Formula (I), the following terms have the meanings set forth below.

“Alkyl” means straight chain, branched or cyclic alkyl group (cycloalkyl), saturated or unsaturated, having from 1 to about 20 carbon atoms (Ci-20 alkyl), and from 3 to 20 carbon atoms in the case of cycloalkyl. Alkyls are typically from 1 to 12 carbons (Ci-12 alkyl) or, in some embodiments, from 1 to 8 carbon atoms (Ci-8 alkyl) or, in some embodiments, from 1 to 4 carbon atoms (Ci-4 alkyl) or, in some embodiments, from 1 to 3 carbon atoms (C1-3 alkyl). Examples 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, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Examples of unsaturated alkyls include alkenyl and alkynyl groups. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range 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.

In one embodiment, a compound is provided having the structure of Formula (I), or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, alkyl is a straight chain or branched saturated alkyl having from 1 to 8 carbon atoms (Ci- 8 alkyl) or, in some embodiments, from 1 to 4 carbon atoms (Ci-4 alkyl) or, in some embodiments, from 1 to 3 carbon atoms (C1-3 alkyl). In a more specific embodiment, alkyl is methyl, ethyl, n-propyl, n-butyl, iso-propyl, iso-butyl, sec-butyl or t-butyl.

In one embodiment, a compound is provided having the structure of Formula (I), or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, wherein alkyl is cycloalkyl having from 3 to 8 ring members or, in some embodiments, 3 to 7, 3 to 6, or 3 to 5 ring member. In more specific embodiments, cycloalklyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

Representative compounds of Formula (I) are listed in Table 1.

Table 1

As mentions above, compounds having the structure of Formula (I) also include pharmaceutically acceptable salts, homologs, hydrates and solvates thereof.

A "salt" as is well known in the art includes an organic compound such as a carboxylic acid, a sulfonic acid, or an amine, in ionic form, in combination with a counterion. For example, acids in their anionic form can form salts with cations such as metal cations, for example sodium, potassium, and the like; with ammonium salts such as NFLri or the cations of various amines, including tetraalkyl ammonium salts such as tetramethylammonium and alkyl ammonium salts such as tromethamine salts, or other cations such as trimethyl sulfonium, and the like. A "pharmaceutically acceptable" or "pharmacologically acceptable" salt is a salt formed from an ion that has been approved for human consumption and is generally non-toxic, such as a chloride salt or a sodium salt. A "zwitterion" is an internal salt such as can be formed in a molecule that has at least two ionizable groups, one forming an anion and the other a cation, which serve to balance each other. For example, amino acids such as glycine can exist in a zwitterionic form. A "zwitterion" is a salt within the meaning herein. The compounds of the present disclosure may take the form of salts. The term "salts" embraces addition salts of free acids or free bases which are compounds of the disclosure. Salts can be "pharmaceutically-acceptable salts." The term "pharmaceutically acceptable salt" refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compounds of the disclosure.

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, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4 hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2 hydroxyethanesulfonic, p toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, b hydroxybutyric, salicylic, galactaric and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, 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 not generally useful as medicaments, such salts may be useful, for example as intermediates in the synthesis of compounds, for example in their purification by recrystallization. All of these salts may be prepared by conventional means from the corresponding compound by reacting, for example, the appropriate acid or base with the compound. The term "pharmaceutically acceptable salts" refers to nontoxic inorganic or organic acid and/or base addition salts, see, for example, Gould etal ., Salt Selection for Basic Drugs (1986), IntJ. Pharm ., 33, 201-217, incorporated by reference herein.

Non-limiting examples of potential salts of this disclosure include but are not limited to hydrochloride, citrate, glycolate, fumarate, malate, tartrate, mesylate, esylate, cinnamate, isethionate, sulfate, phosphate, diphosphate, nitrate, hydrobromide, hydroiodide, succinate, formate, acetate, dichloroacetate, lactate, p-toluenesulfonate, pamitate, pidolate, pamoate, salicylate, 4-aminosalicylate, benzoate, 4-acetamido benzoate, glutamate, aspartate, glycolate, adipate, alginate, ascorbate, besylate, camphorate, camphorsulfonate, camsylate, caprate, caproate, cyclamate, laurylsulfate, edisylate, gentisate, galactarate, gluceptate, gluconate, glucuronate, oxoglutarate, hippurate, lactobionate, malonate, maleate, mandalate, napsylate, napadisylate, oxalate, oleate, sebacate, stearate, succinate, thiocyanate, undecylenate, and xinafoate.

A "homolog" of a compound of the disclosure is a compound having one or more atoms of the compound replaced by an isotope of such atom. For example, homologs include compounds with deuterium in place of one or more hydrogen atoms of the compound such as compounds of the disclosure in which the methyl groups of the isopropoxy moiety of Formulas I-R and I-S are fully or partially deuterated ( e.g ., (D3C)2CHO-). Isotopic substitutions which may be made in the formation of homologs of the disclosure include non-radioactive (stable) atoms such as deuterium and carbon 13, as well as radioactive (unstable) atoms such as tritium, carbon 14, iodine 123, iodine 125, and the like. A "hydrate" is a compound that exists in a composition with water molecules. The composition can include water in stoichiometric quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. As the term is used herein a "hydrate" refers to a solid form, i.e., a compound in water solution, while it may be hydrated, is not a hydrate as the term is used herein.

A "solvate" is a similar composition except that a solvent other that water replaces the water. For example, methanol or ethanol can form an "alcoholate", which can again be stoichiometric or non-stoichiometric. As the term is used herein a "solvate" refers to a solid form, i.e., a compound in solution in a solvent, while it may be solvated, is not a solvate as the term is used herein.

The compound disclosed herein can be prepared by techniques known to one skilled in the art, as well as by the procedures disclosed in the following Examples.

EXAMPLES

General Methods of Synthesis ¾ NMR (400 MHz) and ¹³ C NMR (100 MHz) were obtained in solution of deuteriochloroform (CDCb), deuteri omethanol (CD3OD) or dimethyl sulfoxide - D6 (DMSO). NMR spectra were processed using Mestrec 5.3.0 and 6.0.1. ¹³ C NMR peaks that are bracketed are two rotomers of the same carbon. Mass spectra (LCMS) were obtained using an Agilent 1100/6110 HPLC system equipped with a Thompson ODS-A, 100A, 5 m (50 X 4.6 mm) column using water with 0.1% formic acid as the mobile phase

A, and acetonitrile with 0.1% formic acid as the mobile phase B. The gradient was 20- 100% with mobile phase B over 2.5 min then held at 100% for 2.5 mins. The flow rate was 1 mL/min. For more hydrophobic compounds, the following gradient was used, denoted as Method 1 : 40-95% over 0.5 min, hold at 95% for 8.5 min, then return to 40% over 2 min, with a flow rate of 1 mL/min. Final compounds were checked for purity using Method : 5% for 1 min, 5-95% over 9 min, then hold at 95% for 5 min, with a flow rate of 1 mL/min. Enantiomeric excess was determined by integration of peaks that were separated on a Chiralpak AD-H, 250 x 4.6 mm column, 5 pm particle size. Flow rate of 1 mL/min and an isocratic mobile phase. Unless otherwise indicated, the chiral data provided uses this method. Alternatively, chiral separations were performed under the following conditions, denoted as Chiral Method 1 : Chiralpak AY-H, 250 x 4.6 mm column, 5 pm particle size. Flow rate of 1 mL/min and an isocratic mobile phase. Chiral Method 2: Chiralcel OZ-3, 250 x 4.6, 3 pm particle size at a flow rate of 0.75 ml/min. The pyridine, dichloromethane (DCM), tetrahydrofuran (THF), and toluene used in the procedures were from Aldrich Sure-Seal bottles kept under nitrogen (N2). All reactions were stirred magnetically and temperatures are external reaction temperatures. Chromatographies were carried out using a Combiflash Rf flash purification system (Teledyne Isco) equipped with Redisep (Teledyne Isco) silica gel (S1O2) columns. Preparative HPLC purifications were done on Varian Pro Star/Prep Star system using water containing 0.05% trifluoroacetic acid as mobile phase A, and acetonitrile with 0.05% trifluoroacetic acid as mobile phase B. The gradient was 10-80% with mobile phase B over 12 min, hold at 80% for 2 min, and then return to 10% over 2 min with flow rate of 22 mL/min. Other methods similar to this may have been employed. Fractions were collected using a Varian Prostar fraction collector and were evaporated using a Savant SpeedVac Plus vacuum pump. Microwave heating was performed using a Biotage Initiator microwave reactor equipped with Biotage microwave vessels. The following abbreviations are used: ethanol (EtOH), carbonyldiimidazole (CDI), isopropanol (IPA), and 4-dimethylaminopyridine (DMAP).

Example 1

Synthesis of Compound No. 1 Step 1 - Synthesis of 3-ethoxy-lH-indene-7-carbonitrile (Int 2):

A stirred mixture of l-oxo-2,3-dihydro-lH-indene-4-carbonitrile (Int 1) (20. Og, 98 wt%, 18.6 assay g, 124.8 mmol) in abs EtOH (20 mL), triethylorthoformate (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 h, GC analysis showed orthoformate consumed and 12.8 area% of Int 1 remaining. A further charge of triethylorthoformate (20 mL, 120.2 mmol) was made and after 45 min GC analysis showed 1.5 area% Int 1. The batch was cooled to ambient temperature and then poured into 1 M aq. K2HPO4 (200 mL) with vigorous stirring while maintaining a quench temperature < 15 °C. The two-phase mixture was vigorously stirred for 10 min. 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 to remove 340 mL distillate. Toluene was added (500 mL) and distilled at atmospheric pressure to remove 500 mL distillate. Total distillation time 3 h, temperature range 80-120 °C. At this point the batch was stored overnight at < 5 °C. Excess orthoformate was removed by chasing with ethyl acetate (100 mL) under reduced pressure until distillation stopped. Another volume of ethyl acetate (100 mL) was added and then concentrated under reduced pressure until distillation stopped. A third volume of ethyl acetate (100 mL) was added and then concentrated under reduced pressure until distillation stopped, after which GC analysis confirmed no orthoformate remaining. The crude was then stirred at 110 °C for 1 h, to convert the intermediate ketal to 3-ethoxy-lH-indene-7-carbonitrile (Int 2). Upon cooling, the crude (mobile oil, 21.34 g) was assayed for Int 2 by 'H NMR employing mesitylene as an internal standard. The oil assayed at 78.1 wt% product = 16.73 assay g, 90.0 mmol = 72.1 % assay yield. The crude oil was then purified by filtration through a silica gel plug eluting with 15% EtO Ac/hexane. The pure fractions were combined and utilized for the next step. 'H NMR (400 MHz, de-DMSO) d 7.78 (d, J= 8.4, 1H), 7.63 (m, 1H), 7.49 (m, 1H), 5.60 (m, 1H), 1.38 (t, J= 6.8 Hz, 1H), 1.19 (t, J= 6.8 Hz, 1H); LRMS: calcd for C12H12NCC [M + H]: 186.2; Found: 186.2. Step 2 - Synthesis of Int 3:

An EtOAc/hexane solution (650 mL) of 3 -ethoxy- lH-indene-7- carbonitrile (Int 2) is concentrated under reduced pressure to ~17 mL and isopropyl alcohol (IP A, 40 mL) was added. The solution was concentrated to ~ 17 mL, and a second volume of IPA (34 mL) was added. To the stirred solution was added aqueous hydroxylamine (50%, 30 mL, 455 mmol). The batch was then warmed at 35-40 °C for 5 h, and then stirred at ambient temperature overnight. The batch was cooled to 0 °C, seeded (50 mg), and stirred for 30 min for a seed bed to develop. Water (250 mL) was then added dropwise over ~ 1.5 h. The batch was stirred for lh at 0-20 °C. The product was isolated by filtration, cake-washed with water (100 mL) and dried on the filter under vacuum and a nitrogen atmosphere, to afford 3-ethoxy-N-hydroxy-lH-indene-7-carboximidamide (Int 3) (20.8 g, 90% yield). ¾NMR (400 MHz, de-DMSO) d 9.61 (s, 1H), 7.43 (m, 1H), 7.32 (m, 2H), 5.77 (s, 1H), 5.41 (s, 1H), 4.08 (q, J= 6.8 Hz, 2H), 3.45 (s, 2H), 1.39 (t, J = 6.8 Hz, 3H); LRMS: calcd for Ci2Hi ₅ N ₂ 02 ⁺ [M + H]: 219.2; Found: 219.1.

Step 3 - Synthesis of N-(Y3-cvano-4-isopropoxybenzovDoxy)-3-ethoxy-lH-indene-7- carboximidamide (Int 4):

A mixture of CDI (16.64 g, 102.6 mmol) and 3-cyano-4-isopropoxyl benzoic 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-lH-indene-7-carboximidamide (Int 3) (20.8 g, 93.3 mmol) in DMF (40 mL) was added through an addition funnel over ~ 5 min. After ~ 30 min the batch became viscous and a further volume of DMF (40 mL) was added to aid stirring. At this point HPLC assay indicated that 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 dried on the filter under nitrogen flow to afford N-((3-cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-lH-indene-7- carboximidamide (Int 4) as an off white solid (34.8 g, 90% yield). ¾ NMR (400 MHz, de-DMSO) d 8.70 (s, 1H), 8.33 (d, J= 6.8 Hz, 1H), 7.45 (m, 4H), 7.10 (m, 2H), 5.49 (s, 1H), 4.94 (m, 1H), 4.10 (q, J= 6.8 Hz, 2H), 3.55 (s, 2H), 1.38 (m, 9H); LRMS: calcd for C23H24N ₃ 04 ⁺ [M + H]: 406.4; Found: 406.2. Step 4 - Synthesis of 5-(3-(3-ethoxy-lH-inden-7-vO-L2.4-oxadiazol-5-vO-2- isopropoxybenzonitrile (Int 5)

N-((3-Cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-lH-indene-7 - carboximidamide (Int 4) (34.8 g, 83.97 mmol) was suspended in toluene (590 mL) and heated to reflux with a Dean-Stark apparatus for 18 h. ~2 mL were collected (theory 1.5 mL). The batch was cooled to ambient temperature, filtered through Celite, and concentrated under vacuum. The crude solid 5-(3-(3-ethoxy-lH-inden-7-yl)-l,2,4- oxadiazol-5-yl)-2-isopropoxybenzonitrile (Int 5) (30 g, 90% yield) is taken as is to the next step. LRMS: calcd for C ₂₃ H ₂₂ N303 ⁺ [M + H]: 388.4; Found: 388.3.

Step 5 - Synthesis 2-isopropoxy-5-(3-(l-oxo-2.3-dihydro-lH-inden-4-vD-L2.4- oxadiazol-5-vDbenzonitrile (Cpd. No. 1):

Int 5 (30 g, 75.57 mmol) is suspended in 4: 1 IPA/H ₂ 0 (300 mL). Catalytic H ₂ S04 (0.1 mL, 0.19 mmol) is added, and the resulting mixture is heated to reflux for 12 h. The slurry is cooled to ambient temperature and stirred for 1 h. The product is isolated by filtration and washed with 4:1 IPA/FbO (100 mL). After drying on the filter for 1 h under vacuum, the wet cake is charged back to the reactor and suspended in EtOAc (300 mL). The mixture is heated to reflux for 3 h, then cooled to ambient temperature and stirred for 1 h. The slurry is filtered, washed with EtOAc (100 mL), and dried on the filter under nitrogen to afford 2-isopropoxy-5-(3-(l-oxo-2,3-dihydro-lH-inden-4-yl)-l,2,4- oxadiazol-5-yl)benzonitrile (Cpd. No. 1) (22 g, 80% yield) as an off-white solid. 'H NMR (400 MHz, de-DMSO) d 8.55 (d, J= 2.0 Hz, 1H), 8.44 (m, 2H), 7.88 (d, J= 7.6 Hz, 1H), 7.69 (t, J= 7.6 Hz, 1H), 7.57 (d, J= 9.2 Hz, 1H), 4.99 (h , J = 12.4 Hz, 1H), 3.46 (dd, 7 i = 5.6, L = 11.2 Hz, 2H), 2.76 (dd, J i = 5.6, J ₂ = 11.2 Hz, 2H), 1.45 (d, J= 12.4 Hz, 6H); ¹³ C NMR (100 MHz, de-DMSO) d 205.9, 173.4, 167.4, 162.6, 154.2, 138.1, 134.7, 134.2, 133.9, 128.2, 125.9, 124.5, 115.8, 115.3, 114.9, 102.5, 72.6, 35.9, 27.3, 21.5; LRMS: calcd for C _2i Hi8N303 ⁺ [M + H]: 360.1; Found: 360.2; C,H,N Analysis: Found: %C: 70.25, %H: 4.69; %N: 11.71; Theory: %C: 70.18; %H: 4.77; %N: 11.69. Example 2

Synthesis of Compound 2

(2-isopropoxy-5-(3-(3-methoxy-l H-inden-7-yl )- 1.2.4-oxadiazol-5-yl ibenzonitrile)

Step 1:

A stirred solution of l-oxo-2,3-dihydro-lH-indene-4-carbonitrile (1 g, 6.37 mmol) in MeOH (1 mL) was added a mixture of trimethylorthoformate (2.8 mL, 25.4 mmol) and methanesulfonic acid (0.06 g, 0.637 mmol) in toluene (4 mL). The reaction mixture was stirred at 45°C. After 2 h stirring, an additional equivalent of trimethylorthoformate was added. After 16 h stirring at 45°C, the reaction mixture was cooled to rt and then poured into 1M K2HPO4 (aq) (10 mL) with vigorous stirring for 10 min. The two phases were separated and the aqueous layer was back extracted with EtOAc (50 mL x 2). The organic phases were combined, dried over Na2S04 and concentrated. The residue was stirred at 110°C (no solvent) for 0.5 h and then LCMS showed more formation of the desired product. Upon cooling, the crude product was purified by ISCO (80g column, 0-100% EtO Ac/Hex) to give the desired product: 3- methoxy-lH-indene-7-carbonitrile (0.6 g, 3.5 mmol, 55%). ESIMS found for C11H9NO: m/z 172.1 (M+l).

Step 2:

NH2OH HC1 (730 mg, 10.51 mmol) in IPA (2mL) was added Et ₃ N (1.46 ml, 10.51 mmol) and the mixture was vigorously stirred at rt for 10 min. The NH2OH HC1 was consumed and the resulting fine white NH4CI salt was filtered to give the IPA filtrate with free NH2OH. To the solution of 3-methoxy-lH-indene-7-carbonitrile (0.3 g, 1.75 mmol) in IPA (3 mL) was added the filtrate and the reaction mixture was stirred at 45°C for 5 h and monitored by LCMS. Upon completion by LCMS, the reaction mixture was concentrated to give a yellowish sticky crude product ((N-hydroxy-3-methoxy-lH- indene-7-carboximidamide) (358 mg, 1.75 mmol) which was used as is. Yield: 100% (crude); ESIMS found for C11H12N2O2: m/z 205.1 (M+l).

Step 3 :

To 3-cyano-4-isopropoxybenzoic acid (60 mg, 0.29 mmol) and HOBt (44.6 mg, 0.33 mmol) in DMF (1 mL) was added EDC (63 mg, 0.33 mmol) at rt and stirred for 1~2 h until complete formation of the HOBt adduct, monitored by LCMS. To the HOBt adduct was added N-hydroxy-3-methoxy-lH-indene-7-carboximidamide (150 mg, 0.73 mmol) in 1 mL of DMF and stirred for another 1 h at rt. Upon completion, the reaction mixture was quenched with water. The resulting ESIMS found for solid was filtered and dried under high vacuum to give N-hydroxy-3-methoxy-lH-indene-7- carboximidamide (100 mg, 0.255 mmol, 43%). The dried solid was used as is in the next step. ESIMS found for C22H21N3O4: m/z 392.1 (M+l).

Step 4:

A mixture of N-hydroxy-3-methoxy-lH-indene-7-carboximidamide (100 mg, 0.128 mmol) and Toluene ( 5 ml) was stirred at 110°C for 18 h. Upon completion by LCMS, the reaction mixture was concentrated and the residue was purified by ISCO (12 g column, 0-50% EtO Ac/Hex) to give the desired product. Fractions are individually assessed for their purity by HPLC, then combined the fractions with over 95% pure fractions into the flask. Then it was concentrated under vacuum, transferred to the vial then freeze-dried (in MeOH/water) under high vacuum overnight to provide the desired product: 2-isopropoxy-5-(3-(3-methoxy-lH-inden-7-yl)-l,2,4-oxadiazol- 5- yl)benzonitrile as a solid (30 mg, 0.08 mmol, 63%) (unstable in HPLC). H-NMR suggests >95% purity. ¾ NMR (400 MHz, CHLOROFORM-d) d ppm 1.47 (s, 6 H), 3.72 (m, 2 H), 4.13 (s, 3 H), 4.82 (m, 1 H), 5.46 (t, J=4 Hz, 1 H), 7.12 (d, J=8 Hz, 1 H), 7.49 (m, 1 H), 7.60 (d, J=8 Hz, 1 H), 8.10 (d, J=8 Hz, 1 H), 8.35 (d, J=8 Hz, 1 H), 8.46 (s, 1 H); ESIMS found for C22H19N3O3: m/z 374.1. Example 3

Synthesis of Compound 3

(5-(3-(3-ethoxy- l H-inden-7-yl )- 1.2.4-oxadiazol-5-yl )-2-isopropoxybenzonitrile)

Step 1:

A stirred mixture of l-oxo-2,3-dihydro-lH-indene-4-carbonitrile (1 g, 6.37 mmol) in abs EtOH (2 mL), triethylorthoformate (4.7 g, 31. 84 mmol) and methanesulfonic acid (0.06 g, 0.64 mmol) in toluene (4 mL) was heated at 43-47°C. After 2 h stirring, an additional equivalent of trimethylorthoformate was added. After 16 h stirring at 45°C, the reaction mixture was cooled to rt and then poured into 1M K2HPO4 (aq) (10 mL) with vigorous stirring for 10 min. The two phases were separated and the aqueous layer was back extracted with EtOAc (50 mL x 2). The organic phases were combined, dried over Na2S04 and concentrated. The residue was stirred at 110°C (no solvent) for 0.5 h and then LCMS showed more formation of the desired product. Upon cooling, the crude product was purified by ISCO (80g column, 0-100% EtO Ac/Hex) to give the desired product: 3-ethoxy-lH-indene-7-carbonitrile (0.6 g, 3.2 mmol, 51%). ¾ NMR (400 MHz, d6-DMSO) d 7.78 (d, J = 8.4 Hz, 1 H), 7.63 (m, 1 H), 7.49 (m, 1H), 5.60 (m, 1 H), 1.38 (t, J = 6.8 Hz, 1 H), 1.19 (t, J = 6.8 Hz, 1 H); LRMS: calcd for C12H12NO+ [M + H]: 186.2; Found: 186.2.

Step 2: 3-ethoxy-lH-indene-7-carbonitrile was prepared in accordance with the procedures described in Example 1, except 3-methoxy-lH-indene-7-carbonitrile was replaced by 3-ethoxy-lH-indene-7-carbonitrile in 71% yield. ESIMS found for C12H11NO: m/z 219.0 (M+l). Step 3:

N-((3-cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-lH-indene-7 - carboximidamide was prepared in accordance with the procedures described in Example 1, except 3-methoxy-lH-indene-7-carbonitrile was replaced by 3 -ethoxy- lH-indene-7- carbonitrile in 82% yield. ESIMS found for C23H23N3O4: m/z 406.1 (M+l).

Step 4:

5-(3-(3-ethoxy-lH-inden-7-yl)-l,2,4-oxadiazol-5-yl)-2-iso propoxy- benzonitrile was prepared in accordance with the procedures described in Example 1, except N-((3-cyano-4-isopropoxybenzoyl)oxy)-3-methoxy-lH-indene-7- carboximidamide was replaced by N-((3-cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy- lH-indene-7-carboximidamide in 52% yield. ¾ NMR (400 MHz, CHLOROFORM-d) d ppm 1.47 (m, 9 H), 3.71 (m, 2 H), 4.13 (m, 2 H), 4.80 (m, 1 H), 5.42 (t, J=4 Hz, 1 H),

7.12 (d, J=8 Hz, 1 H), 7.49 (m, 1 H), 7.60 (d, J=8 Hz, 1 H), 8.10 (d, J=8 Hz, 1 H), 8.35 (d, J=8 Hz, 1 H), 8.46 (s, 1 H); ESIMS found for C23H21N3O3: m/z 388.4 (M+l).

Example 4 Synthesis of Compound 4

(2-isopropoxy-5-(3-(3-isopropoxy- l H-inden-7-yl )- l oxadiazol-5-yl ibenzonitrile) 10mol% MsOH iPrOH/toluene

35oC

2. K2HP04, toluene 110oC A stirred mixture of 2-isopropoxy-5-(3-(l-oxo-2,3-dihydro-lH-inden-4- yl)-l,2,4-oxadiazol-5-yl)benzonitrile (100 mg, 0.28 mmol) in iPrOH (1 ml), triisopropylorthoformate (30 eqv) and methanesulfonic acid (3 eqv) in toluene (1 ml) was heated at 100°C. After 1 h, LCMS check and 20% of [M+l] 402 observed, continued heating for 3 h at 100°C and LCMS check: 30% of [M+l] 402 observed; 50% SM remained and other peaks. Reaction was stop at this time. All solvent was removed and the residue was directly loaded on column and purified (using 0-30% EtO Ac/Hexane) to provide the desire product: 2-isopropoxy-5-(3-(3-isopropoxy-lH-inden-7-yl)-l,2,4- oxadiazol-5-yl)-benzonitrile (15 mg, 0.037 mmol, 13.4 %). ¾ NMR (400 MHz,

CHLOROF ORM- ) d ppm 1.43 (d, J=8Hz, 6H), 1.48 (d, J=8Hz, 6 H), 3.70 (m, 2 H), 4.51 (m, 1 H), 4.78 (m, 1 H), 5.38 (t, J=4 Hz, 1 H), 7.12 (d, J= 8 Hz, 1 H), 7.49 (m, 1 H), 7.60 (d, J=8Hz, 1 H), 8.10 (d, J=8 Hz, 1 H), 8.35 (d, J=8 Hz, 1 H), 8.46 (s, 1 H); ESIMS found for C24H23N3O3 : m!z 402.1 (M+l).

Example 5

In Vitro Biological Assays

GTPyS binding assay

Binding assays for [ ³⁵ S]-GTPyS were performed in 96-well non-binding surface plates with a final volume of 200 pL. The test compounds were serial diluted in DMSO and added to assay plates using a Tecan D300E digital printer with a total volume of 0.4 pL. The control sphingosine-1 -phosphate (SIP) was prepared separately by preparing a 400 pM stock solution from a 100 nmol pellet of SIP in 10 mM Na2C03 with 2% b-cyclodextrin. The serial dilution of SIP was done using complete assay buffer (20 mM HEPES, 10 mM MgCb, 100 mM NaCl, 1 mM EDTA, 0.1% fatty acid free bovine serum albumin [BSA], and 30 pg/mL saponin, pH7.4) and transferred to wells already containing 0.4 pL DMSO. All the wells were then loaded to a total volume of 40 pL of complete assay buffer, except the non-specific binding (NSB) wells. For NSB wells, 40 pL/well of 50 pM GTPyS (Sigma Aldrich, cat# G8634, St. Louis, MO) was added to wells containing 0.4 pL of DMSO. The assay was started by the addition of 120 pL/well of CHO-S1P receptor membrane solution containing 40 pg/mL of membrane protein, 16.67 pM guanosine diphosphate (GDP; Sigma Aldrich, cat# G7127, St. Louis, MO), and 2.5 mg/mL of WGA PVT SPA beads in complete buffer. Assay plates were then sealed and incubated at room temperature with gentle agitation for 30 minutes. Next, 40 pL/well of 1 nM of [ ³⁵ S]-GTPyS (PerkinElmer, cat# NEG030X250UC, Waltham, MA) in basic assay buffer (20 mM HEPES, 10 mM MgCb, 100 mM NaCl, and 1 mM EDTA, pH7.4) was added to the assay plates to yield a final concentration of 200 pM and the plates were further incubated for 40 minutes at room temperature with gentle agitation. The assay was terminated by centrifugation of the plates at 1000 rpm for 3 minutes using an Eppendorf 581 OR centrifuge (Eppendorf, Hamburg, Germany) and G protein bound radioactivity was quantitated using a MicroBeta2 microplate scintillation counter (PerkinElmer, Waltham, MA).

The data for representative compounds assayed by the above technique are presented in Table 2.

Table 2

Example 6

In Vivo Biological Assays

Determination of absolute oral bioavailabilitv in rats.

Pharmacokinetic studies are conducted in non-fasted male Sprague- Dawely rats (Simonsen Laboratories or Harlan Laboratories). Rats are housed in an ALAAC accredited facility and the research approved by the facilities Institutional Animal Care and Use Committee (IACUC). The animals are acclimated to the laboratory for at least 48 h prior to initiation of experiments.

Compounds are formulated in 5%DMSO/5%Tween20 and 90% purified water (intravenous infusion) or 5%DMSO/5%Tween20 and 90% 0.1N HCL (oral gavage). The concentration of the dosing solutions is verified by HPLC-UV. For intravenous dosing, compounds were administered by an infusion pump into the jugular vein over one minute to manually restrained animals (n=4 rats/compound). Oral dosing is by gavage using a standard stainless steel gavage needle (n=2-4 rats/compound). For both routes of administration, blood is collected at eight time-points after dosing with the final sample drawn 24 h post dose. Aliquots of the blood samples are transferred to polypropylene 96-well plate and frozen at -20°C until analysis.

After thawing the blood samples at room temperature, 5pL of DMSO is added to each well. Proteins are precipitated by adding 150 pL acetonitrile containing 200 nM internal standard (4-hydroxy-3-(alpha-iminobenzyl)-l-methyl-6- phenylpyrindin-2-( 1 //)-one) and 0.1% formic acid. Plates are mixed for 1 min on a plate shaker to facilitate protein precipitation and then centrifuged at 3,000 rpm for 10 min to pellet protein. The supernatant is transferred to a clean plate and centrifuged at 3,000 rpm for 10 min to pellet any remaining solid material prior to LC/MS/MS analysis. Calibration curve standards are prepared by spiking 5pL compound stock in DMSO into freshly collected EDTA rat blood. An eight point standard curve spanning a range of 5 nM to 10,000 nM is included with each bio-analytical run. The standards are processed identically to the rat pharmacokinetic samples.

Concentrations in the rat pharmacokinetic samples are determined using a standardized HPLC-LC/MS/MS method relative to the eight point standard curve. The system consists of a Leap CTC Pal injector, Agilent 1200 HPLC with binary pump coupled with an Applied Biosystems 3200 QTrap. Compounds are chromatographed on a Phenomenex Synergy Fusion RP 20x2mm 2um Mercury Cartridge with Security Guard. A gradient method is 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 from 0.7 to 0.8 mL/min. Ions are generated in positive ionization mode using an electrospray ionization (ESI) interface. Multiple reaction monitoring (MRM) methods are developed specific to each compound. The heated nebulizer is set at 325°C with a nebulizer current of 4.8 mA. Collision energies are used to generate daughter ions ranged between 29 and 39 V. Peak area ratios are obtained from MRM of the mass transitions specific for each compound used for quantification. The limit of quantification of the method is typically 5 nM. Data are collected and analyzed using Analyst software version 1.4.2.

Blood concentration versus time data are analyzed using non- compartmental methods (WinNonlin version 5.2; model 200 for oral dosing and model 202 for intravenous infusion). Absolute oral bioavailability (%) is calculated using the following expression: (Oral AUC ^c IV Dose)/(IV AUC ^c Oral Dose)x 100.

Lymphopenia

In mice: Female C57BL6 mice (Simonsen Laboratories, Gilroy CA) are housed in an ALAAC accredited facility and the research was approved by the facilities Institutional Animal Care and Use Committee (IACUC). The animals are acclimated to the laboratory for at least 5 days prior to initiation of experiments. Mice (n=3/compound/time-point) are dosed by oral gavage with 1-30 mg/kg compound formulated in a vehicle consisting of 5%DMSO/5%Tween 20 and 90% 0.1N HC1. Control mice are dosed PO with the vehicle. Terminal whole blood samples are collected from isoflurane anesthetized mice by cardiac puncture into EDTA. Whole blood is 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 Fluor647-Rat anti-mouse CD4 (BD #557681) for 30 min on ice. Red blood cells are lysed using BD Pharm Lyse Lysing buffer (#555899) and white blood cells were analyzed by FACS. Lymphopenia is expressed as the % of white blood cells that were CD4 or CD8 positive T cells. The overall lymphopenia response over 24 h is estimated by calculating the area under the effect curve (AUEC) using the linear trapezoidal rule.

In rats: Male rats (Simonsen Laboratories, Gilroy CA) are housed in an ALAAC accredited facility and the research was approved by the facilities Institutional Animal Care and Use Committee (IACUC). The animals are acclimated to the laboratory for at least 5 days prior to initiation of experiments. Rats (n=3/compound/time-point) are dosed by oral gavage with 1-30 mg/kg compound formulated in a vehicle consisting of 5%DMSO/5%Tween 20 and 90% 0. IN HCL. Control rats are dosed PO with the vehicle. Whole blood is collected from isoflurane anesthetized rats via the retro-orbital sinus and terminal samples were collected by cardiac puncture into EDTA. Whole blood is 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) for 30 minutes on ice. Red blood cells are lysed using BD Pharm Lyse Lysing buffer (#555899) and white blood cells are analyzed with a BD FACSArray. Lymphopenia is expressed as the % of white blood cells that were CD4 or CD8 positive T cells. The overall lymphopenia response over 24 h is estimated by calculating the area under the effect curve (AUEC) using the linear trapezoidal rule.

Lymphopenia

In mice: Female C57BL6 mice (Simonsen Laboratories, Gilroy CA) are housed in an ALAAC accredited facility and the research was approved by the facilities Institutional Animal Care and Use Committee (IACUC). The animals are acclimated to the laboratory for at least 5 days prior to initiation of experiments. Mice (n=3/compound/time-point) are dosed by oral gavage with 1 mg/kg compound formulated in a vehicle consisting of 5%DMSO/5%Tween 20 and 90% 0.1N HC1. Control mice are dosed PO with the vehicle. Terminal whole blood samples are collected from isoflurane anesthetized mice by cardiac puncture into EDTA. Whole blood is 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 Fluor647-Rat anti-mouse CD4 (BD #557681) for 30 min on ice. Red blood cells are lysed using BD Pharm Lyse Lysing buffer (#555899) and white blood cells were analyzed by FACS. Lymphopenia is expressed as the % of white blood cells that were CD4 or CD8 positive T cells. The overall lymphopenia response over 24 h is estimated by calculating the area under the effect curve (AUEC) using the linear trapezoidal rule.

In rats: Female rats (Simonsen Laboratories, Gilroy CA) are housed in an ALAAC accredited facility and the research was approved by the facilities Institutional Animal Care and Use Committee (IACUC). The animals are acclimated to the laboratory for at least 5 days prior to initiation of experiments. Rats (n=3/compound/time-point) are dosed by oral gavage with 1 mg/kg compound formulated in a vehicle consisting of 5%DMSO/5%Tween 20 and 90% 0. IN HCL. Control rats are dosed PO with the vehicle. Whole blood is collected from isoflurane anesthetized rats via the retro-orbital sinus and terminal samples were collected by cardiac puncture into EDTA. Whole blood is 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) for 30 minutes on ice. Red blood cells are lysed using BD Pharm Lyse Lysing buffer (#555899) and white blood cells are analyzed with a BD FACSArray. Lymphopenia is expressed as the % of white blood cells that were CD4 or CD8 positive T cells. The overall lymphopenia response over 24 h is estimated by calculating the area under the effect curve (AUEC) using the linear trapezoidal rule.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. These and other changes 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 the 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 63/001,090, filed March 27, 2020 and U.S. Provisional Application 63/018,347, filed April 30, 2020 are incorporated herein by reference, in their entirety.