Cross-Reference to Related Applications

[0001] This application claims the benefit of priority to United States Provisional Application serial number 62/188,207. The contents of the aforementioned application are incorporated herein in their entirety.

Summary of the Invention

[0002] Disclosed and claimed are tercyclic Sl P ₃ -sparing, S I Pi receptor agonists, as well as processes for making and methods of using the same.

Background of the Invention

[0003] The sphingosine-1 -phosphate (SI P) receptors are a family of five related G-protein coupled receptors (S l Pi-s) with affinity for the endogenous ligand S I P. Rosen, H.; Goetzl, E.J. Nat. Rev. Immunol. 2005, 5, 560. The S I P] receptor is expressed on lymphocytes and neuroglia and plays a role in immunomodulation as well as in central nervous system (CNS) function. Rivera, J.; Proia, R.L.; Olivera A. Nat. Rev. Immunol. 2008, 8, 753. (b) Blaho, V. A.; Hla, T. J. Lipid Res. 2014, 55, 1596. (c) Chiba, .; Matsuyuki, H.; Maeda, Y.; Sugahara, . Cell. Mol. Immunol. 2006, 3, 1 1-19. (d) Pritchard, A. J.; Dev, . K. Future Neurol. 2013, 8, 569. Small molecule agonists of the S I Pi receptor act as functional antagonists by inducing internalization and degradation of the receptor. On lymphocytes, S I P] receptor degradation results in the sequestration of the cells in the secondary lymph tissue, thereby preventing their migration to sites of inflammation. The pharmacological effects of fingolimod (also known as Gilenya® or FTY720, Table 1), a drug approved for the treatment of relapsing-remitting multiple sclerosis, are thought to be partly mediated through this mechanism. iuchi, M.; Adachi, .; ohara, T.; Minoguchi, M.; Hanano, T.; Aoki, Y.; Mishina, T.; Arita, M.; Nakao, N.; Ohtsuki, M; Hoshino, Y.; Teshima, .; Chiba, K.; Sasaki, S.; Fujita, T. J. Med. Chem. 2000, 43, 2946. Kappos, L.; Radue, E.; O'Conner, P.; Polman, C; Hohlfeld, R.; Calabresi, P.; Salmaj, K.; Agoropoulou, C; Layk, M; Auberson, L.; Burtin, P. N. Eng. J. Med. 2010, 362, 387.

[0004] Fingolimod is an orally administered prodrug that is metabolized in vivo to form an active phosphate metabolite (FTY720-P, Table 1), which is a potent agonist of four of the five S IP receptor isoforms (S l P| _j3,4>5 ) Hale, J.; Yan, L.; Neway, W.; Hajdu, R.; Bergstrom, J.; Milligan, J.; Shei, G.; Chrebet, G.; Thornton, R.; Card, D.; Rosenbach, M.; Rosen, H.; Mandala, S. Bioorg. Med. Chem. Lett. 2004, 12, 4803. Cross-agonism with S 1P ₃ is considered disadvantageous since this isoform has been associated with adverse effects including bradycardia, vasoconstriction, hypertension, and fibrosis in both in vitro and animal models. While S lP3-mediated bradycardia has been demonstrated to be operative only in rodents, the other effects impact the overall safety profile of non-selective S I P receptor agonists such as fingolimod. Sanna, M.G.; Liao, J.; Jo, E.; Alfonso, C; Ahn, M.Y.; Peterson, M.S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H. J. Biol. Chem. 2004, 279, 13839. Fryer, R. M.; Muthukumarana, A.; Harrison, P. C; Nodop Mazurek, S.; Chen, R. R.;

Harrington, K. E.; Dinallo, R. M.; Horan, J. C; Patnaude, L.; Modis, L. K.; Reinhart, G. A. PLoS ONE 2012, 7, e52985. Salomone, S.; Yoshimura, S.-I.; Reuter, U.; Foley, M.; Thomas, S. S.; Moskowitz, M. A.; Waeber, C. Eur. J. Pharmacol. 2003, 469, 125. (d) Sobel, .;

Menyhart, K.; Killer, N.; Renault, B.; Bauer, Y.; Studer, R.; Steiner, B.; Bolli, M. H.; Nayler, O.; Gatfield, J. J. Biol. Chem. 2013, 288, 14839. Gergely, P.; WallstrSm, E.; Nuesslein- Hildesheim, B.; Bruns, C; Zecri, F.; Cooke, N.; Traebert, M.; Tuntland, T.; Rosenberg, M.; Saltzman, M. Mult. Scler. 2009, 15, S 125. Hamada, M.; Nakamura, M.; Kiuchi, M.;

Marukawa, K.; Tomatsu, A.; Shimano, K.; Sato, N.; Sugahara, K.; Asayama, M.; Takagi, K.; Adachi, K. J. Med. Chem. 2010, 53, 3154.

[0005] This work describes the late-stage optimization of S 1 P ₃ -sparing, S I Pi agonists with the goal of identifying next-generation compounds for the treatment of autoimmune disorders such as multiple sclerosis. Towards this goal, compounds 1 and 2 were identified as advanced leads with excellent S1 P| selectivity. Although this series of compounds shares a common tercyclic scaffold with other known S I Pi agonists (notably PF-991 , Table 1), this core was derived originally from a high-throughput screening campaign. Compounds from this series showed very good potency and selectivity and were orally efficacious in animal models of immune disease. Deng, G.; Meng, Q.; Liu, Q.; Xu, X.; Xu, Q.; Ren, F.; Guo, T. B.; Lu, H.; Xiang, J.-N.; Elliott, J. D.; Lin, X. Bioorg. Med. Chem. Lett. 2012, 22, 3973. (b) Bolli, M. H.; Abele, S.; Birker, M.; Bravo, R.; Bur, D.; de Kanter, R.; Kohl, C; Grimont, J.; Hess, P.; Lescop, C; Mathys, B.; Miiller, C; Nayler, O.; Rey, M.; Scherz, M.; Schmidt, G.; Seifert, J.; Steiner, B.; Velker, J.; Weller, T. J. Med. Chem. 2014, 57, 1 10. Bannen, L.C.; Chan, D.S.; Mac, M.B.; Ng, S.; Nuss, J.M.; Wang, Y.; Xu, W. World Patent WO2010117662, 2010. Although 1 possesses a polar amino alcohol side chain similar to FTY720, this compound was not converted to the corresponding phosphate when dosed in vivo, and is believed to exert its pharmacological action directly, instead of as a prodrug.

[0006] The liabilities of this class of compounds are their low solubility (kinetic solubility < 0.1 μg/mL in pH 7.4 buffer for both 1 and 2) as well as the potential risk of phospholipidosis and hERG channel inhibition associated with the cationic amphiphilic nature of these structures Gleeson, M.P. J. Med. Chem. 2008, 57, 817. (b) Ratcliffe, A. J. Curr. Med. Chem. 2009, 16, 2816. The risk associated with phospholipidosis was confirmed by a positive response in the LipidTox phospholipidosis assay for compound 2. Nioi, P.; Perry, B. K.; Wang, E.-J.; Gu, Y.-Z.; Snyder, R. D. Toxicol. Sci. 2007, 99, 162. However, potential hERG inhibition could not be adequately assessed for either 1 or 2 due to the limited solubility of this class of compounds in the hERG assay buffer.

Table 1. S l Pi agonists

EC50S reported are geometric means

[0007] With these liabilities identified, a second round of optimization was initiated with the goal of finding compounds that met the following criteria:

1. Thermodynamic solubility > 10 μg/mL;

2. A neutral polar head group (i.e. no aliphatic amines or carboxylic acids);

3. Topological polar surface area (TPS A) < 120 A ² ;

5. S I P3 / Sl Pi EC ₅₀ selectivity ratio > 200-fold.

Summary of the Invention

[0008] In one aspect, the invention is directed to a compound of formula I: or a pharmaceutically acceptable salt thereof, wherein Ring A, Ring B, and Ring C and Z have the definitions provided in Tables 2-5.

[0009] In another aspect, the invention is the invention is directed to a compound of formula I as depicted above or a pharmaceutically acceptable salt thereof, wherein Ring A, Ring B, and Ring C and Z have the definitions provided in Tables 2-5, having one, two, three, four or five of the following characteristics:

1. Thermodynamic solubility > 10 μg/mL;

2. A neutral polar head group (i.e. no aliphatic amines or carboxylic acids);

3. Topological polar surface area (TPSA) < 120 A ² ;

4. S IP, EC ₅₀ < 50 nM; and

5. S 1 P ₃ / S 1 P i EC ₅₀ selectivity ratio > 200-fold.

[0010] In another aspect, the invention is directed to a pharmaceutical composition comprising a compound of Tables 2-5 and a pharmaceutically acceptable excipient.

[0011] In a further aspect the invention is directed to a method of treating an SIP-mediated disease or disorder, comprising administering a S I P receptor agonist as described herein to a patient in need thereof.

[0012] In a further aspect, the invention is directed to a method of treating an autoimmune disorder, comprising administering a S I P receptor agonist as described herein to a patient in need thereof. In one embodiment of this aspect, the autoimmune disease is mutiple sclerosis, psoriasis, inflammatory bowel disease, graft-versus-host disease, autoimmune-induced inflammation, or rheumatoid arthritis. In a further embodiment, the autoimmune disease is multiple sclerosis.

Detailed Description of the Invention

[0013] The following only summarizes certain aspects of the invention and is not intended to be limiting in nature. These aspects and other aspects and embodiments are described more fully below. All references cited in this specification are hereby incorporated by reference in their entirety. In the event of a discrepancy between the express disclosure of this specification and the references incorporated by reference, the express disclosure of this specification shall control. Compounds

[0014] The strategy to improve solubility centered on decreasing logP through the removal of halogen atoms and introduction of nitrogen atoms into one or more aromatic rings. Leach, A. G.; Jones, H. D.; Cosgrove, D. A.; Kenny, P. W.; Ruston, L.; MacFaul, P.; Wood, J. M.; Colclough, N.; Law, B. J. Med. Chem. 2006, 49, 6672.1 1 The design of neutral head groups focused on capping or eliminating the basic nitrogen in this region. This was thought to be a straightforward way to simultaneously reduce concern about hERG and phospholipidosis - since the canonical pharmacophores for these issues both contain an aliphatic amine. It was recognized, however, that adopting a neutral head group would negatively impact solubility and increase the challenge of meeting the required solubility criterion. The prohibition of carboxylic acids as well as the TPSA threshold was established as a way to proactively circumvent cell permeability issues. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.12 Lastly, since the tercyclic scaffold was known to provide excellent S 1P3 selectivity, no specific strategy was required to meet the selectivity criterion.

Table 2 Compounds

[0015] Initial work on improving solubility targeted changes to the A-ring for both the oxadiazole and thiadiazole B-rings (for data and ring nomenclature see Table 2). These analogues were prepared according to the synthetic route shown in Scheme 1, infra. The data from this panel of compounds identified three A-ring modifications that improved kinetic solubility (l Od, 6g, and 6h). Kinetic solubility was used as a more efficient method to obtain initial solubility information; compounds with favorable overall profiles were later submitted to an equilibrium assay for a more relevant measure of solubility. Kerns, E. H.; Di, L. In Drug-like Properties: Concepts, Structure Design and Methods; Solubility Methods; Academic Press: Burlington, MA, 2008; pp. 276.13 Unsurprisingly, the improvement in solubility correlated with the expected basicity of the A-ring; however, no consistent trend was observed with regard to the relative solubilities of the thiadiazole versus the oxadiazole B-rings. The potency data showed that introduction of a nitrogen atom decreased potency compared to the parent phenyl ring (c.f. 6b and 1), while 2-cyano substitution improved potency by 10-fold compared to 2-chloro substitution (cf. 10a and 2; l Oe and 10b). In addition, compounds 6g-6h and lOg-lOh demonstrated that the 4-position substitution could be moved to the 5-position while maintaining acceptable S 1 P1 potency. With the exception of compound 6h, the oxadiazoles were consistently more potent than the thiadiazoles. The potency disconnect observed for 6h might be explained if 6h was converted to a more potent phosphate metabolite in the cellular assay milieu (phosphate metabolites are frequently more potent for S I PI than the parent compound: see FTY720 in Table 1 and Horan, J. C; Sanyal, S.; Choi, Y.; Hill-Drzewi, M.; Patnaude, L.; Anderson, S.; Fogal, S.; Mao, C; Cook, B. N.; Gueneva-Boucheva, K.; Fisher, M. B.; Hickey, E.; Pack, E.; Bannen, L. C; Chan, D. S.; Mac, M. B.; Ng, S. M.; Wang, Y.; Xu, W.; Modis, L. .; Lemieux, R. M. Bioorg. Med. Chem. Lett, 2014, 24, 4807. reference 14). However, this hypothesis was not explored since phosphate metabolites often exhibited reduced selectivity over S 1 P3 and were therefore of lower interest as potential drug leads. To assess the effect of increased polarity on phospholipidosis, compounds 10a, 6g, and 6h were tested in the LipidTox phospholipidosis assay: similar to 2, all three compounds gave a positive response. This result further highlighted the need to switch from a basic to a neutral polar head group.

a

Table 2. A-ring modifications

B-Ring Ή

[0016] In one embodiment, Ring A, Ring B, and Ring C and Z of formula I have the definitions provided in Table 2.

is embodiment of formula I Ring A is selected from the group consisting of

, wherein " " indicates the point of attachment.

[0018] In this embodiment of formula I, Ring B is selected from the group consisting of

N— N Q— N

6 B-Rins s' _an( j i»: 8-Rins w " indicates points of attachment.

[0019] In this embodiment, Ring C is " indicates points of attachment.

[0020] In this embodiment of formula I, Z is wherein "- " indicates the point of attachment.

[0021] In a further embodiment, the compound of formula I is a compound of formula 1-2

1-2

or a pharmaceutically acceptable salt thereof, wherein Rings A and B have the definitions provided in Table 2. Table 3 Compounds

[0022] In parallel to the work exploring the SAR of the A-ring, efforts were pursued to find a neutral replacement for the aminoalcohol head group. Given that the pharmacophore for S 1 P1 binding contains an interaction between the basic amine of S IP-like ligands and the Glul21 residue of the receptor, most replacements were designed to preserve an N-H or O-H group which could still form a hydrogen bond with this residue. Lim, H.-S.; Park, J.-J.; o, K.; Lee, M.-H.; Chung, S.- . Bioorg. Med. Chem. Lett. 2004, 14, 2499. (b) Hanson, M. A.; Roth, C. B.; Jo, E.; Griffith, M. T.; Scott, F. L.; Reinhart, G.; Desale, H.; Clemons, B.; Cahalan, S. M.; Schuerer, S. C; Sanna, M. G.; Han, G. W.; Kuhn, P.; Rosen, H.; Stevens, R. C. Science 2012, 335, 851. Using the synthesis shown in Scheme 1 infra, over 100 analogues were synthesized across both the thiadiazole and oxadiazole scaffolds. As a result of the promising solubility improvement of 6g (Table 2), head group modifications were primarily synthesized using an aminopyridine A-ring and the 2-chloro-5-fluorophenyl C-ring. A selection of these compounds is shown in Table 3. It was again observed that the thiadiazoles were consistently less potent than the corresponding oxadiazoles - with very few thiadiazoles, but most oxadiazoles, meeting the criterion threshold for S I PI potency. Among the oxadiazoles, the most potent head groups contained at least one hydrogen bond donor in addition to a 5- or 6-membered ring. To assess the effect of replacing the basic side chain on phospholipidosis, a panel of neutral compounds, including compound l Oi, was tested in the LipidTox assay; gratifyingly, all of the neutral compounds tested gave a negative response. As expected, replacement of the aminoalcohol head group with a neutral moiety resulted in a loss in solubility - with most compounds exhibiting a kinetic solubility of < 0.1 μg/mL in pH 7.4 buffer. These results demonstrated that while it was possible to identify potent agonists with neutral head groups, the needed solubility improvements would have to come from modifications to the A- and C-rings.

Table 3. Neutral head roup SAR ^a

reported so ubility is kinetic solubility in pH 7.4 buffer; EC50S reported are geometr c for n > 2

[0023] In one embodiment, Ring A, Ring B, and Ring C and Z of formula I have the definitions provided in Table 3.

[0024] In this embodiment of formula I, Ring A is , wherein " ^«/ v° ^> indicates the point of attachment.

[0025] In this embodiment of formula I, Ring B is selected from the group consisting of

N-N O-

6 Β- ⁱ ng s· _anc j io 8-R« ₃ N ^ _wnere j _n " . /ν\ " indicates points of attachment.

[0026] In this embodiment, Ring C is , wherein "> ^/ wvr " indicates points of attachment.

[0027] In this embodiment of formula I, Z is R ¹ and R ¹ is selected from the group consisting indicates the point of attachment.

[0028] In a further embodiment, the compound of formula I is a compound of formula 1-3:

1-3

or a pharmaceutically acceptable salt thereof, wherein Ring A, Ring B, and Ring C have the definitions provided in Table 3. Z is defined as R ¹ in Table 3.

Table 4 Compounds

[0029] With initial SAR completed for both the A-ring and the head group, the next effort involved identifying more soluble replacements for the 2-chloro-5-fluorophenyl C-ring. The strategy used was the same as that used for the A-ring, namely decreasing logP through the removal of halogen atoms and introduction of one or more nitrogen atoms into the aromatic ring (Table 4). The A-rings from 6g and 6h, as well as the carbamate head group from lOi were selected as reference groups for this effort. The B-ring was constrained to the oxadiazole since the existing data showed no clear path towards identifying potent compounds with a thiadiazole B-ring.

[0030] Removal of one or both of the halogens from the C-ring did not have a measurable effect on solubility. However, introduction of one or more nitrogens was shown to improve solubility. Introduction of a nitrogen at the 2-position (pyridine l Ot) contributed a modest increase in solubility, while adding an additional 3- or 5-methyl substituent provided a further improvement (lOu-Ι Οχ). Bolli, M.; Lescop, C; Mathys, B.; Mueller, C; Nayler, O.; Steiner, B. World Patent WO2009109907, 2009. (b) Bolli, M.; Lescop, C; Mathys, B.; Mueller, C; Nayler, O.; Steiner, B.; Velker, J. World Patent WO2009024905, 2009.16 Similar analogues incorporating a 3-pyridine ring (l Oy-I Oz) did not show improvements of the same magnitude. Adding one additional nitrogen to provide pyrazines lOaa-lOac also gave a marked improvement in solubility, however these compounds failed to meet the S 1P1 potency threshold. Given the favorable potency and kinetic solubility of compounds lOu and lOv, the equilibrium solubility of these compounds was measured (4 and 7 μg/mL respectively); however, these compounds did not meet the required solubility criterion. Tabl 4. C-ring modifications

a reported solubility is kinetic solubility in pH 7.4 buffer; EC ₅₀ s reported are geometric means for n > 2

[0031] In one embodiment, Ring A, Ring B, and Ring C and Z of formula I have the definitions provided in Table 4.

[0032] In one embodiment, Ring A is , wherein X is selected from NH and

CH ₂ and "ΛΛΛ " indicates the point of attachment.

[0033] In another embodiment, Ring B is wherein "-ΛΛΛΛ" indicates points of attachment. In another embodiment, Ring C is selected from the group consisting of , _{, wherei} „ indicates points of attachment.

[0035] In another embodiment, Z is , indicates the point of attachment.

[0036] In a further embodiment, the compound of formula I is a compound of formula 1-4:

1-4

or a pharmaceutically acceptable salt thereof, wherein Ring A, Ring B, and Ring C and Z have the definitions provided in Table 4.

Table 5 Compounds

[0037] A final synthetic effort to discover compounds with overall balanced profiles involved synthesizing combination molecules that incorporated two or more of the favorable moieties identified from the previous SAR efforts. These combination compounds were deliberately selected to ensure that TPSA did not exceed 120 A2. From this effort, four compounds were identified that met all of the specified criteria (Table 5). Compound lOad was a direct combination molecule from the existing SAR. Compound l Oae incorporated a new dimethylpyridine C-ring - combining features from l Ov and l Ox (Table 4). The last two compounds (l Oaf and l Oag) utilized a new pyrimidine A-ring that was designed as a hybrid of the A-rings from compounds l Od and l Oh (Table 2). Due to the emerging body of evidence that suggests CNS exposure and S1 P5 activity are important for the treatment of multiple sclerosis with S I P receptor agonists, the S 1 P5 activity as well as total brain/plasma (B/P) exposure ratio were measured for a subset of the optimized compounds. Foster, C. A.; Howard, L. M.; Schweitzer, A.; Persohn, E.; Hiestand, P. C; Balatoni, B.; Reuschel, R.; Beerli, C; Schwartz, M.; Billich, A. J. Pharmacol. Exp. Ther. 2007, 323, 469. Choi, J. W.; Gardell, S. E.; Herr, D. R.; Rivera, R.; Lee, C.-W.; Noguchi, K.; Teo, S. T.; Yung, Y. C; Lu, M.; Kennedy, G.; Chun, J. P. Natl. Acad. Sci. USA 2011, 108, 751. Groves, A.; Kihara, Y.; Chun, J. J. Neurol. Sci. 2013, 328, 9. Jackson, S. J.; Giovannoni, G.; Baker, D. J. Neuroinflammation 2011, 8, 76. While all of these compounds showed potent agonism of S 1 P5 (ranging from 7 to 30-fold of their respective S I PI EC50s), the B/P ratio for both compounds lOaf and lOag was 0.1 - indicating low penetration into the CNS and suggesting that this class of compounds would be more appropriate for the treatment of peripheral autoimmune disorders (e.g. psoriasis, Crohn's disease) than for the treatment of multiple sclerosis.

Table 5. Combination compounds with balanced profiles ^a

" EC50S reported are geometric means for n > 2; kinetic solubility measured in pH 7.4 buffer; equilibrium solubility measured in pH 6.8 buffer

[0038] In one embodiment, Ring A, Ring B, and Ring C and Z of formula I have the definitions provided in Table 5.

[0039] In one embodiment, Ring A is , wherein X is selected from NH and CH ₂ ,

Y is N or CH, and "^ΛΛΛ" indicates the point of attachment.

[0040] In another embodiment, Ring B is , wherein "ΛΛΛΟ" indicates points of attachment.

[0041] In another embodiment, Ring C is selected from the group consisting of

wherein " »ΛΛΛΛ " indicates the point of attachment.

[0043] In a further embodiment, the compound of formula I is a compound of formula 1-5:

or a pharmaceutically acceptable salt thereof, wherein Ring A, Ring B, and Ring C and X, Y, Z have the definitions provided in Table 5.

[0044] In another embodiment the compound of formula I is selected from compounds lOad, lOae, lOaf, and lOag as defined in Table 5.

[0045] In summary, a tercyclic class of S l P3-sparing, S I PI receptor agonists was optimized to balance potency, selectivity, solubility and overall molecular charge. Focused SAR studies revealed favorable structural modifications, that when combined, produced compounds with balanced potency, selectivity, solubility and physical property profiles.

General Administration

[0046] In one aspect, the invention provides pharmaceutical compositions comprising tercyclic S 1 P ₃ -sparing, S I P] receptor agonists according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent. In certain other specific embodiments, administration is by the oral route. Administration of the compounds of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration or agents for serving similar utilities. Thus, administration can be, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermal ly, intravaginally, intravesically, intracistemally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, specifically in unit dosage forms suitable for simple administration of precise dosages.

[0047] The compositions will include a conventional pharmaceutical carrier or excipient and a compound of the invention as the/an active agent, and, in addition, may include carriers and adjuvants, etc.

[0048] Adjuvants include preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0049] If desired, a pharmaceutical composition of the invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.

[0050] The choice of formulation depends on various factors such as the mode of drug administration (e.g., for oral administration, formulations in the form of tablets, pills or capsules) and the bioavailability of the drug substance. Recently, pharmaceutical formulations have been developed especially for drugs that show poor bioavailability based upon the principle that bioavailability can be increased by increasing the surface area i.e., decreasing particle size. For example, U.S. Pat. No. 4, 107,288 describes a pharmaceutical formulation having particles in the size range from 10 to 1 ,000 nm in which the active material is supported on a crosslinked matrix of macromolecules. U.S. Pat. No. 5,145,684 describes the production of a pharmaceutical formulation in which the drug substance is pulverized to nanoparticles (average particle size of 400 nm) in the presence of a surface modifier and then dispersed in a liquid medium to give a pharmaceutical formulation that exhibits remarkably high bioavailability.

[0051] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0052] One specific route of administration is oral, using a convenient daily dosage regimen that can be adjusted according to the degree of severity of the disease-state to be treated.

[0053] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, cellulose derivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, magnesium stearate and the like (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

[0054] Solid dosage forms as described above can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain pacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0055] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.

[0056] Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

[0057] Compositions for rectal administrations are, for example, suppositories that can be prepared by mixing the compounds of the present invention with for example suitable non- irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.

[0058] Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.

[0059] Compressed gases may be used to disperse a compound of this invention in aerosol form. Inert gases suitable for this purpose are nitrogen, carbon dioxide, etc.

[0060] Generally, depending on the intended mode of administration, the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, and 99% to 1 % by weight of a suitable pharmaceutical excipient. In one example, the composition will be between about 5% and about 75% by weight of a compound(s) of the invention, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.

[0061] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 18th Ed.,

(Mack Publishing Company, Easton, Pa., 1990). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease-state in accordance with the teachings of this invention.

[0062] The compounds of the invention, or their pharmaceutically acceptable salts or solvates, are administered in a therapeutically effective amount which will vary depending upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease-states, and the host undergoing therapy. The compounds of the present invention can be administered to a patient at dosage levels in the range of about 0.1 to about 1 ,000 mg per day. For a normal human adult having a body weight of about 70 kilograms, a dosage in the range of about 0.01 to about 100 mg per kilogram of body weight per day is an example. The specific dosage used, however, can vary. For example, the dosage can depend on a number of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to one of ordinary skill in the art.

[0063] If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described above and the other pharmaceutically active agent(s) within its approved dosage range. Compounds of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a combination formulation is inappropriate.

Embodiments

[0064] Embodiment 1. A compound of formula 1 -2, 1-3, 1-4 as defined in Tables 2, 3, 4 and 5:

" -AW" indicates the point of attachment;

Ring B is selected from the group consisting of ^{6 ftins s'} and ^{68 Rin3 'Ν'} , wherein / o" indicates points of attachment.

Ring C is " indicates points of attachment; and

NH>

X -0„

Z is , wherein " " indicates the point of attachment.

For the Compound of Formula 1-3:

Ring A is indicates the point of attachment;

N-N O-N

Ring B is selected from the group consisting of ^{6 B Rin} 3 ^'s' and * ^{e R} "^ ^Ν' , wherein " ^j w ¹ " indicates points of attachment;

, wherein ">AW" indicates points of attachment;

indicates the point of attachment;

For the Compound of formula 1-4:

Ring A is , wherein X is selected from NH and CH ₂ and ^/ νν indicates the point of attachment;

Ring Bis °~ ^N , wherein ν/ " indicates points of attachment; Ring C is selected from the group consisting of ^r ^ _t vO ^" ^ vO ^" ^ wherein " ^» indicates points of attachment;

Z is , wherein " ΛΛ " indicates the point of attachment;

For the Compound of Formula 1-5:

\ AW

Ring A is , wherein X is selected from NH and CH ₂ , Y is N or CH, and

" ^ν Λ w " indicates the point of attachment;

Ri in g C is selected from the group consisting of and

wherein " " indicates the point of attachment.

[0065] Embodiment 2. The compound of embodiment 1 having one, two, three, four, or five of the following characteristics:

1. Thermodynamic solubility > 10 μg/mL;

2. A neutral polar head group (i.e. no aliphatic amines or carboxylic acids);

3. Topological polar surface area (TPS A) < 120 A ² ;

4. S 1 Pi EC ₅₀ < 50 nM; and

5. S I P3 / S I P1 EC ₅₀ selectivity ratio > 200-fold.

[0066] Embodiment 3. The compound of embodiment 1 having one of the following characteristics:

1. Thermodynamic solubility > 10 μg/mL·,

2. A neutral polar head group (i.e. no aliphatic amines or carboxylic acids);

3. Topological polar surface area (TPS A) < 120 A ² ; 4. S1P| EC ₅₀ < 50 nM; and

5. S1P ₃ / SIP] EC ₅₀ selectivity ratio > 200-fold.

[0067] Embodiment 4. The compound of embodiment 1 having two of the following characteristics:

1. Thermodynamic solubility > 10 μg/mL;

2. A neutral polar head group (i.e. no aliphatic amines or carboxylic acids);

3. Topological polar surface area (TPSA) < 120 A ² ;

4. SI Pi EC50 < 50 nM; and

5. SIP3 / SI Pi EC ₅₀ selectivity ratio > 200-fold.

[0068] Embodiment 5. The compound of embodiment 1 having three of the following characteristics:

1. Thermodynamic solubility > 10 μg/mL;

2. A neutral polar head group (i.e. no aliphatic amines or carboxylic acids);

3. Topological polar surface area (TPSA) < 120 A ² ;

4. SIP, EC ₅₀ <50nM; and

5. SIP3 / SIP, EC ₅₀ selectivity ratio > 200-fold.

[0069] Embodiment 6. The compound of embodiment 1 having four of the following characteristics:

1. Thermodynamic solubility > 10 μg/mL;

2. A neutral polar head group (i.e. no aliphatic amines or carboxylic acids);

3. Topological polar surface area (TPSA) < 120 A ² ;

4. SI Pi EC ₅₀ <50nM; and

5. SIP3 / SIP] EC ₅₀ selectivity ratio > 200-fold.

[0070] Embodiment 7. The compound of embodiment 1 having the following characteristics:

1. Thermodynamic solubility > 10 μg/mL;

2. A neutral polar head group (i.e. no aliphatic amines or carboxylic acids);

3. Topological polar surface area (TPSA) < 120 A ² ;

4. SIP, EC ₅₀ <50nM; and

5. SIP3 / SIP, EC ₅₀ selectivity ratio > 200-fold.

[0071] Embodiment 8. The compound of embodiment 1 which is a compound of formula I-

2, wherein:

Ring A is selected from the group consisting of wherein

" ^ν ΛΛΛ " indicates the point of attachment; -N O-N

Ring B is selected from the group consisting of ^{6 B"Riruj s'} and ^{10 8"Rir¾f N>} , wherein "./ r " indicates points of attachment. " indicates points of attachment; and indicates the point of attachment.

O-N

[0072] Embodiment 9. The compound of embodiment 6, wherein the B ring is ^{1β a} - ^Rir « ^N' , [0073] Embodiment 10. The compound of embodiment l , which is a compound of formula I-3, wherein:

Ring A is , wherein " ν/ν/νν " indicates the point of attachment;

N-N O-N

Ring B is selected from the group consisting of ^{6 B} - ^R,n n ^'s' and ^{10 B"R} "^ ^'Ν' , wherein " ΛΛΛΓ" indicates points of attachment;

Ring C is

Z is R ¹ and R ¹ is selected from the group consisting of _s , wherein

' ^WVP " indicates the point of attachment

O-N

10: 8-Rir¾: 'Ν'

[0074] Embodiment 1 1. The compound of embodiment 10, wherein the B ring is [0075] Embodiment 12. The compound of embodiment 1 , which is a compound of formula 1-4, wherein:

Ring A is

indicates the poin

Ring B is , wherein "ΆΛ Γ" indicates oints of attachment;

Rin C is selected from the group consisting of , wherein " ΛΛΛΛ " indicates points of attachment; and

Z is indicates the point of attachment.

[0076] Embodiment 13. The compound of embodiment 1 , which is a compound of formula 1-5, wherein:

Ring A is , wherein X is selected from NH and CH ₂ , Y is N or CH, and "

>ΛΛΛΛ" indicates the point of attachment;

Ring wherein " »ΛΛΛ " indicates points of attachment.; ing C is selected from the group consisting of _an d wherein " >ΛΛΛ " indicates points of attachment; and

indicates the point of attachment.

[0077] Embodiment 14. The compound of embodiment 13, which is a compound of Table 5: or a pharmaceutically acceptable salt thereof.

[0078] Embodiment 15. A pharmaceutical composition comprising a compound of embodiments 1-14 and a pharmaceutically acceptable excipient.

[0079] Embodiment 16. A method of treating an S I P-mediated disease or disorder, comprising administering a S I P receptor agonist to a patient in need thereof a compound of embodiments 1 -14 or a pharmaceutical composition of embodiment 15.

[0080] Embodiment 17. The method of embodiment 16, wherein the S I P-mediated disease or disorder is an autoimmune disease.

Examples

Synthesis of Compounds

[0081] Compounds were prepared as depicted in Scheme 1.

Scheme 1

[0082] Scheme 1. Reagents and conditions: (a) anhydrous hydrazine (2 equiv), MeOH, r.t. (73%); (b) R1C02H, EDCI (1 equiv), Et3N (1 equiv), DMF, r.t. (15-58%); (c) Lawesson's or Belleau's reagent (1.5 equiv), 2-MeTHF, reflux (20-84%); (d) R20H (2 equiv), HMDS (2 equiv), THF, 0 °C→ r.t. (27-87%); (e) optional deprotection step for R2 groups containing a BOC or acetonide: HC1 (10 equiv; 4 M in 1 ,4-dioxane), MeOH, r.t. (17-91 %); (f) NH20H (50% in water; 5 equiv), MeOH, reflux. (18-96%); (g) R1C02H, EDCl (1 equiv), HOBt (1 equiv), Et3N (1 equiv), DMA, r.t.→ 90 °C (13-70%); (h) R20H (2 equiv), KHMDS (2 equiv), THF, 0 °C→ r.t. (17-79%) or R20H (2 equiv), NaH (60% in mineral oil; 2 equiv), DMF, 0 °C→ r.t. (7-94%); Note: step 'd' can be performed before step 'a', and step 'h' can be performed before step 'f , to accommodate earlier installation of the -OR2 group.

β-Arrestin assay protocol:

[0083] Tango™ EDG l -bla U20S cells (Human S 1 P1), Tango™ EDG3-bla U20S cells (human S 1 P3) and Tango™ EDG8-bla U20S cells (Human S 1 P5) cell lines were purchased from Life Technologies and cultured according to the manufacturer's instructions, β-arrestfn recruitment assays for S I PI , S 1P3 and S 1 P5 (Tango™) were performed according to the manufacturer's instructions with the following modifications. Agonists were diluted in assay media so that the final DMSO concentration in the assay was 1 %. The agonists were routinely incubated on the cells for 18 hours prior to addition of the GenBlazer substrate. Ratio values for each well were compared to 10 μΜ S I P (POC). EC50 values were calculated with XLfit using a 4-parameter fit.

Kinetic solubility assay protocol:

[0084] Kinetic solubility was measured using the μ8ο1 Evolution instrument from pION incorporated (http://www.pion-inc.com). The assay was run according to the manufacturer's instructions. To summarize: in a 96-well plate format, 6 of a 10 mM DMSO stock solution of compound was added to 600 μΐ. of pH 7.4 buffer (final DMSO concentration -1 %). The plate was incubated at room temperature for 16-19 h and then the sample wells were vacuum filtered to remove any precipitate. The concentration of the compound in the filtrate was measured by UV absorbance (250-498 nm) and compared to the spectra of a precipitation-free reference solution using the pION Evolution software. Each reference solution was prepared by dissolving 10 of a 10 mM DMSO stock solution of compound in 190 μΐ, of n-propanol.

Equilibrium solubility assay protocol:

[0085] To a glass vial containing 2 mg of sample was added 1 mL of pH 6.8 buffer (prepared by the dissolution of 0.476 g citric acid monohydrate and 2.753 g Na2HP04 dihydrate in 100 ml of deionized water). The vial was capped, covered with aluminum foil and allowed to rotate for 24 h. The vial was centrifuged for 10 min at 14000 rpm and the supernatant was filtered using a 0.45 μη Nylon syringe filter to provide the sample filtrate. A reference solution was prepared by dissolving 1 mg of sample in 1 : 1 acetonitrile:water in a 10 mL volumetric flask. The sample filtrate and the reference solution were separately analyzed using a gradient reverse phase HPLC method monitoring at a suitable wavelength. The concentration of sample in the filtrate was calculated from the resulting HPLC peak areas using the following relationship:

[Sample] = ^{Peak ea} * vie _{χ [Reference}

LipidTox phopholipidosis assay protocol:

[0086] HepG2 cells were seeded into 96-well plates at a density of 5x 104 cells per well and allowed to attach overnight. To each well was added 50 μί of the HCS LipidTOXTM red reagent (Life Technologies; diluted 1 :500 in normal growth medium) and 50 μί of the test article or control (diluted in normal growth medium). After 48 h, cells were fixed in 4% formaldehyde in PBS containing 10 μg/ml Hoechst 33341 for 30 min at 37 °C. Following washing, fluorescence was measured using a Arrayscan VTi high content analysis reader (Thermo Scientific). For each assay, amiodarone (Sigma) was used as a positive control and aspirin was used as a negative control. Compounds and controls were typically assayed at 9 concentrations ranging from 0.1 to 200 DM (depending on solubility). Compounds showing a dose-responsive LipidTOX fluorescence intensity that is equal or greater than 2.5x of a concurrent vehicle control at non-cytotoxic concentrations (>75% viability of concurrent vehicle control), is considered a positive response in this assay.

[0087] The foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention. It will be obvious to one of skill in the art that changes and modifications can be practiced within the scope of the appended claims. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled. All references cited herein are incorporated by reference in their entirety.