Enantiomers that inhibit fatty acid binding protein 5

Abstract

Disclosed herein are purified enantiomers isolated from racemic 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2-methoxyphenyl)cyclobutane-1-carboxylic acid useful for treating pain, neurological diseases, anxiety, arthritis, skin disorders, spinal cord injuries, psoriasis, metabolic disorders, and cancer.

Description

ENANTIOMERS THAT INHIBIT FATTY ACID BINDING PROTEIN 5FIELD OF THE DISCLOSURE

[0001] The present disclosure relates to inhibitors of fatty acid binding protein 5 (FABP5), particularly, optically active forms of 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4- di(2-methoxyphenyl)cyclobutane-l -carboxylic acid. The optically active compounds inhibit FABP5 and are useful for treating pain, dermatological conditions, arthritis, psoriatic arthritis, osteoarthritis, spinal cord injury, metabolism disorders including inflammation, neurological diseases, such as anxiety, PTSD, multiple sclerosis, Parkinson’s disease, glaucoma, and cancers.BACKGROUND OF THE DISCLOSURE

[0002] Fatty acid binding protein 5 (FABP5) plays a critical role in regulating lipid transport, homeostasis, and metabolism (Xu, B, et al., Front. Cell Dev. Biol., 04 April 2022). Studies have shown that fatty acid binding proteins are involved in metabolism disorders and abnormal cell proliferation (Maeda, K., et al. (2005), Cell Metab 1 (2), 107-119; Iso. T., et al.. (2013). Arterioscler. Thromb. Vase. Biol. 33 (11). 2549-2557). Previous data indicates that FABP5 inhibition may have therapeutic utility for treating pain, inflammation, and cancer.

[0003] The analgesic effects of these compounds involve reduced degradation of endocannabinoids. FABP5 delivers endocannabinoids to fatty acid amide hydrolase (FAAH) for metabolism and the effects of FABP5 inhibitors can be blocked by antagonists of CB1, TRPV1, and PPARa (Peng X, Studholme K, Kanjiya MP, et al., Molecular Pain; 2017, Jan;13: 1744806917697007; Kaczocha M, Rebecchi MJ, Ralph BP, et al., PLoS ONE; 2014 Apr 4;9(4):e94200). Inhibiting FABPs reduces degradation of endocannabinoids. Thus, inhibiting FABPs decreases anandamide. oleoylethanolamide and. palmitoylethanolamide levels, and the activation of CB1, TPV1 and PPARα (Yan S., et al., Eur J Med Chem., 2018, Jun25; 154:233-252; Berger et al., PLoS One, 2012; 7(12): e50968. doi: 10. 1371 / journal. pone.0050968; Peng et al. 2017; and Kazocha, et al. 2014). FABP5 inhibition is effective in inflammatory pain models, where inhibition decreases pro-inflammatory prostaglandins and cytokines in addition to direct analgesic effects (Bogdan D, Falcone J, Kanjiya MP, et al.. Journal of Biological Chemistry 2018;293:5295-306).

[0004] FABP5 inhibition may have relevance in treating cancers, such as prostate, breast, liver, brain cancers, and squamous cell carcinoma (William George Warren, Myles Osbom, Andy Yates, and Saoirse E. O’Sullivan; Drug Discovery Today, 2023;28(7): 1-15). FABP5 has been shown to be overexpressed in prostate cancer (Morgan EA, Forootan SS, Adamson J, et al. International Journal of Oncology 2008;32:767-75). In addition, pharmacological inhibition ofFABP5 decreases tumor size in mice injected with prostate cancer cells and decreases the spread of the cancer to other areas such as the liver and lungs (Al-Jameel W, Gou X, Forootan SS, et al..Oncotarget 2017;8:31041-56; Carbonetti G, Converso C, Clement T, et al.. Prostate 2020;80:88- 98).BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Fig. 1A-B: 1A shows that enantiomer 2 (denoted as “ART26.12) significantly reverses mechanical allodynia in oxaliplatin treated animals. Von Frey thresholds for vehicle and test compound treated groups. #p<0.05; ##p<0.01; ###p<0.0001 indicate significant decrease in Von Frey threshold when compared to the respective group’s baseline values. *0.05, **<0.01, and ***<0.001 indicate significant reversal of mechanical allodynia by treatment when compared to the respective group’s neuropathic baseline values. Values are presented as mean ±s.e.m. (n=8-l 1). IB shows terminal plasma samples and terminal brain samples of enantiomer 2.

[0006] Fig. 2A-2D: 2A shows that on day 5 / 6 animals treated with 25 mg / kg p.o. of enantiomer 2 did not show a significant reduction in mechanical allodynia after oxaliplatin treatment, whereas on day 15. animals treated with enantiomer 2 did not display significant mechanical allodynia compared to baseline. 2B shows that prophylactic daily treatment with 25 mg / kg p.o. enantiomer 2 significantly reversed cold allodynia latencies on day 15 similar to pregabalin. 2C shows that prophylactic treatment with enantiomer 2 prevented transient weight loss induced by oxaliplatin treatment in the vehicle or pregabalin groupies. 2D shows that terminal plasma levels of enantiomer 2 corresponded to analgesic efficacy.

[0007] Fig. 3-Molecular structure, with displacement ellipsoids shown at 50% probability for non-H atoms. Only one of the two crystallographically independent molecules is shown.

[0008] Fig. 4-Overlay of the crystallographically independent molecules A (gray) and B (black), showing different orientations of the substituent groups relative to the cyclobutene core.

[0009] Fig. 5- Shows thaPIAs3 and SOCS3 were significantly downregulated by ART26.12

[0010] Fig. 6- shows the procedure used for Imiquimod-induced skin inflammation in male Balb / c mice.

[0011] Fig. 7- shows that ART26. 12 reduced PASI on days 6 and 7.

[0012] Fig. 8- shows that ART26.12 reduced skin thickness and reduced scaling.SUMMARY OF THE INVENTION

[0013] One aspect of the present disclosure is the purified enantiomeric forms, i.e.,Enantiomer 1 and Enantiomer 2, of racemic 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2- methoxyphenyl)cyclobutane-l-carboxylic acid) (Compound I)Enantiomer 1 and enantiomer 2 are optically active and have chemical structures represented byFormula I and Formula II, respectively.Formula I (enantiomer 1) Formula II (enantiomer 2)

[0014] Another aspect of the disclosure is a method for resolving the racemic Compound I into the purified enantiomers, enantiomer 1, and enantiomer 2. The method comprises contacting racemic 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2- methoxyphenyl)cyclobutene-l-carboxylic acid and a chiral amine in a solvent to form a salt. The salt is isolated and treated with aqueous acid to form the optically active compound of Formula 1. The filtrate obtained from the step of isolating the salt can be treated with aqueous acid to form the optically active compound of Formula II.

[0015] Still another aspect of the disclosure is a pharmaceutical composition of the optically the active compound of Formula I. or Formula II, or mixtures of Formula I and Formula II at different ratios or a pharmaceutically acceptable salt, ester, prodrug, hydrate, or tautomer thereof.

[0016] In still yet another aspect, described herein are methods of treating cancer, such as, without limitation, prostate, breast, liver, brain cancers, and squamous cell carcinoma in asubject in need of treatment by administering a pharmaceutical composition of Formula I, or Formula II or mixtures thereof to the subject.

[0017] In still yet another aspect, described herein are methods of treating pain in a subject in need of treatment by administering a pharmaceutical composition of Formula I, or Formula II or mixtures thereof to the subject. In some embodiments, the method can be used for treating pain, for example, pain caused by chemotherapy -induced neuropathy.

[0018] In still yet another aspect, disclosed herein are methods of treating a dermatological condition, such as, without limitation, psoriasis, radiation-induced skin damage, atopic dermatitis, atopic march, UV -induced skin damage, and skin lesions in a subject in need of treatment by administering a pharmaceutical composition of enantiomer 2 to the subject.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] Racemic 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2- methoxyphenyl)cyclobutane-l-carboxylic acid (Compound I) and its preparation are disclosed in International Application No. PCT / US2021 / 054174, filed on October 8, 2021.

[0020] The term “enantiomeric excess" is herein defined as % of the major enantiomer minus % of the minor enantiomer. For example, a mixture composed of 86% of one enantiomer and 14% of the other enantiomer would have an enantiomeric excess of 72%.

[0021] In an embodiment disclosed herein, the optically active compounds, have the structures represented by Formula I and Formula II:The compounds of Formula I and Formula II are also referred to as “enantiomer 1” and “enantiomer 2”, respectively, and can be obtained by chiral resolution of Compound I. The compound of Formula I is dextrorotatory, and the compound of Formula II is levorotatory, with respect to plane polarized light.

[0022] In embodiments disclosed herein, the compound of Formula I can have an enantiomeric excess of about 70% -100%, or about 55%, or about 55% - to 80%, or about 55%- toabout 90% or about 55% to 95%, or about 70% - 100% or about 60%- 90%, or about 60%-92%, or about 60% to 95%. or about 60%-97%, or about 60%-98%. or 60%-100%.S

[0023] In another embodiment, disclosed herein are methods for preparing an optically active compound of Formula I. The method involves optically resolving the racemic compound (Compound I) using a chiral amine. In the first step, racemic 3-(2,3-dihydro-1H-inden-2- yloxycarbonyl)-2,4-di(2-methoxyphenyl)cyclobutene-l-carboxylic acid and a chiral amine are combined in a solvent to form an acid-base salt. The salt is isolated, for example, by filtration, and then treated with an aqueous acid, such as aqueous HO, to liberate the free carboxylic acid group and provide the optically active compound of Formula I.

[0024] A variety of chiral amines can be used. Non-limiting examples of useful chiral amines include one or more members selected from the group consisting of (S)-(-)-l- phenylethylamine, (S)-l-(4-methoxyphenyl)ethylamine, (S)-(-)-l-(4-bromophenyl)ethylamine, (S)- (-)-N,N-Dimethyl-l-phenylethylamine, (-)-bis[(S)-l-phenylethyl]amine, d-valinol, d-alaninol, (R)- l-aminopropan-2-ol, quinine, (-)-cinchonidine, (D)-(-)-l-(l-naphthyl)ethylamine, (R)-(-)-2- phenylglycinol, and L-phenylalaninol. In some embodiments, (R)-(-)-2-phenylglycinolis used.

[0025] Any solvent that can aid in formation of the solid salt intermediate product can be used. In an embodiment, a liquid alkyl acetate can be used. Some examples of liquid alkyl acetates that can be used include, without limitation, methyl acetate, ethyl acetate, iso-propyl acetate, n-butyl acetate, and combinations thereof.

[0026] As described in the Experimental Section, a variety of resolving agents and solvents were evaluated with the chiral amine (R)-(-)-2-phenylglycinol in ethyl acetate providing the best results.

[0027] The methods described herein can be used for obtaining the optically active compound of Formula I at an enantiomeric excess of about 70% - about 100%. or at least about 51%, or about 51% - to about 80%, or about 51 %- to about 90% or about 51% to about 92%, or about 51% to about 95% or about 51% to about 100% or about 70% - to about 100%.

[0028] Another embodiment of the disclosure is a method of obtaining a an optically active compound of Formula I from racemic 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2- methoxyphenyl)cyclobutene-l-carboxylic acid by a process comprising: (i) contacting racemic 3- (2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2-methoxyphenyl)cyclobutene-l-carboxylic acid and a chiral amine in a solvent to form a salt, (ii) isolating the salt, and (iii) treating the salt with aqueous acid to form the optically active compound of Formula I.

[0029] In another embodiment of the disclosure is a method of obtaining an optically active compound of Formula II from racemic 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2- methoxyphenyl)cyclobutene-l-carboxylic acid by a process comprising: (i) contacting racemic 3-(2, 3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2-methoxyphenyl)cyclobutene-l-carboxylic acid and a chiral amine in a solvent to form a salt, (ii) isolating the salt, and (iii) treating the salt with aqueous acid to form the optically active compound of Formula I. In another embodiment, disclosed herein is a method of obtaining an optically active compound of Formula II which is obtained from the filtrate from the step of isolating the salt with aqueous acid to form the optically active compound having Formula II.

[0030] The optically active compound of Formula I or Formula II, or a pharmaceutically acceptable salt, ester, prodrug, hydrate, or tautomer thereof can be formulated into a composition suitable for therapeutic applications. In another aspect, the pharmaceutical composition can further comprise at least one pharmaceutically acceptable excipient.

[0031] In an aspect disclosed herein, the optically active compounds of Formula I or Formula II and compositions thereof are useful for treating, without limitation, pain, dermatological conditions, arthritis, psoriatic arthritis, osteoarthritis, spinal cord injury, metabolism disorders including inflammation, neurological diseases, such as anxiety. PTSD, multiple sclerosis, Parkinson’s disease, glaucoma, and cancers.

[0032] In some embodiments, the method of treatment comprises administering to a subject in need thereof a therapeutically effective amount of one or more of the pharmaceutical compositions described herein.

[0033] Suitable subjects / patients according to the present disclosure include mammalian subjects. Mammals according to the present disclosure include, but are not limited to, humans, canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, and the like, and encompass mammals in utero. In one embodiment, humans are suitable subjects.

[0034] In embodiments, the pharmaceutical composition can comprise the optically active compound of Formula I that has an enantiomeric excess of at least 50%, or 50% to 80%, or 70% to 100%. Included in this range is any range or singular numerical value that falls between the specified upper and lower range limits. In other embodiments, the pharmaceutical compositioncan comprise the optically active compound of Formula II having an enantiomeric excess of at least 50%, or 50% to 80%. or 70% to 100%.

[0035] Studies described herein provide evidence that purified enantiomer 2 is useful in treating chemotherapy-induced peripheral neuropathy (CIPN). CIPN affects 30% to 40 % of all cancer patients undergoing chemotherapy. Symptoms include numbness, pins and needles, stabbing and burning pain, and sensitivity to touch. Prevalence depends on the chemotherapy agent used, e.g., paclitaxel and oxaliplatin, dose and dosing duration, and many other risk factors. At present there are no approved medications for CIPN, but Duloxetine, antidepressants, anticonvulsants, and analgesics are used. CIPN impairs daily activities, increases the risk of falls and hospitalizations, and can give cause for subjects to reduce or discontinue chemotherapy.

[0036] As described herein, studies were conducted to examine whether the purified optically active compound of Formula II (i.e. , enantiomer 2) could ameliorate Oxaliplatin-induced pain in a rodent model. As shown in Fig. 1, repeated dosing p.o. with enantiomer 2 significantly reverses mechanical allodynia in oxaliplatin treated animals (similar efficacy to pregabalin). Mean plasma levels of about 5μM-13μM of enantiomer 2 corresponded to efficacy. Twice daily dosing was better than once daily dosing and may be due to more prolonged drug exposure. Low levels of enantiomer 2 found in the brain suggests a possible peripheral mechanism of action. Unlike pregabalin, enantiomer 2 had no sedating effects.

[0037] In another study described herein, depicted in Fig. 2A. enantiomer 2 was tested to determine if it could prophylactically prevent the induction of Oxaliplatin-induced allodynia in a rodent model. Oxaliplatin was administered on day 0 and Enantiomer 1 was dosed from day 0 to day 14. Neuropathic measurements were taken on day 2 / 3, day 5 / 6, and day 14. On day 5 / 6, animals treated with 25 mg / kg p.o. did not show a significant reduction in mechanical allodynia after oxaliplatin treatment. On day 15, animals treated with either dose of enantiomer 2 did not display significant mechanical allodynia compared to baseline. Prophylactic daily treatment with 25 mg / kg p.o. enantiomer 2 significantly reversed cold allodynia latencies on day 15 (similar to pregabalin). Prophylactic treatment with purified enantiomer 2 prevented the transient weight loss induced by oxaliplatin treatment in the vehicle or pregabalin groups suggesting a protective effect (Fig. 2C). Mean plasma levels of about 5 - 10 μM enantiomer 2 corresponded to analgesic efficacy (Fig. 2D).

[0038] Binding assays show that Enantiomer 2 has a tenfold selectivity for FABP5 over FABP3 and FABP7 (see, Table 10)

[0039] In another embodiment, the disclosure comprises the use of one or more compounds disclosed herein for the preparation of a medicament for the treatment of the diseases / conditions recited herein.

[0040] The compounds disclosed herein may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The active compounds and compositions, for example, may be administered orally, rectally, parenterally, or topically (e.g., intranasal or ophthalmic).

[0041] Other carrier materials and modes of administration known in the pharmaceutical art may also be used. Pharmaceutical compositions disclosed herein may be prepared by any of the well-known techniques of pharmacy, such as effective formulation and administration procedures. The above considerations in regard to effective formulations and administration procedures are well known in the art and are described in standard textbooks. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, Pa.. 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0042] The compounds disclosed herein can be used, alone or in combination with other therapeutic agents, in the treatment of various conditions or disease states. The compound(s) disclosed herein and other therapeutic agent(s) may be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially.

[0043] The administration of two or more compounds “in combination” means that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The two or more compounds may be administered simultaneously, concurrently or sequentially. Additionally, simultaneous administration may be carried out by mixing the compounds prior to administration or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration.

[0044] The phrases “concurrent administration,” “co-administration,” “simultaneous administration,” and “administered simultaneously” mean that the compounds are administered in combination.

[0045] In some embodiments enantiomer 1 or enantiomer 2 inhibit the activity of a Fatty Acid Binding Protein such as FABP3, FABP5 and / or FABP7, wherein the enantiomers inhibit binding of a FABP ligand to the FABP.

[0046] As used herein, the term "fatty acid binding protein" or “FABP” refers to fatty acid binding proteins (FABPs) that function as intracellular carriers that shuttle endocannabinoids (and by extension fatty acid amides (FAAs)) to FAAH where cannabinoids are hydrolyzed and degraded. Further, uptake of endocannabinoids (and by extension FAAs) by the cell and the subsequent hydrolysis of endocannabinoids (and by extension FAAs) are enhanced by FABPs, and inhibiting the interaction of endocannabinoids (and by extension FAAs) with FABPs reducesendocannabinoid (and by extension FAA) uptake and hydrolysis. FABPS include, for example, fatty acid binding protein 1 (FABP 1). fatty acid binding protein 2 (FABP 2). fatty acid binding protein 3 (FABP 3), fatty acid binding protein 4 (FABP 4), fatty acid binding protein 5 (FABP 5), fatty acid binding protein 6 (FABP 6), fatty acid binding protein 7 (FABP 7), fatty acid binding protein 8 (FABP 8), fatty' acid binding protein 9 (FABP 9), fatty' acid binding protein 10 (FABP 10), fatty acid binding protein 11 (FABP 11), fatty’ acid binding protein 5-like (FABP 5-like 1), fatty acid binding protein 5-like 2 (FABP 5-like 2), fatty acid binding protein 5-like 3 (FABP 5-like 3), fatty acid binding protein 5-like 4 (FABP 5-like 4), fatty' acid binding protein 5-like 5 (FABP 5- like 5), fatty' acid binding protein 5-like 6 (FABP 5-like 6), and fatty' acid binding protein 5-like 7 (FABP 5-like 7) (see Chmurzynska et al. 2006 and PCT International Application Publication No. WO 2010 / 083532 Al, the contents of each of which are hereby incorporated by reference).Treatment Indications

[0047] The optically active compounds of Formula I and II are useful for treating, ameliorating, or preventing, without limitation, a neurological disorder, such as without limitation, pain, neuropathic pain, inflammatory pain, cancer, and dermatological conditions, such as psoriasis.

[0048] In some embodiments the optically active compounds, enantiomer 1 or enantiomer 2, disclosed herein are useful for treating cancer. In some embodiments, the cancer treated is selected from prostate, breast, liver, brain and squamous cell carcinoma. In some embodiments the cancer treated is a metastatic cancer. In other embodiments the cancer treated is a drug resistant cancer.

[0049] In some embodiments, the compounds described herein are given in combination with other compounds, biologies, and other treatments known in the art and used in the treatment, amelioration, and prevention of cancers, neurological disorders, pain, neuropathic pain, inflammatory' pain, or dermatological conditions.

[0050] Enantiomer 2 reduces inflammation in human epidermis; increases antimicrobial peptides; reduces chemokines and cytokines; reduces JAK / STAT; reduces the psoriasis area and severity index scores (PASI) of imiquimod-induced skin inflammation in male Balb / c mice, reduces scaling and thickness, and reduces histopathological markers of damage.

[0051] For the treatment of the conditions referred to above, the compounds disclosed herein can be administered as compound per se.

[0052] Alternatively, pharmaceutically acceptable salts are suitable for medical applications because of their greater aqueous solubility relative to the parent compound.

[0053] In another embodiment, the present disclosure comprises pharmaceutical compositions. Such pharmaceutical compositions comprise a compound disclosed herein presented with a pharmaceutically acceptable carrier. The carrier can be a solid, a liquid, or both, and may be formulated with the compound as a unit-dose composition, for example, a tablet, which can contain from 0.05% to 95% by weight of the active compounds. A compound disclosed herein may be coupled with suitable polymers as targetable drug carriers. Other pharmacologically active substances can also be present.Formulations

[0054] In another embodiment, the present disclosure comprises the use of one or more of the optically active compounds of Formula I or Formula II for the preparation of a medicament for the treatment of the conditions recited herein.

[0055] The compounds disclosed herein may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed, by which the compound enters the blood stream directly from the mouth.

[0056] Oral administration of a solid dose form may be. for example, presented in discrete units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the present disclosure. In another embodiment, the oral administration may be in a powder or granule form. In another embodiment, the oral dose form is sub-lingual, such as, for example, a lozenge. In such solid dosage forms, the compounds of the present disclosure are ordinarily combined with one or more adjuvants. Such capsules or tablets may contain a controlled-release formulation. In the case of capsules, tablets, and pills, the dosage forms also may comprise buffering agents or may be prepared with enteric coatings.

[0057] In another embodiment, oral administration may be in a liquid dose form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (e.g., water). Such compositions also may comprise adjuvants, such as wetting, emulsifying, suspending, flavoring (e.g., sweetening), and / or perfuming agents.

[0058] In another embodiment, the compounds of the disclosure may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrastemal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

[0059] In another embodiment, the present disclosure comprises a parenteral dose form. “Parenteral administration" includes, for example, subcutaneous injections, intravenous injections, intraperitoneal injections, intramuscular injections, intracistemal injections, and infusion. Injectable preparations (i.e., sterile injectable aqueous or oleaginous suspensions) may be formulated according to the known art using suitable dispersing, wetting, and / or suspending agents, and include depot formulations.

[0060] In another embodiment, the compounds disclosed herein may also be formulated as a topical dose form such that administration topically to the skin or mucosa (i.e., dermally or transdermally) leads to systemic absorption of the compound. “Topical administration” includes, for example, transdermal administration, such as via transdermal patches or iontophoresis devices, intraocular administration, or intranasal or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments, and creams. A topical formulation may include a compound that enhances absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of this disclosure are administered by a transdermal device, administration will be accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated — see, for example, Finnin and Morgan, J. Pharm. Sci., 88 (10), 955-958 (1999).

[0061] Formulations suitable for topical administration to the eye include, for example, eye drops wherein the compound of this disclosure is dissolved or suspended in a suitable carrier. A typical formulation suitable for ocular or aural administration may be in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

[0062] For intranasal administration or administration by inhalation, the active compounds of the disclosure are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant. Formulations suitable for intranasal administration are ty pically administered in the form of a dry powder (either alone; as a mixture, for example, in a dry blend with lactose; or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electro-hydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1, 1,2, 3, 3, 3- heptafluoropropane. For intranasal use, the powder may comprise a bio-adhesive agent, for example, chitosan or cyclodextrin.

[0063] In another embodiment, the present disclosure comprises a rectal dose form. Such rectal dose form may be in the form of, for example, a suppository . Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

[0064] In another embodiment, the compounds of the disclosure may be formulated such that administration vaginally leads to systemic absorption of the compound.

[0065] The dosage regimen for the compounds and / or compositions containing the compounds is based on a variety of factors, including the type, age, weight, sex, and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the compound employed. Thus, the dosage regimen may vary widely. Dosage levels of the order from about 0.01 mg to about 100 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions. In one embodiment, the total daily dose of a compound disclosed herein (administered in single or divided doses) is ty pically from about 0.01 to about 100 mg / kg. In another embodiment, the total daily dose of a compound disclosed herein is from about 0. 1 to about 50 mg / kg, and in another embodiment, from about 0.5 to about 30 mg / kg (i.e., mg compound of the disclosure per kg body weight). In one embodiment, dosing is from 0.01 to 10 mg / kg / day . In another embodiment, dosing is from 0. 1 to 1.0 mg / kg / day. Dosage unit compositions may contain such amounts or submultiples thereof to make up the daily dose. In many instances, the administration of the compound will be repeated a plurality of times in a day (typically no greater than 4 times). Multiple doses per day typically may be used to increase the total daily dose, if desired.

[0066] For oral administration, the compositions may be provided in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 75.0, 100, 125, 150, 175, 200, 250 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage tothe patient. A medicament ty pically contains from about 0.01 mg to about 500 mg of the active ingredient, or in another embodiment, from about 1 mg to about 100 mg of the active ingredient. Intravenously, doses may range from about 0. 1 to about 10 mg / kg / minute during a constant rate infusion.

[0067] Oral administration of a solid dose form may be, for example, presented in discrete units, such as hard or soft capsules, pills, cachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the present disclosure. In another embodiment, the oral administration may be in a powder or granule form. In another embodiment, the oral dose form is sub-lingual, such as, for example, a lozenge. In such solid dosage forms, the compounds of the present disclosure are ordinarily combined with one or more adjuvants. Such capsules or tablets may contain a controlled-release formulation. In the case of capsules, tablets, and pills, the dosage forms also may comprise buffering agents or may be prepared with enteric coatings.

[0068] Suitable subjects / patients according to the present disclosure include mammalian subjects. Mammals according to the present disclosure include, but are not limited to, canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, and the like, and encompass mammals in utero. In one embodiment, humans are suitable subjects. Human subjects may be of either gender and at any stage of development. Definitions and Examples:

[0069] As used throughout this application, including the claims, the following terms have the meanings defined below, unless specifically indicated otherwise. The plural and singular should be treated as interchangeable, other than the indication of number:

[0070] The embodiments disclosed herein are also meant to encompass all pharmaceutically acceptable compounds of enantiomer 1 and enantiomer 2 including isotopically labeled compounds in which one or more atoms can be replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2H, 3H, 13C, 14C, 13N, 15N, 150, 170, 180, 31P, 32P, 35S, 18F, 36C1, 1231, and 1251. These radiolabeled compounds could be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically- labeled compounds of enantiomer 1 and enantiomer 2 for example, those incorporating a radioactive isotope, may be useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.. 3H. and carbon-14, i.e., 14C, may particularly be useful for this purpose in view of their ease of incorporation and ready means of detection.

[0071] Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability. For example, in vivo half-life may increase, or dosage requirements may be reduced. Thus, heavier isotopes may be preferred in some circumstances.

[0072] Substitution with positron emitting isotopes, such as C, F, O and N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds of Enantiomer 1 and Enantiomer 2 can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Examples as set out below using an appropriate isotopically labeled reagent in place of the non-labeled reagent previously employed.

[0073] The methods, compositions, kits and articles of manufacture provided herein use or include the compounds of Formula I, or Formula II, or pharmaceutically acceptable salts, prodrugs, or solvates thereof, in which from 1 to n hydrogen atoms attached to a carbon atom may be replaced by a deuterium atom or D, where "n" is the number of hydrogen atoms in the molecule. As known in the art, the deuterium atom is a non-radioactive isotope of the hydrogen atom. Such compounds may increase resistance to metabolism, and thus may be useful for increasing the half- life of compounds or pharmaceutically acceptable salts, prodrugs, or solvates thereof, when administered to a mammal. See, e.g., Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism" , Trends Pharmacol. Sci., 5(12):524-527 (1984). Such compounds are synthesized by- means well known in the art, for example, by employing starting materials in which one or more hydrogen atoms have been replaced by deuterium.

[0074] The embodiments disclosed herein are also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the embodiments disclosed herein include compounds produced by a process comprising administering a compound according to the embodiments disclosed herein to a mammal for a duration sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound according to the embodiments disclosed herein in a detectable dose to an animal, such as rat. mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood, or other biological samples. “Stable compound" and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0075] ‘‘Mammal” includes humans and both domestic animals such as laboratory' animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and nondomestic animals such as wildlife and the like.

[0076] “Pharmaceutically acceptable excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0077] "Pharmaceutically acceptable salt" refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2 -hydroxy ethanesulfonic acid, benzenesulfonic acid, 4- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N methylglucamine and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality', salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. The term "pharmaceutically acceptable cation" refers to an acceptable cationic counter-ion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like. See, e.g., Berge, et al., J. Pharm. Sci. (1977) 66(1): 1-79.

[0078] For therapeutic use, salts of active ingredients of the compounds disclosed herein will typically be pharmaceutically acceptable, i.e., they will be salts derived from a physiologically acceptable acid or base. However, salts of acids or bases which are notpharmaceutically acceptable may also find use, for example, in the preparation or purification of enantiomer 1 and enantiomer 2 or another compound of the embodiments disclosed herein. All salts, whether or not they are derived from a physiologically acceptable acid or base, are within the scope of the embodiments disclosed herein.

[0079] In some embodiments, the pharmaceutically acceptable salt of enantiomer 1 or enantiomer 2 is ethylenediamine salt, or diethanolamine salt

[0080] Often crystallizations produce a solvate of a compound of the embodiments disclosed herein. As used herein, the term “solvate” refers to an aggregate that comprises one or more molecules of a compound of the embodiments disclosed herein with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the embodiments disclosed herein may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compounds of the embodiments disclosed herein may be true solvates, while in other cases, a compound of the embodiments disclosed herein may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

[0081] Also, within the scope of the present disclosure are so-called “prodrugs” of the compounds disclosed herein. Thus, certain derivatives of the compounds disclosed herein that may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into the compounds of the disclosure having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as “prodrugs.” Further information on the use of prodrugs may be found in “Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and “Bioreversible Carriers in Drug Design,” Pergamon Press. 1987 (ed. E. B. Roche. American Pharmaceutical Association). Prodrugs in accordance with the disclosure can, for example, be produced by replacing appropriate functionalities present in the compounds of the present disclosure with certain moieties known to those skilled in the art as “pro- moieties” as described, for example, in “Design of Prodrugs” by H. Bundgaard (Elsevier, 1985).

[0082] A “pharmaceutical composition” refers to a formulation of a compound of the embodiments disclosed herein, and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e g., humans. Such a medium includes all pharmaceutically acceptable excipients. “Effective amount” or “therapeutically effective amount” refers to an amount of a compound according to the embodiments disclosed herein, which when administered to a patient in need thereof, is sufficient to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by aresearcher or clinician. The amount of a compound according to the embodiments disclosed herein which constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of the treatment, the type of disease-state or disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds of the embodiments disclosed herein, and the age, body weight, general health, sex and diet of the patient. Such a therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure.

[0083] ‘‘Effective amount’' or “therapeutically effective amount” refers to an amount of a compound according to the embodiments disclosed herein, which when administered to a patient in need thereof, is sufficient to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by a researcher or clinician. The amount of a compound according to the embodiments disclosed herein which constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of the treatment, the type of disease-state or disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds of the embodiments disclosed herein, and the age, body weight, general health, sex and diet of the patient. Such a therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure.

[0084] The term “treatment” as used herein is intended to mean the administration of a compound or composition according to the present embodiments disclosed hereinto alleviate or eliminate symptoms of the conditions described herein.

[0085] The compounds of the embodiments disclosed herein, or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-. or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable opticallypure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherw ise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0086] A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0087] A “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any said compounds.EXAMPLESAnalytical MethodsChiral HPLC method used to separate racemic Compound I is shown in Table 1 below:Table 1ART~1621 Chiral Purity by HPLC CHIRAL PURITY BY HPLCReaction Scheme:

[0088] Chiral resolution screening of racemic-3-(2,3-dihydro-1H-inden-2- yloxycarbonyl)-2,4-di(2-methoxyphenyl)cyclobutane-l -carboxylic acid (Compound I)Compound I was performed with 13 chiral amines (Table 2) and 11 solvents on a 50 mg scale. The initial screening was carried out using 0.5 eq of each chiral amine and 10 vol of each solvent. Only 3 systems produced a salt with an enrichment in chiral purity. (A)-(-)-2-Phenylglycinol produced salts where the organic group in the organoammonium moiety had the desired chirality and enriched in the desired enantiomer, that is, Enantiomer 1, having the R configuration. When (S )-(- )-l-(4-bromophenyl)ethylamine was used as the resolving agent, the enantiomer having opposite chirality, i.e.. Enantiomer 2, having S configuration was obtained, however, its chiral purity was low. In some cases, the racemic acid (starting material) precipitated out of solution withoutforming the corresponding salt. When the chiral resolution was attempted using 0.5 eq. of the chiral amine, in a majority of cases, suspensions resulted where the predominant component was only the racemic carboxylic acid, not the salt form.

[0089] Table 2 Chiral amines used in the screening tests.

[0090] Due to the low number of salts formed, the screening was then repeated with 1.0 eq of the chiral amine and varying the solvent. The results from these experiments led to the identification of six systems that afforded the salt form where the organoammonium group of the salt form had an enrichment in optical purity, i.e., high enantiomeric excess values (% ee). These are shown in Table 2. The screening runs revealed that use of (R)-(-)-2-phenylglycinol as the chiral resolving agent generally provided the salt form where the organoammonium group was enriched in the desired R-configuration. Interestingly, use of (S)-(-)-l-(4-bromophenyl)ethylamine as the chiral resolving agent in acetonitrile solvent resulted in formation of the salt form w here the organoammonium group w as enriched in the S-configuration, i.e., opposite to what was seen with (R)-(-)-2-phenylglycinol as the chiral resolving agent. Very high optical enrichment (high % enantiomeric excess (ee) values) was obtained from the salts formed using 1.0 eq of (R )-(-)-2-phenylglycinol in ethyl acetate or isopropyl acetate as the solvent.

[0091] Table 3 - Chiral resolution of Compound 1 using 1.0 equivalent of a chiral amine.pureSalt form of optically active compound of Formula I (Enantiomer 1 Salt):

[0092] The purified salt form of Enantiomer 1 was prepared for use as seed in scaled-up reactions.

[0093] 100 mg of pure Enantiomer 1 (99.6% e.e.) was reacted with (R)-(-)-2-Phenylglycinol (1.0 eq) in ethyl acetate from which 52 mg (40% yield) of the desired salt form of Enantiomer was isolated as the product. The salt had an e.e. of 99.52%.

[0094] Scaled up preparation of the salt form of optically active compound of Formula I (Enantiomer 1 Salt):

[0095] It was found from the 50 mg screening reactions that (R)-(-)-2-phenylglycinol was the only chiral amine to form a salt with Compound I, which led to isolation of the desired Enantiomer 1 of Formula I in high enantiomeric excess. Also, use of the acetate solvents, EtOAc. i-PrOAc, or n-BuOAc gave the salt form in high e.e. ’s and recoveries of greater than 14 mg. Therefore, these conditions were used in the scaled-up reaction using 500 mg of Compound I and using both 0.5 eq. and 1.0 eq of the amine and 5 vol of solvent. If no suspension formed after being heated to 80°C and then cooled to room temperature, the salt of pure Enantiomer 1, obtained as described above, was used as seed material to aid in precipitating the desired product from solution.

[0096] The above procedure was repeated, but with the following modification. After heating to 80°C and stirring, the batch was cooled to 50°C and seeded with the pure Enantiomer 1 salt. If a suspension did not form, the batch was re-heated to 50°C and seeded again with the pure Enantiomer 1 salt to aid in the precipitation. The Tables 3-6 below summarize the results.

[0097] Table 4. Using 0.5 eq of (R)-(-)-2-phenylglycinol.

[0098] Table 5. Using 1.0 eq of (R)-(-)-2-phenylglycinol.* = seeded if no suspension formed

[0099] Table 6. Using 0.5 eq of (R)-(-)-2-phenylglycinol.

[0100] Table 7. Using 1.0 eq of (R)-(-)-2-phenylglycinol** = ** seeded twice

[0101] The enantiomeric excess values were lower than expected in the 500 mg scale reactions as compared to the 50 mg trials. 5 vol of solvent was sufficient on a 50 mg scale, however, in the 500 mg scale reactions, the material afforded a thick precipitate. It is believed that this led to inefficient mixing, and therefore, filtration and washing led to isolated solids havinglower chiral purity. For this reason, in subsequent scaled-up reactions, a higher volume of solvent was used to improve the stability, consistency and isolation of the product.Reactions conducted on 5 g and 20 g Scale:

[0102] Ethyl acetate was chosen as the solvent for the 5 g scale reaction, because overall it gave the salt form of Enantiomer 1 having the best chiral purity. (R)-(-)-2-phenylglycinol (1.0 eq) and the racemic Compound I (5 g) were reacted in EtOAc (8 vol), heated to 50°C, seeded, and cooled to room temperature. 2.47 g (76% yield) of the salt product having an ee of 89.74% was isolated. To improve the % ee, 1 g of the Enantiomer 1 salt was slurried in EtOAc (8 vol), which gave a recovery of 640 mg (64% recovery) and an improved ee of 99.62%.

[0103] The slurry in EtOAc produced quite a low yield, so 1 g of the original Enantiomer 1 salt was slurried in iPrOAc. This gave a similar recovery of 66% (657 mg) and an ee of 99.5%. As iPrOAc did not provide a significant increase in yield over EtOAc. the latter was used in future reactions with a lower isolation temperature to improve the yield.

[0104] The salt formation reaction was then further scaled up to 20 g. scale. 10. 14 g (78% yield, 91 .14% ee) of the Enantiomer 1 salt was isolated at 20°C. NMR analysis of the isolated salt confirmed the presence of the amine and the carboxylic acid components in a 0.99: 1 equiv ratio, i.e., essentially 1: 1 ratio. The material was then slurried in EtOAc (8 vol) and the slurry was cooled to 0-5°C from which 9.39 g (73% yield, 99.3% ee) of the desired salt form of Enantiomer 1 was isolated.

[0105] Conversion of the salt form of Enantiomer 1 to the free chiral carboxylic acid form.

[0106] After the synthesis of the salt form of the Enantiomer 1 in high yield and % ee, a salt break procedure was developed to isolate Enantiomer 1, i.e., the free carboxylic acid form (see reaction above). The salt break procedure was originally tried on a 100 mg scale. A salt from the previous 500 mg trials having 61.9% ee was treated with IM HC1 (10 vol) in EtOAc (10 vol) toafford the free acid. This process gave the free acid in a yield of 77 mg (96% yield) and a % ee of 64.12%.

[0107] The procedure was then scaled up using the salt from the 20 g batch (99.3% ee) described previously. 5 g of the salt was reacted with 8 vol of IM HC1 to give 3.45 g (65% yield overall) of enantiomer 1 having a % ee of 99.54%.Intermediate scale run:

[0108] The procedure established as previously developed was used to further scale-up the formation and isolation of the (R)-(-)-2-phenylglycinol salt with Enantiomer 1. 200 g of Enantiomer 1 was charged to a 5 L jacketed vessel with (R)-(-)-2-phenylglycinol (1.0 eq.) and EtOAc (8 vol). The batch was heated to 50°C and seeded with purified Enantiomer 1 salt. At this stage of the process, in the previous scale-up reactions, both reagents dissolved in EtOAc. However, in this batch, a brown cloudy solution was observed. The batch was agitated for 50 minutes at 50°C, and then cooled to room temperature, and stirred overnight. The brown cloudy solution persisted, with no white suspension being formed.

[0109] NMR and HPLC analysis were taken of the batch, starting materials, and isolated solid from the batch. The starting matenals were as expected; how ever, it was found that the solid formed in the batch was an acid salt of phenylglycinol, not the target active pharmaceutical ingredient (API) salt.

[0110] Due to the issues encountered, it was decided to re-isolate Enantiomer 1 as the free acid. A salt break procedure was then undertaken using IM HC1 (8 vol) to isolate 145 g of Enantiomer 1. This yield was much lower than the expected 200 g recovery, therefore analysis of the aqueous layers w as performed. This only showed trace levels of the API related material Trial Reaction:

[0111] Because of the issues with the first batch as described above, a 20 g trial reaction using the re-processed racemic Compound I was earned out. The same method as described previously w as used. The reaction was heated to 50°C, and the two reagents w ent into solution as expected (no haziness observed). The batch w as seeded, agitated, and cooled to RT. The procedure worked as expected, forming a white suspension. After filtration, NMR analysis confirmed the salt had formed. An ethyl acetate slurry of the salt form was prepared, followed by the salt break step with IM HC1, to give 5.64 g (56%) of Enantiomer 1 having a % ee of 99.68%. NMR analysis confirmed that the isolated product w as the desired Enantiomer 1, the chiral free acid. The yield for the overall process w as slightly lower than the initial trial (56% compared to 65%) but still deemed acceptable for further scale-up.Scale-up reaction:

[0112] 126 g of racemic Compound I was reacted with (R )-(-)-2-phenylglycinol (1.0 eq.) in EtOAc (8 vol). Both reagents went into solution as expected, and upon cooling, an off- white suspension formed from which 51.8 g (63% yield) of the salt was isolated, with NMR analysis confirming the identity of the salt form. The solid was then slurried in EtOAc, which resulted in 55.7 g of the salt in solvent-wet form. Analysis by chiral HPLC showed a % ee of 99.24% for the first batch and 99.84% for the re-slurried material. The yield for this batch was lower than that obtained in the development (76-78%) run, however, the initial chiral purity was significantly higher (% e.e. of 99. 1% as compared to 91%). This suggests that the second EtOAc slurry may not be necessary and that an IPC for chiral purity should be introduced before the slurry to confirm whether it is necessary.

[0113] The salt break procedure was then performed using IM HC1 (8 vol) and EtOAc (8 vol). After being dried and concentrated in vacuo, the solid was slurried in heptane (20 vol) to form a cry stalline solid. 26.0 g of enantiomer 1 from Compound I was isolated (41% yield), with a % ee of 99.82%. The slightly lower than expected yield was due to some mechanical losses incurred during vacuum release in the oven-drying step. Some material from the oven was recovered, dissolved in EtOAc, filtered, and concentrated to give an additional 3.6 g. The product from the main batch (26 g), the test batch (5.64 g) and the material recovered from the oven (3.6 g) were blended. They were dissolved in EtOAc (4 vol), combined and concentrated. The batch was slurried in heptane (20 vol) to give 34.8 g of the free acid as a white crystalline solid having a % ee of 99.66 %.Stress Tests: Several stress tests were carried out at different stages of the procedure. The results from these are shown in Table 8 below.Table 8Key: S = solid, L = liquor, A = aqueous. O = organicProcess Experimentals.Stage 1.

[0114] To a 5 L jacketed vessel under nitrogen was added racemic Compound I (126 g, 0.27 mol), (R)-(-)-2-phenylglycinol (36.6 g, 0.27 mol) and ethyl acetate (1008 mL, 8 vol). The batch was heated to 50°C, seeded with the pure enantiomer 1 salt (126 mg, 0.27 mmol) and stirred for 30 minutes. Racemic Compound I went into solution to form a brown, transparent solution. The batch was slowly cooled to room temperature (RT) and stirred overnight. A white suspension formed which was filtered and washed with ethyl acetate (252 mL, 2 vol) to form a white solid. Recovery and yield: 51.8 g, 0.084 mol, 62%. NMR analysis confirmed salt formation. Chiral HPLC: 99.24% ee.

[0115] The solid was re-slurried in ethyl acetate (1008 mL, 8 vol) and heated to 50°C and stirred for 30 minutes. The batch was cooled to 0-5°C and stirred for another 30 minutes. It was filtered and washed with cold (0-5°C) ethyl acetate (252 mL, 2 vol) to give a white solid. Recovery: 55.7 g, (5% EtOAc) Chiral HPLC: 99.82 % ee.Stage 2:

[0116] The stage 1 salt (55.7 g, 0.091 mol), ethyl acetate (1008 mL, 8 vol) and IM HC1 (1008 mL, 8 vol) were charged to the vessel and stirred at RT for 10 minutes. The layers were separated, and the organic layer washed with IM HC1 (630 mL, 5 vol) and H2O (630 mL, 5 vol). The organic layer was dried with MgSO4, filtered, and concentrated in vacuo. The product was slurried in heptane (20 vol) for 1 hour to afford a crystalline white solid. Recovery and yield: 26 g, 41%. NMR analysis: >95%. Chiral HPLC: 99.82% ee. XRPD data showed that the material was crystalline.

[0117] The product from this batch (26.0 g). the test batch (5.64 g) and the material recovered from the oven (3.6 g) were blended by dissolving in EtOAc (4 vol). They were combined, concentrated and then slurried in heptane (20 vol) to afford a white solid. Recovery: 34.8 g. NMR analysis: >95%. Chiral HPLC: 99.66 % ee. XRPD data showed that the material was crystalline.Analytical Methods.Optical Properties of enantiomers of Compound 1.

[0118] Optical rotation was determined using a Rudolph Research Analytical Autopol I Automatic Polarimeter. The sample cell was washed with acetone (~ 7 mL) and air-dried before use. Acetone was used as the blank sample to calibrate the background signals at 589nm. The cell was emptied and dried before adding the completely dissolved sample solution (~10mg in 7 mL), fulfilling the cylinder region of the chamber. The optical rotation was measured at room temperature (20°C).

[0119] The optical rotation of Enantiomer 1 and Enantiomer 2 were determined by a polarimeter. The specific rotation was calculated based on the corresponding solution concentration. Enantiomer 1 was determined as (+)-a-3-(2.3-dihydro-1H-inden-2-yloxycarbonyl)- 2,4-di(2-methoxyphenyl)cyclobutane-l-carboxylic acid, while enantiomer 2 was determined as (-)- a-3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2-methoxyphenyl)cyclobutane-l -carboxylic acid (Table 9).Table 9. The optical rotations of Enantiomer 1 and Enantiomer 2. The values represent an average ±S.E. of at least three independent experiments.Optical rotation values representan average +S.E. of at least three independent experiments.Biological Properties:Affinity (Ki) of (±)-a-3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2,4-di(2- methoxyphenyl)cyclobutane-l-carboxylic acid enantiomers and racemate to FABP3, FABP5 and FABP7.

[0120] Enantiomer 1, Enantiomer 2. and Compound I, were subjected to a fluorescence displacement assay to determine the in vitro binding affinity (Ki) of these compounds to FABP3, FABP5 and FABP7 (Table 10). Enantiomer 1 was found to have higher affinities to FABPs than Enantiomer 2Table 10. Affinity of Enantiomer 1 and Enantiomer 2 towards FAB3, FAB 5, and FAB 7 (Ki,gM).Ki values represent an average ± S.E. of at least three independent experiments.Example 2- Determination of Single-Crystal X-Ray Structure: Formula I and Formula II:

[0121] Samples labeled RH-0210-031-S4 (enantiomer II) or RH-0210-031-S5(enantiomer I) that contained crystalline material in heptane / ethyl acetate were obtained. In each case, the crystalline material was transferred to a drop of inert poly fluoroether oil and a selected crystal was stuck on a MiTeGen crystal mount using the same oil. The crystal from RH-0210-031- S4 was plate-like (dimensions 0. 12 x 0. 12 x 0.03 mm) and the crystal from RH-0210-031-S5 was needle-like (dimensions 0.22 x 0.06 x 0.06 mm).

[0122] An analyses was carried out under an Oxford Cryosyslems open-flow N2 Cryostream operating at 180(2) K. X-ray data were collected on a Bruker D8-QUEST diffractometer, equipped with an Incoatec IμS Cu microsource (γ = 1.5418 A) and a PHOTON-III detector operating in shutterless mode. The control and processing software was Bruker APEX4 (ver. 2022.1-1). Lattice parameters were determined from a 180°scan around the Φ axis, with 1° image exposures. The Bravais lattice (C) and unit cell were selected on the basis of the initial scan, and the data collection strategy was set up assuming a non-centrosymmetric point group (2) for the diffracted intensities. Image exposure times were variable for different detector positions, with a maximum of 12 sec / image at the highest diffraction angles. The diffraction images w ere integrated using SAINT within APEX4 to a resolution of 0.84 A, and a multi-scan correction was applied using SADABS. In both cases, the crystals diffracted strongly and the data quality was good.Structure solution and refinement (including absolute structure determination:

[0123] The structures were solved using SHELXT and refined using SHELXL-2019 / 1. Summary tables of the cry stal data and structure refinement information are given below. All nonFI atoms of the main molecules were refined with anisotropic displacement parameters. H atoms bound to C atoms were placed in idealized positions and allowed to ride on their parent C atoms with C-H = 0.95-1.00 A, Uiso(H)= 1.2 or 1.5 Ueq(C). Both cry stals produced effectively the same result.

[0124] The principal interest was to determine of the absolute structure. This was challenging because the structure contains only light CHNO atoms, which means that the anomalous scattering signal required to determine the absolute structure from the X-ray data is weak. The Flack parameter is obtained using the quotient method of Parsons [S. Parsons, H. D. Flack, T. Wagner, Acta Cryst. (2013), B69, 249-259], as implemented in SHELXL. The results were:RH-0210-031-S5: Flack parameter = -0.02(10) RH-0210-031-S4: Flack parameter = 0.08(12)

[0125] In both cases, the value of the Flack parameter is zero within one standard uncertainty, which suggests that the assigned absolute structure is correct. Both samples produce the same absolute structure. The confidence in each result is indicated by the associated standard uncertainty. For a crystal that is known to be enantiopure, a common guideline for satisfactory determination of the absolute structure is that the standard uncertainty should be < 0. 1 [H. D. Flack, G. Bemardinelli, J. Appl. Cryst. (2000), 33, 1143-1148], Hence, both structure determinations are individually at the borderline of a satisfactory absolute structure determination. However, the fact that tw o separate structure determinations from two different cry stal batches produced the same result, in each case at the borderline of a satisfactory individual result, adds confidence to the overall conclusion. The collective evidence strongly suggests that the absolute structure determination is correct.Molecular structure and absolute configuration:

[0126] Tables of bond distances and angles are given in the Appendices. The crystal structure contains two independent molecules in the asymmetric unit (not related by any symmetry’ operators of the space group), with atom labels given suffix “A” or "‘B” (Fig. 3). The two independent molecules have the same absolute configuration. According to the core Cahn-Ingold- Prelog (CIP) rules, atoms Cl and C3 have two attached groups with identical priority' (due to identical connectivity’), so it is not clear that R / S descriptors can be applied to these atoms. Atoms C2 and C4 can be described using core CIP rules: both have the R configuration. The conformations of molecules A and B are different, with the substituent groups adopting different orientations relative to the cyclobutane core (Fig. 4).

[0127] Table Al shows the cry stal data and structure refinement information.Table AlTable A2. Bond lengths [A] (values from enantiomer 1; enantiomer 2 is essentially identical)Table A3. Bond angles [°] (values from enantiomer 2; enantiomer 1 is essentially identical)Table A4. Torsion angles [°] (values from enantiomer 2; enantiomer 1 is essentially identical)Table A4 (Continued). Torsion angles [°] (values from enantiomer 2; enantiomer 1 is essentially identical)

Claims

WHAT IS CLAIMED IS:

1. An optically active compound having Formula I or Formula II:Formula I Formula II.

2. The optically active compound of Claim 1, wherein the compound of Formula I has an enantiomeric excess of at least 50%.

3. The optically active compound of Claim 1, wherein the compound of Formula I has an enantiomeric excess of 50% - 80%.

4. The optically active compound of Claim 1 , wherein the compound of Formula I has an enantiomeric excess of 70% - 100%.

5. The optically active compound of Claim 1, wherein the compound of Formula II has an enantiomeric excess of at least 50%.

6. The optically active compound of Claim 1 , wherein the compound of Formula II has an enantiomeric excess of 50% - 80%.

7. The optically active compound of Claim 1, wherein the compound of Formula II has an enantiomeric excess of 70% - 100%.

8. The optically active compound of Claim 1. wherein the compound of Formula I is dextrorotatory.

9. The optically active compound of Claim 1, wherein the compound of Formula II is levorotatory.

10. A pharmaceutical composition comprising the optically active compound of Formula I, or Formula II, or a pharmaceutically acceptable salt, ester, prodrug, hydrate, or tautomer thereof.

11. A method for preparing an optically active compound, the method comprising: contacting racemic 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2.4-di(2- methoxyphenyl)cy cl obutene-1 -carboxylic acid and a chiral amine in a solvent to form a salt. isolating the salt, and treating the salt with aqueous acid to form the optically active compound, wherein the optically active compound has Formula IFormula I.

12. The method of Claim 11, further comprising treating the filtrate obtained from the step of isolating the salt with aqueous acid to form the optically active compound having Formula IIFormula II.

13. The method of Claim 11, wherein the chiral amine is selected from the group consisting of (S)-(-)-l -phenylethylamine, (S)-l-(4-methoxyphenyl)ethylamine. (S)-(-)-l-(4- bromophenyl)ethylamine, (S)-(-)-N,N-Dimethyl-l -phenyl ethyl amine. (-)-bis[(S)-l- phenylethyl] amine, d-valinol, d-alaninol, (R)-l-aminopropan-2-ol, quinine, (-)-cinchonidine, (D)-(- )-l-(l-naphthyl)ethylamine, (R)-(-)-2-phenylglycinol, L-phenylalaninol, and combinations thereof.

14. The method of Claim 11. wherein the chiral amine is (R)-(-)-2-phenylglycinol.

15. The method of Claim 1 1. wherein the solvent comprises a liquid alkyl acetate.

16. The method of Claim 13, wherein the alkyl acetate is selected from the group consisting of methyl acetate, ethyl acetate, iso-propyl acetate, n-butyl acetate, and combinations thereof.

17. The method of Claim 11, wherein the optically active compound of Formula I has an enantiomeric excess of at least 50%.

18. The method of Claim 11, wherein the optically active compound of Formula I has an enantiomeric excess of at least 50% - 80%.

19. The method of Claim 11, wherein the optically active compound of Formula I has an enantiomeric excess of at least 70% - 100%.

20. The method of Claim 11, wherein the optically active compound of Formula I has an enantiomeric excess of at least 50%.

21. The method of Claim 11, wherein the optically active compound of Formula I has an enantiomeric excess of at least 50% - 80%.

22. The method of Claim 11, wherein the optically active compound of Formula I has an enantiomeric excess of at least 70% - 100%.

23. An optically active compound obtained from racemic 3-(2.3-dihydro-1H-inden-2- yloxycarbonyl)-2,4-di(2-methoxyphenyl)cyclobutene-l -carboxylic acid by a process, the process comprising: contacting racemic 3-(2,3-dihydro-1H-inden-2-yloxycarbonyl)-2.4-di(2- methoxyphenyl)cyclobutene-l-carboxylic acid and a chiral amine in a solvent to form a salt. isolating the salt, and treating the salt with aqueous acid to form the optically active compound, wherein the optically active compound has Formula IFormula I.

24. A method of treating cancer in a subject in need of treatment, the method comprising administering the pharmaceutical composition of Claim 10 to the subject.

25. A method of treating pain in a subject in need of treatment, the method comprising administering the pharmaceutical composition of Claim 10 to the subject.

26. The method of claim 25, wherein the pain is caused by chemotherapy -induced neuropathy.

27. The method of claim 25, wherein the pain is caused by osteoarthritis.

28. A method of treating skin disorders in a subject in need of treatment, the method comprising administering the pharmaceutical composition of Claim 10 to the subject.

29. The method of claim 28. wherein the skin disorder treated is selected from one or more of radiation-induced skin damage, atopic dermatitis, atopic march, UV -induced skin damage, and skin lesions.

29. A method of treating psoriasis in a subject in need of treatment, the method comprising administering the pharmaceutical composition of Claim 10 to the subject.

30. A method of treating a dermatological condition in a subject in need of treatment, the method comprising administering the pharmaceutical composition of claim 10 to the subject.

31. A method of treating arthritis in a subject in need of treatment, the method comprising administering the pharmaceutical composition of claim 10 to the subject.

32. The method of claim 31. wherein the arthritis is selected from one or more of psoriatic arthritis, osteoarthritis.

33. A method of treating a metabolic disorder in a subject in need of treatment, the method comprising administering the pharmaceutical composition of claim 10 to the subject.

34. The method of claim 33, wherein the metabolic disorder is atherosclerosis.

35. A method of treating a neurologic disorder in a subject in need of treatment, the method comprising administering the pharmaceutical composition of claim 10 to the subject.

36. The method of claim 35, wherein the neurological is selected from one or more of anxiety, PTSD, multiple sclerosis, Parkinson's disease and glaucoma.

37. A method of treating a spinal cord injury in a subject in need of treatment, the method comprising administering the pharmaceutical composition of claim 10 to the subject.