US00885955OB2

(12) United States Patent (10) Patent No.: US 8,859,550 B2 Chen et al. (45) Date of Patent: *Oct. 14, 2014

(54) HETEROCYCLIC INHIBITORS OF FOREIGN PATENT DOCUMENTS RECEPTORS FOR THE TREATMENT OF DISEASE Wo 2004:36 A 358 (75) Inventors: Pingyun Chen, Chapel Hill, NC (US); W. SS: A. 33. Stephen A. R. Carino, Del Mar, CA WO 2009098320 A2 8, 2009 (US); Ricky Wayne Couch, Durham, WO 2010030785 A2 3, 2010 NC (US); Daniel S. Kinder, Del Mar, W. 38H 73 A. 338 sS); Beth A. Norton, Del Mar, CA WO 20110894.00 A1 7, 2011 (73) Assignee: Kalypsys, Inc., Del Mar, CA (US) OTHER PUBLICATIONS c Khankari et al. Thermochimica Acta 1995, 248, 61-79. (*) Notice: Subject to any disclaimer, the term of this Conalty, M.L. et akl., “Anticancer agents—XII. Pyridazine and patent is extended or adjusted under 35 Benzodiazine derivatives.” Proceedings of the Royal Irish Academy, U.S.C. 154(b) by O days. Section B: Biological, Geological and Chemical Science (1976), 76(10), 151-63. This patent is Subject to a terminal dis- Kim, HS et al., “Synthesis of tetrazolo 1.5-aquinoxalines with anti Ca10. microbial activity,” Journal of the Korean Chemical Society (2001), 45(4), 325-333. (21) Appl. No.: 13/606.947 Smits R. A. et al., “Major advances in the developments of histamine H4 ligands.” Discovery Today, vol. 14, No. 15/16, Aug. (22) Filed: Sep. 7, 2012 2009. Beres M. et al., “Ring Opening of 1.2.3Triazolo 1.5- (65) Prior Publication Data alpha pyrazinium Salts: Synthesis and Some Transformations of a US 2013/0245001A1 Sep. 19, 2013 Novel9393-94.00. Type of 2-Aza-1,3-butadienes.” Tetrahedron 53 (1997) 27. Makata S. et al., “Reduction of 4, 7-Diphenyl-1,2,5- O O thia(oxa)diazolo 3,4-cpyridines Affording 2,5-Diphenyl-3,4- Related U.S. Application Data diaminopyridines and Ring Closure of the Diamines to Fluorescent (60) Provisional application No. 61/533,455, filed on Sep. AZaheterocycles,” Journal of Heterocyclic Chemistry vol. 19, Issue 12, 2011. 6, pp. 1481-1488, 1982. s Paparin JL et al., “Synthesis of 2,5-bis-Arylpyridines by 4 + 2 (51) Int. Cl Cycloaddition of 1,4-bisAryl-2-aza-1,3-butadienes with Electron cond is 7/04 2006.O1 Poor Dienophiles,” J. Heterocyclic Chem. 37, 411 (2000). ( .01) Tetrazolopyrazines Registry Records Not Indexed in CAS subset A6 IK9/00 (2006.01) from STN (accessed approx. Jul. 20, 2009). A6 IK3I/4985 (2006.01) WO2010030785A2 Int'l Search Report, May 10, 2010. A6IP37/08 (2006.01) WO2010030785A2 Written Opinion of the Int'l Searching Author C07C309/04 (2006.01) ity, May 10, 2010. CD7C 53/26 (2006.01) WO2010030785A2 Int’l Preliminary Report on Patentability, Mar. C07C 55/10 (2006.01) 24, 2011. C07C 59/245 (2006.01) EP2324029 Extended European Search Report, Aug. 17, 2011. (52) U.S. Cl. Sarges R. et al., “4-Amino 1.2.4 triazolo 4.3-a Iquinoxalines. A CPC ...... C07D 487/04 (2013.01); C07C309/04 Novel Class of Potent Adenosine Receptor Antagonists and Potential (2013.01); A619/00 (2013.01); CD7C 53/26 Rapid-Onset .” Journal of Medicinal Chemistry (2013.01);s C07C 55/10 (2013.01);s C07C ( 1990), 33(8), 2240-54. 59/245 (2013.01); C70C59/255 (2013.01); (Continued) C07B 2.200/13 (2013.01) USPC ...... 514/249; 544/350:544/359: 548/953; 549/59 Primary Examiner — Sreeni Padmanabhan (58) Field of Classification Search Assistant Examiner — Irina Neagu CPC ..... C07D 487/04; A61K9/00; C07B 2200/13 (74) Attorney, Agent, or Firm — Dennis A. Bennett; Cynthia USPC ...... 514/249; 544/350, 359: 548/953; Hathaway 549/59 See application file for complete search history. (57) ABSTRACT (56) References Cited The present invention relates to salts, hydrates, and polymor U.S. PATENT DOCUMENTS phs of bicyclic heteroaryl compounds, and pharmaceutical compositions thereof, which may be useful as inhibitors of 8,569,300 B2 * 10/2013 Borchardt et al...... 514,249 2008/O139572 A1 6/2008 Wang et al. HR for the treatment or prevention of diseases including 2010, 0120741 A1 5, 2010 Borchardt allergic rhinitis. 2010/0249142 A1 9/2010 Deleuze-Masquefa et al. 2011/0237565 A1 9, 2011 Borchardt et al...... 514,210.21 2011/0237599 A1 9, 2011 Borchardt 59 Claims, 32 Drawing Sheets US 8,859,550 B2 Page 2

(56) References Cited ture-Activity Relationships, and Biological Studies,” J. Med. Chem. 1999, 42,4362-4379. OTHER PUBLICATIONS EP11754043 Extended European Search Report, Jun. 20, 2013. WO2011 112687 International Search Report and Written Opinion of Nagarajan K et al., “Displacement reactions of 2,3-dichloro-6- the Int'l Searching Authority, Nov. 25, 2011. nitroquinoxaline: Synthesis of s-Triazolo 3,4-aquinoxaline.” Indian WO2011 112766 International Search Report and Written Opinion of Journal of Chemistry, Section B: Organic Chemistry Including the Int'l Searching Authority, Nov. 25, 2011. Medicinal Chemistry (1986), 25B(7), 739-40. Badran MM et al., “Part I: Novel quinoxaline derivatives of biologi Non-final rejection dated Jan. 2, 2013 in U.S. Appl. No. 13/044,661. cal interest.” Bulletin of Pharmaceutical Sciences, Assiut University filed Mar. 10, 2011 (publ. No. US2011/0237565A1, now patent No. (2001), 24(2), 135-144. 8,569,300). Campiani G. et al., “Pyrroloquinoxaline Derivatives as High-Affinity and Selective 5-HT3 Receptor : Synthesis, Further Struc * cited by examiner U.S. Patent Oct. 14, 2014 Sheet 1 of 32 US 8,859,550 B2

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US 8,859,550 B2 1. 2 HETEROCYCLIC INHIBITORS OF reduce the risk of gastrointestinal (GI) bleeding associated HISTAMINE RECEPTORS FOR THE with severe upper GI ulcers and GI stress in the inpatient TREATMENT OF DISEASE Setting. The HR is primarily found in the CNS and peripheral This application claims the benefit of priority of U.S. pro 5 nerves innervating cardiac, bronchial, and GI tissue. HR visional Application No. 61/533,455, filed Sep. 12, 2011, the signaling regulates the release of multiple neurotransmitters, contents of which are hereby incorporated by reference as if Such as acetylcholine, dopamine, serotonin, and histamine written herein in its entirety. itself (where it acts as a CNS autoreceptor). In the CNS, HR Disclosed herein are new compounds, and salts, hydrates, participates in the processes of cognition, memory, sleep, and 10 feeding behaviors. HR antagonists may be used potentially and polymorphs thereof, of heterocyclic compounds, as well for treating cognition disorders (such as Alzheimer's dis as compositions comprising them, and their application as ease), sleep and wakefulness disorders, attention disorders, pharmaceuticals for the treatment of disease. and metabolic disorders (especially related to obesity). Histamine, a low molecular weight , is a Existence of the HR was predicted in the early 1990s, but potent chemical mediator of normal and pathological physi 15 its cloning by multiple groups was not reported until 2000. In ology. Histamine functions as a secreted signal in immune contrast to the other histamine receptors, the HR has a dis and inflammatory responses, as well as a neurotransmitter. tinctly selective expression profile in bone marrow and on The functions of histamine are mediated through four distinct certain types of hematopoietic cells. HR signaling modu cell surface receptors (H.R., HR, HR and HR). Histamine lates the function of mast cells, eosinophils, dendritic cells, receptors vary in expression, signaling, function and hista and subsets of T cells. The HR appears to control multiple mine affinity, and therefore have different potential therapeu behaviors of these cells, such as activation, migration, and tic applications (Zhang M. Thurmond RL, and Dunford PJ cytokine and chemokine production (Zhang M. Thurmond R & Therapeutics. 2007). L., and Dunford PJ Pharmacology & Therapeutics. 2007). All four histamine receptors are G protein-coupled recep Of the 4 known histamine receptors, HR, HR and HR tors (GPCRs). Upon histamine or other binding, they 25 have been shown clearly to affect inflammation and other activate distinct signaling pathways through differentheterot immune responses and are proposed therapeutic targets for rimeric G proteins. The HR couples to the G family of G treating immune and inflammatory disorders (Jutel et al., proteins, whose primary signaling cascade induces second 2002; Akdis & Simons, 2006). The HR was the first messenger calcium mobilization from intracellular stores, described , and ligands targeting this 30 receptor were initially developed in the 1930s and in wide followed by multiple downstream effects. HR can also spread use by the 1940s. Common HR antagonist increase cyclic GMP (cGMP) production and activate NFKB, currently approved for use include systemic agents such as a potent, positive transcriptional regulator of inflammation. (Benadryl, also used topically), The HR couples to the G family of G proteins and increases (Zyrtec), (Allegra), (Claritin) and cyclic AMP (cAMP) formation by stimulating adenylate 35 (ClarineX), and topical agents such as olopata cyclase, although it can also induce calcium mobilization in dine (Patanol, Pataday, Patanase), , (Opti Some cell types. The HR mediates its function through G, var, Astelin) and (Elestat). Traditional uses have proteins and decreases cAMP formation by inhibiting adeny included allergic diseases and reactions such as asthma, rhini late cyclase. Like other G-coupled receptors, HR also acti tis, and other chronic obstructive pulmonary disorders, ocular Vates the mitogen-activated protein/extracellular-signal regu 40 disorders such as allergic conjunctivitis, and pruritis of vary lated protein (MAP/ERK) kinase pathway. HR has also been ing etiologies. demonstrated to couple to G proteins, with canonical inhi However, H receptor antagonists have certain deficiencies bition of cAMP formation and MAP kinase activation. How as therapeutic agents in the treatment of diseases where his ever, HR also couples to calcium mobilization in certain cell tamine is an important mediator. First, their effects are often types. In fact, HR signaling in mast cells is primarily through 45 only moderate and reduce allergic symptoms by only 40 to calcium mobilization with little to no impact on cAMP for 50%. In particular, H receptor antagonists, especially sys mation. temic agents, have little to no effect in relieving nasal con The HR is expressed in many cell types, including endot gestion. In allergic asthma, despite the fact that histamine helial cells, most Smooth muscle cells, cardiac muscle, central levels rapidly increase in the airways and in plasma (corre nervous system (CNS) neurons, and lymphocytes. HR sig 50 lating with disease severity), H receptor antagonists have naling causes Smooth muscle contraction (including bron largely failed as a therapeutic strategy, though some effect is choconstriction), vasodilation, and increased vascular perme seen with administration during the priming phase as opposed ability, hallmarks of allergic and other immediate to the challenge phase (Thurmond R L et al., Nat Rev Drug hypersensitivity reactions. In the CNS, HR activation is Discov, 2008, 7:41-53). Additionally, although the efficacy of associated with wakefulness. Its activation is also associated 55 H receptor antagonists against pruritus in acute urticarias, with pruritus and nociception in skin and mucosal tissues. For associated with hives and insect stings, and in chronic idio many years, the anti-allergic and anti-inflammatory activities pathic urticaria is well proven, HR antagonists are mostly of HR antagonists have been utilized to treat acute and ineffective in the treatment of atopic dermatitis-associated chronic allergic disorders and other histamine-mediated pruritus, with the only modest benefits derived from some pathologies, such as itch and hives. 60 first-generation compounds likely a consequence of their The HR is expressed similarly to the HR, and can also be sedative properties (Sharpe, G. R. & Shuster, S. Br: I Derma found in gastric parietal cells and neutrophils. HR is best tol. 1993, 129:575-9). Finally, sedation caused by HR known for its central role in gastric acid secretion but has also antagonists that cross the blood-brain barrier, among other been reported to be involved in increased vascular permeabil side effects, limits the utility of many HR antagonists in ity and airway mucus production. Antagonists of HR are 65 diseases for which they would otherwise be efficacious. widely used in treating peptic ulcers and gastroesophageal These deficiencies render HR antagonists amenable to reflux disease. These drugs are also used extensively to replacement by or Supplementation with other agents. US 8,859,550 B2 3 4 Consequently, attention has focused on the more recently example in a tablet or capsule, or alternatively as a solution for discovered H receptor as a therapeutic target. Given the intranasal delivery, for the treatment of allergic disorders. ability of HR to modulate the cellular function of eosino Novel salts and polymorphs of compounds which have phils, mast cells, dendritic cells and T cells (M. Zhang et al., been found to inhibit the histamine type-4 receptor (HR) are Pharmacol Ther 2007), it is natural to speculate that the HR herein disclosed, together with pharmaceutical compositions may be involved in various inflammatory diseases, and that comprising them, methods of synthesizing, characterizing, HRantagonists would have therapeutic potential (Juteletal. and using the salts and polymorphs including methods for the 2006). Indeed, both in vitro and in vivo evidence has been treatment of histamine receptor-mediated diseases in a demonstrated for the utility of HR antagonists as anti-in patient by administering the salts and polymorphs. flammatory agents in inflammatory bowel disease (IBD) 10 (Sander L. E et al., Gut 2006:55:498-504). The finding that H. Provided herein are salts and polymorphs which comprise receptor antagonists inhibit histamine-induced migration of compounds of structural Formula (I) together with a counte mast cells and eosinophils in vitro and in vivo, both of which 1O. are important effector cells in the allergic response, raises the possibility that this class of compounds could reduce the 15 allergic hyper-responsiveness developed upon repeated expo (I) Sure to antigens, which is characterized by an increase in the number of mast cells and otherinflammatory cells in the nasal and bronchial mucosa (Fung-Leung W P et al., Curr Opin Inves Drugs, 2004.5:11 1174-1182). In contrast to some of the HR antagonists, HR antagonists given during the allergen challenge phase of a mouse model of asthma are equally effective to those given during sensitization (Thurmond RL et al., Nat Rev Drug Discov, 2008, 7:41-53). In two recent wherein: mouse studies, a selective HaRagonist was shown to induce 25 the ring comprising X-X is aromatic; itch, whereas these responses, and those of histamine, were X' and X are independently chosen from C, CH and N: blocked by pretreatment with HR antagonists. Similarly, X is chosen from C(R)(R), NR, O and S: histamine or Ha receptor agonist-induced itch was markedly X is chosen from)(C(R)(R'), NR'', O, and S: attenuated in H receptor-deficient animals (Dunford, P. J. et X is chosen from C(R)(R'), NR'', O and S; al., J. Allergy Clin. Immunol, 2007, 119:176-183). The pres 30 X is chosen from CR'' and N: ence of the HR in nasal tissue was first discovered by Nakaya X7 is chosen from CR'' and N: et al. (Nakaya, M. et al., Ann Otol Rhinol Laryngol, 2004, Y' is chosen from a bond, lower alkyl, lower alkoxy, OR', 113:552-557). In addition, a more recent finding showed that NR'R'', and lower aminoalkyl: there is a significant increase in the level of HR in human Y is chosen from a bond, lower alkyl, lower alkoxy, OR, nasal polyp tissue taken from patients with chronic rhinosi 35 NR'R'', S, C(O)NH, C(O)NHR, C(O)NRR and nusitis (infection of the nose and nasal cavities) when com lower aminoalkyl, pared to normal nasal mucosa. Jókiti et al. suggest that the R" is selected from the group consisting of: administration of HRantagonists might be a new way to treat aryl, heterocycloalkyl, cycloalkyl, and heteroaryl, any of nasal polyps and chronic rhinosinusitis. The administration of which may be optionally substituted, when Y is a bond; and HR antagonists may prevent the accumulation of eosino 40 null, when Y is chosen from OR', NR'R'', lower alkyl, phils as a result of impaired cell chemotaxis toward polypous lower alkoxy, or lower aminoalkyl; tissue (J6kiti, A. et al., Cell Biol Int, 2007, 31: 1367). R is chosen from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, Although scientific data on the role of the HR in rhinitis is heterocycloalkyl, and heteroaryl, any of which may be limited, at present, it is the only indication for which an HR optionally substituted; (CZC-13788) is reported to be in preclinical 45 R. R. R. R. R. R. R', and Rare independently development (Hale, R. A. et al., Drug News Perspect, 2007, chosen from null, hydrogen, alkyl, alkenyl, alkynyl, het 20:593-600). eroalkyl, alkoxy, halogen, haloalkyl, perhaloalkyl, amino, Additional reports have shown potential for HR antago aminoalkyl, amido, carboxyl, acyl, hydroxy, cyano, nitro, nists in the treatment of metabolic disorders such as obesity aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, (Jorgensen E et al., Neuroendocrinology. 2007: 86(3):210-4), 50 heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl, any of vascular or cardiovascular diseases such as atherosclerosis which may be optionally substituted; (Tanihide A et al., TCM 2006: 16(8): 280-4), inflammation R. R'', and R'' are independently chosen from null, and pain (Coruzzi Get al., Eur J Pharmacol. 2007 Jun. 1: hydrogen, alkyl, heteroalkyl, alkoxy, haloalkyl, perhaloalkyl, 563(1-3):240-4), rheumatoid arthritis (Grzybowska-Kowalc aminoalkyl, C-amido, carboxyl, acyl, hydroxy, aryl, aryla ZykAet al., Inflamm Res. 2007 April; 56 Suppl 1:S59-60) and 55 lkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy other inflammatory and autoimmune diseases including sys cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which temic lupus erythematosus (Zhang M. Thurmond R L. and may be optionally substituted; Dunford PJ Pharmacology & Therapeutics. 2007). What is R. R. R', and R are independently chosen from clear is that a need still exists in the art for improved and aminoalkyl, alkylaminoalkyl, aryl, arylalkyl, cycloalkyl, varied for the treatment of disease, and that 60 cycloalkylalkyl, ether, heterocycloalkyl, lower alkylamino compounds with HR antagonist activity may fill this need. heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and het At least some selective HR antagonists have been previ eroarylalkyl, any of which may be optionally substituted; ously disclosed. Nevertheless, a need still exists for com R7 and R’ are independently chosen from hydrogen, ami pounds with HR antagonist activity which have properties noalkyl, alkylaminoalkyl aryl, arylalkyl, cycloalkyl, making them Suitable for development as drugs, such as the 65 cycloalkylalkyl, ether, heterocycloalkyl, lower alkylamino ability to be isolated in a solid (preferably crystalline) form heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and het and appropriate solubility for formulation and delivery, for eroarylalkyl, any of which may be optionally substituted; and US 8,859,550 B2 5 6 R and R2 are independently chosen from hydrogen, FIG.10: FT-Raman Spectrum of 8-(4-Methylpiperazin-1- alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, halogen, yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate haloalkyl, perhaloalkyl, perhaloalkoxy, amino, aminoalkyl, Monohydrate Form 1, also known as Compound 1, Mesylate amido, carboxyl, acyl, hydroxy, cyano, nitro, aryl, arylalkyl, Salt, Form 1. In the figure, the horizontal axis is the Raman cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy shift in units of cm. cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which FIG. 11: DSC/TGA Analysis of 8-(4-Methylpiperazin-1- may be optionally substituted. yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate Salts and polymorphs disclosed herein may possess useful Monohydrate Form 1, also known as Compound 1, Mesylate histamine receptor inhibitory activity, and may be used in the Salt, Form 1. treatment or prophylaxis of a disease or condition in which 10 FIG. 12: GVS Isotherm Plot of 8-(4-Methylpiperazin-1- HR plays an active role. Thus, in broad aspect, certain yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate embodiments also provide pharmaceutical compositions Monohydrate Form 1, also known as Compound 1, Mesylate comprising one or more compounds, salts, hydrates, or poly Salt, Form 1. morphs disclosed herein together with a pharmaceutically FIG. 13: ORTEP drawing of the 8-(4-methylpiperazin-1- acceptable carrier, as well as methods of making and using the 15 yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate compounds and compositions. Certain embodiments provide Monohydrate Form 1 cation showing the atom naming and methods for inhibiting H.R. Other embodiments provide numbering scheme. Ellipsoids are at the 50% probability methods for treating an HR-mediated disorder in a patient in level and hydrogenatoms were drawn with arbitrary radii for need of Such treatment, comprising administering to said clarity. The disordered sulfur atom is depicted with an open patient a therapeutically effective amount of a compound, ellipsoid and open bonds. salt, hydrate, or polymorph or a composition comprising it. FIG. 14: ORTEP drawing of the 8-(4-methylpiperazin-1- Also provided is the use of certain compounds, salts, yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate hydrates, or polymorphs disclosed hereinforuse in the manu Monohydrate Form 1 anion showing the atom naming and facture of medicaments for the treatment of a disease or numbering scheme. Ellipsoids are at the 50% probability condition ameliorated by the inhibition of HR. 25 level and hydrogenatoms were drawn with arbitrary radii for clarity. The disordered component is drawn with open ellip BRIEF DESCRIPTION OF THE DRAWINGS soids and bonds. FIG. 15: PXRD diffractogram of 8-(4-Methylpiperazin-1- FIG. 1: PXRD diffractogram of 8-(4-Methylpiperazin-1- yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Hemi 30 Form 2, also known as Compound 1 Mesylate Salt Form 2. hydrate Form 1 also known as Compound 1 Tartrate Salt FIG. 16: FT-Raman Spectrum of 8-(4-Methylpiperazin-1- Form 1. yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate FIG. 2: FT-Raman Spectrum of 8-(4-Methylpiperazin-1- Form 2, also known as Compound 1 Mesylate Form 2. In the yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Hemi figure, the horizontal axis is the Raman shift in units of cm'. hydrate Form 1, also known as Compound 1, Tartrate Salt, 35 FIG. 17: PXRD diffractogram of 8-(4-Methylpiperazin-1- Form 1. In the figure, the horizontal axis is the Raman shift in yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate units of cm'. Form 3, also known as Compound 1 Mesylate Form 3. FIG. 3: DSC/TGA Analysis of 8-(4-Methylpiperazin-1- FIG. 18: FT-Raman Spectrum of 8-(4-Methylpiperazin-1- yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Hemi yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate hydrate Form 1, also known as Compound 1, Tartrate Salt, 40 Form 3, also known as Compound 1 Mesylate Form 3. In the Form 1. figure, the horizontal axis is the Raman shift in units of cm'. FIG. 4: GVS Isotherm Plot of 8-(4-Methylpiperazin-1-yl)- FIG. 19: DSC/TGA Analysis of 8-(4-Methylpiperazin-1- 5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Hemihy yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate drate Form 1, also known as Compound 1, Tartrate Salt, Form Form 3, also known as Compound 1, Mesylate Salt, Form 3. 1. In the figure legend, “Cycle 1 S' means Cycle 1 Sorption, 45 FIG. 20: PXRD diffractogram of N-methyl-1-(5- “Cycle 1 DS' means Cycle 1 Desorption, and so on. (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- FIG. 5: PXRD diffractogram of 8-(4-Methylpiperazin-1- amine Citrate Form 1, also known as Compound 2 Citrate Salt yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Form 1. Monohydrate Form 2, also known as Compound 1 Tartrate FIG.21: FTIR Spectrum of N-methyl-1-(5-(thiophen-2-yl) Salt Form 2. 50 tetrazolo 1.5-apyrazin-8-yl)azetidin-3-amine Citrate Form FIG. 6: FT-Raman Spectrum of 8-(4-Methylpiperazin-1- 1, also known as Compound 2 Citrate Salt Form 1. yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate FIG.22: DSC/TGA Analysis of N-methyl-1-(5-(thiophen Monohydrate Form 2, also known as Compound 1, Tartrate 2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-amine Citrate Salt, Form 2. In the figure, the horizontal axis is the Raman Form 1, also known as Compound 2 Citrate Salt Form 1. shift in units of cm'. 55 FIG. 23: GVS Isotherm Plot of N-methyl-1-(5-(thiophen FIG. 7: DSC/TGA Analysis of 8-(4-Methylpiperazin-1- 2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-amine Citrate yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Form 1, also known as Compound 2 Citrate Salt Form 1. The Monohydrate Form 2, also known as Compound 1, Tartrate last six data points of cycle 2 desorption were aberrant due to Salt, Form 2. balance failure and not plotted. FIG. 8: GVS Isotherm Plot of 8-(4-Methylpiperazin-1-yl)- 60 FIG. 24: PXRD diffractogram of N-methyl-1-(5- 5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Monohy (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- drate Form 2, also known as Compound 1, Tartrate Salt, Form amine Hydrochloride Form 1, also known as Compound 2 2. Hydrochloric Salt Form 1. FIG. 9: PXRD diffractogram of 8-(4-Methylpiperazin-1- FIG.25: FTIR Spectrum of N-methyl-1-(5-(thiophen-2-yl) yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate 65 tetrazolo 1.5-apyrazin-8-yl)azetidin-3-amine Hydrochlo Monohydrate Form 1, also known as Compound 1, Mesylate ride Form 1, also known as Compound 2 Hydrochloride Salt Salt, Form 1. Form 1. US 8,859,550 B2 7 8 FIG. 26: DSC/TGA Analysis of N-methyl-1-(5-(thiophen cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which 2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-amine Hydro may be optionally Substituted; and chloride Form 1, also known as Compound 2 Hydrochloride R. R'', and R'' are independently chosen from null, Salt Form 1. hydrogen, alkyl, heteroalkyl, alkoxy, haloalkyl, perhaloalkyl, FIG. 27: PXRD diffractogram of N-methyl-1-(5- aminoalkyl, C-amido, carboxyl, acyl, hydroxy, aryl, aryla (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- lkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy amine Hydrochloride Form 2, also known as Compound 2 cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which Hydrochloric Salt Form 2. may be optionally substituted. FIG.28: FTIR Spectrum of N-methyl-1-(5-(thiophen-2-yl) In certain embodiments provided herein, tetrazolo 1.5-apyrazin-8-yl)azetidin-3-amine Hydrochlo 10 X7 is CR; ride Form 2, also known as Compound 2 Hydrochloride Salt X is N; and Form 2. Y' and Y are each independently a bond. FIG. 29: PXRD diffractogram of N-methyl-1-(5- Also provided herein are salts and polymorphs which com (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- 15 prise compounds of structural Formula (III) together with a amine Mesylate Form 1, also known as Compound 2 Mesy counterion late Salt Form 1. FIG.30: FTIR Spectrum of N-methyl-1-(5-(thiophen-2-yl) tetrazolo 1.5-alpyrazin-8-yl)aZetidin-3-amine Mesylate (III) Form 1, also known as Compound 2 Mesylate Salt Form 1. FIG.31: DSC/TGA Analysis of N-methyl-1-(5-(thiophen 2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-amine Mesylate Form 1, also known as Compound 2 Mesylate Salt Form 1. FIG. 32: GVS Isotherm Plot of N-methyl-1-(5-(thiophen 2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-amine Mesylate 25 Form 1, also known as Compound 2 Mesylate Salt Form 1. The last two data points of cycle 2 desorption were aberrant wherein: due to balance failure and not plotted. X' and X are independently chosen from C and N: In certain embodiments, compounds, salts, hydrates, and X is chosen from C(R)(R), and NR; polymorphs of compounds disclosed herein are solid. 30 X is chosen from C(R)(R'), and NR'': In further embodiments, compounds, salts, hydrates, and X is chosen from C(R)(R'), and NR'': polymorphs of compounds disclosed herein are crystalline. R" is chosen from aryl, heterocycloalkyl, cycloalkyl, and heteroaryl, any of which may be optionally substituted; In certain embodiments provided herein, at least two of R is chosen from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, X-X’ are ring heteroatoms. 35 In certain embodiments provided herein, heterocycloalkyl and heteroaryl, any of which may be option ally substituted; X7 is N: R. R. R. R', R', and R' are independently chosen X is CR'; and from null, hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, Y' and Y are each independently a bond. alkoxy, halogen, haloalkyl, perhaloalkyl, amino, aminoalkyl, Also provided herein are salts and polymorphs which com 40 amido, carboxyl, acyl, hydroxy, cyano, nitro, aryl, arylalkyl, prise compounds of structural Formula (II) together with a cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy counterion cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which may be optionally Substituted; and R. R'', and R'' are independently chosen from null, (II) 45 hydrogen, alkyl, heteroalkyl, alkoxy, haloalkyl, perhaloalkyl, aminoalkyl, C-amido, carboxyl, acyl, hydroxy, aryl, aryla lkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which may be optionally substituted. 50 In certain embodiments, X7 is CR; X is CR'; and wherein: Y' and Y are each independently a bond. X' and X are independently chosen from C and N: Also provided herein are salts and polymorphs which com X is chosen from C(R)(R), and NR; 55 X is chosen from C(R)(R'), and NR'': prise compounds of structural Formula (IV) together with a X is chosen from C(R)(R'), and NR'': counterion R" is chosen from aryl, heterocycloalkyl, cycloalkyl, and heteroaryl, any of which may be optionally substituted; (IV) R is chosen from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, 60 heterocycloalkyl and heteroaryl, any of which may be option ally substituted; R. R. R. R', R', and R' are independently chosen from null, hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, halogen, haloalkyl, perhaloalkyl, amino, aminoalkyl, 65 amido, carboxyl, acyl, hydroxy, cyano, nitro, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy US 8,859,550 B2 9 10 wherein: X' and X are independently chosen from C and N: 3 (Va) X is chosen from C(R)(R7), and NR; X2a2X X is chosen from C(R)(R'), and NR'': 2 h x X is chosen from C(R)(R'), and NR; R XSs? R" is chosen from aryl, heterocycloalkyl, cycloalkyl, and heteroaryl, any of which may be optionally substituted; R is chosen from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, R18 als, heterocycloalkyl and heteroaryl, any of which may be option ally substituted; 10 wherein: R. R. R. R', R', and R' are independently chosen from null, hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, X' and X are independently chosen from C and N: alkoxy, halogen, haloalkyl, perhaloalkyl, amino, aminoalkyl, X is chosen from C(R)(R), NR, O and S: amido, carboxyl, acyl, hydroxy, cyano, nitro, aryl, arylalkyl, 15 X is chosen from)(C(R)(R'), and NR'': cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy X is chosen from C(R)(R'), NR'', O and S; cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which may be optionally Substituted; and R" is chosen from aryl, heterocycloalkyl, cycloalkyl, and R. R', and R'' are independently chosen from null, heteroaryl, any of which may be optionally substituted; hydrogen, alkyl, heteroalkyl, alkoxy, haloalkyl, perhaloalkyl, R’ is chosen from aryl, cycloalkyl, heterocycloalkyl, and aminoalkyl, C-amido, carboxyl, acyl, hydroxy, aryl, aryla heteroaryl, any of which may be optionally substituted; lkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy R. R. R. R', and R' are independently chosen from cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which null, hydrogen, alkyl, alkynyl, heteroalkyl, alkoxy, halogen, may be optionally substituted. haloalkyl, perhaloalkyl, amino, aminoalkyl, amido, carboxyl, In certain embodiments, 25 acyl, hydroxy, cyano, nitro, aryl, arylalkyl, cycloalkyl, X is chosen from)(C(R)(R'), and NR'': R" is chosen from null, hydrogen, and lower alkyl; and cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, R" and R' independently chosen from null, lower alkyl, heteroaryl, and heteroarylalkyl, any of which may be option and hydrogen. ally substituted; Also provided herein are salts and polymorphs which com 30 R" is chosen from null, hydrogen, and lower alkyl: prise compounds of structural Formula (V) together with a R. R'', and R'' are independently chosen from null, counterion hydrogen, alkyl, heteroalkyl, alkoxy, haloalkyl, perhaloalkyl, aminoalkyl, C-amido, carboxyl, acyl, hydroxy, aryl, aryla lkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy (V) 35 cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which XaX may be optionally Substituted; and R2 {N: x' R" is chosen from lower alkyl and hydrogen. In certain embodiments: 40 Nals R1 R" is chosen from phenyl, monocyclic 4- to 7-membered heterocycloalkyl, monocyclic 4- to 7-membered cycloalkyl, wherein: and monocyclic 5- to 6-membered heteroaryl, any of which X' and X are independently chosen from C and N: may be optionally substituted; X is chosen from C(R)(R7), and NR; 45 R’ is chosen from phenyl, monocyclic 5- to 7-membered X is chosen from)(C(R)(R'), and NR'': cycloalkyl, monocyclic 5- to 7-membered heterocycloalkyl, X is chosen from C(R)(R'), and NR; monocyclic 5- to 6-membered heteroaryl, and heteroarylalkyl R" is chosen from aryl, heterocycloalkyl, cycloalkyl, and any of which may be optionally substituted. heteroaryl, any of which may be optionally substituted; In certain embodiments, R. R. R', R', R', and R'' are R is chosen from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, 50 independently chosen from null and hydrogen. heterocycloalkyl and heteroaryl, any of which may be option ally substituted; In certain embodiments, compounds of Formula I have a R. R. R. R', R', and R' are independently chosen structure chosen from: from null, hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, halogen, haloalkyl, perhaloalkyl, amino, aminoalkyl, 55 amido, carboxyl, acyl, hydroxy, cyano, nitro, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy Naa 2 cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which Y2 1 Y2 1 may be optionally Substituted; and /'N' / R. R', and R'' are independently chosen from null, 60 R N4 -R. R. N4 -R'. hydrogen, alkyl, heteroalkyl, alkoxy, haloalkyl, perhaloalkyl, etN aminoalkyl, C-amido, carboxyl, acyl, hydroxy, aryl, aryla - \ lkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocy N 2 -- N cloalkylalkyl, heteroaryl, and heteroarylalkyl, any of which as? 2Y&N may be optionally substituted. 65 2 2 In certain embodiments, compounds have structural for R N4 -R, R els mula (Va): US 8,859,550 B2 11 12

-continued -continued R25 N N-N V | W N N Y2 N Y2 N

Rifle,N y11 fullN y11 R25 p-( O 10 \ | \V wherein: N 2 R" is chosen from phenyl, monocyclic 4- to 7-membered YS YS heterocycloalkyl, monocyclic 4- to 7-membered cycloalkyl, R2 R 4 R, R2 R 4 R, 15 and monocyclic 5- to 6-membered heteroaryl, any of which N y11 N y11 may be optionally substituted; R is chosen from alkyl, monocyclic cycloalkyl, monocy clic heterocycloalkyl and monocyclic heteroaryl, any of which may be optionally substituted; R" and Rare independently chosen from null, hydrogen, alkyl, alkynyl, heteroalkyl, alkoxy, halogen, haloalkyl, per haloalkyl, amino, aminoalkyl, amido, carboxyl, acyl, hydroxy, cyano, nitro, aryl, arylalkyl, cycloalkyl, cycloalky lalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, 25 and heteroarylalkyl, any of which may be optionally substi wherein: tuted; and R’ is chosen from hydrogen, alkyl, alkenyl, alkynyl, het R’ is chosen from hydrogen, alkyl, alkenyl, alkynyl, het eroalkyl, alkoxy, halogen, haloalkyl, perhaloalkyl, perha eroalkyl, alkoxy, halogen, haloalkyl, perhaloalkyl, perha loalkoxy, amino, aminoalkyl, amido, carboxyl, acyl, hydroxy, loalkoxy, amino, aminoalkyl, amido, carboxyl, acyl, hydroxy, cyano, nitro, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, het 30 cyano, nitro, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, het erocycloalkyl, heterocycloalkylalkyl, heteroaryl, and het erocycloalkyl, heterocycloalkylalkyl, heteroaryl, and het eroarylalkyl, any of which may be optionally substituted; and eroarylalkyl, any of which may be optionally substituted. all other groups are as disclosed in Formula I. In certain embodiments, compounds of Formula I have a structure chosen from: In certain embodiments, compounds of Formula I have a 35 structure chosen from:

N e I N 2 N R2 NN NN 2

R2 N 4. p-V R2 N N

2 US 8,859,550 B2 13 14

-continued -continued |-y

r 2 2 R2, and N r R27 10 wherein wherein R is chosen from alkyl, monocyclic cycloalkyl, monocy A is chosen from a monocyclic heterocycloalkyl and a clic heterocycloalkyl and monocyclic heteroaryl, any of 15 monocyclic heteroaryl, either of which is attached through a which may be optionally substituted; ring nitrogen to the core and either of which may be option R’ is chosen from hydrogen, alkyl, alkenyl, alkynyl, het ally substituted; eroalkyl, alkoxy, halogen, haloalkyl, perhaloalkyl, perha loalkoxy, amino, aminoalkyl, amido, carboxyl, acyl, hydroxy, R is chosen from alkyl, phenyl, monocyclic cycloalkyl, cyano, nitro, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, het monocyclic heterocycloalkyl and monocyclic heteroaryl, any erocycloalkyl, heterocycloalkylalkyl, heteroaryl, and het of which may be optionally substituted; and eroarylalkyl, any of which may be optionally substituted; and R’ is chosen from hydrogen, alkyl, alkenyl, alkynyl, het R° and R7 are independently chosen from hydrogen, eroalkyl, alkoxy, halogen, haloalkyl, perhaloalkyl, perha alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, halogen, 25 loalkoxy, amino, aminoalkyl, amido, carboxyl, acyl, hydroxy, haloalkyl, perhaloalkyl, amino, aminoalkyl, amido, acyl, cyano, nitro, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, het hydroxy, cyano, nitro, aryl, arylalkyl, cycloalkyl, cycloalky lalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, erocycloalkyl, heterocycloalkylalkyl, heteroaryl, and het and heteroarylalkyl, any of which may be optionally substi eroarylalkyl, any of which may be optionally substituted. tuted; or Rand R7 together with the nitrogen to which they 30 In certain embodiments, A is four- to seven-membered. are attached may combine to form heterocycloalkyl or het eroaryl, either of which is attached through a ring nitrogen to In certain embodiments, R is chosen from hydrogen and the core and either of which may be optionally substituted. methyl. In certain embodiments, compounds of Formula I have a In certain embodiments: structure chosen from: 35 X is chosen from C(R)(R), and NR; X is chosen from C(R)(R'), and NR'': R’ is chosen from monocyclic 5- to 7-membered hetero cycloalkyl, phenyl, and monocyclic 5- to 6-membered het 40 eroaryl, any of which may be optionally substituted; and R" is chosen from methyl and hydrogen. In certain embodiments, R is chosen from phenyl, furan, 45 thiophene, pyrrole, pyrroline, pyrrolidine, oxazole, thiazole, imidazole, pyrazole, imidazoline, imidazolidine, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, thiadiazole, triazole, pyran, pyridine, piperidine, morpholine, thiomor pholine, piperazine, pyridazine, pyrimidine, and pyrazine. 50 In certain embodiments, R' is optionally substituted mono cyclic 4- to 7-membered heterocycloalkyl. In certain embodiments,

55 X' is C; X2 is NR; X* is NR; X is N; and 60 R is chosen from null, hydrogen, alkyl, alkoxy, halogen, haloalkyl, acyl perhaloalkyl, amino, aminoalkyl, hydroxy, cyano, any of which may be optionally substi tuted.

65 Also provided herein are salts and polymorphs which com prise compounds of structural Formula (VI) together with a counterion US 8,859,550 B2 16 In certain embodiments, (VI) R" is chosen from piperazine and azetidine, either of which may be optionally substituted with one to three substitu ents chosen from lower alkyl and lower amino; R is chosen from phenyl, furan, thiophene, and thiazole, any of which may be optionally substituted with one to three Substituents chosen from halogen, lower alkyl, lower haloalkyl, lower alkoxy, lower haloalkoxy, cyano, 10 lower amino, hyrdoxy, and nitro. wherein: Also provided herein are salts and polymorphs which com R" is optionally substituted monocyclic 4- to 7-membered prise compounds of structural Formula (VIII) together with a heterocycloalkyl; and counterion R is chosen from monocyclic 5- to 7-membered hetero 15 cycloalkyl, phenyl, and monocyclic 5- to 6-membered heteroaryl, any of which may be optionally substituted; (VIII) and R is chosen from null, hydrogen, and lower alkyl. In certain embodiments, X is N: X is chosen from C(R)(R7), and NR; X is NR; X is C; R" is chosen from phenyl, monocyclic 4- to 7-membered 25 heterocycloalkyl, monocyclic 4- to 7-membered wherein: cycloalkyl, and monocyclic 5- to 6-membered het eroaryl, any of which may be optionally substituted; R" is optionally substituted monocyclic 4- to 7-membered R" is optionally substituted monocyclic 4- to 7-membered heterocycloalkyl; heterocycloalkyl; and 30 R’ is chosen from monocyclic 5- to 7-membered hetero R is chosen from monocyclic 5- to 7-membered hetero cycloalkyl, phenyl, and monocyclic 5- to 6-membered het cycloalkyl, phenyl, and monocyclic 5- to 6-membered eroaryl, either of which may be optionally substituted with heteroaryl, either of which may be optionally substi one to three Substituents chosen from halogen, lower alkyl, tuted. 35 lower haloalkyl, lower alkoxy, lower haloalkoxy, cyano, Also provided herein are salts and polymorphs which com lower amino, hyrdoxy, and nitro; and prise compounds of structural Formula (VII) together with a counterion R’ is chosen from hydrogen, alkyl, alkoxy, halogen, haloalkyl, perhaloalkyl, amino, aminoalkyl, hydroxy, cyano, 40 any of which may be optionally substituted. (VII) Also provided herein are salts and polymorphs which com prise compounds of structural Formula (IX) together with a counterion

45 (IX) NetN 2 I N R Na ? wherein: 50 R" is chosen from phenyl, monocyclic 4- to 7-membered CCN R1 heterocycloalkyl, monocyclic 4- to 7-membered cycloalkyl, and monocyclic 5- to 6-membered het wherein: eroaryl, any of which may be optionally substituted; 55 R is chosen from phenyl, furan, thiophene, pyrrole, pyr R" is optionally substituted monocyclic 4- to 7-membered roline, pyrrolidine, oxazole, thiazole, imidazole, pyra heterocycloalkyl; and Zole, imidazoline, imidazolidine, pyrazoline, pyrazoli R’ is chosen from monocyclic 5- to 7-membered hetero dine, isoxazole, isothiazole, oxadiazole, thiadiazole, cycloalkyl, phenyl and monocyclic 5- to 6-membered het triazole, pyran, pyridine, piperidine, morpholine, thio 60 morpholine, piperazine, pyridazine, pyrimidine, and eroaryl, either of which may be optionally substituted with pyrazine, any of which may be optionally Substituted; one to three Substituents chosen from halogen, lower alkyl, and lower haloalkyl, lower alkoxy, lower haloalkoxy, cyano, R is chosen from hydrogen, lower alkyl, lower alkoxy, lower amino, hyrdoxy, and nitro. halogen, lower haloalkyl, lower amino, lower ami 65 Also provided herein are salts and polymorphs which com noalkyl, hydroxy, cyano, any of which may be optionally prise compounds of structural Formula (X) together with a Substituted. counterion US 8,859,550 B2 17 18 Also provided herein are compounds, or salts, hydrates or polymorphs thereof, of structural Formula (XII) 2 I e N R N 2 (XII) NetN ? 2 / I N N R.)N S Na ? 10 CC wherein: 1N-- A is chosen from a monocyclic 4- to 7-membered hetero (\Xs cycloalkyl and a monocyclic 5- to 6-membered heteroaryl, either of which is attached through a ring nitrogen to the core 15 and either of which may be optionally substituted; and or a salt, hydrate, or polymorph thereof, wherein: R is chosen from phenyl, furan, thiophene, pyrrole, pyr X is chosen from CH and N: roline, pyrrolidine, oxazole, thiazole, imidazole, pyrazole, m and n are each an integer chosen from 1 and 2: imidazoline, imidazolidine, pyrazoline, pyrazolidine, isox R is chosen from lower amino, lower alkylamino, and azole, isothiazole, oxadiazole, thiadiazole, triazole, pyran, lower alkyl: pyridine, piperidine, morpholine, thiomorpholine, pipera Y is chosen from a counterion and null; and Zine, pyridazine, pyrimidine, and pyrazine, any of which may Z is a chosen from a hydrate and null. be optionally substituted. In certain embodiments, provided herein are compounds, Also provided herein are salts and polymorphs which com or salts, polymorphs, or hydrates thereof, in which: prise compounds of structural Formula (XI) together with a 25 Y is a counterion, if the compound of Formula I is a salt, or counterion Y is chosen from a counterion and null, if the compound of Formula I is a polymorph. In certain embodiments, provided herein are compounds, N (XI) or salts, polymorphs, or hydrates thereof, in which: Ne 30 X is N: | \ m and n are each 2; and R is lower alkyl. In further embodiments, R is methyl. r 2 In certain embodiments, the compound is 8-(4-Methylpip N N ) pi 35 erazin-1-yl)-5-(thiophen-2-yl)tetraZolo 1.5-alpyrazine, or a (\), \ YR24 salt, hydrate, or polymorph thereof. In other embodiments, provided herein are compounds, or salts, polymorphs, or hydrates thereof, in which: wherein: X is CH: X is chosen from CH and N: 40 m and n are each 1; m and n are each an integer chosen from 1 and 2: R is lower alkylamino. R is chosen from phenyl, furan, thiophene, and thiazole, In certain embodiments, R is lower alkylamino. any of which may be optionally substituted with one to three In further embodiments, R is lower methylamino. Substituents chosen from halogen, lower alkyl, lower In certain embodiments, the compound is N-methyl-1-(5- haloalkyl, lower alkoxy, lower haloalkoxy, cyano, lower 45 (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- amino, hyrdoxy, and nitro; and amine, or a salt, hydrate, or polymorph thereof. R is chosen from hydrogen, amino, and lower alkyl. In certain embodiments, provided herein are compounds, In certain embodiments, or salts, polymorphs, or hydrates thereof, in which: X is CH: Y is a counterion, if the compound of Formula XII is a salt, m and n are each 1; and 50 O R" is chosen from hydrogen, amino, and lower alkyl. Y is chosen from a counterion and null, if the compound of In certain embodiments, R is amino. Formula XII is a polymorph. In certain embodiments, R is NHCH. In certain embodiments, Y is a counterion and the com In certain embodiments, pound is a salt, or a polymorph or hydrate thereof. X is N: 55 In further embodiments, Y is a counterion chosen from m and n are each 2; and acetate, citrate, Sulfate, phosphate, hydrochloride, aspartate, R" is chosen from hydrogen and lower alkyl. mesylate, malate, tartrate, Stearate, and Succinate. In certain embodiments, R is chosen from hydrogen and In further embodiments, the counterion is chosen from methyl. tartrate, mesylate, citrate, and hydrochloride. In certain embodiments, R is methyl. 60 In certain embodiments, Z is chosen from a stoichiometric In certain embodiments provided herein, hydrate and a nonstoichiometric hydrate, or a polymorph R is chosen from phenyl, furan, thiophene, pyrrole, pyr thereof. roline, pyrrolidine, oxazole, thiazole, imidazole, pyrazole, In further embodiments, Z is chosen from a monohydrate imidazoline, imidazolidine, pyrazoline, pyrazolidine, isox and a hemihydrate. azole, isothiazole, oxadiazole, thiadiazole, triazole, pyran, 65 In certain embodiments, provided herein are compounds, pyridine, piperidine, morpholine, thiomorpholine, pipera or salts, polymorphs, or hydrates thereof, having structural Zine, pyridazine, pyrimidine, and pyrazine. Formula (XIII) US 8,859,550 B2 19 20 at least 80%, at least 85%, at least 90%, at least 95%, at least (XIII) 96%, at least 97%, at least 98%, or at least 99% 8-(4-Meth eN ylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine ( \, mesylate tartrate monohydrate (Form 2). S NSZ In certain embodiments, provided herein are compounds, or salts, polymorphs, or hydrates thereof, having structural 2 N r Formula (XIV) N-so 10 (XIV) eN 7 V or a salt, polymorph or hydrate thereof, wherein: N Y is a counterion chosen from tartrate and mesylate; and S 2 Zis a chosen from a monohydrate, a hemihydrate, and null. 15 In certain embodiments, provided herein is the salt 8-(4- methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a 1 pyrazine mesylate, or a hydrate or polymorph thereof. N In certain embodiments, provided herein is a hydrate of the H oYoz. salt, which is 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl) tetrazolo 1.5-alpyrazine mesylate monohydrate, or a poly or a polymorph or hydrate thereof, wherein: morph thereof. Y is a counterion chosen from citrate, hydrochloride, In certain embodiments, provided herein is a polymorph of mesylate; and the hydrate, which is 8-(4-methylpiperazin-1-yl)-5- Zis a chosen from a monohydrate, a hemihydrate, and null. (thiophen-2-yl)tetrazolo 1.5-alpyrazine meSylate monohy 25 In certain embodiments, provided herein is the salt N-me drate Form 1. In certain embodiments, the polymorph com thyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azeti prises at least 75% 8-(4-Methylpiperazin-1-yl)-5-(thiophen 2-yl)tetrazolo 1.5-alpyrazine mesylate monohydrate Form 1. din-3-amine citrate, or a hydrate or polymorph thereof. In certain embodiments, the polymorph comprises at least In further embodiments, provided herein is a polymorph of 80% 8-(4-Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 30 the salt, which is N-methyl-1-(5-(thiophen-2-yl)tetrazolol, 1.5-alpyrazine mesylate monohydrate Form 1. In certain 5-alpyrazin-8-yl)azetidin-3-amine citrate Form 1. In certain embodiments, the polymorph comprises at least 90% 8-(4- embodiments, the polymorph comprises at least 75%, at least Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a 80%, at least 85%, at least 90%, at least 95%, at least 96%, at pyrazine mesylate monohydrate Form 1. In certain embodi least 97%, at least 98%, or at least 99% N-methyl-1-(5- ments, the polymorph comprises at least 95% 8-(4- 35 (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a amine citrate Form 1. pyrazine mesylate monohydrate Form 1. In certain In other embodiments, provided herein is the salt N-me embodiments, the polymorph comprises at least 96% 8-(4- thyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azeti Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a din-3-amine hydrochloride, or a hydrate or polymorph pyrazine mesylate monohydrate Form 1. In certain embodi 40 thereof. ments, the polymorph comprises at least 97% 8-(4- In further embodiments, provided herein is a polymorph of Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a the salt, which is N-methyl-1-(5-(thiophen-2-yl)tetrazolol, pyrazine mesylate monohydrate Form 1. In certain 5-alpyrazin-8-yl)azetidin-3-amine hydrochloride Form 1. In embodiments, the polymorph comprises at least 98% 8-(4- certain embodiments, the polymorph comprises at least 75% Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a 45 N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) pyrazine mesylate monohydrate Form 1. In certain embodi azetidin-3-amine hydrochloride Form 1. In certain embodi ments, the polymorph comprises at least 99% 8-(4- ments, the polymorph comprises at least 80% N-methyl-1- Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a (5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- pyrazine mesylate monohydrate Form 1. amine hydrochloride Form 1. In certain embodiments, the In certain embodiments, the polymorph is 8-(4-methylpip 50 erazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-apyrazine polymorph comprises at least 90% N-methyl-1-(5-(thiophen mesylate Form 3 2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-amine hydro In certain embodiments, provided herein is a salt, which is chloride Form 1. In certain embodiments, the polymorph 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- comprises at least 95% N-methyl-1-(5-(thiophen-2-yl)tetra apyrazine tartrate, or a hydrate or polymorph thereof. 55 Zolo 1.5-alpyrazin-8-yl)aZetidin-3-amine hydrochloride In certain embodiments, provided herein is a polymorph of Form 1. In certain embodiments, the polymorph comprises at the salt, which is 8-(4-methylpiperazin-1-yl)-5-(thiophen-2- least 96% N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a yl)tetrazolo 1.5-alpyrazine tartrate hemihydrate (Form 1). In pyrazin-8-yl)azetidin-3-amine hydrochloride Form 1. In cer certain embodiments, the polymorph comprises at least 75%, tain embodiments, the polymorph comprises at least 97% at least 80%, at least 85%, at least 90%, at least 95%, at least 60 N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) 96%, at least 97%, at least 98%, or at least 99% 8-(4-Meth azetidin-3-amine hydrochloride Form 1. In certain embodi ylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine ments, the polymorph comprises at least 98% N-methyl-1- mesylate tartrate hemihydrate (Form 1). (5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- In other embodiments, provided herein is a polymorph of amine hydrochloride Form 1. In certain embodiments, the the salt, which is 8-(4-methylpiperazin-1-yl)-5-(thiophen-2- 65 polymorph comprises at least 99% N-methyl-1-(5-(thiophen yl)tetrazolo 1.5-alpyrazine tartrate monohydrate (Form 2). In 2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-amine hydro certain embodiments, the polymorph comprises at least 75%, chloride Form 1. US 8,859,550 B2 21 22 In yet other embodiments, provided herein is the salt N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) N (XV) aZetidin-3-amine mesylate, or a hydrate or polymorph thereof. 7 - In further embodiments, provided herein is a polymorph of 5 S N,N-2 the salt, which is N-methyl-1-(5-(thiophen-2-yl)tetrazolol, 5-alpyrazin-8-yl)azetidin-3-amine mesylate Form 1. In cer 2 tain embodiments, provided herein is a polymorph of the N n hydrate, which is N-methyl-1-(5-(thiophen-2-yl)tetrazolol, N-N 5-alpyrazin-8-yl)azetidin-3-amine mesylate Form 1. In cer OH O tain embodiments, the polymorph comprises at least 75% N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) HO aZetidin-3-amine mesylate Form 1. In certain embodiments, OH 0.5 H2O the polymorph comprises at least 80% N-methyl-1-(5- O OH (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- 15 amine mesylate Form 1. In certain embodiments, the poly morph comprises at least 90% N-methyl-1-(5-(thiophen-2- in a crystalline form. yl)tetrazolo 1.5-alpyrazin-8-yl)aZetidin-3-amine mesylate In certain embodiments, the crystalline form of 8-(4-Me Form 1. In certain embodiments, the polymorph comprises at thylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyra least 95% N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a zine Tartrate Hemihydrate is characterized in that said form pyrazin-8-yl)aZetidin-3-amine mesylate Form 1. In certain has at least one of the following characteristics: embodiments, the polymorph comprises at least 96% N-me an X-ray powder diffraction pattern with peaks at 10.2, thyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azeti 13.4, 17.4, 18.1, 23.5 and 26.0 degrees two theta (+0.2 din-3-amine mesylate Form 1. In certain embodiments, the 25 degree) (CuKO. =1.54059A), or an X-ray powder diffraction polymorph comprises at least 97% N-methyl-1-(5-(thiophen pattern substantially in accordance with that shown in FIG.1; 2-yl)tetrazolo 1.5-alpyrazin-8-yl)aZetidin-3-amine mesylate and Form 1. In certain embodiments, the polymorph comprises at an FT-Raman spectrum with the bands at 1339, 1356, 1524 least 98% N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a and 1589 cm or an FT-Raman spectrum substantially in pyrazin-8-yl)aZetidin-3-amine mesylate Form 1. In certain accordance with that shown in FIG. 2. embodiments, the polymorph comprises at least 99% N-me In certain embodiments, provided herein is 8-(4-Meth thyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azeti ylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine din-3-amine mesylate Form 1. Tartrate Monohydrate, having the structural formula (XVI) In certain embodiments, provided herein is a compound of Formula XII as recited above, or a salt, hydrate, or polymorph thereof, which forms a crystalline solid which has a solubility, (XVI) NetNe in aqueous media at about pH 3.5 to about pH 5.0, of at least 7 I V 2.5 mg/mL. S Nna N In further embodiments, the compound of Formula XII as 40 recited above, or a salt, hydrate, or polymorph thereof, has a solubility, in aqueous media at about pH 3.5 to about pH 5.0, 2 of at least 5.0 mg/mL. N r In further embodiments, the compound of Formula XII as Nu N recited above, or a salt, hydrate, or polymorph thereof, has a 45 OH O solubility, in aqueous media at about pH 3.5 to about pH 5.0, HO of at least 10.0 mg/mL. OH HO In further embodiments, the compound of Formula XII as recited above, or a salt, hydrate, or polymorph thereof, has a O OH solubility, in aqueous media at about pH 3.5 to about pH 5.0, 50 of at least 20.0 mg/mL. in a crystalline form. In further embodiments, the compound of Formula XII as recited above, or a salt, hydrate, or polymorph thereof, has a In certain embodiments, the crystalline form of 8-(4-Me solubility, in aqueous media at about pH 3.5 to about pH 5.0, thylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyra of at least 30.0 mg/mL. 55 zine Tartrate Monohydrate is characterized in that said form In certain embodiments, compound is a polymorph and Y has at least one of the following characteristics: is null. an X-ray powder diffraction pattern with peaks at 11.4. In certain embodiments, the compound is a polymorph of 16.2, 17.1 19.0, 23.8 and 24.1 degrees two theta (+0.2 degree) (CuKO. = 1.54059A), or an X-ray powder diffraction pattern 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- 60 apyrazine. substantially in accordance with that shown in FIG. 5; and In certain embodiments, the compound is a polymorph of an FT-Raman spectrum with the bands at 1350, 1422, N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) 1494, 1522 and 1593 cm or an FT-Raman spectrum sub aZetidin-3-amine. stantially in accordance with that shown in FIG. 6. In certain embodiments, provided herein is 8-(4-Meth 65 In certain embodiments, provided herein is 8-(4-Meth ylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine ylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Hemihydrate, having the structural formula (XV) Mesylate Monohydrate, having the structural formula (XVII) US 8,859,550 B2 23 24

(XVII) (XIX) NetN eN (i \, ( \, S Nna S N 2 O O Né HO 2 2 / Yon N N N1 10 H O OH O O in crystalline form. HO OH In certain embodiments, the crystalline form of 8-(4-Me 15 OH thylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyra zine Mesylate Monohydrate is characterized in that said form has at least one of the following characteristics: in a crystalline form. In certain embodiments, the crystalline form of N-methyl an X-ray powder diffraction pattern with peaks at 5.3, 10.5, 1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- 15.1, 20.4, 21.2, 21.8 and 23.0 degrees two theta (+0.2 amine Citrate is characterized in that said form has at least one degree) (CuKO. =1.54059A), or an X-ray powder diffraction of the following characteristics: pattern substantially in accordance with that shown in FIG.9; an X-ray powder diffraction pattern with peaks at 9.4, 12.4. and 25 18.9, 19.9, 26.2 and 28.6 degrees two theta (+0.2 degree) an FT-Raman spectrum with the bands at 1299, 1313, (CuKO. = 1.54059A), or an X-ray powder diffraction pattern 1354, 1422, 1494, 1523 and 1590 cm or an FT-Raman substantially in accordance with that shown in FIG. 20; and spectrum substantially in accordance with that shown in FIG. an FTIR spectrum with the bands at 731, 844, 1110, 1199, 10 1209, 1310, 1448 and 1564 cm or an FTIR spectrum sub 30 In certain embodiments, provided herein is 8-(4-Meth stantially in accordance with that shown in FIG. 21. ylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine In certain embodiments, provided herein is N-methyl-1-(5- Mesylate, having the structural formula (XVIII) (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- amine Hydrochloride, having the structural formula (XX) 35

(XVIII) ( eN\, S N 2 O O 40 HCI C2 X/ Yo,

45 1. N

in a crystalline form. 50 in a crystalline form; In certain embodiments, the crystalline form of 8-(4-Me thylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyra In certain embodiments, the crystalline form of N-methyl zine Mesylate is characterized in that said form has at least 1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- one of the following characteristics: amine Hydrochloride is characterized in that said form has at 55 least one of the following characteristics: an X-ray powder diffraction pattern with peaks at 10.4, an X-ray powder diffraction pattern with peaks at 6.0, 10.8, 16.3, 17.2, 17.8, 22.8, 24.0 and 27.8 degrees two theta (+0.2 12.7, 18.1, 20.2, 25.1 and 25.4 degrees two theta (+0.2 degree) (CuKO. =1.54059A), or an X-ray powder diffraction degree) (CuKO. =1.54059A), or an X-ray powder diffraction pattern substantially in accordance with that shown in FIG. pattern substantially in accordance with that shown in FIG. 17; and 60 24; and an FT-Raman spectrum with the bands at 1346, 1423, an FTIR spectrum with the bands at 632, 715, 850, 1196, 1493, 1524 and 1589 cm or an FT-Raman spectrum sub 1421, 1551 and 2689 cm oran FTIR spectrum substantially stantially in accordance with that shown in FIG. 18. in accordance with that shown in FIG. 25. In certain embodiments, provided herein is N-methyl-1-(5- 65 In certain embodiments, provided herein is N-methyl-1-(5- (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- amine Citrate, having the structural formula (XIX) amine Mesylate, having the structural formula (XXI) US 8,859,550 B2 25 26 In further embodiments, the pharmaceutical composition (XXI) comprises N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a pyrazin-8-yl)azetidin-3-amine hydrochloride Form 1. together with a pharmaceutically acceptable carrier. ( A In certain embodiments, the pharmaceutical composition comprises N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a S C \,2” pyrazin-8-yl)aZetidin-3-amine mesylate or a polymorph 4N / Yon thereof, together with a pharmaceutically acceptable carrier. In further embodiments, the pharmaceutical composition As 10 comprises N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a pyrazin-8-yl)aZetidin-3-amine mesylate Form 1, together with a pharmaceutically acceptable carrier. in a crystalline form. Also provided herein is a method of treatment of an HR In certain embodiments, the crystalline form of N-methyl mediated disease comprising the administration, to a patient 1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- 15 in need thereof, of a therapeutically effective amount of a amine Mesylate is characterized in that said form has at least compound as disclosed herein. In further embodiments, the one of the following characteristics: method comprises the administration, to a patient in need an X-ray powder diffraction pattern with peaks at 11.6, thereof, of a therapeutically effective amount of a compound 15.0, 17.7, 21.0, 24.3 and 25.2 degrees two theta (+0.2 of Formula XII, disclosed above. All methods disclosed degree) (CuKO. =1.54059A), or an X-ray powder diffraction below also encompass the equivalent medical use of the com pattern substantially in accordance with that shown in FIG. pounds, including the compounds for use in the manufacture 29; and of medicaments for the treatment of disease. an FTIR spectrum with the bands at 733,781, 1038, 1140, In certain embodiments provided herein, said treatment is 1199 and 1557 cm or an FTIR spectrum substantially in systemic. accordance with that shown in FIG. 30. 25 In certain embodiments, said administration is topical. In certain embodiments, provided herein is a pharmaceu In certain embodiments, said disease is chosen from an tical composition comprising a compound, or a salt, poly inflammatory disease, an autoimmune disease, an allergic morph or hydrate thereof, as disclosed herein, together with a disorder, and an ocular disorder. pharmaceutically acceptable carrier. In certain embodiments, disease is chosen from pruritus, 30 eczema, atopic dermatitis, asthma, chronic obstructive pull In certain embodiments, the compound has structural For monary disease (COPD), allergic rhinitis, non-allergic rhini mula XII, disclosed above, together with a pharmaceutically tis, rhinosinusitis, nasal inflammation, nasal congestion, acceptable carrier. sinus congestion, otic inflammation dry eye, ocular inflam Also provided herein is a pharmaceutical composition mation, allergic conjunctivitis, Vernal conjunctivitis, Vernal comprising a compound chosen from 8-(4-methylpiperazin 35 keratoconjunctivitis, and giant papillary conjunctivitis. 1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine and N-me In further embodiments, the disease is chosen from pruri thyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azeti tis, allergic rhinitis, non-allergic rhinitis, rhinosinusitis, nasal din-3-amine, or a salt, polymorph or hydrate thereof. inflammation, nasal congestion, sinus congestion, nasal poly In certain embodiments, the compound is a salt and Y is posis, asthma, COPD, allergic conjunctivitis, dry eye, and chosen from tartrate, mesylate, citrate, and hydrochloride. 40 otic inflammation. Also provided herein is a pharmaceutical composition In certain embodiments, the compound of Formula XII is a comprising a salt, or a polymorph thereof, chosen from salt and Y is chosen from tartrate, mesylate, citrate, and 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- hydrochloride. apyrazine tartrate hemihydrate; In certain embodiments, the compound of Formula XII is a 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- 45 salt, or a polymorph thereof, chosen from apyrazine tartrate monohydrate; 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- apyrazine tartrate hemihydrate; apyrazine mesylate monohydrate; 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) apyrazine tartrate monohydrate; aZetidin-3-amine citrate; 50 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) apyrazine mesylate monohydrate; aZetidin-3-amine hydrochloride; and N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) aZetidin-3-amine citrate; aZetidin-3-amine mesylate; N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) together with a pharmaceutically acceptable carrier. 55 aZetidin-3-amine hydrochloride; and In certain embodiments, the pharmaceutical composition N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) comprises 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tet aZetidin-3-amine mesylate. razolo 1.5-alpyrazine mesylate monohydrate or a polymorph In certain embodiments, the method comprises the admin thereof, together with a pharmaceutically acceptable carrier. istration of a compound of Formula XII is chosen from 8-(4- In further embodiments, the pharmaceutical composition 60 methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a comprises 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tet pyrazine mesylate monohydrate Form 1, N-methyl-1-(5- razolo 1.5-alpyrazine mesylate monohydrate Form 1. (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- together with a pharmaceutically acceptable carrier. amine hydrochloride Form 1, and N-methyl-1-(5-(thiophen In certain embodiments, the pharmaceutical composition 2-yl)tetrazolo 1.5-alpyrazin-8-yl)aZetidin-3-amine mesylate comprises N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a 65 Form 1. pyrazin-8-yl)azetidin-3-amine hydrochloride, together with a In certain embodiments, the administration is oral. pharmaceutically acceptable carrier. In certain embodiments, the administration is intravenous. US 8,859,550 B2 27 28 In certain embodiments, the administration is topical. In certain embodiments, the other therapeutic agent is an In certain embodiments, the topical administration is intra HR antagonist. nasal, otic, dermal, ophthalmic, or by inhalation. In certain embodiments, the HRantagonist is chosen from In certain embodiments, said topical administration is to , alcaftadine, , azelastine, , the skin. , cetirizine, chlorpheniramine, , In certain embodiments, said topical administration is to desloratidine, diphenhydramine, , , the eye. , epinastine, feXofenadine, hydroxy Zine, keto In certain embodiments, the topical administration is intra tifen, , , loratidine, , nasal. , , , and . In certain embodiments, the topical administration is intra 10 nasal and the compound is chosen from 8-(4-methylpiper In certain embodiments, the other therapeutic agent is an azin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine mesy HR antagonist. late monohydrate Form 1, N-methyl-1-(5-(thiophen-2-yl) In certain embodiments, the other therapeutic agents are an tetrazolo 1.5-apyrazin-8-yl)azetidin-3-amine hydrochloride 15 HR antagonist and an HR antagonist. Form 1, and N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a In certain embodiments, the other therapeutic agent is an pyrazin-8-yl)aZetidin-3-amine mesylate Form 1. intranasal corticosteroid. Further provided herein is a method of treatment of allergic rhinitis comprising the topical, intranasal administration, to a In certain embodiments, the intranasal corticosteroid is patient in need thereof, of a therapeutically effective amount chosen from fluticaSone, budesonide, beclomethasone, of 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo.1, mometasone and ciclesonide. 5-alpyrazine mesylate monohydrate, or a polymorph thereof. Also provided herein is a method of treatment of an HR In further embodiments, the 8-(4-methylpiperazin-1-yl)-5- mediated disease comprising the administration, to a patient (thiophen-2-yl)tetrazolo 1.5-alpyrazine meSylate monohy in need thereof, of a therapeutically effective amount of a drate is the Form 1 polymorph. 25 compound, or a salt, hydrate, or polymorph thereof, as recited Also provided herein is a method of treatment of allergic herein. rhinitis comprising the topical, intranasal administration, to a Also provided herein is a method of treatment of an HR patient in need thereof, of a therapeutically effective amount of N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8- mediated disease comprising the administration of yl)azetidin-3-amine hydrochloride, or a polymorph thereof. 30 a) a therapeutically effective amount of a salt or polymorph In further embodiments, the N-methyl-1-(5-(thiophen-2- as recited herein; and yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-amine hydrochlo b) another therapeutic agent. ride is the Form 1 polymorph. Examples of other therapeutic agents and combinations are Also provided herein is a method of treatment of the pain or disclosed herein. inflammation resulting from eye surgery, comprising deliver 35 ing to a patient in need of Such treatment with a therapeuti Also provided herein is a method for achieving an effect in cally effective amount of a salt or polymorph as disclosed a patient comprising the administration of a therapeutically herein. In certain embodiments, the eye surgery is chosen effective amount of a salt or polymorph as recited herein to a from cataract Surgery and refractive Surgery (including, e.g., patient, wherein the effect is chosen from reduction in the Laser Assisted In-Situ Keratomileusis (LASIK), Laser 40 number of mast cells, inhibition of inflammatory cell (e.g., Assisted Sub-Epithelium Keratomileusis (LASEK), photore granulocytes including eosinophils, basophils, and neutro fractive keratectomy, and various types of keratotomy and phils, mast cells, lymphocytes, and dendritic cells) migration keratoplasty). In further embodiments, the disease is an aller optionally to the nasal mucosa, the ear, the eye, or the wound gic disorder. In further embodiments, the method comprises site, reduction in inflammatory markers, reduction in inflam the administration, to a patient in need thereof. ofatherapeu 45 matory cytokines, reduction in Scratching, relief of symptoms tically effective amount of a compound of Formula XII, dis and/or signs of nasal congestion from allergic and non-aller closed above. gic causes, decreased watering or redness of the eyes, and Also provided herein is a method of inhibition of HR signaling comprising contacting HR with a compound, or a reduction in ocular pain. salt, hydrate, or polymorph thereof, as disclosed herein. 50 As used herein, the terms below have the meanings indi Also provided herein is a compound, or a salt, hydrate, or cated. polymorph thereof, as recited in Claim 1 for use as a medi The compound 8-(4-Methylpiperazin-1-yl)-5-(thiophen cament. In certain embodiments, the compound has structural 2-yl)tetrazolo 1.5-alpyrazine Monomethanesulfonate Formula XII, disclosed above. Also provided herein is the use of a compound, or a salt, 55 hydrate, or polymorph thereof, as disclosed herein in the NeN manufacture of a medicament for the prevention or treatment / VN of a disease or condition ameliorated by the inhibition of HR. 2 In certain embodiments, the compound has structural For S \,2” mula XII, disclosed above. 60 Also provided herein is a pharmaceutical composition 4. n / Yoh comprising: a. a compound, or a salt, hydrate, or polymorph thereof, as Non recited herein; b. another therapeutic agent; and 65 c. one or more pharmaceutically acceptable carriers or is also referred to as Compound 1 Monomethanesulfonate adjuvants. and Compound 1 Mesylate. US 8,859,550 B2 29 30 The compound 8-(4-Methylpiperazin-1-yl)-5-(thiophen- -continued 2-yl)tetrazolo 1.5-alpyrazine Monomethanesulfonate mono OH O hydrate HO OH 0.5 H2O 5 O OH 7 NeN S NaNa is also referred to as Compound 1 Tartrate Hemihydrate. & a' The compound N-methyl-1-(5-(thiophen-2-yl)tetrazolo.1, S HO 5-alpyrazin-8-yl)azetidin-3-amine Hydrochloride N 2 N ~ /NOH N N / INetN N 15 S Nna is also referred to as Compound 1 Monomethanesulfonate Monohydrate and Compound 1 Mesylate Monohydrate. 2 HCI The compound 8-(4-Methylpiperazin-1-yl)-5-(thiophen- N \\ 2-yl)tetrazolo 1.5-alpyrazine Tartrate 2O N1 H

7 NetN is also referred to as Compound 2 Hydrochloride. N OH O The compound N-methyl-1 -(5 -(thiophen-2-yl)tetrazolol, S 2 25 5-alpyrazin-8-yl)azetidin-3-amine Monomethanesulfonate HO 4. N OH N ~ O OH / lNe V NS 30 S NNZ is also referred to as Compound 1 Tartrate. C.As / OH The compound 8-(4-Methylpiperazin-1-yl)-5-(thiophen 2-yl)tetrazolo 1.5-alpyrazine Tartrate Monohydrate 35 N

is also referred to as Compound 2 Monomethanesulfonate / N=N and Compound 2 Mesylate. 40 The compound N-methyl-1-(5-(thiophen-2-yl)tetrazolo1, S 2 5-alpyrazin-8-yl)azetidin-3-amine Citrate

4. N 45 ON /S N -2 N OH O HO 4N OH H2O 50 As 1. O OH N O OH O O is also referred to as Compound 1 Tartrate Monohydrate. The compound 8-(4-Methylpiperazin-1-yl)-5-(thiophen- 55 HO OH 2-yl)tetrazolo 1.5-alpyrazine Tartrate Hemihydrate OH is also referred to as Compound 2 Citrate. Compounds Disclosed N 60 When ranges of values are disclosed, and the notation 2 “from n . . . to n is used, where n and n are the numbers, then unless otherwise specified, this notation is intended to 2 ~ include the numbers themselves and the range between them. N N This range may be integral or continuous between and includ Nu 65 ing the end values. By way of example, the range “from 2 to N 6 carbons' is intended to include two, three, four, five, and six carbons, since carbons come in integer units. Compare, by US 8,859,550 B2 31 32 way of example, the range “from 1 to 3 uM (micromolar).” singly or doubly oxidized. Examples of suitable alkylthioet which is intended to include 1 uM, 3 uM, and everything in her groups include methylthio, ethylthio, n-propylthio, iso between to any number of significant figures (e.g., 1.255uM. propylthio, n-butylthio, iso-butylthio, sec-butylthio, tert-bu 2.1 uM, 2.9999 uM, etc.). tylthio, methanesulfonyl, ethanesulfinyl, and the like. The term “about as used herein, is intended to qualify the The term “alkynyl as used herein, alone or in combina numerical values which it modifies, denoting Such a value as tion, refers to a straight-chain or branched chain hydrocarbon variable within a margin of error. When no particular margin group having one or more triple bonds and containing from 2 of error, such as a standard deviation to a mean value given in to 20 carbon atoms. In certain embodiments, said alkynyl a chart or table of data, is recited, the term “about should be group comprises from 2 to 6 carbonatoms. In further embodi understood to mean that range which would encompass the 10 recited value and the range which would be included by ments, said alkynyl group comprises from 2 to 4 carbon rounding up or downto that figure as well, taking into account atoms. The term “alkynylene' refers to a carbon-carbon triple significant figures. bond attached at two positions such as ethynylene (—C::: C , —C=C- ). Examples of alkynyl groups include ethy The term “acyl, as used herein, alone or in combination, nyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pen refers to a carbonyl attached to an alkenyl, alkyl, aryl, 15 cycloalkyl, heteroaryl, heterocycle, or any other moiety tyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like. where the atom attached to the carbonyl is carbon. An Unless otherwise specified, the term “alkynyl' may include “acetyl group refers to a —C(O)CH group. An “alkylcar “alkynylene' groups. bonyl or "alkanoyl group refers to an alkyl group attached The terms "amido” and “carbamoyl.” as used herein, alone to the parent molecular moiety through a carbonyl group. or in combination, refer to an amino group as described below Examples of Such groups include methylcarbonyl and ethyl attached to the parent molecular moiety through a carbonyl carbonyl. Examples of acyl groups include formyl, alkanoyl group, or vice versa. The term “C-amido” as used herein, and aroyl. alone or in combination, refers to a —C(=O)—NR group The term “alkenyl, as used herein, alone or in combina with Ras defined herein. The term "N-amido” as used herein, tion, refers to a straight-chain or branched-chain hydrocarbon 25 alone or in combination, refers to a RC(=O)NH group, group having one or more double bonds and containing from with R as defined herein. The term “acylamino” as used 2 to 20 carbon atoms. In certain embodiments, said alkenyl herein, alone or in combination, embraces an acyl group will comprise from 2 to 6 carbon atoms. The term “alk attached to the parent moiety through an amino group. An enylene' refers to a carbon-carbon double bond system example of an “acylamino' group is acetylamino (CHC(O) attached at two or more positions such as ethenylene 30 NH ). (—CH=CH-),(—C::C ). Examples of suitable alkenyl The term "amino as used herein, alone or in combination, groups include ethenyl, propenyl, 2-methylpropenyl, 1,4- refers to NRR", wherein Rand Rare independently chosen butadienyl and the like. Unless otherwise specified, the term from hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, het “alkenyl may include “alkenylene' groups. eroaryl, and heterocycloalkyl, any of which may themselves The term “alkoxy.” as used herein, alone or in combination, 35 be optionally substituted. Additionally, R and R' may com refers to an alkyl ether group, wherein the term alkyl is as bine to form heterocycloalkyl, either of which may be option defined below. Examples of suitable alkyl ether groups ally substituted. include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, The term “aryl, as used herein, alone or in combination, iso-butoxy, sec-butoxy, tert-butoxy, and the like. means a carbocyclic aromatic system containing one, two or The term “alkyl, as used herein, alone or in combination, 40 three rings wherein such polycyclic ring systems are fused refers to a straight-chain or branched-chain alkyl group con together. The term “aryl” embraces aromatic groups such as taining from 1 to 20 carbon atoms. In certain embodiments, phenyl, naphthyl, anthracenyl, and phenanthryl. said alkyl group will comprise from 1 to 10 carbon atoms. In The term “arylalkenyl or “aralkenyl, as used herein, further embodiments, said alkyl group will comprise from 1 alone or in combination, refers to an aryl group attached to the to 6 carbonatoms. Alkyl groups may be optionally substituted 45 parent molecular moiety through an alkenyl group. as defined herein. Examples of alkyl groups include methyl, The term “arylalkoxy' or “aralkoxy.” as used herein, alone ethyl, n-propyl, isopropyl. n-butyl, isobutyl, sec-butyl, tert or in combination, refers to an aryl group attached to the butyl, pentyl, iso-amyl, hexyl, octyl, noyl and the like. The parent molecular moiety through an alkoxy group. term “alkylene, as used herein, alone or in combination, The term “arylalkyl or “aralkyl, as used herein, alone or refers to a saturated aliphatic group derived from a straight or 50 in combination, refers to an aryl group attached to the parent branched chain saturated hydrocarbon attached at two or molecular moiety through an alkyl group. more positions, such as methylene (—CH2—). Unless other The term “arylalkynyl or “aralkynyl, as used herein, wise specified, the term “alkyl may include “alkylene’ alone or in combination, refers to an aryl group attached to the groups. parent molecular moiety through an alkynyl group. The term “alkylamino, as used herein, alone or in combi 55 The term “arylalkanoyl or “aralkanoyl or “aroyl, as nation, refers to an alkyl group attached to the parent molecu used herein, alone or in combination, refers to an acyl group lar moiety through an amino group. Suitable alkylamino derived from an aryl-substituted alkanecarboxylic acid such groups may be mono- or dialkylated, forming groups such as, as benzoyl, naphthoyl phenylacetyl, 3-phenylpropionyl (hy for example, N-methylamino, N-ethylamino, N,N-dimethy drocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlo lamino, N,N-ethylmethylamino and the like. 60 rohydrocinnamoyl, and the like. The term “alkylidene, as used herein, alone or in combi The term aryloxy as used herein, alone or in combination, nation, refers to analkenyl group in which one carbonatom of refers to an aryl group attached to the parent molecular moiety the carbon-carbon double bond belongs to the moiety to through an oxy. which the alkenyl group is attached. The terms “benzo' and “benz, as used herein, alone or in The term “alkylthio.” as used herein, alone or in combina 65 combination, refer to the divalent group CH= derived from tion, refers to an alkylthioether (R—S—) group wherein the benzene. Examples include benzothiophene and benzimida term alkyl is as defined above and wherein the sulfur may be Zole. US 8,859,550 B2 33 34 The term “carbamate, as used herein, alone or in combi The term "heteroalkyl, as used herein, alone or in combi nation, refers to an ester of carbamic acid ( NHCOO—) nation, refers to a stable straight or branched chain, or cyclic which may be attached to the parent molecular moiety from hydrocarbon group, or combinations thereof, fully saturated either the nitrogen or acid end, and which may be optionally or containing from 1 to 3 degrees of unsaturation, consisting substituted as defined herein. of the stated number of carbon atoms and from one to three The term “O-carbamylas used herein, alone or in combi heteroatoms chosen from O, N, and S, and wherein the nitro nation, refers to a —OC(O)NRR' group, with R and R' as gen and Sulfur atoms may optionally be oxidized and the defined herein. nitrogen heteroatom may optionally be quaternized. The het The term “N-carbamylas used herein, alone or in combi eroatom(s) O, N and S may be placed at any interior position nation, refers to a ROC(O)NR' group, with R and R' as 10 defined herein. of the heteroalkyl group. Up to two heteroatoms may be The term “carbonyl, as used herein, when alone includes consecutive, such as, for example, —CH2—NH-OCH. formyl —C(O)H and in combination is a —C(O)—group. The term "heteroaryl, as used herein, alone or in combi The term “carboxyl or “carboxy, as used herein, refers to nation, refers to a 3 to 7 membered unsaturated heteromono —C(O)CH or the corresponding “carboxylate' anion, such 15 cyclic ring, or a fused monocyclic, bicyclic, or tricyclic ring as is in a carboxylic acid salt. An "O-carboxy' group refers to system in which at least one of the fused rings is aromatic, a RC(O)O— group, where R is as defined herein. A “C-car which contains at least one atom chosen from O, S, and N. In boxy' group refers to a C(O)OR groups where R is as certain embodiments, said heteroaryl will comprise from 5 to defined herein. 7 carbon atoms. The term also embraces fused polycyclic The term "cyano.” as used herein, alone or in combination, groups wherein heterocyclic rings are fused with aryl rings, refers to CN. wherein heteroaryl rings arefused with other heteroaryl rings, The term “cycloalkyl, or, alternatively, “carbocycle.” as wherein heteroaryl rings are fused with heterocycloalkyl used herein, alone or in combination, refers to a Saturated or rings, or wherein heteroaryl rings are fused with cycloalkyl partially saturated monocyclic, bicyclic or tricyclic alkyl rings. Examples of heteroaryl groups include pyrrolyl pyr group wherein each cyclic moiety contains from 3 to 12 25 rolinyl, imidazolyl pyrazolyl pyridyl, pyrimidinyl, pyrazi carbon atom ring members and which may optionally be a nyl, pyridazinyl, triazolyl pyranyl, furyl, thienyl, oxazolyl, benzo fused ring system which is optionally Substituted as isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, defined herein. In certain embodiments, said cycloalkyl will indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, comprise from 5 to 7 carbon atoms. Examples of such isoquinolyl, quinoxalinyl, quinazolinyl, indazolyl, benzotria cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopen 30 Zolyl, benzodioxolyl, benzopyranyl, benzoxazolyl, benzoxa tyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, indanyl. diazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, ben octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl and Zothienyl, chromonyl, coumarinyl, benzopyranyl, the like. “Bicyclic' and “tricyclic” as used herein are intended tetrahydroquinolinyl, tetraZolopyridazinyl, tetrahydroiso to include both fused ring systems, such as decahydronaph quinolinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl thalene, octahydronaphthalene as well as the multicyclic 35 and the like. Exemplary tricyclic heterocyclic groups include (multicentered) saturated or partially unsaturated type. The carbazolyl, benzidolyl, phenanthrolinyl, dibenzofuranyl, latter type of isomer is exemplified in general by bicyclo1, acridinyl, phenanthridinyl, Xanthenyl and the like. 1.1 pentane, camphor, adamantane, and bicyclo3.2.1]oc The terms "heterocycloalkyl and, interchangeably, “het tane. erocycle as used herein, alone or in combination, each refer The term “ester, as used herein, alone or in combination, 40 to a saturated, partially unsaturated, or fully unsaturated refers to a carboxy group bridging two moieties linked at monocyclic, bicyclic, or tricyclic heterocyclic group contain carbon atoms. ing at least one heteroatom as a ring member, wherein each The term “ether, as used herein, alone or in combination, said heteroatom may be independently chosen from nitrogen, refers to an oxy group bridging two moieties linked at carbon oxygen, and Sulfur. In certain embodiments, said heterocy atOmS. 45 cloalkyl will comprise from 1 to 4 heteroatoms as ring mem The term “halo,” or “halogen, as used herein, alone or in bers. In further embodiments, said heterocycloalkyl will combination, refers to fluorine, chlorine, bromine, or iodine. comprise from 1 to 2 heteroatoms as ring members. In certain The term “haloalkoxy.” as used herein, alone or in combi embodiments, said heterocycloalkyl will comprise from 3 to nation, refers to a haloalkyl group attached to the parent 8 ring members in each ring. In further embodiments, said molecular moiety through an oxygen atom. 50 heterocycloalkyl will comprise from 3 to 7 ring members in The term “haloalkyl, as used herein, alone or in combina each ring. In yet further embodiments, said heterocycloalkyl tion, refers to an alkyl group having the meaning as defined will comprise from 5 to 6 ring members in each ring. “Het above wherein one or more hydrogens are replaced with a erocycloalkyl and "heterocycle” are intended to include sul halogen. Specifically embraced are monohaloalkyl, diha fones, Sulfoxides, N-oxides of tertiary nitrogen ring mem loalkyl and polyhaloalkyl groups. A monohaloalkyl group, 55 bers, and carbocyclic fused and benzo fused ring systems; for one example, may have an iodo, bromo, chloro or fluoro additionally, both terms also include systems where a hetero atom within the group. Dihalo and polyhaloalkyl groups may cycle ring is fused to an aryl group, as defined herein, or an have two or more of the same halo atoms or a combination of additional heterocycle group. Examples of heterocycle different halo groups. Examples of haloalkyl groups include groups include aziridinyl, aZetidinyl, 1,3-benzodioxolyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, 60 dihydroiSoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl, dichloromethyl, trichloromethyl, pentafluoroethyl, hep dihydrobenzodioxinyl, dihydro1.3oxazolo 4,5-b]pyridi tafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, nyl, benzothiazolyl, dihydroindolyl, dihy-dropyridinyl, 1,3- difluoroethyl, difluoropropyl, dichloroethyl and dichloropro dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, mor pyl. "Haloalkylene' refers to a haloalkyl group attached at pholinyl, piperazinyl, pyrrolidinyl, tetrahydropyridinyl, two or more positions. Examples include fluoromethylene 65 piperidinyl, thiomorpholinyl, and the like. The heterocycle ( CFH ), difluoromethylene ( CF ), chloromethylene groups may be optionally Substituted unless specifically pro (—CHCl ) and the like. hibited. US 8,859,550 B2 35 36 The term “hydrazinyl as used herein, alone or in combi The term “perhaloalkoxy' refers to analkoxy group where nation, refers to two amino groups joined by a single bond, all of the hydrogen atoms are replaced by halogen atoms. i.e., —N-N-. The term “perhaloalkyl as used herein, alone or in com The term “hydroxy.” as used herein, alone or in combina bination, refers to an alkyl group where all of the hydrogen tion, refers to —OH. atoms are replaced by halogen atoms. The term “hydroxyalkyl, as used herein, alone or in com The terms "sulfonate.” “sulfonic acid, and “sulfonic, as bination, refers to a hydroxy group attached to the parent used herein, alone or in combination, refer to the SOH molecular moiety through an alkyl group. group and its anion as the Sulfonic acid is used in Salt forma The term “imino, as used herein, alone or in combination, tion. refers to —N—. 10 The term "sulfanyl.” as used herein, alone or in combina tion, refers to —S—. The term “iminohydroxy.” as used herein, alone or in com The term "sulfinyl, as used herein, alone or in combina bination, refers to —N(OH) and —N O—. tion, refers to —S(O)—. The term "isocyanato” refers to a NCO group. The term “sulfonyl, as used herein, alone or in combina The term “isothiocyanato” refers to a NCS group. 15 tion, refers to —S(O) . The term “lower” as used herein, alone or in a combina The term “N-sulfonamido” refers to a RS(=O)NR'— tion, where not otherwise specifically defined, means con group with R and R' as defined herein. taining from 1 to and including 6 carbon atoms. The term “S-sulfonamido” refers to a S(=O)NRR', The term “lower aryl, as used herein, alone or in combi group, with R and R as defined herein. nation, means phenyl or naphthyl, which may be optionally The terms “thia' and “thio.” as used herein, alone or in substituted as provided. combination, refer to a —S— group or an ether wherein the The term “lower heteroalkyl as used herein, alone or in oxygen is replaced with sulfur. The oxidized derivatives of the combination, refers to a stable straight or branched chain, or thio group, namely Sulfinyl and Sulfonyl, are included in the cyclic hydrocarbon group, or combinations thereof, fully definition of thia and thio. saturated or containing from 1 to 3 degrees of unsaturation, 25 The term “thiol, as used herein, alone or in combination, consisting of one to six atoms in which one to three may be refers to an —SH group. heteroatoms chosen from O, N, and S, and the remaining The term “thiocarbonyl as used herein, when alone atoms are carbon. The nitrogen and Sulfur atoms may option includes thioformyl —C(S)H and in combination is a ally be oxidized and the nitrogen heteroatom may optionally —C(S)—group. be quaternized. The heteroatom(s) O, N and S may be placed 30 The term “N-thiocarbamyl” refers to an ROC(S)NR' at any interior or terminal position of the heteroalkyl group. group, with R and R as defined herein. Up to two heteroatoms may be consecutive, such as, for The term "O-thiocarbamyl” refers to a OC(S)NRR example, —CH NH-OCH. group with R and R' as defined herein. The term “lower heteroaryl, as used herein, alone or in The term “thiocyanato” refers to a —CNS group. combination, means either 1) monocyclic heteroaryl com 35 Any definition herein may be used in combination with any prising five or six ring members, of which between one and other definition to describe a composite structural group. By four said members may be heteroatoms chosen from O, S, and convention, the trailing element of any such definition is that N, or 2) bicyclic heteroaryl, wherein each of the fused rings which attaches to the parent moiety. For example, the com comprises five or six ring members, comprising between posite group alkylamido would represent an alkyl group them one to four heteroatoms chosen from O, S, and N. 40 attached to the parent molecule through an amido group, and The term “lower cycloalkyl, as used herein, alone or in the term alkoxyalkyl would represent an alkoxy group combination, means a monocyclic cycloalkyl having between attached to the parent molecule through an alkyl group. three and six ring members. Lower cycloalkyls may be unsat When a group is defined to be “null.” what is meant is that urated. Examples of lower cycloalkyl include cyclopropyl. said group is absent. cyclobutyl, cyclopentyl, and cyclohexyl. 45 The term “optionally substituted” means the anteceding The term “lower heterocycloalkyl, as used herein, alone or group may be substituted or unsubstituted. When substituted, in combination, means a monocyclic heterocycloalkyl having the substituents of an “optionally substituted group may between three and six ring members, of which between one include, without limitation, one or more Substituents indepen and four may be heteroatoms chosen from O, S, and N. dently selected from the following groups or a particular Examples of lower heterocycloalkyls include pyrrolidinyl, 50 designated set of groups, alone or in combination: lower imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, and alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower morpholinyl. Lower heterocycloalkyls may be unsaturated. heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lower The term “lower amino, as used herein, alone or in com haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower bination, refers to —NRR", wherein R and R' are indepen perhaloalkoxy, lower cycloalkyl, phenyl, aryl, aryloxy, lower dently chosen from hydrogen, lower alkyl, and lower het 55 alkoxy, lower haloalkoxy, Oxo, lower acyloxy, carbonyl, car eroalkyl, any of which may be optionally substituted. boxyl, lower alkylcarbonyl, lower carboxyester, lower car Additionally, the RandR' of a lower amino group may com boxamido, cyano, hydrogen, halogen, hydroxy, amino, lower bine to form a five- or six-membered heterocycloalkyl, either alkylamino, arylamino, amido, nitro, thiol, lower alkylthio. of which may be optionally substituted. lower haloalkylthio, lower perhaloalkylthio, arylthio, sul The term “mercaptyl” as used herein, alone or in combi 60 fonate, sulfonic acid, trisubstituted silyl N, SH, SCHC(O) nation, refers to an RS-group, where R is as defined herein. CH, COCH CO.H. pyridinyl, thiophene, furanyl, lower The term "nitro, as used herein, alone or in combination, carbamate, and lower urea. Two Substituents may be joined refers to NO. together to form a fused five-, six-, or seven-membered car The terms “oxy' or “oxa.” as used herein, alone or in bocyclic or heterocyclic ring consisting of Zero to three het combination, refer to —O—. 65 eroatoms, for example forming methylenedioxy or ethylene The term “oxo, as used herein, alone or in combination, dioxy. An optionally Substituted group may be unsubstituted refers to —O. (e.g., —CH2CH), fully Substituted (e.g., —CFCF), mono US 8,859,550 B2 37 38 substituted (e.g., —CHCHF) or substituted at a levelany “disorder and “condition' (as in medical condition), in that where in-between fully substituted and monosubstituted all reflect an abnormal condition of the human oranimal body (e.g., —CHCF). Where substituents are recited without or of one of its parts that impairs normal functioning, is qualification as to Substitution, both Substituted and unsub typically manifested by distinguishing signs and symptoms, stituted forms are encompassed. Where a Substituent is quali and causes the human or animal to have a reduced duration or fied as “substituted, the substituted form is specifically quality of life. intended. Additionally, different sets of optional substituents The term “combination therapy’ means the administration to a particular moiety may be defined as needed; in these of two or more therapeutic agents to treat a therapeutic con cases, the optional substitution will be as defined, often dition or disorder described in the present disclosure. Such immediately following the phrase, “optionally substituted 10 administration encompasses co-administration of these with therapeutic agents in a Substantially simultaneous manner, The term R or the term R', appearing by itself and without Such as in a single capsule having a fixed ratio of active a number designation, unless otherwise defined, refers to a ingredients or in multiple, separate capsules for each active moiety chosen from hydrogen, alkyl, cycloalkyl, heteroalkyl, ingredient. In addition, Such administration also encom aryl, heteroaryl and heterocycloalkyl, any of which may be 15 passes use of each type of therapeutic agent in a sequential optionally substituted. Such RandR' groups should be under manner. In either case, the treatment regimen will provide stood to be optionally substituted as defined herein. Whether beneficial effects of the drug combination in treating the an R group has a number designation or not, every R group, conditions or disorders described herein. including R, R and R' where n=(1, 2, 3, . . . n), every sub The term “inhibition' (and by extension, “inhibitor') as stituent, and every term should be understood to be indepen used herein encompasses all forms of functional protein (en dent of every other in terms of selection from a group. Should Zyme, kinase, receptor, channel, etc., for example) inhibition, any variable, Substituent, or term (e.g. aryl, heterocycle, R, including neutral antagonism, inverse agonism, competitive etc.) occur more than one time in a formula or generic struc inhibition, and non-competitive inhibition (such as allosteric ture, its definition at each occurrence is independent of the inhibition). Inhibition may be phrased in terms of an ICso definition at every other occurrence. Those of skill in the art 25 defined below. Compounds disclosed herein may be HR will further recognize that certain groups may be attached to allosteric antagonists that are non competitive with hista a parent molecule or may occupy a position in a chain of mine. Additionally, compounds disclosed herein may be ago elements from either end as written. Thus, by way of example nists in one species and antagonists in another. Methods are only, an unsymmetrical group Such as —C(O)N(R)—may be known in the art, and are disclosed herein and can be adapted attached to the parent moiety at either the carbon or the 30 by those of skill in the art, to ascertain whethera compound is, nitrogen. for example, a suitable HRantagonistina species of interest. Asymmetric centers exist in the compounds disclosed In certain embodiments, “HR inhibitor” is used herein to herein. These centers are designated by the symbols “R” or refer to a compound that exhibits an ICs with respect to the “S” depending on the configuration of Substituents around histamine type-1 receptor of no more than about 100 uMand the chiral carbonatom. It should be understood that the inven 35 more typically not more than about 50 LM, as measured in the tion encompasses all Stereochemical isomeric forms, includ in vitro histamine receptor cell-based assays described gen ing diastereomeric, enantiomeric, and epimeric forms, as erally hereinbelow. well as d-isomers and 1-isomers, and mixtures thereof. Indi Similarly, “HR inhibitor” is used herein to refer to a com vidual stereoisomers of compounds can be prepared syntheti pound that exhibits an ICso with respect to the histamine cally from commercially available starting materials which 40 type-3 receptor of no more than about 100 uM and more contain chiral centers or by preparation of mixtures of enan typically not more than about 50 LM, as measured in the in tiomeric products followed by separation Such as conversion vitro histamine receptor cell-based assays described gener to a mixture of diastereomers followed by separation or ally hereinbelow. recrystallization, chromatographic techniques, direct separa Also similarly, “HR inhibitor is used herein to refer to a tion of enantiomers on chiral chromatographic columns, or 45 compound that exhibits an ICso with respect to the histamine any other appropriate method known in the art. Starting com type-4 receptor of no more than about 100 uM and more pounds of particular Stereochemistry are either commercially typically not more than about 50 LM, as measured in the in available or can be made and resolved by techniques known in vitro histamine receptor cell-based assays described gener the art. Additionally, the compounds disclosed herein may ally hereinbelow. exist as geometric isomers. The present invention includes all 50 A “H/H inhibitor is used herein to refer to a compound cis, trans, syn, anti, entgegen (E), and Zusammen (Z) isomers that exhibits an ICs with respect to both the histamine type-1 as well as the appropriate mixtures thereof. Additionally, receptor and the histamine type-4 receptor of no more than compounds may exist as tautomers; all tautomeric isomers about 100 uM and more typically not more than about 50 uM, are provided by this invention. Additionally, the compounds as measured in the in vitro histamine receptor cell-based disclosed herein can exist in unsolvated as well as Solvated 55 assays described generally hereinbelow; the amount of inhi forms with pharmaceutically acceptable solvents such as bition need not be equivalent at each receptor, but should not water, ethanol, and the like. In general, the Solvated forms are be negligible. considered equivalent to the unsolvated forms. In certain embodiments, such as, for example, in the case of The term “bond refers to a covalent linkage between two an in vitro ligand-binding assay protocol. “ICso is that con atoms, or two moieties when the atoms joined by the bond are 60 centration of compound which is required to displace a natu considered to be part of larger substructure. A bond may be ral ligand or reference standard to a half-maximal level. In single, double, or triple unless otherwise specified. A dashed other embodiments, such as, for example, in the case of cer line between two atoms in a drawing of a molecule indicates tain cellular or in vivo protocols which have a functional that an additional bond may be present or absent at that readout, “ICso is that concentration of compound which position. 65 reduces the activity of a functional protein (e.g., HR and/or The term “disease' as used herein is intended to be gener HR) to a half-maximal level. In either of these scenarios, the ally synonymous, and is used interchangeably with, the terms term “ECs” may also be used. In vitro or in vivo, “ECs” US 8,859,550 B2 39 40 refers to the concentration of a compound required to achieve drug), but then is metabolically hydrolyzed to the carboxylic half of the maximal effect in an assay or protocol, typically as acid, the active entity. Additional examples include peptidyl compared to a reference standard. derivatives of a compound. Certain compounds disclosed herein have been discovered Suitable salts include pharmaceutically acceptable salts to exhibit inhibitory activity against H.R. In certain embodi formed with counterions deriving from both organic and inor ments, compounds will exhibit an ICso with respect to HR of ganic acids as well as metals. Such acid addition salts will no more than about 10 uM; in further embodiments, com normally be pharmaceutically acceptable. However, salts of pounds will exhibit an ICs with respect to HR of no more non-pharmaceutically acceptable salts may be of utility in the than about 5uM; in yet further embodiments, compounds will preparation and purification of the compound in question. exhibit an ICs with respect to HR of not more than about 1 10 Basic addition salts may also be formed and be pharmaceu uM; in yet further embodiments, compounds will exhibit an tically acceptable. For a more complete discussion of the ICs with respect to HR of not more than about 200 nM, as preparation and selection of salts, refer to Pharmaceutical measured in an HR assay Such as that described herein. Salts. Properties, Selection, and Use (Stahl, P. Heinrich. The phrase “therapeutically effective' is intended to Wiley-VCHA, Zurich, Switzerland, 2002). qualify the amount of active ingredients used in the treatment 15 The term “therapeutically acceptable salt, as used herein, of a disease or disorder. This amount will achieve the goal of represents salts or Zwitterionic forms of the compounds dis reducing or eliminating the said disease or disorder. closed herein which are water or oil-soluble or dispersible and The term “therapeutically acceptable' refers to those com therapeutically acceptable as defined herein. The salts can be pounds (or salts, prodrugs, tautomers, Zwitterionic forms, prepared during the final isolation and purification of the etc.) which are suitable for use in contact with the tissues of compounds or separately by reacting the appropriate com patients without undue toxicity, irritation, and allergic pound in the form of the free base with a suitable acid. response, are commensurate with a reasonable benefit/risk Representative acid addition salts include acetate, adipate, ratio, and are effective for their intended use. alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate As used herein, reference to “treatment of a patient is (besylate), bisulfate, butyrate, camphorate, camphorsul intended to include prophylaxis. The term “patient’ means all 25 fonate, citrate, digluconate, formate, fumarate, gentisate, glu mammals including humans. Examples of patients include tarate, glycerophosphate, glycolate, hemisulfate, heptanoate, humans, cows, dogs, cats, goats, sheep, pigs, and rabbits. hexanoate, hippurate, hydrochloride, hydrobromide, Preferably, the patient is a human. hydroiodide, 2-hydroxyethanSulfonate (isethionate), lactate, The term “compound, as used herein, includes salts, Sol maleate, malonate, DL-mandelate, mesitylenesulfonate, Vates and polymorphs of the compound, as well as the free 30 methanesulfonate (including monomethanesulfonate, or base. In certain embodiments, the Solvate is a hydrate. A mesylate), naphthylenesulfonate, nicotinate, 2-naphthalene solvate is a stable, solid or semi-solid form of a compound sulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenyl that comprises either a non-stoichiometric or a stoichimetric proprionate, phosphonate, picrate, pivalate, propionate, pyro equivalent of solvent. If the solvent is water, the solvate is a glutamate. Succinate, Sulfonate, tartrate, L-tartrate, hydrate. In certain embodiments, the hydrate has a stoichi 35 trichloroacetate, trifluoroacetate, phosphate, glutamate, metric equivalent of water chosen from about 0, about 0.5, bicarbonate, para-toluenesulfonate (p-tosylate), and unde and about 1 H2O: that is, the hydrate is anhydrous, a hemi canoate. Also, basic groups in the compounds disclosed hydrate, or a monohydrate. Non-stoichiometric hydrates and herein can be quaternized with methyl, ethyl, propyl, and stoichiometric hydrates are both contemplated. As further butyl chlorides, bromides, and iodides; dimethyl, diethyl, discussed below, a polymorph is a distinct crystalline form of 40 dibutyl, and diamyl Sulfates; decyl, lauryl, myristyl, and a compound. A compound may be, for example, a polymorph steryl chlorides, bromides, and iodides; and benzyl and phen ofa free base, a polymorph of a salt, a polymorph of a hydrate, ethyl bromides. Examples of acids which can be employed to or a polymorph of a hydrate of a salt of a compound, and so form therapeutically acceptable addition salts include inor forth. ganic acids such as hydrochloric, hydrobromic, Sulfuric, and The term “prodrug” refers to a compound that is made 45 phosphoric, and organic acids such as oxalic, maleic. Suc more active in vivo. Certain compounds disclosed herein may cinic, and citric. Salts can also be formed by coordination of also exist as prodrugs, as described in Hydrolysis in Drug and the compounds with an alkali metal or alkaline earth ion. Prodrug Metabolism. Chemistry, Biochemistry, and Enzy Hence, the present invention contemplates Sodium, potas mology (Testa, Bernard and Mayer, Joachim M. Wiley sium, magnesium, and calcium salts of the compounds dis VHCA, Zurich, Switzerland 2003). Prodrugs of the com 50 closed herein, and the like. pounds described herein are structurally modified forms of Salts disclosed herein may combine in 1:1 molar ratios, and the compound that readily undergo chemical changes under in fact this is often how they are initially synthesized. How physiological conditions to provide the compound. Addition ever, it will be recognized by one of skill in the art that the ally, prodrugs can be converted to the compound by chemical stoichiometry of one ion in a salt to the other may be other or biochemical methods in an ex vivo environment. For 55 wise. Salts shown herein may be, for the sake of convenience example, prodrugs can be slowly converted to a compound in notation, shown in a 1:1 ratio; all possible Stoichiometric when placed in a transdermal patch reservoir with a suitable arrangements are encompassed by the scope of the present enzyme or chemical reagent. Prodrugs are often useful invention. because, in Some situations, they may be easier to administer When the phrase “Y is a counterion' is used in structural than the compound, or parent drug. They may, for instance, be 60 formulas herein, and neither the compound northe counterion bioavailable by oral administration whereas the parent drug is is drawn showing explicit ionic character, such ionic charac not. The prodrug may also have improved solubility in phar ter may be inferred and a corresponding charges on each maceutical compositions over the parent drug. A wide variety moiety be assumed to be present or absent. For example, if X of prodrug derivatives are known in the art, Such as those that is a monovalent cation such as Mg(OH), it may be inferred rely on hydrolytic cleavage or oxidative activation of the 65 that the coupled compound has lost a proton to form an ionic prodrug. An example, without limitation, of a prodrug would bond with X, despite Formula I being drawn to explicitly be a compound which is administered as an ester (the “pro show all protons in place. Similarly, when X is an anion, the US 8,859,550 B2 41 42 coupled compound takes on cationic character. The notation cal microscopy, Scanning electron microscopy (SEM), elec is left intentionally ambiguous as to placement and ratios of tron crystallography and quantitative analysis, particle size charges since without extensive physical characterization, analysis (PSA), Surface area analysis, solubility studies and such as X-ray crystal diffraction, it is often difficult to know dissolution studies. with certainty where on a compound a counterion has bound. The term, “solvate, as used herein, refers to a crystal form Additionally, counterions and compounds may combine in of a substance which contains solvent. The term “hydrate' uneven molar ratios to form Solid salts. refers to a solvate wherein the solvent is water. The terms, “polymorphs” and “polymorphic forms and The term, "desolvated solvate, as used herein, refers to a related terms herein refer to crystal forms of the same mol crystal form of a substance which can only be made by ecule. Polymorphs may beformed from both the free bases or 10 removing the solvent from a solvate. free acids of compounds, or of salts of compounds, or of The term “amorphous form,” as used herein, refers to a hydrates of compounds or their salts. Different polymorphs noncrystalline form of a Substance. may have different physical properties such as, for example, The term "solubility” is generally intended to be synony melting temperatures, heats of fusion, solubilities, dissolu mous with the term “aqueous solubility.” and refers to the tion rates and/or vibrational spectra as a result of the arrange 15 ability, and the degree of the ability, of a compound to dis ment or conformation of the molecules in the crystal lattice. Solve in water or an aqueous solvent or buffer, as might be The differences in physical properties exhibited by polymor found under physiological conditions. Aqueous solubility is, phs affect pharmaceutical parameters such as storage stabil in and of itself, a useful quantitative measure, but it has ity, compressibility and density (important informulation and additional utility as a correlate and predictor, with some limi product manufacturing), and dissolution rates (an important tations which will be clear to those of skill in the art, of oral factor in bioavailability). Differences in stability can result bioavailability. In practice, a soluble compound is generally from changes in chemical reactivity (e.g. differential oxida desirable, and the more soluble, the better. There are notable tion, such that a dosage form discolors more rapidly when exceptions; for example, certain compounds intended to be comprised of one polymorphthan when comprised of another administered as depot injections, if stable over time, may polymorph) or mechanical changes (e.g. tablets crumble on 25 actually benefit from low solubility, as this may assist in slow storage as a kinetically favored polymorph converts to ther release from the injection site into the plasma. Solubility is modynamically more stable polymorph) or both (e.g., tablets typically reported in mg/mL, but other measures, such as g/g, of one polymorph are more Susceptible to breakdown at high may be used. Solubilities typically deemed acceptable may humidity). As a result of solubility/dissolution differences, in range from 1 mg/mL into the hundreds or thousands of the extreme case, Some polymorphic transitions may result in 30 mg/mL. lack of potency or, at the other extreme, toxicity. In addition, Solubility may be measured under varying conditions. For the physical properties of the crystal may be important in example, it may be measured under conditions similar to processing, for example, one polymorph might be more likely those found in the body, Such as at gastric pH, physiologic or to form solvates or might be difficult to filter and wash free of near-physiologic pH, or nasal mucosa pH. "Gastric pH as impurities (i.e., particle shape and size distribution might be 35 used herein means about pH 1. “Near-physiologic pH, as different between polymorphs). used herein refers to the typical pH of bodily tissues and Polymorphic forms described herein may include forms fluids, such as blood and plasma, or cytoplasm, generally Such as Form 1. Form 2, form 3, amorphous, and the like. about 7.4. “Nasal mucosa pH as used herein means from These terms encompass polymorphs that are substantially about pH 3.5 to about pH 7.6. similar to those described herein. In this context, “substan 40 As used herein, “solid when referring to a salt form means tially similar means that one of skill in the art would recog relatively solid, at room temperature, and/or containing a nize the polymorphs differing insignificantly from those Substantial amount of Solids. A solid may be amorphous in polymorphs as physically characterized herein, or those poly form and/or be a solvated solid with some quantity of residual morphs having one or more properties described herein. By or coordinated of solvent molecules. A crystalline salt is an way of example, a polymorph encompassed by the term Form 45 example of a solid. By way of example, a wax could be 1 could have an X-ray powder diffraction (PXRD) spectrum considered a solid, whereas an oil would not be. which is at least 70%, at least 80%, at least 90%, or at least A “solid composition” as used herein includes a salt of a 95% identical to that shown in the PXRD for Form 1. For compound, or a polymorph or amorphous solid form thereof. example, the encompassed polymorph might have at least While it may be possible for the salts or polymorphs dis 80% of the peaks in common with the disclosed Form 1. 50 closed hereinto be administered as the raw chemical, it is also Alternatively, if the PXRD spectrum is identified by only a possible to present them as a pharmaceutical formulation. few major peaks, the encompassed polymorph might have Accordingly, provided herein are pharmaceutical formula major peaks at least 80% identical to those shown in a PXRD tions which comprise one or more of certain salts or polymor spectrum. Alternatively, the encompassed polymorph might phs disclosed herein, together with one or more pharmaceu have an aqueous solubility which is within 80 to 120% that 55 tically acceptable carriers thereof and optionally one or more shown herein. other therapeutic ingredients. The carrier(s) must be “accept Polymorphs of a molecule can be obtained by a number of able' in the sense of being compatible with the other ingre methods, as known in the art. Such methods include, but are dients of the formulation and not deleterious to the recipient not limited to, melt recrystallization, melt cooling, Solvent thereof. Proper formulation is dependent upon the route of recrystallization, desolvation, rapid evaporation, rapid cool 60 administration chosen. Any of the well-known techniques, ing, slow cooling, vapor diffusion and Sublimation. carriers, and excipients may be used as Suitable and as under Techniques for characterizing polymorphs include, but are stood in the art; e.g., in Remington’s Pharmaceutical Sci not limited to, differential scanning calorimetry (DSC), X-ray ences. The pharmaceutical compositions disclosed herein powder diffractometry (PXRD), thermal gravimetric analysis may be manufactured in any manner known in the art, e.g., by (TGA), dynamic vapor sorption/desorption (DVS), single 65 means of conventional mixing, dissolving, granulating, dra crystal X-ray diffractometry, vibrational spectroscopy, e.g. gee-making, levigating, emulsifying, encapsulating, entrap IR and Raman spectroscopy, solid state NMR, hot stage opti ping or compression processes. US 8,859,550 B2 43 44 The formulations include those suitable for oral, parenteral etary, such in the case of the filler PROSOLVR) (available (including Subcutaneous, intradermal, intramuscular, intrave from JRS Pharma). PROSOLV is a proprietary, optionally nous, intraarticular, and intramedullary), intraperitoneal, high-density, silicified microcrystalline cellulose composed transmucosal, transdermal, rectal and topical (including der of 98% microcrystalline cellulose and 2% colloidal silicon mal, buccal, Sublingual, ocular, intranasal, and intraocular) 5 dioxide. Silicification of the microcrystalline cellulose is administration although the most Suitable route may depend achieved by a patented process, resulting in an intimate asso upon for example the condition and disorder of the recipient. ciation between the colloidal silicon dioxide and microcrys The formulations may conveniently be presented in unit dos talline cellulose. ProSolv comes in different grades based on age form and may be prepared by any of the methods well particle size, and is a white or almost white, fine or granular known in the art of pharmacy. Typically, these methods 10 powder, practically insoluble in water, acetone, ethanol, tolu include the step of bringing into association a salt or poly ene and dilute acids and in a 50 g/l solution of sodium hydrox morph as disclosed herein (“active ingredient') with the car ide. rier which constitutes one or more accessory ingredients. In Examples of disintegrants for use in oral pharmaceutical general, the formulations are prepared by uniformly and inti formulations such as capsules and tablets include, without mately bringing into association the active ingredient with 15 limitation, sodium starch glycolate, sodium carboxymethyl liquid carriers or finely divided solid carriers or both and then, cellulose, calcium carboxymethyl cellulose, croScarmellose if necessary, shaping the product into the desired formulation. Sodium, povidone, crospovidone (polyvinylpolypyrroli Formulations of the salts or polymorphs disclosed herein done), methyl cellulose, microcrystalline cellulose, pow Suitable for oral administration may be presented as discrete dered cellulose, low-substituted hydroxy propyl cellulose, units such as capsules, cachets or tablets each containing a starch, pregelatinized starch, and sodium alginate. predetermined amount of the active ingredient; as a powder or Additionally, glidants and lubricants may be used in oral granules; as a solution or a suspension in an aqueous liquid or pharmaceutical formulations to ensure an even blend of a non-aqueous liquid; or as an oil-in-water liquid emulsion or excipients upon mixing. Examples of lubricants include, a water-in-oil liquid emulsion. The active ingredient may also without limitation, calcium Stearate, glyceryl monostearate, be presented as a bolus, electuary or paste. 25 glyceryl palmitostearate, hydrogenated vegetable oil, light Pharmaceutical preparations which can be used orally mineral oil, magnesium Stearate, mineral oil, polyethylene include tablets, push-fit capsules made of gelatin, as well as glycol, Sodium benzoate, Sodium lauryl Sulfate, sodium soft, sealed capsules made of gelatin and a plasticizer, such as Stearyl fumarate, Stearic acid, talc, and Zinc Stearate. glycerol or sorbitol. Tablets may be made by compression or Examples of glidants include, without limitation, silicon molding, optionally with one or more accessory ingredients. 30 dioxide (SiO2), talc cornstarch, and poloxamers. Poloxamers Compressed tablets may be prepared by compressing in a (or LUTROL(R), available from the BASF Corporation) are suitable machine the active ingredient in a free-flowing form A-B-A block copolymers in which the A segment is a hydro Such as a powder or granules, optionally mixed with binders, philic polyethylene glycol homopolymer and the B segment inert diluents, or lubricating, Surface active or dispersing is hydrophobic polypropylene glycol homopolymer. agents. Molded tablets may be made by molding in a Suitable 35 Examples of tablet binders include, without limitation, machine a mixture of the powdered compound moistened acacia, alginic acid, carbomer, carboxymethyl cellulose with an inert liquid diluent. The tablets may optionally be Sodium, dextrin, ethylcellulose, gelatin, guar gum, hydroge coated or scored and may beformulated so as to provide slow nated vegetable oil, hydroxyethyl cellulose, hydroxypropyl or controlled release of the active ingredient therein. All for cellulose, hydroxypropylmethyl cellulose, copoly Vidone, mulations for oral administration should be in dosages Suit 40 methyl cellulose, liquid glucose, maltodextrin, poly able for Such administration. The push-fit capsules can con methacrylates, povidone, pregelatinized starch, Sodium algi tain the active ingredients in admixture with filler such as nate, starch, Sucrose, tragacanth, and Zein. lactose, binders such as starches, and/or lubricants such as The salts or polymorphs may be formulated for parenteral talc or magnesium Stearate and, optionally, stabilizers. In soft administration by injection, e.g., by bolus injection or con capsules, the active ingredients may be dissolved or Sus 45 tinuous infusion. Formulations for injection may be presented pended in Suitable liquids, such as fatty oils, liquid paraffin, or in unit dosage form, e.g., in ampoules or in multi-dose con liquid polyethylene glycols. In addition, stabilizers may be tainers, with an added preservative. The compositions may added. Dragee cores are provided with Suitable coatings. For take Such forms as Suspensions, solutions or emulsions in oily this purpose, concentrated Sugar Solutions may be used, or aqueous vehicles, and may contain formulatory agents which may optionally contain gum arabic, talc, polyvinyl 50 Such as Suspending, stabilizing and/or dispersing agents. The pyrrolidone, carbopol gel, polyethylene glycol, and/or tita formulations may be presented in unit-dose or multi-dose nium dioxide, lacquer Solutions, and Suitable organic solvents containers, for example sealed ampoules and vials, and may or solvent mixtures. Dyestuffs or pigments may be added to be stored in powder form or in a freeze-dried (lyophilized) the tablets or dragee coatings for identification or to charac condition requiring only the addition of the sterile liquid terize different combinations of active compound doses. 55 carrier, for example, saline or sterile pyrogen-free water, Examples of fillers or diluents for use in oral pharmaceu immediately prior to use. Extemporaneous injection solu tical formulations such as capsules and tablets include, with tions and Suspensions may be prepared from sterile powders, out limitation, lactose, mannitol. Xylitol, dextrose, Sucrose, granules and tablets of the kind previously described. Sorbitol, compressible Sugar, microcrystalline cellulose Formulations for parenteral administration include aque (MCC), powdered cellulose, cornstarch, pregelatinized 60 ous and non-aqueous (oily) sterile injection solutions of the starch, dextrates, dextran, dextrin, dextrose, maltodextrin, salts or polymorphs which may contain antioxidants, buffers, calcium carbonate, dibasic calcium phosphate, tribasic cal bacteriostats and solutes which render the formulation iso cium phosphate, calcium sulfate, magnesium carbonate, tonic with the blood of the intended recipient; and aqueous magnesium oxide, poloxamers such as polyethylene oxide, and non-aqueous sterile Suspensions which may include Sus and hydroxypropyl methyl cellulose. Fillers may have com 65 pending agents and thickening agents. Suitable lipophilic plexed solvent molecules, such as in the case where the lac Solvents or vehicles include fatty oils such as sesame oil, or tose used is lactose monohydrate. Fillers may also be propri synthetic fatty acid esters, such as ethyl oleate or triglycer US 8,859,550 B2 45 46 ides, or liposomes. Aqueous injection Suspensions may con ingredient. In preferred embodiments, the excipients to be tain Substances which increase the Viscosity of the Suspen included in the formulations are typically selected on the Sion, such as Sodium carboxymethyl cellulose, Sorbitol, or basis of their inertness towards the active ingredient compo dextran. Optionally, the Suspension may also contain Suitable nent of the formulations. stabilizers or agents which increase the solubility of the salts Relative to ophthalmic, otic, and nasal formulations, Suit or polymorphs to allow for the preparation of highly concen able tonicity-adjusting agents include, but are not limited to, trated Solutions. mannitol, dextrose, Sodium chloride, glycerin, Sorbitol and In addition to the formulations described previously, the the like. Suitable buffering agents include, but are not limited salts or polymorphs may also be formulated as depot prepa to, phosphates, citrates, borates, acetates and the like. Suit rations. Such long acting formulations may be administered 10 by implantation (for example subcutaneously or intramuscu able Surfactants include, but are not limited to, ionic and larly) or by intramuscular injection. Thus, for example, the nonionic Surfactants (though nonionic Surfactants are pre salts or polymorphs may be formulated with suitable poly ferred), polysorbate 80, RLM 100, POE20 cetylstearylethers meric or hydrophobic materials (for example, as an emulsion such as Procol(R) CS20 and poloxamers such as Pluronic R. in an acceptable oil) or ion exchange resins, or as sparingly 15 F68. Formulations may contain substances which increase soluble derivatives, for example, as a sparingly soluble salt. the Viscosity of the Solution or Suspension, such as Sodium For buccal or Sublingual administration, the compositions carboxymethyl cellulose, hypromellose, micro crystalline may take the form of tablets, lozenges, pastilles, or gels cellulose, sorbitol, or dextran. Optionally, the formulation formulated in conventional manner. Such compositions may may also contain Suitable stabilizers or agents which increase comprise the active ingredient in a flavored basis Such as the solubility of the salts or polymorphs to allow for the Sucrose and acacia or tragacanth. preparation of highly concentrated Solutions, including but The salts or polymorphs may also be formulated in rectal not limited to ethanol, benzyl alcohol, polyethylene glycol, compositions such as Suppositories or retention enemas, e.g., phenylethyl alcohol and glycerin. containing conventional Suppository bases such as cocoa but The formulations set forth herein may comprise one or ter, polyethylene glycol, or other glycerides. 25 more preservatives. Examples of Such preservatives include Salts or polymorphs disclosed herein may be administered benzalkonium chloride, p-hydroxybenzoic acid ester, sodium topically, that is by non-systemic administration. This perborate, Sodium chlorite, alcohols such as chlorobutanol, includes the application of a salt or polymorph disclosed benzyl alcohol or phenyl ethanol, guanidine derivatives Such herein externally to the epidermis or the buccal cavity and the as polyhexamethylene biguanide, sodium perborate, instillation of Such a salt or polymorph into the ear, eye and 30 nose. Such that the salt or polymorph does not significantly polyguaternium-1, amino alcohols such as AMP-95, or sorbic enter the blood stream. In contrast, systemic administration acid. In certain embodiments, the formulation may be self refers to oral, intravenous, intraperitoneal and intramuscular preserved so that no preservation agent is required. administration. For ophthalmic, otic, or nasal administration, the formula Formulations suitable for topical administration include 35 tion may be a solution, a Suspension, or a gel. In preferred liquid or semi-liquid preparations suitable for penetration aspects, the formulations are for topical application to the eye, through the skin to the site of inflammation Such as gels, or ear are in aqueous Solution or Suspension in the form of liniments, lotions, creams, ointments or pastes, and drops drops. Formulations for topical application to the nose in suitable for administration to the eye, ear or nose. The active aqueous solution or Suspension are in the form of drops, spray ingredient for topical administration may comprise, for 40 or aerosol. The term “aqueous typically denotes an aqueous example, from 0.001% to 10% w/w (by weight) of the for formulation wherein the formulation is >50%, more prefer mulation. In certain embodiments, the active ingredient may ably >75% and in particular >90% by weight water. The comprise as much as 10% w/w. In other embodiments, it may drops, spray or aerosol may be delivered from a single dose comprise less than 5% w/w. In certain embodiments, the ampoule which may preferably be sterile and thus render active ingredient may comprise from 2% w/w to 5% w/w. In 45 bacteriostatic components of the formulation unnecessary. other embodiments, it may comprise from 0.1% to 2% w/w of Alternatively, the drops, spray or aerosol may be delivered the formulation. from a multi-dose bottle which may preferably comprise a Topical ophthalmic, otic, and nasal formulations may com device which extracts any preservative from the formulation prise excipients in addition to the active ingredient. Excipi as it is delivered, such devices being known in the art. Solu ents commonly used in Such formulations include, but are not 50 tion and Suspension formulations may be nasally adminis limited to, tonicity agents, preservatives, chelating agents, tered using a nebulizer. Intranasal delivery as a solution, buffering agents, and Surfactants. Other excipients comprise Suspension or dry powder may also facilitated by propellant solubilizing agents, stabilizing agents, comfort-enhancing based aerosol systems, which include but are not limited to agents, polymers, emollients, pH-adjusting agents and/or hydrofluoroalkane-based propellants. Alternatively the active lubricants. Any of a variety of excipients may be used in 55 pharmaceutical ingredient may be delivered in the form of a formulations disclosed herein including water, mixtures of dry powder. water and water-miscible solvents, such as C1-C7-alkanols, For ophthalmic disorders, components may be delivered to vegetable oils or mineral oils comprising from 0.5 to 5% the eye as a concentrated gel or a similar vehicle, or as dis non-toxic water-soluble polymers, natural products, such as solvable inserts that are placed beneath the eyelids. alginates, pectins, tragacanth, karaya gum, guar gum, Xan 60 The formulations disclosed herein that are adapted for than gum, carrageenin, agar and acacia, starch derivatives, topical administration to the eye are preferably isotonic, or Such as starch acetate and hydroxypropyl Starch, and also slightly hypotonic in order to combat any hypertonicity of other synthetic products such as polyvinyl alcohol, polyvi tears caused by evaporation and/or disease. This may require nylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, a tonicity agent to bring the osmolality of the formulation to preferably cross-linked polyacrylic acid and mixtures of 65 a level at or near 210-320 milliosmoles per kilogram (mCsm/ those products. The concentration of the excipient is, typi kg). These formulations generally have an osmolality in the cally, from 1 to 100,000 times the concentration of the active range of 220-320 mOsm/kg, and preferably have an osmola US 8,859,550 B2 47 48 lity in the range of 235-300 mOsm/kg. The ophthalmic for Lotions include those suitable for application to the skin or mulations will generally be formulated as Sterile aqueous eye. An eye lotion may comprise a sterile aqueous solution Solutions. optionally containing a bactericide and may be prepared by In certain ophthalmic embodiments, the compositions dis methods similar to those for the preparation of drops. Lotions closed herein are formulated with one or more tear substi or liniments for application to the skin may also include an tutes. A variety of tear substitutes are known in the art and agent to hasten drying and to cool the skin, Such as an alcohol include, but are not limited to: monomeric polyols. Such as, oracetone, and/or a moisturizer Such as glycerol oran oil Such glycerol, propylene glycol, and ethylene glycol; polymeric as castor oil or arachis oil. polyols such as polyethylene glycol, cellulose esters such Creams, ointments or pastes are semi-solid formulations of hydroxypropylmethyl cellulose, carboxy methylcellulose 10 Sodium and hydroxy propylcellulose; dextrans such as dext the active ingredient for external application. They may be ran 70; vinyl polymers, such as polyvinyl alcohol; and car made by mixing the active ingredient in finely-divided or bomers, such as carbomer 934P. carbomer 941, carbomer 940 powdered form, alone or in Solution or Suspension in an and carbomer 974P. Certain formulations disclosed herein aqueous or non-aqueous fluid, with the aid of Suitable may be used with contact lenses or other ophthalmic prod 15 machinery, with a greasy or non-greasy base. The base may uctS. comprise hydrocarbons such as hard, Soft or liquid paraffin, Preferred formulations are prepared using a buffering sys glycerol, beeswax, a metallic Soap; a mucilage; an oil of tem that maintains the formulation at a pH of about 4.5 to a pH natural origin such as almond, corn, arachis, castor or olive of about 8. A most preferred formulation pH is from 5.5 to 7.5. oil; wool fat or its derivatives or a fatty acid such as stearic or In certain embodiments, a formulation disclosed herein is oleic acid together with an alcohol Such as propylene glycol administered once a day. However, the formulations may also or a macrogel. The formulation may incorporate any Suitable be formulated for administration at any frequency of admin Surface active agent such as an anionic, cationic or non-ionic istration, including once a week, once every 5 days, once Surfactant such as a Sorbitan ester or a polyoxyethylene every 3 days, once every 2 days, twice a day, three times a day, derivative thereof. Suspending agents such as natural gums, four times a day, five times a day, six times a day, eight times 25 cellulose derivatives or inorganic materials such as silica a day, every hour, or any greater frequency. Such dosing ceous silicas, and other ingredients such as lanolin, may also frequency is also maintained for a varying duration of time be included. depending on the therapeutic regimen. The duration of a Drops or sprays may comprise sterile aqueous or oily solu particular therapeutic regimen may vary from one-time dos tions or Suspensions and may be prepared by dissolving the ing to a regimen that extends for months or years. The for 30 mulations are administered at varying dosages, but typical active ingredient in a Suitable aqueous solution of a bacteri dosages are one to two drops at each administration, or a cidal and/or fungicidal agent and/or any other suitable pre comparable amount of a gel or other formulation. One of servative, and, in certain embodiments, including a Surface ordinary skill in the art would be familiar with determining a active agent. The resulting solution may then be clarified by therapeutic regimen for a specific indication. 35 filtration, transferred to a suitable container which is then Gels for topical or transdermal administration may com sealed and sterilized by autoclaving or maintaining at prise, generally, a mixture of Volatile solvents, nonvolatile 98-100° C. for half an hour. Alternatively, the solution may be Solvents, and water. In certain embodiments, the Volatile sol sterilized by filtration and transferred to the container by an vent component of the buffered solvent system may include aseptic technique. Examples of bactericidal and fungicidal lower (C1-C6) alkyl alcohols, lower alkyl glycols and lower 40 agents suitable for inclusion in the drops are phenylmercuric glycol polymers. In further embodiments, the volatile solvent nitrate or acetate (0.002%), benzalkonium chloride (0.01%) is ethanol. The Volatile solvent component is thought to act as and chlorhexidine acetate (0.01%). Suitable solvents for the a penetration enhancer, while also producing a cooling effect preparation of an oily Solution include glycerol, diluted alco on the skinas it evaporates. The nonvolatile solvent portion of hol and propylene glycol. the buffered solvent system is selected from lower alkylene 45 Formulations for topical administration in the mouth, for glycols and lower glycol polymers. In certain embodiments, example buccally or Sublingually, include lozenges compris propylene glycol is used. The nonvolatile solvent slows the ing the active ingredient in a flavored basis Such as Sucrose evaporation of the volatile solvent and reduces the vapor and acacia or tragacanth, and pastilles comprising the active pressure of the buffered solvent system. The amount of this ingredient in a basis such as gelatin and glycerin or Sucrose nonvolatile solvent component, as with the Volatile solvent, is 50 and acacia. determined by the active ingredient or drug being used. When For administration by inhalation, salts or polymorphs may too little of the nonvolatile solvent is in the system, the drug be conveniently delivered from an insufflator, nebulizer pres may crystallize due to evaporation of volatile solvent, while Surized packs or other convenient means of delivering an an excess may result in a lack of bioavailability due to poor aerosol spray. Pressurized packs may comprise a suitable release of drug from solvent mixture. The buffer component 55 propellant such as hydrofluoroalkane, dichlorodifluo of the buffered solvent system may be selected from any romethane, trichlorofluoromethane, dichlorotetrafluoroet buffer commonly used in the art; in certain embodiments, hane, carbon dioxide or other Suitable gas. In the case of a water is used. A common ratio of ingredients is about 20% of pressurized aerosol, the dosage unit may be determined by the nonvolatile solvent, about 40% of the volatile solvent, and providing a valve to deliver a metered amount. Alternatively, about 40% water. There are several optional ingredients 60 for administration by inhalation or insufflation, the formula which can be added to the topical composition. These include, tion may take the form of a dry powder composition, for but are not limited to, chelators and gelling agents. Appropri example a powder mix of the salt or polymorph and a suitable ate gelling agents can include, but are not limited to, semi powder base Such as lactose or starch. The powder composi synthetic cellulose derivatives (such as hydroxypropylmeth tion may be presented in unit dosage form, in for example, ylcellulose) and synthetic polymers, galactomannan 65 capsules, cartridges, gelatin or blister packs from which the polymers (such as guarand derivatives thereof) and cosmetic powder may be administered with the aid of an inhalator or agents. insufflator. US 8,859,550 B2 49 50 Preferred unit dosage formulations are those containing an nists; FLAP inhibitors; tissue kallikreins; PI3 kinase inhibi effective dose, as herein below recited, or an appropriate tors; secretory phospholipase A2 (SPLA2) inhibitors; gluco fraction thereof, of the active ingredient. corticoid receptor agonists; immunomodulators; leukotriene It should be understood that in addition to the ingredients D4 antagonists; LT/TX dual inhibitors; mast cell inhibitors: particularly mentioned above, the formulations described tyrosine kinase inhibitor, and inverse agonists of inflamma above may include other agents conventional in thearthaving tion-related GPCRs. regard to the type of formulation in question, for example In certain embodiments, a compound disclosed herein is those Suitable for oral or intranasal administration may combined with an HR antagonist, an HR antagonist, an include flavoring agents. HR/HR antagonist, a leukotriene inhibitor, an alpha-2 ago Salts or polymorphs may be administered orally or via 10 nist, a PDE4 inhibitor, or an intranasal corticosteroid. In injection at a dose of from 0.1 to 500 mg/kg per day. The dose further embodiments, a compound disclosed herein is com range for adult humans is generally from 5 mg to 2 g/day. bined with an HR antagonist, a leukotriene inhibitor, an Tablets or other forms of presentation provided in discrete alpha-2 agonist, a PDE4 inhibitor, or an intranasal corticos units may conveniently contain an amount of one or more teroid. In further embodiments, a compound disclosed herein salts or polymorphs which is effective at Such dosage or as a 15 is combined with an HRantagonist oran intranasal corticos multiple of the same, for instance, units containing 5 mg to teroid. In further embodiments, a compound disclosed herein 500 mg, usually around 10 mg to 200 mg. is combined with an HRantagonist. In other embodiments, a The amount of active ingredient that may be combined compound disclosed herein is combined with an intranasal with the carrier materials to produce a single dosage form will corticosteroid. vary depending upon the host treated and the particular mode Specific, non-limiting examples of possible combination of administration. therapies include use of compounds disclosed herein with: The salts or polymorphs can be administered in various H Rantagonists such as acrivastine, alcaftadine, andolast, modes, e.g. orally, topically, or by injection. The precise antazoline, azelastine, , , bro amount of compound administered to a patient will be the mazine, brompheniramine, cetirizine, chlorphe responsibility of the attendant physician. The specific dose 25 niramine, clemastine, desloratidine, diphenhydramine, level for any particular patient will depend upon a variety of diphenylpyraline, ebastine, emedastine, epinastine, fex factors including the activity of the specific salt or polymorph ofenadine, , ketotifen, levocabastine, levo employed, the age, body weight, general health, sex, diets, cetirizine, loratidine, methdilazine, mizolastine, time of administration, route of administration, rate of excre promethazine, olopatadine, and triprolidine; tion, drug combination, the precise disorder being treated, 30 Intranasal corticosteroids such as fluticasone, budesonide, and the severity of the indication or condition being treated. beclomethasone, mometasone, ciclesonide, and triamci Also, the route of administration may vary depending on the nolone: condition and its severity. HR/HR antagonists Such as GSK835726 and In certain instances, it may be appropriate to administer at GSK1004723; least one of the salts or polymorphs described herein in com 35 CRTh2 antagonists such as septipiprant; bination with another therapeutic agent. By way of example Leukotriene inhibitors such as monteleukast (Singulair); only, if one of the side effects experienced by a patient upon alpha-2 agonists such as oxymetazoline (Afrin); receiving one of the salts or polymorphs herein is inflamma PDE4 inhibitors such as GSK256066 (6-3-(Dimethy tion, then it may be appropriate to administer an anti-inflam lamino)carbonylphenylsulfonyl-4-(3-methoxyphe matory agent in combination with the initial therapeutic 40 nyl)amino-8-methyl-3-quinolinecarboxamide); agent. Or, by way of example only, the therapeutic effective Monoclonal antibodies such as anti-CCR4 monoclonal ness of one of the salts or polymorphs described herein may antibodies (e.g., mogamulizumab), interleukin-13 (IL be enhanced by administration of an adjuvant (i.e., by itself 13) monoclonal antibodies, and anti-eotaxin-1 mono the adjuvant may only have minimal therapeutic benefit, but clonal antibodies (e.g., bertilimumab); in combination with another therapeutic agent, the overall 45 Inhibitors of PI3K such as wortmannin (irreversible inhibi therapeutic benefit to the patient is enhanced). Or, by way of tor), demethoxyviridin, and LY294.002 (reversible example only, the benefit of experienced by a patient may be inhibitor); and increased by administering one of the salts or polymorphs Other therapies, such as iodinated contrast agents (e.g. described herein with another therapeutic agent (which also ASP-1001), IG-RD-001 (Leucet, Indigene Pharma includes a therapeutic regimen) that also has therapeutic ben 50 ceuticals), and EWO1. efit. By way of example only, in a treatment for allergic In certain embodiments, compounds disclosed herein are rhinitis involving administration of one of the salts or poly used in combination with an immunomodulator. The immu morphs described herein, increased therapeutic benefit may nomodulator may be a liquid mixture of allergen extracts (e.g. result by also providing the patient with another therapeutic Sublivac), a Sublingual allergen tablet (e.g., Actair, Oralair, agent for allergic rhinitis. In any case, regardless of the dis 55 MK-7243 (SCH 697243), MK-3641 (SCH 039641), and ease, disorder or condition being treated, the overall benefit MK-8237 (SCH 900237)), or an aluminium hydroxide-ad experienced by the patient may simply be additive of the two sorbed allergoid preparation of pollen allergens. The immu therapeutic agents or the patient may experience a synergistic nomodulator may be a mix of allergen extracts or a single benefit. agent such as rBet V1 (birch pollen). Non-limiting examples of possible combination therapies 60 In certain embodiments, compounds disclosed herein are include use of certain compounds disclosed herein with his used in combination with a preparation of Lactobacillus aci tamine receptor antagonists, including HR antagonists, HR dophilus, Such as En-Lac (encapsulated Lactobacillus acido antagonists, and HR/HR antagonists; leukotriene inhibi philus L-92). tors; alpha-2 agonists; PDE4 inhibitors; intranasal corticos The present compounds may also be used in co-therapies, teroids; CRTh2 antagonists; prostaglandin D2 (PGD2) recep 65 partially or completely, in place of other conventional anti tor antagonists, including PGD2-1 antagonists; toll-like inflammatory therapies, such as together with steroids, receptor agonists, including TLR7 agonists and TLR8 ago NSAIDs, decongestants, COX-2 selective inhibitors, 5-li US 8,859,550 B2 51 52 poxygenase inhibitors, LTBantagonists and LTA hydrolase vided herein is a combination therapy comprising Compound inhibitors. The compounds disclosed herein may also be used 2 Mesylate Form 1 and chlorpheniramine. Also provided to prevent tissue damage when therapeutically combined with herein is a combination therapy comprising Compound 2 antibacterial or antiviral agents. Mesylate Form 1 and bepotastine. Also provided herein is a In certain embodiments, Compound 1 Mesylate Monohy combination therapy comprising Compound 2 Mesylate drate Form 1 is used in combination with an orally-adminis Form 1 and bilastine. tered chosen from cetirizine (Zyrtec), levoce In certain embodiments, Compound 1 Mesylate Monohy tirizine (Xyzal), loratadine (Claritin), desloratidine drate Form 1 is used in combination with a nasally-adminis (Clarinex), fexofenadine (Allegra), chlorpheniramine, bepo tered H1 antagonist chosen from bepotastine (Bepomax or tastine (Talion), and bilastine (2-4-(2-4-1-(2-Ethoxy 10 Beposone), loratadine, olopatadine (Patanase), azelastine ethyl)-1H-benzimidazol-2-yl)-1-piperidinyl)ethyl)phenyl (Astelin), and Andolast (CR-2039, 4-(1H-tetrazol-5-yl)-N- 2-methylpropanoic acid). Provided herein is a combination (4-1H-tetrazol-5-yl)phenylbenzamide)). Provided herein is therapy comprising Compound 1 Mesylate Monohydrate a nasal spray formulation comprising Compound 1 Mesylate Form 1 and cetirizine. Also provided herein is a combination Monohydrate Form 1 and bepotastine. Also provided herein therapy comprising Compound 1 Mesylate Monohydrate 15 is a nasal spray formulation comprising Compound 1 Mesy Form 1 and levocetirizine. Also provided herein is a combi late Monohydrate Form 1 and loratadine. Also provided nation therapy comprising Compound 1 Mesylate Monohy herein is a nasal spray formulation comprising Compound 1 drate Form 1 and loratadine. Also provided herein is a com Mesylate Monohydrate Form 1 and olopatadine. Also pro bination therapy comprising Compound 1 Mesylate vided herein is a nasal spray formulation comprising Com Monohydrate Form 1 and desloratidine. Also provided herein pound 1 Mesylate Monohydrate Form 1 and azelastine. Also is a combination therapy comprising Compound 1 Mesylate provided herein is a nasal spray formulation comprising Monohydrate Form 1 and fexofenadine. Also provided herein Compound 1 Mesylate Monohydrate Form 1 and andolast. is a combination therapy comprising Compound 1 Mesylate In certain embodiments, Compound 2 Hydrochloride Monohydrate Form 1 and chlorpheniramine. Also provided Form 1 is used in combination with a nasally-administered herein is a combination therapy comprising Compound 1 25 H1 antagonist chosen from bepotastine (Bepomax or Mesylate Monohydrate Form 1 and bepotastine. Also pro Beposone), loratadine, olopatadine (Patanase), azelastine vided herein is a combination therapy comprising Compound (Astelin), and Andolast (CR-2039, 4-(1H-tetrazol-5-yl)-N- 1 Mesylate Monohydrate Form 1 and bilastine. (4-1H-tetrazol-5-yl)phenylbenzamide)). Provided herein is In certain embodiments, Compound 2 Hydrochloride a nasal spray formulation comprising Compound 2 Hydro Form 1 is used in combination with an orally-administered 30 chloride Form 1 and bepotastine. Also provided herein is a H1 antagonist chosen from cetirizine (Zyrtec), levocetirizine nasal spray formulation comprising Compound 2 Hydrochlo (XyZal), loratadine (Claritin), desloratidine (Clarinex), fex ride Form 1 and loratadine. Also provided herein is a nasal ofenadine (Allegra), chlorpheniramine, bepotastine (Talion), spray formulation comprising Compound 2 Hydrochloride and bilastine (2-4-(2-4-1-(2-Ethoxyethyl)-1H-benzimida Form 1 and olopatadine. Also provided herein is a nasal spray Zol-2-yl)-1-piperidinyl)ethyl)phenyl)-2-methylpropanoic 35 formulation comprising Compound 2 Hydrochloride Form 1 acid). Provided herein is a combination therapy comprising and azelastine. Also provided herein is a nasal spray formu Compound 2 Hydrochloride Form 1 and cetirizine. Also pro lation comprising Compound 2 Hydrochloride Form 1 and vided herein is a combination therapy comprising Compound andolast. 2 Hydrochloride Form 1 and levocetirizine. Also provided In certain embodiments, Compound 2 Mesylate Form 1 is herein is a combination therapy comprising Compound 2 40 used in combination with a nasally-administered H1 antago Hydrochloride Form 1 andloratadine. Also provided herein is nist chosen from bepotastine (Bepomax or Beposone), lora a combination therapy comprising Compound 2 Hydrochlo tadine, olopatadine (Patanase), azelastine (Astelin), and ride Form 1 and desloratidine. Also provided herein is a Andolast (CR-2039, 4-(1H-tetrazol-5-yl)-N-(4-1H-tetrazol combination therapy comprising Compound 2 Hydrochlo 5-yl)phenylbenzamide)). Provided herein is a nasal spray ride Form 1 and fexofenadine. Also provided herein is a 45 formulation comprising Compound 2 Mesylate Form 1 and combination therapy comprising Compound 2 Hydrochlo bepotastine. Also provided herein is a nasal spray formulation ride Form 1 and chlorpheniramine. Also provided herein is a comprising Compound 2 Mesylate Form 1 and loratadine. combination therapy comprising Compound 2 Hydrochlo Also provided herein is a nasal spray formulation comprising ride Form 1 and bepotastine. Also provided herein is a com Compound 2 Mesylate Form 1 and olopatadine. Also pro bination therapy comprising Compound 2 Hydrochloride 50 vided herein is a nasal spray formulation comprising Com Form 1 and bilastine. pound 2 Mesylate Form 1 and azelastine. Also provided In certain embodiments, Compound 2 Mesylate Form 1 is herein is a nasal spray formulation comprising Compound 2 used in combination with an orally-administered H1 antago Mesylate Form 1 and andolast. nist chosen from cetirizine (Zyrtec), levocetirizine (Xyzal), In certain embodiments, Compound 1 Mesylate Monohy loratadine (Claritin), desloratidine (Clarinex), fexofenadine 55 drate Form 1 is used in combination with a nasally-adminis (Allegra), chlorpheniramine, bepotastine (Talion), and bilas tered corticosteroid chosen from fluticasone proprionate tine (2-4-(2-4-1-(2-Ethoxyethyl)-1H-benzimidazol-2-yl)- (Flonase), fluticasone furoate (Veramyst, GSK685698), 1-piperidinyl)ethyl)phenyl-2-methylpropanoic acid). Pro mometasone furoate monohydrate (Nasonex), ciclesonide vided herein is a combination therapy comprising Compound (Omnaris), budesonide (Rhinocort), triamcinolone acetonide 2 Mesylate Form 1 and cetirizine. Also provided herein is a 60 acetonide (Nasacort), and beclomethasone dipropionate combination therapy comprising Compound 2 Mesylate dipropionate (Beconase, Clenil). Provided herein is a nasal Form 1 and levocetirizine. Also provided herein is a combi spray formulation comprising Compound 1 Mesylate Mono nation therapy comprising Compound 2 Mesylate Form 1 and hydrate Form 1 and fluticasone proprionate. Also provided loratadine. Also provided herein is a combination therapy herein is a nasal spray formulation comprising Compound 1 comprising Compound 2 Mesylate Form 1 and desloratidine. 65 Mesylate Monohydrate Form 1 and fluticasone furoate. Also Also provided herein is a combination therapy comprising provided herein is a nasal spray formulation comprising Compound 2 Mesylate Form 1 and fexofenadine. Also pro Compound 1 Mesylate Monohydrate Form 1 and mometa US 8,859,550 B2 53 54 Sone furoate monohydrate. Also provided herein is a nasal combination therapy comprising Compound 1 Mesylate spray formulation comprising Compound 1 Mesylate Mono Monohydrate Form 1 and oxymetazoline. Also provided hydrate Form 1 and ciclesonide. Also provided herein is a herein is a nasal spray formulation comprising Compound 1 nasal spray formulation comprising Compound 1 Mesylate Mesylate Monohydrate Form 1 and oxymetazoline. Also pro Monohydrate Form 1 and budesonide. Also provided herein is vided herein is a nasal spray formulation comprising Com a nasal spray formulation comprising Compound 1 Mesylate pound 2 Hydrochloride Form 1 and oxymetazoline. Also Monohydrate Form 1 and triamcinolone acetonide. Also pro provided herein is a nasal spray formulation comprising vided herein is a nasal spray formulation comprising Com Compound 2 Mesylate Form 1 and oxymetazoline. pound 1 Mesylate Monohydrate Form 1 and beclomethasone In certain embodiments, Compound 1 Mesylate Monohy dipropionate. 10 drate Form 1 is used in combination with a PDE4 inhibitor, In certain embodiments, Compound 2 Hydrochloride such as GSK256066 (6-3-(Dimethylamino)carbonylphe Form 1 is used in combination with a nasally-administered nylsulfonyl)-4-(3-methoxyphenyl)amino-8-methyl-3- corticosteroid chosen from fluticaSone proprionate (Flonase), quinolinecarboxamide). Provided herein is a combination fluticasone furoate (Veramyst, GSK685698), mometasone therapy comprising Compound 1 Mesylate Monohydrate furoate monohydrate (Nasonex), ciclesonide (Omnaris), 15 Form 1 and GSK256066. Provided herein is a combination budesonide (Rhinocort), triamcinolone acetonide (Nasacort), therapy comprising Compound 1 Mesylate Monohydrate and beclomethasone dipropionate (Beconase, Clenil). Pro Form 1 and GSK256066. Also provided herein is a nasal vided herein is a nasal spray formulation comprising Com spray formulation comprising Compound 2 Hydrochloride pound 2 Hydrochloride Form 1 and fluticasone proprionate. Form 1 and GSK256066. Also provided herein is a nasal Also provided herein is a nasal spray formulation comprising spray formulation comprising Compound 2 Mesylate Form 1 Compound 2 Hydrochloride Form 1 and fluticasone furoate. and GSK256066. Also provided herein is a nasal spray formulation comprising In certain embodiments, Compound 1 Mesylate Monohy Compound 2 Hydrochloride Form 1 and mometasone furoate drate Form 1 is used in combination with a CRTh2 antago monohydrate. Also provided herein is a nasal spray formula nists such as septipiprant. Provided herein is a combination tion comprising Compound 2 Hydrochloride Form 1 and 25 therapy comprising Compound 1 Mesylate Monohydrate ciclesonide. Also provided herein is a nasal spray formulation Form 1 and septipiprant. Also provided herein is a nasal spray comprising Compound 2 Hydrochloride Form 1 and budes formulation comprising Compound 2 Hydrochloride Form 1 onide. Also provided herein is a nasal spray formulation com and septipiprant. Also provided herein is a nasal spray formu prising Compound 2 Hydrochloride Form 1 and triamcino lation comprising Compound 2 Mesylate Form 1 and septip lone acetonide. Also provided herein is a nasal spray 30 iprant. formulation comprising Compound 2 Hydrochloride Form 1 In any case, the multiple therapeutic agents (at least one of and beclomethasone dipropionate. which is a salt or polymorph disclosed herein) may be admin In certain embodiments, Compound 2 Mesylate Form 1 is istered in any order or even simultaneously. If simulta used in combination with a nasally-administered corticoster neously, the multiple therapeutic agents may be provided in a oid chosen from fluticaSone proprionate (Flonase), flutica 35 single, unified form, or in multiple forms (by way of example sone furoate (Veramyst, GSK685698), mometasone furoate only, either as a single pill or as two separate pills). One of the monohydrate (Nasonex), ciclesonide (Omnaris), budesonide therapeutic agents may be given in multiple doses, or both (Rhinocort), triamcinolone acetonide (Nasacort), and may be given as multiple doses. If not simultaneous, the beclomethasone dipropionate (Beconase, Clenil). Provided timing between the multiple doses may be any duration of herein is a nasal spray formulation comprising Compound 2 40 time ranging from a few minutes to four weeks. Mesylate Form 1 and fluticasone proprionate. Also provided Thus, in another aspect, certain embodiments provide herein is a nasal spray formulation comprising Compound 2 methods for treating HR-mediated disorders in a human or Mesylate Form 1 and fluticasone furoate. Also provided animal Subject in need of Such treatment comprising admin herein is a nasal spray formulation comprising Compound 2 istering to said Subject an amount of a salt or polymorph Mesylate Form 1 and mometasone furoate monohydrate. 45 disclosed herein effective to reduce or prevent said disorder in Also provided herein is a nasal spray formulation comprising the Subject, optionally in combination with at least one addi Compound 2 Mesylate Form 1 and ciclesonide. Also pro tional agent for the treatment of said disorder that is known in vided herein is a nasal spray formulation comprising Com the art. pound 2 Mesylate Form 1 and budesonide. Also provided In a related aspect, certain embodiments provide therapeu herein is a nasal spray formulation comprising Compound 2 50 tic compositions comprising at least one salt or polymorph Mesylate Form 1 and triamcinolone acetonide. Also provided disclosed herein, optionally in combination with one or more herein is a nasal spray formulation comprising Compound 2 additional agents for the treatment of HR-mediated disor Mesylate Form 1 and beclomethasone dipropionate. ders. Specific diseases to be treated by the salts, polymorphs, In certain embodiments, compounds disclosed herein are compositions, and methods disclosed herein include inflam used in combination with a leukotriene inhibitor such as 55 mation and related diseases, including autoimmune diseases. monteleukast (Singulair). Provided herein is a combination The salts and polymorphs are useful to treat arthritis, includ therapy comprising Compound 1 Mesylate Monohydrate ing but not limited to rheumatoid arthritis, spondyloarthropa Form 1 and monteleukast. Also provided herein is a combi thies, gouty arthritis, osteoarthritis, Systemic lupus erythema nation therapy comprising Compound 2 Hydrochloride Form tosus, juvenile arthritis, acute rheumatic arthritis, 1 and monteleukast. Also provided herein is a combination 60 enteropathic arthritis, neuropathic arthritis, psoriatic arthritis, therapy comprising Compound 2 Mesylate Form 1 and mon and pyogenic arthritis. The salts and polymorphs are also teleukast. useful in treating osteoporosis and other related bone disor In certain embodiments, Compound 1 Mesylate Monohy ders. drate Form 1 is used in combination with an alpha-2 agonist These salts and polymorphs can also be used to treat gas such as oxymetazoline (Afrin). Provided herein is a combi 65 trointestinal conditions such as reflux esophagitis, diarrhea, nation therapy comprising Compound 1 Mesylate Monohy inflammatory bowel disease, Crohn's disease, gastritis, irri drate Form 1 and oxymetazoline. Also provided herein is a table bowel syndrome and ulcerative colitis. US 8,859,550 B2 55 56 The salts and polymorphs may also be used in the treatment ischemia, retinitis, retinopathies, uveitis, ocular photophobia, of allergic disorders and disorders related to nasal and upper and of inflammation and pain associated with acute injury to respiratory inflammation, such as, but not limited to, seasonal the eye tissue. The salts and polymorphs can also be used to allergic rhinitis, non-seasonal allergic rhinitis, acute non-al treat post-operative inflammation or pain as from ophthalmic lergic rhinitis, chronic non-allergic rhinitis, Sampter's triad, Surgery Such as cataract Surgery and refractive Surgery. In non-allergic rhinitis with eosinophilia syndrome, nasal certain embodiments, the salts and polymorphs disclosed inflammation, nasal congestion, sinus congestion, nasal poly herein are used to treat an allergic eye disease chosen from posis, atrophic rhinitis, hypertrophic rhinitis, membranous allergic conjunctivitis; Vernal conjunctivitis; Vernal kerato rhinitis, vasomotor rhinitis, rhinosinusitis, chronic rhinophar conjunctivitis; and giant papillary conjunctivitis. yngitis, rhinorrhea, occupational rhinitis, hormonal rhinitis, 10 Salts and polymorphs disclosed herein are useful in treat drug-induced rhinitis, gustatory rhinitis, as well as pulmonary ing patients with inflammatory pain Such as reflex sympa inflammation, Such as that associated with viral infections thetic dystrophy/causalgia (nerve injury), peripheral neur and cystic fibrosis. In addition, compounds disclosed herein opathy (including diabetic neuropathy), and entrapment are also useful in organ transplant patients either alone or in neuropathy (carpel tunnel syndrome). The salts and polymor combination with conventional immunomodulators. 15 Moreover, salts and polymorphs disclosed herein may be phs are also useful in the treatment of pain associated with used in the treatment of tendonitis, bursitis, skin-related con acute herpes Zoster (shingles), postherpetic neuralgia (PHN), ditions such as psoriasis, allergic dermatitis, atopic dermatitis and associated pain syndromes Such as ocular pain. Pain and other variants of eczema, allergic contact dermatitis, irri indications include, but are not limited to, pain resulting from tant contact dermatitis, Seborrhoeic eczema, nummular dermal injuries and pain-related disorders such as tactile allo eczematous dermatitis, autosensitization dermatitis, Lichen dynia and hyperalgesia. The pain may be somatogenic (either Simplex Chronicus, dyshidrotic dermatitis, neurodermatitis, nociceptive or neuropathic), acute and/or chronic. Stasis dermatitis, generalized ordinary urticaria, acute allergic Besides being useful for human treatment, certain salts and urticaria, chronic allergic urticaria, autoimmune urticaria, polymorphs and formulations disclosed herein may also be chronic idiopathic urticaria, drug-induced urticaria, cholin 25 useful for veterinary treatment of companion animals, exotic ergic urticaria, chronic cold urticaria, dermatographic urti animals and farm animals, including mammals, rodents, and caria, Solar urticaria, urticaria pigmentosa, mastocytosis, the like. More preferred animals include horses, dogs, and acute or chronic pruritis, including pruritis associated with Cats. skin-localized or systemic diseases and disorders such as All references, patents or applications, U.S. or foreign, pancreatitis, hepatitis, burns, Sunburn, and vitiligo. 30 cited in the application are hereby incorporated by reference Further, the salts and polymorphs disclosed herein can be as if written herein in their entireties. Where any inconsisten used to treat respiratory diseases, including therapeutic meth cies arise, material literally disclosed herein controls. ods of use in medicine for preventing and treating a respira tory disease or condition including: asthmatic conditions METHODS FOR PREPARING COMPOUNDS including allergen-induced asthma, exercise-induced asthma, 35 pollution-induced asthma, cold-induced asthma, and viral AND EXAMPLES induced-asthma; chronic obstructive pulmonary disease (COPD) including chronic bronchitis with normal airflow, The following schemes can be used to practice the present chronic bronchitis with airway obstruction (chronic obstruc invention. A person skilled in the art may adapt the schemes tive bronchitis), emphysema, asthmatic bronchitis, and 40 to synthesis of compounds other than those they may specifi bullous disease; and other pulmonary diseases involving cally depict. The invention is further illustrated by the follow inflammation including bronchioectasis cystic fibrosis, ing compound examples, which may be made my methods pigeon fancier's disease, farmer's lung, acute respiratory dis known in the art and/or as shown below. tress syndrome, pneumonia, aspiration or inhalation injury, fat embolism in the lung, acidosis inflammation of the lung, 45 acute pulmonary edema, acute mountain sickness, acute pull SCHEME 1 monary hypertension, persistent pulmonary hypertension of the newborn, perinatal aspiration syndrome, hyaline mem t brane disease, acute pulmonary thromboembolism, heparin N C N NH 50 n NHNH2 n NaNO protamine reactions, sepsis, status asthamticus and hypoxia. 3a -- The salts and polymorphs disclosed herein are also useful 2 EtOH 2 HOAc in treating tissue damage in Such diseases as vascular dis N C N C eases, periarteritis nodosa, thyroiditis, Sclerodoma, rheu NetN / V matic fever, type I diabetes, neuromuscular junction disease l N HN N including myasthenia gravis, white matter disease including 55 NSZ \ / multiple Sclerosis, sarcoidosis, nephritis, nephrotic Syn CHCl2 drome, Behcet’s syndrome, polymyositis, gingivitis, peri 2 odontis, hypersensitivity, and Swelling occurring after injury. N C The compounds disclosed herein can be used in the treat NetN ment of otic diseases and otic allergic disorders, including 60 I V otic inflammation and eustachian tube itching. N 2 N NBS The compounds disclosed herein can be used in the treat He ment of ophthalmic diseases, such as ophthalmic allergic 2 CH3CO2H disorders, including allergic conjunctivitis, Vernal conjunc tivitis, Vernal keratoconjunctivitis, and giant papillary con 65 junctivitis, dry eye, glaucoma, glaucomatous retinopathy, diabetic retinopathy, retinal ganglion degeneration, ocular US 8,859,550 B2 57 58 -continued Step 2

NetN OH Br N a S \ OH 2 (C6H5)3P2PdCl2, N r (CH3)2CH2NCH2CH3, Nu CH3CN/H2O, 45-50° C. 10 N 8-Chlorotetrazolo 1.5-alpyrazine NetN 15 2-Chloro-3-hydrazinylpyrazine (440 g, 3.0 mol) was sus (S Naa\, pended in acetic acid (500 mL) and cooled to 10° C. A solution of sodium nitrite (220 g, 3.2 mol) in water (200 mL) 2 at 10°C. was added. The resulting mixture was stirred at 10° N r C. for 1 h, during which time a crystalline solid precipitated. N The precipitate was collected by filtration, washed with etha N- N nol (200 mL) and dried to afford the desired product as a red Compound 1 solid (350 g, 73% yield). Step 3 25

Example 1 NetN | \ NZN 8-(4-Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetra 30 Zolo 1.5-alpyrazine C 2 N r N 35 N- N 7 - S NN2 8-(4-Methylpiperazin-1-yl)tetrazolo 1.5-alpyrazine 2 40 8-Chlorotetrazolo 1.5-alpyrazine (100 g, 0.643 mol. 1 eq.) was suspended in methylene chloride (650 ml) and cooled to 3-7°C. under nitrogen. 1.-Methylpiperazine (156g, 1.56 mol, CHSOH, H2O 2.42 eq) was added at Such a rate that the temperature of the 45 reaction mixture was maintained at 3-7°C. The mixture is stirred at approximately 10° C. for 15 minutes after which Step 1 time conversion to product was a 99%. Celite 545 (40 g) and water (260 g) were added and the pH of the suspension adjusted to 9.5-9.8 by the addition of 27% sodium hydroxide, t 50 with the temperature maintained range at 3-7°C. The suspen N NH sion was filtered on a pad of Celite 545, and the filter cake washed with methylene chloride. The filtrate was separated 2 into an organic and aqueous phase. The aqueous phase was NDC C 55 washed with methylene chloride. The organic phases are washed twice with aqueous sodium hydroxide. The organic phases were combined, and dried over magnesium Sulfate 2-Chloro-3-hydrazinylpyrazine prior to addition of activated carbon (pH 8-10). The mixture was filtered on a pad of Celite 545 and the filtered cake 60 washed with methylene chloride. The filtrate was concen 2,3-dichloropyrazine (1000 g. 6.7 mol) and hydrazine trated at reduced pressure and crystallization induced by slow monohydrate (700 g, 14 mol) were dissolved in absolute addition of heptane, with further concentration and seeding as ethanol (2 L) and refluxed under N, overnight. A crystalline necessary. The resulting crystal Suspension is cooled to precipitate formed, which was collected by filtration, washed 65 10-15°C., filtered, washed with n-heptane and dried under with ethanol (1L), and dried to afford the desired produced as vacuum at 27-33°C. to afford the product as a buff coloured a yellow solid (880 g, 90% yield). solid (114.6 g., 81% yield). US 8,859,550 B2 60 Step 4 water (10-12°C.), and dried in vacuum at 25-35°C. to afford the title compound as a solid (100.27g, 83% yield). H NMR (300 MHz, CDC1,) 8:8.00 (m, 2H), 7.45 (dd, J=5.1, 1.2 Hz, 1H), 7.20 (dd, J=5.1, 3.6 Hz, 1H), 4.39 (br, 4H), 2.60 (t, J=5.1 Hz, 4H), 2.37 (s.3H). MS m/z. 302 (M+H").

SCHEME 2 10

N C N NHt n -NH-NH2 - || N HeNaNO 5-Bromo-8-(4-methylpiperazin-1-yl)tetrazolo 1.5-a 2 EtOH 2 HOAc pyrazine 15 N C N C 8-(4-Methylpiperazin-1-yl)tetrazolo 1.5-alpyrazine (100 NetN / I \ HN N g, 0.456 mol. 1.0 eq.) was suspended in acetic acid (110 ml) N N V under nitrogen and stirred at 35° C. until a solution was 2 Boc obtained. The solution was cooled to 16° C. and N-Bromo EtN, EtOH 2 Succinimide (82.14g, 0.461 mol, 1.01 eq.) is added at Such a N C rate that the temperature of the reaction was maintained at 14-18°C. The reaction was monitored and additional aliquots NetN of N-bromosuccinimide added as necessary to ensure conver 25 I V sion was >99.5% after 2-3 hours stirring at 20° C. stirring. N,N-2 NBS Water was added, and the solution is cooled to 13°C. The pH -e- of the suspension is adjusted to 8.3-8.5 by addition of triethy 2 DMF lamine. The resulting Suspension was filtered, washed with 30 water, and dried to afford the desired product (136.02g, 100% ty yield). Step 5 Bocr 35 N=N / \ OH I N b/ Br N a S \ OH 2 Pd(PPh3)4, Cs2CO3, 40 N NAs dioxane/H2O, 80° C.

Boc

45 8-(4-Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetra NetN Zolo 1.5-alpyrazine (S NaNa\, TFA 5-Bromotetrazolo 1.5-alpyrazin-8-yl)piperazine (120 g, -e- 0.402 mol, 1.00 eq.) was suspended in acetonitrile (1275 ml) 50 2 CHCl2 then 2-thiophene boronic acid (72.10 g, 0.564 mol, 1.4.0 eq.), purified water (110 ml), and N,N-diisopropylethylamine \ 181.8g, 1.407 mol, 3.50 eq) added. The resulting suspension Boc is heated to 35-38°C. to form a clear solution, the pressure 55 was lowered to the point that reflux was established (approx. NetN 250 mbar) and then maintained under refluz at 35-38°C. for an additional 15 minutes. The pressure is released with nitro (S Na Z\, gen, and the reaction mixture maintained under nitrogen 60 throughout the rest of the procedure. Bis(triphenylphosphine) 2 palladium (II) dichloride (14.1 g, 0.020 mol, 0.05 eq.) was added, and the reaction mixture stirred at 45-50° C. overnight. \ N1 The product precipitates during the reaction. When conver H sion achieved a minimum 99.0% (HPLC), the reaction mix 65 Compound 2 ture was cooled to 10-12°C. and stirred at 10-12°C. for 2-4 hours. The precipitate was isolated, washed with purified US 8,859,550 B2 61 62 Example 2 Step 4

N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a NetN pyrazin-8-yl)aZetidin-3-amine I \ Br NN a t 2 10 N \\ 1 Boc

15 1. tert-Butyl (1-(5-bromotetrazolo 1.5-alpyrazin-8-yl) N azetidin-3-yl)(methyl)carbamate

Step 1-2 A 3 L round bottom flask was charged with tert-butyl methyl(1-(tetrazolo 1.5-alpyrazin-8-yl)azetidin-3-yl)car bamate (200 g, 0.66 mol) and DMF (1 L). To the above was added N-bromosuccinimide (117 g. 0.66 mol) in portions at 25 10° C. The resulting mixture was stirred at 10° C. for 0.5 h. Work-up: the reaction mixture was poured into water (3 L). The resulting crystalline solid was collected by filtration, washed with water (500 mL), and dried to afford 200 g (79%) 30 of the product as a white solid. Step 5 8-Chlorotetrazolo 1.5-alpyrazine

35 The title compound was prepared as described in Example 1 steps 1-2. Step 3 40

45

50 tert-Butyl methyl(1-(5-(thiophen-2-yl)tetrazolo 1.5- apyrazin-8-yl)aZetidin-3-yl)carbamate tert-Butyl methyl(1-(tetrazolo 1.5-alpyrazin-8-yl) A 3 L round bottom flask was charged with tert-butyl aZetidin-3-yl)carbamate 55 (1-(5-bromotetrazolo 1.5-alpyrazin-8-yl)azetidin-3-yl)(me thyl)carbamate (50 g., 0.13 mol), thiophene-2-boronic acid A 2 L round bottom flask was charged with 8-chlorotetra (22 g, 0.17 mol), tetrakis(triphenylphosphine)palladium(0) Zolo 1.5-alpyrazine (100 g, 0.64 mol), triethylamine (195g, (5.0 g, 4.3 mmol), CsCO (50 g., 0.15 mol), 1,4-dioxane (1.5 1.93 mol) and ethanol (1L). To the above was added tert-butyl 60 L) and water (500 mL). After the air was purged by bubbling azetidin-3-yl(methyl)carbamate hydrochloride (14.6 g. 0.66 N into the solution, the resulting solution was stirred at 80° mol) at 25°C. The resulting mixture was stirred at 25°C. for C. under N for 14 h. Work-up: the reaction mixture was 1 h. Work-up: the resulting crystalline solid was collected by concentrated in vacuo. The residue was purified by flash filtration, washed with ethanol (200 mL), and dried to afford 65 column chromatography on silica gel with 0-25% ethyl 176 g (91%) of the product as a white solid. MS m/z. 306 acetate in CH2Cl and then crystallized from methanol, to (M+H"). afford 35 g (70%) of the product as a yellow solid. US 8,859,550 B2 63 64 Step 6 prepared at room temperature (15-25°C.) to 4°C. over 24 hours, followed by cooling to -15°C., and slow evaporation of Saturated solutions at room temperature. Vapor diffusion etN experiments were also conducted, typically at room tempera ( \, ture, with solvent systems selected based on API solubility, S Na ? and the miscibility and boiling points of Solvents and anti solvents. Typically a crystal form screen employed 48 solvent 2 systems and in excess of 150 experimental conditions. N \\ N1 10 Salt and Solid State Form Characterization H Salt and solid state forms were characterized by one or more standard techniques including, but not limited to Dif N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a ferential Scanning calorimetry (DSC), Fourier Transform pyrazin-8-yl)aZetidin-3-amine 15 Infrared (FTIR) spectroscopy, Fourier Transform Raman (FT-Raman) spectroscopy, Gravimetric Vapor Sorption A 2 L round bottom flask was charged with tert-butyl (GVS), High Performance Liquid Chromatography (HPLC), methyl(1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) Nuclear Magnetic Resonance (NMR), Polarized Light azetidin-3-yl)carbamate (50 g, 0.13 mol) and dichlo Microscopy (PLM), Powder X-Ray Diffraction (PXRD), romethane (500 mL). To the solution was added trifluoroace Single Crystal X-Ray Diffraction (SCXRD), ThermoGravi tic acid (100 mL). The resulting slurry was stirred at room metric Analysis (TGA) and ThermoGravimetric-InfraRed temperature for 2.5 h. Work-up: the reaction mixture was Analysis (TGA-IR). concentrated in vacuo. The residue was suspended in water DSC: DSC was conducted with a TA Instruments Q100 (500 L) and treated with solid NaCO (pH 10-11, there was 25 differential scanning calorimeter equipped with an autosam un-dissolved NaCO, remaining). The solid was collected by pler and a refrigerated cooling system under 40 mL/min N. filtration, re-suspended in water (500 mLx2) with stirring to purge. DSC thermograms were obtained at 15° C./min in remove NaCO. It was further washed with EtOH (500 mL), crimped Al pans. and dried to afford 27g (73%) of the product as a yellow solid. FTIR: IR spectra were collected with a Nicolet 6700 spec H NMR (400 MHz, DMSO-d) 8: 8.19 (s, 1H), 7.96 (dd. 30 trometer (Thermo Electron) equipped with a DTGS detector J=4.0, 0.8 Hz, 1H), 7.76 (dd, J=48, 0.8 Hz, 1H), 7.27 (dd, and a DuraScope. All spectra were acquired at 4 cm reso J=4.8, 4.0 Hz, 1H), 4.60(br, 2H), 4.18 (br, 2H), 3.73 (m. 1H), lution, 64 scans, using Happ-Cenzel apodization function and 2.43 (br. 1H), 2.29 (s.3H). MS m/z: 288 (M+H"). 2-level Zero-filling. Example 3 35 FT-Raman: Raman spectra were collected with a Nicolet NXR9650 or NXR 960 spectrometer (Thermo Electron) equipped with 1064 nm Nd:YVO4 excitation laser, aliquid Salt Formation and Solid State Form Selection N. cooled Ge detector, and a MicroStage. All spectra were An in-situ Salt Screen was conducted to identify pharma acquired at 4 cm' resolution, 64-128 scans, using Happ ceutically acceptable salts with high aqueous solubility, e.g. 40 Genzel apodization function and 2-level Zero-filling. 10 mg/ml. The in situ salt screen was performed by the GVS: GVS experiments were conducted on a SurfaceMea method described by Tong & Whitesell (Tong W-Q and Whi surement Systems DVS-HT at 25° C. The instrument was tesell G Pharmaceutical Development and Technology, 1998, operated in step mode and the relative humidity (RH) was 3:215-223. Acid counterions were selected partly on the basis increased in 10% RH increments from 40% RH to 90% RH, of having pKa two pH units lower that the cognate base and 45 then decreased from 90% RH to 0% RH, then increased a aqueous formulation precedent as an FDA approved nasal second time from 0% RH to 90% RH, then decreased from spray. Preferred salts demonstrated solubility compatable 90% RH to 0% RH. An extra step at 75% RH was included in with delivery as a solution formulation with a target concen each cycle. The mass equilibrium criterion was set at 0.005% tration of 25 mg/mL at pH >4, are crystalline and are easily change in mass overtime (dm/dt). A minimum step time of 10 and reproducibly prepared. 50 minutes and a maximum step time of 240 minutes were speci Crystal form screens were performed on preferred salts fied. identified on the basis of in-situ Salt Screening. The crystal HPLC: HPLC was performed using a HP1100 HPLC sys form screen was specifically designed to explore diverse crys tem equipped with a G1131 Quad pump, G1367A autosam tallization conditions. Solvents and aqueous mixtures were pler, and G 1315B diode array detector. The Column com initially selected based on a diverse range of properties, 55 prised Phenomenex Luna CN 100A (50x4.6 mm, 3 Lum) at 40° including dielectric constant, boiling points and H-bonding, C. and a mobile phase of A) 0.05% TFA in water and B) which are important to crystallization and crystal-form dis 0.05%TFA in acetonitrile. The gradient was 0% B to 95% B covery. The initial list was then modified to include water and over 8 min (2 min re-equilibration), the flow rate: 1 mL/min, aqueous mixtures of high wateractivity to ensure the discov and the injection Volume 1 uL. Detection was done at 214 and ery of potential hydrates, which are particularly important in 60 345 nm. the context of a nasal spray Solution or Suspension in water NMR: NMR spectra were acquired using a Varian Unity formulation. The solvent mixture list was then further modi Inova 500 MHz instrument, equipped with VNMR 6.1C soft fied to assess compatibility with API manufacturing process ware at 25°C., unless otherwise noted. Chemical shifts are chemistry. Crystallization experiments were conducted in reported in ppm relative to a TMS reference. Coupling con preferred solvents and aqueous mixtures employing known 65 stants are reported in HZ. methods such as temperature-cycled ripening of API slurries PLM: Residual solid filtrants isolated from salt screening between 5-40°C. for 48 hours, cooling of saturated solutions experiments and salt candidates were assessed by PLM, with US 8,859,550 B2 65 66 observations being recorded as “Crystalline' or “Not Crys The results indicate that Compound 1 citrate, sulfate, phos talline'. On occasion, the appearance of solid state forms was phate and hydrochloride salts have solubility of <5 mg/mL, captured photographically. whereas Compound 1 malate and tartrate salts have solubility PXRD: PXRD diffractograms were acquired using a of >5 mg/mL under these conditions. PANalytical XPert Pro diffractometer on Si Zero-back 5 In certain embodiments, initially promising compounds ground wafers. Diffractograms were collected using a mono are those which formed crystalline salts having high solubil chromatic Cu KO. (45 kV/40 mA) radiation and a step size of ity in or near the range of pH 4-5. 0.02° 20 unless noted otherwise. On the basis of the data presented in Table 1, Compound 1 Tartrate and Compound 1 Mesylate were selected for more SCXRD: X-ray measurements were made on a Bruker 10 Nonius Kappa Axis X8 Apex2 diffractometerata temperature detailed assessment. of 110K. Crystal were mounted on a Mitegen polyimide Compound 1 Tartrate Salt micromount with a small amount of Paratone Noil. Structure was solved by direct methods using the XS program (Bruker 15 Crystallization experiments targeting the preparation of AXS, XS version 2009.9). Structure refinement was effected Compound 1 tartrate salt were conducted in which the effect using the XL program from SHELXTL. Graphic plots were of solvent (acetone, acetonitrile, tetrahydrofuran, methanol, produced using the NRCVAX crystallographic program isopropyl alcohol, water/1% DMSO, ethyl acetate, dichloro Suite. ethane, MTBE, Toluene, MIBK and dioxane) was surveyed. TGA: TGA thermograms were obtained with a TA Instru A single hemihydrate form designated Compound 1 Tar ments Q500 thermogravimetric analyzer under 40 mL/min trate Form 1 was produced in nine of twelve crystallization N purge at 15°C/min in Pt or Al pans. experiments performed. A stable monohydrate form, desig TGA-IR: TGA-IS was conducted with a TA Instruments nated Compound 1 Tartrate Form 2 was encountered during Q5000 thermogravimetric analyzer interfaced to a Nicolet scale-up experiments. A competitive ripening experiment 6700 FT-IR spectrometer (Thermo Electron) equipped with 25 conducted between Compound 1 Tartrate salt Form 1 and an external TGA-IR module with a gas flow cell and DTGS Compound 1 Tartrate salt, Form 2 demonstrated that Com detector. TGA was conducted with 60 mL/min N. flow and pound 1 Tartrate Form 2 is more stable than Compound 1 heating rate of 15°C./min in Pt or Al pans. IR spectra were Tartrate Form 1 in water at 23° C. collected at 4 cm resolution and 32 scans at each time point. 30 Evidence for possible additional forms and/or salts of dif ferent stoichiometry was discovered. Thus equilibrium solu Compound 1 bility experiments of Compound 1 Tartrate in acetate buffer (pH5) resulted in a new solid state which NMR analysis Salt Formation and Solid State Forms revealed to possess Compound 1: Tartrate stoichiometry of 35 In situ Salt formation screening was performed by the 1:0.8. An attempt to scale up of the in situ Compound 1 method of Tong & Whitesell (Tong W-Q and Whitesell G Tartrate form failed, but ultimately yielded a pure sample of Pharmaceutical Development and Technology, 1998, 3:215 Compound 1 Tartrate Form 2. 223. Compound 1 was combined with 0.1M aqueous solu Preparation of Compound 1 Tartrate Salt, Form 1 tions of twelve acids at ambient temperature, reaction mix 40 tures monitored, recharged with Compound 1 where Compound 1 (101.5 mg) was added to 3.0 mL of methanol appropriate and slurries temperature cycled between 5-40°C. (30 vol). The slurry was stirred and heated to 60° C. L-tartaric for 48 hours. Reaction mixtures were then filtered and filtrant acid (3M in water, 1.0 eq.) was added to the slurry at 60° C. residual solids and filtrates analyzed as shown in Table 1. 45 The solids dissolved quickly. This solution was mixed for 15 minutes at 60° C. during which time crystallization occurred TABLE 1. to produce a thick slurry. The slurry was mixed and tempera Results of In-Situ Salt Formation Screen for Compound 1 ture-cycled from 40 to 5°C. over 18 hours then equilibrated to 23°C. Crystalline solids were isolated by vacuum filtration to Com- Filtrate 50 yield the tartrate salt (144 mg.93%). pound Filtrant Solubility Compound 1 Tartrate Salt Form 1 was characterized as follows: 1 (mg) Counterion PLM FTIR pH (mg/mL) i)"H NMR: (500 MHz, DMSO-d) 8:8.27 (s, 1H), 7.99 (d. 25 Acetic Acid Crystalline Free Base 4.02 7.99 55 J=3.5 Hz, 1H), 7.79 (d. J=5 Hz, 1H), 7.28 (m, 1H), 4.27, 4.20 25 Citric Acid Crystalline Salt 3.11 1.75 25 Sulfuric Acid Crystalline Salt 1.39 O.S6 (ss, 5.6H*), 2.58 (m, 6.7H**), 2.504 (s, H**), 2.29 (s.2.85H) 25 Phosphoric Acid Crystalline Salt 1.76 O.87 *** (where * indicates singlet coincident with water: ** 25 Hydrochloric Crystalline Salt 1.25 O.99 Acid indicates aliphatic coincident with DMSO: *** indicates 25 L-Aspartic Acid Crystalline Free Base 4.20 8.64 unable to determine tartrate salt stoichiometry due to over 25 L-Glutamic Acid Crystalline Free Base 4.28 6.75 60 lapping signals.) 40 L-Malic Acid Crystalline Salt 4.15 13.4 25 L-Tartaric Acid Crystalline Salt 3.48 8.69 ii) PXRD: The PXRD diffractogram of Compound 1, Tar 40 Mesic Acid No Solids Isolated 1.49 2O.O trate Salt, Form 1 is depicted in FIG. 1; the associated PXRD 25 Stearic Acid Crystalline Free Base 8.00 ND Peaks are listed in Table 2 (where * indicates +0.2° and ** 25 Succinic Acid Crystalline Free Base 4.13 10.4 65 indicates that the relative intensity for each peak is deter ND: Note Detectable mined by normalizing its intensity to that of the strongest peak at 18.1° angle as 100). US 8,859,550 B2 67 68 TABLE 2 thick slurry. The slurry was cooled from 60°C. to 5°C. over 2 hours then stirred at 5° C. for 12 hours. Crystalline solids PXRD Peaks of Compound 1, Tartrate Salt, Form 1 were isolated by vacuum filtration to yield 69.6 mg of Com PXRDPeaks of Compound 1 Tartrate Forn 1 (Hemihydrate pound 1 Tartrate Form 2. 5 Compound 1 Tartrate Salt Form 2 was characterized as Two-Theta Angle Relative Intensity** follows: (degree) (arbitrary) H NMR: (500 MHz, DMSO-d) 8: 8.28 (s, 1H), 8.00 (d. S.1 15.1 J=3.0 Hz, 1H), 7.80 (d. J=5 Hz, 1H), 7.29, 7.28 (ABq, J-4 10.2 99.1 HZ, 1H), 4.29 (s., H2O), 4.25 (s. 2H), 2.64 (m, 7.5H), 2.33 (s, 13.4 76.2 10 3H**)*** (where indicates * used for stoichiometric deter 14.1 22.0 mination of tartrate, * indicates used for stoichiometric 16.6 29.2 determination of Compound 1, and *** indicates the 'H 17.4 88.9 NMR spectrum confirms that the ratio of Compound 1:Tar 18.1 1OOO trate is 1:1). 19.1 93.1 ii) PXRD: The PXRD diffractogram of Compound 1, Tar 22.1 27.6 15 22.9 45.4 trate Salt, Form 2 is depicted in FIG. 5; the associated PXRD 23.5 88.8 Peaks are listed in Table 4. 24.4 33.6 25.3 35.5 26.0 88.3 TABLE 4 27.0 S4.O 29.8 48.2 PXRD Peaks of Compound 1, Tartrate Salt, Form 2 30.7 19.9 PXRD Peaks of Compound 1 Tartrate Form 2 (Monohydrate 36.7 12.6 38.2 23.0 Two-Theta Angle Relative Intensity** (degree) (arbitrary)

25 5.7 8.3 iii) FT-Raman: The FT-Raman Spectrum of Compound 1. 11.4 22.0 Tartrate Salt, Form 1 is depicted in FIG. 2; the associated 12.7 2.3 FT-Raman Peaks are listed in Table 3. 14.5 2.4 15.3 5.2 15.7 6.6 TABLE 3 16.2 14.3 30 17.1 1OOO FT-Raman Peaks of Compound 1, Tartrate Salt, Form 1 19.0 1O.S FT-Raman Peaks of 19.3 3.5 Compound 1 Tartrate Forn 1 (Hemihydrate 23.8 11.8 24.1 35.7 Position 26.2 6.9 (cm) Intensity 35 27.2 6.1 27.6 7.1 624.3 31.4 28.1 4.5 649.8 15.3 29.2 6.6 1089.3 15.7 31.7 4.8 1229.0 31.8 34.7 3.4 1295.4 30.2 40 35.1 3.3 1339.3 119.9 1356.4 219.9 1419.7 84.2 **The relative intensity for each peak is determined by normalizing its intensity to that of the 1497.3 85.8 strongest peak at 17.1° angle as 100 1524.4 1776 1551.2 42.5 iii) FT-Raman: The FT-Raman Spectrum of Compound 1. 1589.4 1OS.O 45 Tartrate Salt, Form 2 is depicted in FIG. 6; the associated FT-Raman Peaks are listed in Table 5. iv) DSC/TGA: The DSC/TGA scans Compound 1, Tartrate Salt, Form 1 is depicted in FIG. 3. The results of DSC abd TABLE 5 TGA-IR suggest that Compound 1 Tartrate Form 1 is a hemi 50 FT-Raman Peaks of Compound 1, Tartrate Salt, Form 2 hydrate (1.6% water). FT-Raman Peaks of Compound 1 Tartrate Forn 2 (Monohydrate v) GVS: The GVS isotherm plot for Compound 1, Tartrate Position Salt Form 1 is depicted in FIG. 4. The GVS curve indicates a (cm) Intensity weight change of 1.5% below 30% Relative Humidity (RH) and 1% between 30-90% RH. These results are consistent 55 622.3 26.2 644.8 41.9 with the thermal analysis and Compound 1 Tartrate Form 1 748.1 22.1 being a hemihydrate. 1229.3 30.1 1295.0 47.0 Preparation of Compound 1 Tartrate Salt, Form 2 1350.4 3.31.8 1376.5 48.5 60 1421.8 123.9 Compound 1 (49.8 mg) was added to 0.5 mL of methanol/ 1432.2 97.3 water (1:1) (10 vol). The slurry was stirred and heated to 60° 1494.4 167.5 C. L-Tartaric acid (3M in water, 1.0 equiv.) was added to the 1522.1 237.2 slurry at 60° C. Most solids dissolved quickly, but a few large 1592.6 152.6 particles required 10 minto completely dissolve. The solution 65 was seeded with ~2 mg of Compound 1 Tartrate Form 2 iv) DSC/TGA: The DSC/TGA scans of Compound 1, Tar crystals at 60° C. and stirred for 105 minutes to produce a trate Salt, Form 2 are depicted in FIG. 7. Compound 1, Tar US 8,859,550 B2 69 70 trate Salt, Form 2 is a stable monohydrate. The dehydration Compound 1 Mesylate Salt Form 5 is an ethanol solvate event occurs between 80 and 130°C., with loss of 3.9% water. (DSC/TGA-IR: 4.1%, 0.4 eq. ethanol) that was obtained from v) GVS: The GVS isotherm plot for Compound 1, Tartrate cooling crystallization of an ethanol solution and as a mixture Salt, Form 2 is depicted in FIG.8. Compound 1, Tartrate Salt, with Compound 1 Mesylate Salt Form 1 from evaporation of Form 2 exhibits a ~1% weight change between 5% and 90% an ethanol solution. Compound 1 Mesylate Salt Form 5 con RH. FT-Raman analysis before and after GVS showed no verted to Compound 1 Mesylate Salt Form 1 in six days in a Form change. The data are consistent with the thermal analy closed vial at ambient temperature. sis and indicate that Compound 1, Tartrate Salt Form 2 is a stable, non-hydroscopic monohydrate. Preparation Compound 1 Mesylate Salt, Form 1 10 Compound 1 Mesylate Salt Compound 1 (2.315 g, 7.68 mmol) was added to 11.58 mL of acetronitrile/5% water (5 vol). The slurry was stirred and Crystallization experiments targeting the preparation of heated to 60° C. Mesic acid (>99.5%, 0.523 mL, 1.05 eq.) was Compound 1 Mesylate Salt were conducted in which the diluted in 1.16 mL of acetronitrile/5% water (0.5 vol) and effect of solvent (acetone, acetonitrile, tetrahydrofuran, 15 added to the slurry at 60°C. This solution was seeded at 60° methanol, isopropyl alcohol, water/1% DMSO, ethyl acetate, C. and stirred for 60 minto produce a thick slurry. The slurry dichloroethane, MTBE, Toluene, MIBK and dioxane) and was cooled from 60 to 5° C. over 2 hours and stirred at 5° C. crystallization methods (temperature cycling, evaporation) for 2 hrs. Crystalline solids were isolated by vacuum filtra were surveyed. tion, rinsed and washed with ~2.3 mL (1 Vol) of acetronitrile/ Two crystal forms of Compound 1 Mesylate Salt were 5% water to yield a 2.585-g wet cake. It was dried at ambient obtained from these experiments. Compound 1 Mesylate temperature under vacuum for 16 hours then exposed to Salt, Form 1 is a monomesylate reversible monohydrate solid ambient conditions for 3 hours to afford Compound 1 Mesy state form that was obtained from ten of the twelve crystalli late Salt Form 1 (2.55g, 80%). zation experiments conducted. Compound 1 Mesylate Salt 25 Compound 1 Mesylate Salt Form 1 was characterized as Form 1 exhibits good physical properties and high aqueous follows: solubility (>100 mg/ml). Compound 1 Mesylate Salt Form 2 i)"H NMR: (500 MHz, DMSO-d) 8:10.11 (brs, 1H), 8.36 was obtained by slow evaporation of gummy Solids at ambi (s, 1H), 8.05 (d, J-3.0 Hz, 1H), 7.85 (d. J=5 Hz, 1H), 7.31 (at, ent temperature in two of twelve screening experiments. J–3.5 Hz, 1H), 5.32 (brs, 2H), 3.66(brs, 2H), 3.36 (brs, 2H), Compound 1 Mesylate Salt Form 2 was obtained as a mixture 30 3.27 (brs, 2H), 2.88 (s.3H). of Form 1 and Form 2 in one of twelve crystallization experi ii) PXRD: The PXRD diffractogram of Compound 1, ments. Compound 1 Mesylate Salt Form 2 was subsequently Mesylate Salt, Form 1 is depicted in FIG. 9; the associated isolated in pure form from a scale up experiment and was PXRDPeaks are listed in Table 6 (where * indicates +0.2° and demonstrated to undergo significant conversion to Com ** indicates the relative intensity for each peak is determined pound 1 Mesylate Salt Form 1 after three days in a closed vial 35 at room temperature. by normalizing its intensity to that of the strongest peak at A Compound 1 Mesylate salt form screen was conducted 21.2° angle as 100). involving about 160 crystallization experiments in 48 sol vents and aqueous mixtures was performed conducted as TABLE 6 previously described (vide supra). Compound 1 Mesylate 40 PXRD Peaks of Compound 1, Mesylate Salt, Form 1 Salt Form 1, the reversible monohydrate initially discovered PXRD Peaks of Compound 1 MeSylate Form 1 (Monohydrate in during the crystallization experiments cited above, was obtained from a variety of solvents and crystallization condi Two-Theta Angle Relative Intensity** tions, including 25 slurry-ripening, six cooling, 16 evapora (degree) (arbitrary) tive and four vapor diffusion experiments. Form 1 was deter 45 5.3 25.4 1O.S 3.8 mined to be the most, and the only, stable crystal form of the 13.5 S.O five discovered mesylate salts under ambient conditions as 15.1 1.8 tested. Samples of Compound 1, Mesylate Salt Forms 2-5 15.9 2.5 converted to Compound 1 Mesylate Salt Form 1 in solid state 18.0 4.8 within one week at ambient conditions. 50 18.3 3.7 19.8 3.1 Compound 1, Mesylate Salt Form 2, described above was 20.4 1.5 not encountered in the Compound 1 Mesylate Salt form 21.2 1OO.O SCC. 21.8 1.7 22.5 2.3 Compound 1 Mesylate Salt Form 3 is a non-solvated form 23.0 8.0 that was obtained from 17 slurry ripening, one cooling and 55 23.3 7.1 one evaporative crystallization experiments. FT-Raman 25.3 8.1 analysis of 8 Compound 1 Mesylate Salt Form 3 screening 26.5 2.9 27.1 3.3 samples showed complete conversion to Compound 1 Mesy 28.0 7.6 late Salt Form 1 in four days on a well plate at ambient 28.5 3.4 conditions. 60 30.6 2.1 Compound 1 Mesylate Salt Form 4 is a non-stoichiometric 33.0 6.0 hydrate that was obtained from one slurry ripening (ni 35.3 2.6 tromethane), four cooling and one vapor diffusion experi 37.5 4.1 ments. The Compound 1 Mesylate Form 4 exhibits a broad dehydration event below 110°C. with loss of 0.9% water and 65 iii) FT-Raman: The FT-Raman Spectrum of Compound 1. converted to Compound 1 Mesylate Salt Form 1 in four days Mesylate Salt, Form 1 is depicted in FIG. 10; the associated on a well plate at ambient conditions. FT-Raman Peaks are listed in Table 7. US 8,859,550 B2 71 72 TABLE 7 distinct, while C1' was coincident with C2, C2' was coinci dent with C1, C3' was coincident with S1 and C4' was coin FT-Raman Peaks of Compound 1, Mesylate Salt, Form 1 FT-Raman Peaks of cident with C4. Difference maps showed a peak of approxi Compound 1 MeSylate Forn 1 (Monohydrate mately 1.3e/A near the C2/C1' site. An attempt to fit this 5 peak to a distinct C1 site did not yield a refinement with Position reasonable even constraints in the least-squares model. The (cm) Intensity final refinements assumed that sites for C2 and C1' were S48.5 11.1 coincident. The normalized occupancy refined to a value of 626.8 15.9 0.7893(14) for the major orientation of the thiophene ring. 794.9 9.3 10 1044.9 10.7 The anion was disordered over 2 orientations. The S2 site 1058.3 9.9 for both orientations was coincident while all other atoms in 1076.3 10.3 1231.5 14.8 the anion occupied distinct sites. The angle between the ori 1299.2 20.1 entation is indicated by the angle C14-S2-C14 which was 1312.9 25.6 15 approximately 23°. The normalized occupancy for the major 1353.6 88.0 1422.2 60.1 orientation refined to a value of 0.892(4). 1494.3 39.7 The hydrogenatoms were introduced at idealized positions 1522.7 75.6 and were treated in a mixed fashion. The hydrogen atoms in 1546.7 14.3 the ordered regions of the structure were allowed to refine 1589.5 69.6 2931.8 11.1 isotropically while the hydrogen atoms in the disordered 30041 1O.O regions were allowed to ride on the parent atom. The hydro gen atom positions on the water oxygen were derived from a difference Fourier map. The structural model was fit to the iv) DSC/TGA: The DSC/TGA curves for Compound 1, data using full matrix least-squares based on F. The calcu Mesylate Salt, Form 1 is depicted in FIG. 11. The DSC curve 25 exhibits a broad dehydration event between 50 to 110° C. lated Structure factors included corrections for anomalous followed by simultaneous melting and decomposition events dispersion from the usual tabulation. The structure was at approximately 230°C. TGA-IR analysis showed a loss of refined using the XL program from SHELXTL, graphic plots 4% water below 100° C. were produced using the NRCVAX crystallographic program suite. Results are shown below in Tables 8-11 and in FIGS. 13 V) GVS: The GVS isotherm plot for Compound 1, Mesy 30 alte Salt Form 1 is depicted in FIG. 12. The water content and 14. (-4.3%) remained relatively constant between 20-90% RH. TABLE 8 There was a loss of -4.30% water below 20% RH. The results are consistent with the thermal analysis and indicate that Summary Crystal Data: Compound 1 MeSylate Monohydrate. Form 1 Compound 1 Mesylate Salt Form 1 is not hydroscopic. FT 35 Raman analysis before and after GVS confirmed that there Identification Code Compound 1 Mesylate was no crystal form change. Formula C14H2N/OS2 vi) X-ray Crystal Structure Determination: Formula Weight (g/mol) 41S.SO Data Collection and Processing. Crystal Dimensions (mm) O.30 x 0.26 x 0.25 Crystal Color and Habit colourless prism A single crystal of 8-(4-Methylpiperazin-1-yl)-5- 40 Crystal System Triclinic (thiophen-2-yl)tetrazolo 1.5-alpyrazine mesylate, monohy Space Group P-1 drate, Form 1 was mounted on a Mitegen polyimide micro Temperature, K 110 mount with a small amount of Paratone N oil. All X-ray 6.706(2) 8.396(2) measurements were made on a Bruker-Nonius Kappa Axis 16.978(4) X8 Apex2 diffractometer at a temperature of 110 K. The unit 45 81.095 (19) cell dimensions were determined from a symmetry con 80.260(14) Strained fit of 99.06 reflections with 5.34°-20<77.18°. The 76.100(13) 908.0(4) data collection strategy was a number of () and (p scans which Number of reflections to determine 99.06 collected data up to 86.42 (20). The frame integration was final unit cell performed using SAINT (Bruker-Nonius, SAINT version 50 Min & Max 20 for cell detn, 5.34, 77.18 2009.9, 2009, Bruker-Nonius, Madison, Wis. 53711, USA). Z. 2 F(000) 436 The resulting raw data was scaled and absorption corrected p (g/cm) using a multi-scan averaging of symmetry equivalent data , A, (MoKo.) 0.71073 using SADABS (Bruker-Nonius, SADABS version 2009.9). I, (cm) O.332 Structure Solution and Refinement. 55 Diffractometer Type Bruker-Nonius Kappa Axis X8 Apex2 The structure was solved by direct methods using the XS Scan Type(s) omega and phi scans program (Bruker-AXS, XS version 2009.9). Most non-hy Max 20 for data collection, 86.42 drogen atoms were obtained from the initial solution. The Measured fraction of data O.984 remaining positions were obtained from Subsequent differ Number of reflections measured 55627 Unique reflections measured 12971 ence Fourier maps. 60 Rmerge The structure exhibits two independent disorders, one in Number of reflections included in 12971 the cation and one on the anion. The cation disorder involves refinement an approximate 180° rotation of the thiophene ring. This Cutoff Threshold Expression >2sigma(I) Structure refined using full matrix least-squares using F. interchanges the position of atom S1 and C3. The disorder Weighting Scheme Calcw = 1/(sigma’ (Fo) + was model by introducing 2 sets of atoms, the major compo 65 (0.0625P)? + 0.0983P nent consisting of S1, C1, C2, C3, C4, and minor component where P = (Fo? + 2Fc)/3 consisting of S1, C1, C2, C3', C4'. The position for S1' was

US 8,859,550 B2 75 76 TABLE 11-continued solution was washed with toluene (2x1 L) and the pH of the remaining aqueous phase adjusted to 9.5-10.5 by addition of Bond Angles for Compound 1 MeSylate Monohydrate. Form 1 sodium hydroxide (27%). The product precipitated during the Bond Angle Bond Angle pHadjustment, was isolated, Suspended intoluene (2.5L) and heated to 55-60° C. The resulting organic solution was N4-C6-C7 34.31(7) N6-C12-H12B 10.8(7) N1-C6-C7 7.79(6) C11-C12-H12B O9.5(7) washed with 1% cysteine solution and the pH of the solution NS-C7-N6 9.37(7) H12A-C12-H12B 07.3(10) is adjusted to 8.8-9.2 by addition of sodium hydroxide (27%). NS-C7-C6 8.67(7) N7-C13-H13A O8.6(9) The separated organic phase was then washed with purified N6-C7-C6 21.89(7) N7-C13-H13B O9.3(8) water at 55.60° C. 3-mercaptopropyl ethyl sulfide silica was C7-NS-C8 20.08(7) H13A-C13-H13B 10.7(12) 10 NS-C8-CS 25.55(7) N7-C13-H13C O8.5(9) added top the separated organic phase and the mixture is NS-C8-H8 4.1(8) H13A-C13-H13C 12.2(12) stirred for 20 minutes at 55-60° C. Activated carbon was C5-C8-H8 20.3(8) H13B-C13-H13C 07.5(11) C7-N6-C12 25.22(7) O1-S2-O3 14.67(9) added and the organic phase stirred for 20 minutes at 55-60° C7-N6-C9 20.22(6) O1-S2-O2 11.86(7) C., then filtered through a pad of Celite. The filter cake is C12-N6-C9 3.19(6) O3-S2-O2 10.16(8) 15 washed with toluene, and the wash combined with the filtrate. N6-C9-C10 0.06(6) O1-S2-C14 06.18(6) To the combined filtrates was added a solution of methane N6-C9-H9A O9.3(8) O3-S2-C14 07.25(7) C1O-C9-H9A 0.0(8) O2-S2-C14 06.18(7) sulfonic acid in purified water, and the mixture stirred until all N6-C9-H9B O8.9(7) H1WA-O1W-H1WB 02.4(15) the product is extracted from the toluene phase into the aque ous phase. The aqueous phase was Subjected to clear filtra tion. The pH of the filtrate was adjusted to 9.5-10.5 by addi tion of 27% aqueous sodium hydroxide. The precipitated free SCHEME 3 base was isolated, and dried in vacuum at 35° C. until the water content in the product does not exceed 21.7%. The NetN partially dried material was suspended in acetonitrile (5.45 / I \ 25 S N 2 CHSO3H, g/g crude material) and purified water (0.278 g/g, crude mate CH3CN/HO rial), heated to 55-60° C., and methanesulfonic acid (0.344 He g/g crude material 1.08 eq) added. The clear Solution was 2 seeded at 60-70° C. and cooled to 50-60° C. The suspension N N was cooled to 5-10°C. and the product isolated, washed with N 30 acetonitrile, dried in vacuum at 30° C. and allowed to equili N brate in a moist atmosphere (92.9 g, 75%). NetN Compound 1, monomethanesulfonate monohydrate exhib (S Na Z\, its >5-fold improvement in solubility over Compound 1 in 35 aqueous acetate buffer (80 mM) at pH3.5-4.5. 2 N N Characterization of Compound 1 Mesylate Salt Form 2 N N- N Compound 1 Mesylate Salt Form 2 was characterized as CHSOH, H2O 40 follows: i) PXRD: The PXRD diffractogram of Compound 1, Mesylate Salt, Form 2 is depicted in FIG. 15: the associated Alternative Preparation Compound 1 Mesylate Salt, PXRD Peaks are listed in Table 12 (where * indicates +0.2° and ** indicates the relative intensity for each peak is deter Form 1 45 mined by normalizing its intensity to that of the strongest peak at 23.4° angle as 100).

TABLE 12 NetN 50 PXRD Peaks of Compound 1 Mesylate Salt Form 2 (S Naa\, PXRD Peaks of Compound 1 MeSylate Form 2 2 Two-Theta Angle Relative Intensity** N N (degree) (arbitrary) 55 N 10.6 24.9 N- N 11.6 9.4 CHSOH, H2O 12.3 2.5 15.1 20.8 15.9 6.7 16.4 69.1 60 17.2 5.3 8-(4-Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetra 17.7 97.9 Zolo 1.5-alpyrazine Mesylate Monohydrate. Form 1 18.7 24.6 21.0 1.1 23.4 1OOO 8-(4-Methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo.1, 24.2 32.1 5-alpyrazine (90g, 0.298 mol 1.0 eq.) purified water (950 ml) 65 2SO 46.8 and methanesulfonic acid (43.05 g 0.448 mol, 1.5 eq) were 25.9 3.9 combined and the resulting solution heated to 50–55°C. The US 8,859,550 B2 77 78 TABLE 12-continued TABLE 14-continued PXRD Peaks of Compound 1 Mesylate Salt Form 2 PXRDPeaks of PXRD Peaks of Compound 1 Mesylate Salt Form 3 Compound 1 MeSylate Form 2 5 PXRD Peaks of Two-Theta Angle Relative Intensity** Compound 1 Mesylate Form 3 (Anhydrous) (degree) (arbitrary) 28.1 12.1 Two-Theta Angle Relative Intensity** 29.3 9.9 (degree) (arbitrary) 10

ii) FT-Raman: The FT-Raman Spectrum of Compound 1. 29.4 8.9 Mesylate Salt, Form 2 is depicted in FIG. 16; the associated 32.9 12.4 FT-Raman Peaks are listed in Table 13. 15 TABLE 13 ii) FT-Raman: The FT-Raman Spectrum of Compound 1. FT-Raman Peaks of Compound 1 Mesylate Salt Form 2 Mesylate Salt, Form 2 is depicted in FIG. 18; the associated FT-Raman Peaks of FT-Raman Peaks are listed in Table 15. Compound 1 MeSylate Form 2

Position TABLE 1.5 (cm) Intensity FT-Raman Peaks of Compound 1, Mesylate Salt, Form 3 626.1 13.6 FT-Raman Peaks of 794.1 8.4 Compound 1 Mesylate Form 3 (Anhydrous 1037.0 11.6 25 1226.5 8.6 Position 1295.9 21.3 1312.5 23.1 (cm) Intensity 1352.2 137.4 555.7 9.4 1423.5 54.4 1493.9 74.5 625.1 20.3 1521.6 82.8 30 774.4 8.2 1541.9 23.9 796.3 10.9 1589.8 92.4 1034.5 11.1 2934.5 12.6 1236.4 12.4 2970.5 9.8 1269.8 11.3 3O11.5 9.6 1293.9 26.7 35 1309.6 25.1 1346.2 135.4 1422.6 89.4 Characterization of Compound 1 Mesylate Salt Form 1493.2 53.5 3 1524.1 116.6 40 1588.9 100.1 Compound 1 Mesylate Salt Form 1 was characterized as 2931.0 14.6 follows: 3011.3 8.5 i) PXRD: The PXRD diffractogram of Compound 1, 3O84.5 9.6 Mesylate Salt, Form 2 is depicted in FIG. 17; the associated PXRD Peaks are listed in Table 14 (where * indicates +0.2° 45 iii) DSC/TGA: The DSC scans Compound 1, Mesylate and * indicates the relative intensity for each peak is deter Salt, Form 3 is depicted in FIG. 19. The DSC curve indicates mined by normalizing its intensity to that of the strongest a melting onset at 223.5° C. followed by immediate recrys peak at 24.0° angle as 100). tallization and subsequent melt/decomposition at 234.7° C. TGA-IR analysis indicated -0.4% water loss below 50°C., TABLE 1.4 50 Suggesting that Compound 1, Mesylate Salt, Form 3 is a PXRD Peaks of Compound 1 Mesylate Salt Form 3 non-Solvated form, PXRDPeaks of Compound 1 MeSylate Forn 3 (Anhydrous Equilibrium Solubility Studies of Compound 1 Two-Theta Angle Relative Intensity** 55 Tartrate Salt Form 1. Compound 1 Tartrate Salt Form (degree) (arbitrary) 2 and Compound 1 Mesylate Salt Form 1 7.8 8.7 10.4 23.1 13.2 8.0 Equilibrium solubility experiments were conducted for 15.1 1.6 Compound 1 Tartrate Salt Form 1, Compound 1 Tartrate Salt 16.3 43.3 60 17.2 27.0 Form 2 and Compound 1 Mesylate Salt Form 1 in water and 17.8 46.0 pH-5 acetate buffer solution The results of the equilibrium 19.6 8.8 solubility study are summarized in Table 16 (where “a” indi 22.8 45.8 24.0 1OOO cates Raman spectra of both samples matched each other, but 26.5 2.6 65 were distinctly different from the known forms of FB or 27.8 55.3 tartrate salt; NMR results showed a non-stoichiometric ratio of Free Base:Tartaric acid (1:0.8)). US 8,859,550 B2 79 80 TABLE 16 TABLE 17 Equilibrium Solubility of Compound 1 Tartrate Salt Form 1, Results of In-Situ Salt Formation Screen for Compound 2. Compound 1 Tartrate Salt Form 2 and Compound 1 MeSylate Salt Form 1 Filtrate 5 Filtrate Cpd. 2 Filtrant Solubility

Concen (mg) Counterion PLM FTIR pH (mg/mL) Salt tration Cpd. 1 Weight Solvent Residual Final (mg/ 55 Acetic Acid No Solids Isolated 541 >24.53 Salt (mg) Solvent (mL) Solid pH mL) 10 25 Citric Acid Crystalline Salt 3.69 4.27 25.1 Sulfuric Acid Crystalline Salt 1.42 O.61 Tartrate 24.6 Water 1.O Tartrate 3.48 8.18 25 Phosphoric Acid Crystalline Salt 4.8 O.74 Form 1 Form 1 55 Hydrochloric Crystalline Salt 2.32 15.25 Acid Tartrate 23.6 O.2M 1.O New 4.53 2.73 40 L-Aspartic Acid No Solids Isolated 4.95 >1845 Form 1 Acetate Form 15 25 L-Glutamic Acid Crystalline Free Base 5.46 10.36 Buffer Salt 40 L-Malic Acid No Solids Isolated S.13 >21.1 (pH5) 25.1 L-Tartaric Acid Crystalline Salt 3.23 1.39 Tartrate 29.1 Water 1.O Tartrate 3.55 4.43 55 Mesic Acid No Solids Isolated 2.28 -25.3 Form 2 Form 2 25.1 Stearic Acid Crystalline Salt 7.16 ND Tartrate 25.6 O.2M 1.O New 4.61 3.09 40 Succinic Acid Crystalline Salt 4.75 O.1 Form 2 Acetate Form Buffer Salt (pH5) Mesylate 50.7 Water O.S Dissolved 3.51 >100 The results indicate that Compound 2 sulfate, phosphate, Form 1 L-tartrate and Succinate salts have solubility of <5 mg/mL, Mesylate 53.1 O.2M O.S Free Base 4.17 58.2 whereas Compound 2 hydrochloride salt has solubility of 10 Form 1 Acetate 25 mg/mL under these conditions. Buffer In certain embodiments, initially promising compounds (pH5) are those which formed crystalline salts having high solubil ity in or near the range of pH4-5. Compound 1 Tartrate Salt Form 1 had solubility values of On the basis of the data presented in Table 17, three salt 8.18 mg/mL in water, but dissolution in 0.2M acetate buffer 30 candidates, namely Compound 2 Citrate, Compound 2 (pH5) resulted in a residual New Form/Salt with lower solu Hydrochloride, and Compound 2 Mesylate were selected for bility of 2.73 mg/mL. more detailed assessment. Compound 1 Tartrate Salt Form 2 had a solubility values of 4.43 mg/mL in water, but dissolution in 0.2M acetate buffer 35 Compound 2 Citrate Salt (pH5) resulted in a residual New Form/Salt with lower solu bility of 3.09 mg/mL. Raman-FTIR spectra of both New Crystallization experiments targeting the preparation of Form/Salt samples matched each other, but were distinctly Compound 2 Citrate Salt were conducted in which the effect different from the known forms of Compound 1 and tartrate of solvent (acetone, acetonitrile, tetrahydrofuran, methanol, salt. NMR results showed a non-stoichiometric ratio of Com 40 isopropyl alcohol, water/1% DMSO, ethyl acetate, dichloro pound 1 Free Base:Tartaric acid (1:0.8). ethane, MTBE, Toluene, MIBK and dioxane) up crystalliza In the case of Compound 1 Mesylate Salt Form 1 in water, tion by temperature cycling was Surveyed. no residual Solids were isolated as the sample completely Multiple crystal forms of Compound 2 Citrate Salt were dissolved. The equilibrium solubility of Compound 1 Mesy obtained from these experiments. Compound 2 Citrate Salt late Salt Form 1 is >100 mg/mL in water. 45 Form 1 is a non-Solvated, monocitrate Solid state form that was obtained from six of the twelve screening experiments. Compound 1 Mesylate Salt Form 1 in 0.2M Acetate Buffer Five other samples in the present study displayed unique (pH5) had equilibrium solubility of 58.2 mg/mL. The residual FTIR & PXRD spectra, indicative of additional solid state solid was identified as Compound 1 on the basis of Raman forms and/or citrate salts of different stoichiometry. Two FTIR analysis. 50 additional unique samples were obtained from equilibrium solubility experiments of Compound 2 Citrate Salt Form 1 in Compound 2 water and 0.2M Acetate buffer, pH 5.

Salt Formation and Solid State Forms 55 Preparation of Compound 2 Citrate Salt, Form 1 Compound 2 (500 mg, 1.74 mmol) was added to 15 mL of In situ Salt formation screening was performed by the acetone and the slurry heated, with stirring, to 40° C. Citric method of Tong & Whitesell (Tong W-Q and Whitesell G acid (1M in water, 1.75 mL, 1.0 eq.) was added to the slurry Pharmaceutical Development and Technology, 1998, 3:215 60 at 40°C. This solution was seeded with ~2 mg of Compound 223. Compound 2 was combined with 0.1M aqueous solu 2 Citrate salt Form 1 at 40° C. and stirred for 60 minutes tions of twelve acids at ambient temperature, reaction mix resulting in a thick slurry. The slurry was cooled from 40°C. tures monitored, recharged with Compound 2 where to 25° C. and then Stirred at 25° C. for 16 hours. The Solids appropriate and slurries temperature cycled between 5-40°C. were isolated by vacuum filtration, and rinsed and washed for 48 hours. Reaction mixtures were then filtered and filtrant 65 with acetone (-5 mL). The product was dried at 50° C. under residual solids and filtrates analyzed as shown in Table 1 vacuum for 3 hours to afford Compound 2 Citrate Salt, Form (where ND indicates Note Detectable). 1 (510 mg, 91% yield). US 8,859,550 B2 81 82 Compound 2 Citrate Salt Form 1 was characterized as weight increase of -0.2% at 90% RH. These results indicate follows: that Compound 2 Citrate Salt Form 1 is not hydroscopic. i) H NMR: (500 MHz, DO) 8: 7.91 (s. 2H), 7.84 (d. J=3 FTIR analysis of the sample before and after GVS confirmed HZ, 1H), 7.68 (d. J=5 Hz, 1H), 7.274, 7.268 (ABq, J3.75 that there was no change in crystal form. HZ, 1H), 4.81 (brd, J=113 Hz, nd), 4.46 (m. 1H), 2.90, 2.87 (s, s, 5H**), 2.78, 2.75 (d. J=15 Hz, 2H) (where * indicates Compound 2 Hydrochloride Salt nNot determined, obscured by DOH, and * indicates com bined). Crystallization experiments targeting the preparation of ii) PXRD: The PXRD diffractogram of Compound 2 Cit Compound 2 Hydrochloride Salt were conducted in which rate Salt Form 1 is depicted in FIG. 20; the associated PXRD 10 counterion Stoichiometry, the effect of solvent (acetone, Peaks are listed in Table 18 (where * indicates +0.2° and ** acetonitrile, tetrahydrofuran, methanol, isopropyl alcohol, indicates the relative intensity for each peak is determined by water/1% DMSO, ethyl acetate, dichloroethane, MTBE, normalizing its intensity to that of the strongest peak at 18.9° Toluene, MIBK and dioxane) and crystallization methods angle as 100). (temperature cycling, evaporation) were Surveyed. 15 A single non-solvated form of Compound 2 Hydrochloride TABLE 18 Salt was obtained from these experiments. Compound 2 Hydrochloride Salt Form 1 is a non-solvated anhydrous PXRD Peaks of Compound 2, Citrate Salt, Form 1 monohydrochloride solid state form that was obtained from PXRDPeaks of all 24 crystallization experiments, regardless of whether one Compound 2 Citrate Form 1 or two stoichiometric equivalents of hydrochloric acid were Two-Theta Angle Relative Intensity** employed. (degree) (arbitrary) A Compound 2 Hydrochloride salt form screen was con 9.4 37.2 ducted involving about 160 crystallization experiments in 48 9.9 9.2 Solvents and aqueous mixtures was performed conducted as 11.1 12.7 25 previously described (vide supra). 12.4 18.0 18.9 1OOO Two crystal forms were identified and characterized in the 19.9 49.1 context of this study. 24.9 10.1 Compound 2 Hydrochloride Form 1 is the predominant 26.2 76.9 form and was obtained from a variety of crystallization con 28.6 744 30 31.4 9.3 ditions in many solvents and aqueous mixtures, including 48 33.4 4.7 slurry-ripening, 16 evaporative, 3 vapor diffusion and 5 sol 36.2 8.0 vent-antisolvent addition experiments. 39.0 6.7 Compound 2 Hydrochloride Form 2 was isolated from four evaporative crystallization experiments from aqueous mix iii) FTIR: The FTIR Spectrum of Compound 2, Citrate 35 tures. Compound 2 Hydrochloride Form 2 is poorly crystal Salt, Form 1 is depicted in FIG.21; the associated FTIRPeaks line and difficult to scale up. The available data is consistent are listed in Table 19. with a putative hydrate. DSC analysis shows a small endot hermic event between 45 and 75°C. TGA was not performed TABLE 19 due to limited sample size. A competitive ripening experi 40 ment conducted between Compound 2 Hydrochloride Salt FTIR Peaks of Compound 2 Citrate Salt Form 1 Form 1 and Compound 2 Hydrochloride Salt Form 2 demon FTIRPeaks of strated that Compound 2 Hydrochloride Salt Form 1 is more Compound 2 Citrate Form 1 stable than Compound 2 Hydrochloride Salt Form 2 in water Position at 23° C. (cm) Intensity 45 Preparation of Compound 2 Hydrochloride Salt, 730.9 O.18 744.3 O.OS Form 1 796.1 O.O7 844.4 O.12 Compound 2 (500 mg, 1.74 mmol) was added to 15.0 mL 902.5 O.08 50 1110.1 O.14 of methanol. The slurry was stirred and heated to 40° C. 1199.2 O16 Hydrochloric acid (3M in water, 0.586 mL, 1.0 eq.) was 1209.3 O.12 added to the slurry at 40°C. The slurry was seeded with ~2 mg 1222.5 O.09 of Compound 2 HCl salt at 40°C. and stirred for 60 minutes, 1309.5 O.13 resulting in a thick slurry. The slurry was cooled from 40°C. 1341.7 O.08 1372.2 O.O7 55 to 25° C. and then Stirred at 25° C. for 16 hours. The Solids 1448.1 O.13 were isolated by vacuum filtration, rinsed and washed with ~5 1564.O O.19 mL methanol. The product was dried at 50° C. under vacuum 1616.6 O.09 for 3 hours to afford Compound 2 Hydrochloride salt Form 1 1710.8 O.O6 (510 mg, 91% yield) as a white crystalline powder. 60 Compound 2 Hydrochloride Salt Form 1 was characterized iv) DSC/TGA: The DSC/TGA scans Compound 2 Citrate as follows: Salt Form 1 is depicted in FIG. 22. DSC analysis indicates a i) PXRD: The PXRD diffractogram of Compound 2, broad melt onset at 171.4°C. TGA indicates neflible weight Hydrochloride Salt, Form 1 is depicted in FIG. 24; the asso loss below 150° C. ciated PXRD Peaks are listed in Table 20 (where * indicates v) GVS: The GVS curve for Compound 2 Citrate salt Form 65 +0.2 and ** indicates the relative intensity for each peak is 1 is depicted in FIG. 23. The water content remained rela determined by normalizing its intensity to that of the stron tively constant between 0-90% RH, reaching a maximum gest peak at 25.1° angle as 100). US 8,859,550 B2 83 84 TABLE 20 SCHEME 4 PXRD Peaks of Compound 2 Hydrochloride Salt Form 1 NetN PXRDPeaks of 5 (S NSZ\, Compound 2 Hydrochloride Form 1 HCI -- 2 MeOH Two-Theta Angle Relative Intensity** (degree) (arbitrary) 10 A N1 H 6.O 6.3.3 Compound 2 9.5 6.9 NetN 10.8 37.1 15 ( \, 12.0 6.3 S Na ? HCI 12.7 82.4 17.2 5.8 2 17.5 12.3 \ 1. N 18.1 99.3 H 19.1 25.4 Compound 2 Monohydrochloride 2O2 6S.O 25.1 1OO.O 25.4 42.7 25 25.7 22.4 Alternative Preparation of Compound 2. 26.4 8.1 Hydrochloride Salt, Form 1 28.1 11.O 30 ii) FTIR: The FTIR Spectrum of Compound 2, Hydrochlo ride Salt, Form 1 is depicted in FIG.25; the associated FTIR Peaks are listed in Table 21. 35 TABLE 21 FTIR Peaks of Compound 2 Hydrochloride Salt Form 1 FTIRPeaks of Compound 2 Hydrochloride Form 1 40 Position N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8- (cm) Intensity yl)azetidin-3-amine (Compound 2, 5.0 g, 17.40 mmol) was added methanol (150 mL) and the slurry was stirred at room 605.9 O.O8 6321 O.15 45 temperature. Aqueous hydrochloric acid (3M in water, 5.8 675.6 O.10 mL, 1.0 eq.) was added, and the slurry was seeded with ~2 mg 692.0 O.11 of Compound 2, Hydrochloric Salt, Form 1 resulting in a 714.9 0.37 849.7 O.14 thick slurry which was stirred for 3 hours at 25° C. The 898.4 O.11 product was isolated by vacuum filtration, washed with 50 1011.5 O.10 methanol (~15 mL) and dried at 50° C. under vacuum for 3 1075.0 O.13 hours to afford Compound 2, Hydrochloride Salt, Form 1 (4.9 1196.2 O.15 g, 92% yield). 1281.9 O.12 1420.8 O.17 1477.9 O.O8 55 Characterization of Compound 2 Hydrochloride Salt 1492.5 O.11 Form 2 1551.2 O.39 1590.1 O.13 2447.9 O.O8 Compound 2 Hydrochloride Salt Form 2 was characterized 2689.3 O.14 60 as follows: i) PXRD: The PXRD diffractogram of Compound 2, iv) DSC/TGA: The DSC scan Compound2, Hydrochloride Hydrochloride Salt, Form 2 is depicted in FIG. 27: the asso Salt, Form 1 is depicted in FIG. 26. Compound 2 Hydrochlo ciated PXRD Peaks are listed in Table 22 (where * indicates ride Form 1 exhibits no endothermic melting event before the 65 +0.2 and ** indicates the relative intensity for each peak is apparent exothermic decomposition near 250°C. TGA indi determined by normalizing its intensity to that of the stron cates negligible weight loss below 200° C. gest peak at 25.1° angle as 100). US 8,859,550 B2 85 86 TABLE 22 resulting in a thick slurry. The slurry was cooled from 40°C. to 25° C. and then Stirred at 25° C. for 16 hours. The Solids PXRD Peaks of Compound 2 Hydrochloride Salt Form 2 PXRDPeaks of were isolated by vacuum filtration, rinsed and washed with 5 Compound 2 Hydrochloride Form 2 mL acetonitrile. The product was dried at 50° C. under 5 vacuum for 3 hours to afford Compound 2 Mesylate Salt, Two-Theta Angle Relative Intensity** Form 1 (590 mg, 88% yield). (degree) (arbitrary) Compound 2 Mesylate Salt Form 1 was characterized as S.O 1OO follows: 13.2 10.9 i)"H NMR: (500 MHz, DMSO-d) 8:9.21 (s. 2H), 8.29 (s. 28.7 11.2 10 1H), 8.031, 8.03 (d. J=3.0 Hz, 1H), 7.30, 7.29 (ABq, J–4 25.6 7.5 HZ, 1H), 4.69 (brd, J=92.5 Hz, 3.75H), 4.29 (m. 1H), 2.68 (s, 3H), 2.33 (s.3H)* (where * indicates 1H integration confirms ii) FTIR: The FTIR Spectrum of Compound 2 Hydrochlo Compound 2: Mesylate stoichiometry to be 1:1). ride Salt Form 2 is depicted in FIG. 28; the associated FTIR ii) PXRD: The PXRD diffractogram of Compound 2, Peaks are listed in Table 23. 15 Mesylate Salt, Form 1 is depicted in FIG. 29; the associated PXRD Peaks are listed in Table 24 (where * indicates +0.2°, TABLE 23 ** indicates the relative intensity for each peak is determined by normalizing its intensity to that of the strongest peak at FTIR Peaks of Compound 2 Hydrochloride Salt Form 2 FTIRPeaks of 15.0° angle as 100). Compound 2 Hydrochloride Form 2 TABLE 24 Position (cm) Intensity PXRD Peaks of Compound 2 Mesylate Salt Form 1 PXRD Peaks of 841.5 O.044 Compound 2 MeSylate Form 1 (Anhydrous 1076.6 O.O39 25 12014 O.044 Two-Theta Angle Relative Intensity** 1287.6 O.044 (degree) (arbitrary) 1335.3 O.O37 1365.2 O.040 11.6 31.6 1421.9 O.O38 12.1 7.7 1464.1 O.O47 30 1S.O 1OOO 1520.1 O.O37 15.4 6.7 1562.9 O.O85 16.5 1O.S 1598.1 O.O63 17.0 6.6 17.4 9.0 17.7 27.6 35 18.7 14.0 Compound 2 Mesylate Salt 19.6 18.2 21.0 23.0 Crystallization experiments targeting the preparation of 22.3 6.2 Compound 2 Mesylate Salt were conducted in which the 23.3 8.3 24.1 18.2 effect of solvent (acetone, acetonitrile, tetrahydrofuran, 24.3 22.0 40 methanol, isopropyl alcohol, water/1% DMSO, ethyl acetate, 25.2 31.9 dichloromethane, MTBE, Toluene, MIBK and dioxane) and 28.7 14.1 crystallization methods (temperature cycling, evaporation) 3O.S 7.1 were surveyed. Two crystal forms of Compound 2 Mesylate salt were iii) FTIR: The FTIR Spectrum of Compound 2, Mesylate obtained from these experiments. Compound 2 Mesylate 45 Salt, Form 1 is depicted in FIG.30; the associated FTIR Peaks Salt, Form 1 is a non-Solvated, monomesylate Solid State form are listed in Table 25. that was obtained from nine of twelve crystallization experi ments. Compound 2 Mesylate Salt Form 1 exhibits good physical properties and high aqueous solubility (>100 TABLE 25 mg/ml). Compound 2 Mesylate Salt Form 2 is a hydrate that 50 FTIR Peaks of Compound 2 Mesylate Salt Form 1 was obtained by slow evaporation of gummy Solids at ambi FTIRPeaks of ent temperature in two of twelve screening experiments. Sev Compound 2 MeSylate Form 1 (Anhydrous eral attempts to reproduce Compound 2 Mesylate Salt Form 2 Position were unsuccessful and produced Compound 2 Mesylate Salt, (cm) Intensity Form 1 instead. 55 733.1 O.34 A competitive ripening experiment was conducted 781.0 O.22 between Compound 2 Mesylate Salt Form 1 and Compound 840.8 O.13 2 Mesylate Salt, Form 2 indicated that Form 1 is more stable 10O8.1 O16 than Form 2 in acetone/10% water at 23°C. 1038.4 O.43 1079.7 O.15 60 1139.9 O.26 Preparation of Compound 2 Mesylate Salt, Form 1 1199.0 O.30 1330.2 O.11 1461.2 O.17 Compound 2 (500 mg, 1.74 mmol) was added to 15 mL of 1518.1 O.12 acetonitrile. The slurry was stirred and heated to 40°C. Mesic 1556.6 O.33 acid (3M in water, 0.586 mL, 1.0 eq.) was added to the slurry 65 1603.5 O.17 at 40°C. The solution was seeded with ~2 mg of Compound 2 Mesylate Salt, Form 1 at 40° C. and stirred for 60 minutes US 8,859,550 B2 87 88 iv) DSC/TGA: The DSC/TGA scans Compound 2 Mesy In Vitro Biological Activity late Salt Form 1 is depicted in FIG. 31. The DSC curve showed no evidence for melting before decomposition at The activity of the compounds in Examples 1-2 as HR approximately 230°C. TGA showed a loss of 0.3% between inhibitors is illustrated in the following assays. Similar com 25° C. and 1759 C. pounds have activity in these assays as well. V) GVS: The GVS isotherm plot for Compound 2, Mesy In vitro Histamine Receptor Cell-Based Assays late Salt Form 1 is depicted in FIG. 32. Water content The cell-based assays utilize an aequorin dependent biolu remained relatively constant between 0-50% RH. Above 50% minescence signal. Doubly-transfected, stable CHO-K1 cell RH there was a small increase in weight reaching 1.6% at lines expressing human H, or H. mitochondrion-targeted 10 aequorin, and (H only) human G protein GC 16 are obtained 90% RH. FTIR analysis of the sample before and after GVS from Perkin-Elmer. Cells are maintained in F12 (Hams) confirmed that there was no change in crystal form. growth medium, containing 10% (vol./vol.) fetal bovine serum, penicillin (100 IU/mL), streptomycin (0.1 mg/mL). Equilibrium Solubility Studies of Compound 2 Zeocin (0.25 mg/mL) and geneticin (0.40 mg/mL). Cell media Citrate Form 1. Compound 2 Hydrochloride Form 1 15 components are from Invitrogen, Inc. One day prior to assay, and Compound 2 Mesylate Form 1 the growth medium is replaced with the same, excluding Zeocin and geneticin. In some assays, cells previously frozen Equilibrium solubility experiments were conducted for at “ready to use density’ are thawed and immediately avail Compound 2 Monohydrochloride Form 1, Compound 2 able for loading with coelenterazine-h dye as described Monomesylate Form 1 and Compound 2 Monocitrate Form 1 below. salts in water and pH-5 acetate buffer solution. The results of For assay preparation, growth medium is aspirated, and the equilibrium solubility study are summarized in Table 26 cells are rinsed with calcium-free, magnesium-free phos (where 'a' indicates FTIR and PXRD spectra of both samples phate-buffered saline, followed by two to three minute incu were distinctly different from the known forms of Compound bation in Versene (Invitrogen, Inc.) at 37°C. Assay medium 2 or Compound 2 Monocitrate salt Form 1). 25 (DMEM:F12 50:50, phenol-red free, containing 1 mg/mL protease-free bovine serum albumin) is added to collect the released cells, which are then centrifuged. The cell pellet is TABLE 26 re-suspended in assay medium, centrifuged once more, and Equilibrium Solubility of Compound 2 Citrate Form 1, Compound 2 re-suspended in assay medium to a final density of 5x10' Hydrochloride Form 1 and Compound 2 MeSylate Form 1 30 cells/mL. Coelenterazine-h dye (500 uM in ethanol) is added to a final concentration of 5uM, and mixed immediately. The Filtrate conical tube containing the cells is then wrapped with foil to Salt Sol- Resid- Concen protect the light-sensitive dye. The cells are incubated for four Weight went ual Final tration hours further at room temperature (approximately 21° C.) Salt (mg) Solvent (mL) Solid pH (mg/mL) 35 with end-over-end rotation to keep them in Suspension. Citrate 25.5 Water 1.O New 3.41 3.4 Just before assay, the dye-loaded cells are diluted to 1.5x Form 1 Form 10° cells/mL (H receptor) or 0.75x10 cells/mL (H recep Citrate 25.6 O.2M 1.O New 4.41 1.5 Form 1 Acetate Form tor) with additional assay medium. Cells are dispensed to Buffer 1536 well micro-titer plates at 3 ul ?well. To assay receptor (pH5) 40 antagonism 60 ml of 100x concentration test compounds in HCI SO.2 Water O.S HC Salt 4.93 18.1 100% dimethylsulfoxide (DMSO) are dispensed to the wells, Form 1 Form 1 HCI S.O.3 O.2M O.S HC Salt 4.81 21.6 one compound per well in concentration response array by Form 1 Acetate Form 1 passive pin transfer, and the plates are incubated for 15 min Buffer utes at room temperature. Assay plates are then transferred to (pH5) 45 a Lumilux bioluminescence plate reader (Perkin-Elmer) Mesylate SO.1 Water O.S None 4.68 >100 Form 1 equipped with an automated 1536 disposable tip pipette. The Mesylate SO.4 O.2M pipette dispenses 3 LL/well of agonist (histamine, at twice the Form 1 Acetate final concentration, where final concentration is a previously Buffer O.S None 4.76 >100 (pH5) determined ECs) in assay medium, with concurrent biolu 50 minescence detection. Potential agonist activity of test com pounds is measured by separate assays that measure response Compound 2 Citrate salt (New Form) had solubility values to test compounds alone, without added histamine agonist. of 3.4 and 1.5 mg/mL in water and in pH 5 buffer, respec CCD image capture on the Lumilux includes a 5 second tively. FTIR spectra of the residual solids from these two baseline read and generally a 40 second read per plate after experiments were each unique, and were different from the 55 agonist (or test compound only in agonist mode assay) addi known forms of Compound 2 or Compound 2 Citrate salt. tion. A decrease in bioluminescence signal (measured either as area-under-the-curve, or maximum signal amplitude Compound 2 Monohydrochloride salt Form 1 had solubil minus minimum signal amplitude) correlates with receptor ity values of 18.1 and 21.6 mg/mL in water and 0.2M acetate antagonism in a dose dependent manner. The negative control buffer (pH5). The FTIR spectra of the residual solids from 60 is DMSO lacking any test compound. For antagonist assays, these two experiments matched the reference spectra for the positive controls are JNJ7777120 (1-(5-Chloro-1H-in Compound 2 Hydrochloride Salt Form 1. dol-2-yl)carbonyl-4-methyl-piperazine, 10M final concen In the case of Compound 2 Mesylate salt Form 1 no tration, H receptor) and diphenhydramine (2-Diphenyl residual Solids were isolated; the sample completely dis methoxy-N,N-dimethylethylamine, 10 uM final solved. The equilibrium solubility of Compound 2 Mesylate 65 concentration, H receptor). For agonist assays, the positive salt Form 1 is >100 mg/mL in water and in 0.2M acetate control is histamine (10 uM final concentration). Efficacy is buffer (pH5). measured as a percentage of positive control activity. US 8,859,550 B2 89 90 TABLE 27 tilator (Flexivent; Scireq, Montreal, Quebec, Canada). After airflow impedance measurement, the study was terminated Biological Activity and animals were euthanized. Exam- H4 Antagonist EC50, H4 Agonist EC50, It is expected that many of these compounds when tested ple "+" indicates s10 IM, "NA" indicates no activity to 100 M 5 No. "-" indicates >10 M "NT" indicates not tested will be active and will have utility similar to those that have been tested. In Table 28 below, entries with a "+" are active 1 -- NA 2 -- NA and statistically significant compared to positive control (based on standard error of the mean). Entries with a "- are 10 either weakly active, or inactive (statistically indistinguish In Vivo Activity able from positive control). Assessment of H. Antagonism—Model of Allergic Rhinits in TABLE 28 Balb/C Mice. 15 In Vivo Activity Animals. WBP, Day 4 Female BALB/c mice, 6-12 weeks of age, were obtained +: increase in RF over positive control from Jackson Laboratories (Bar Harbor, Me...). All experimen Example # Dosage -: no increase in RF over positive control tal animals used in this work were under a protocol approved 1 0.1%, wiv -- by the Institutional Animal Care and Use Committee of the 2 0.1%, wiv -- National Jewish Medical and Research Center, Denver, Colo. Induction and Measurement of Allergic Rhinitis. From the foregoing description, one skilled in the art can The assay protocol is similar to that described in Miyahara, easily ascertain the essential characteristics of this invention, S. etal. (2005),JAllergy Clin Immunol., 116:1020-1027. The 25 and without departing from the spirit and scope thereof, can role of the H4 receptor in this model has been validated make various changes and modifications of the invention to Shiraishi, Y. et al. (2009), JAllergy Clin Immunol., 123:556). adapt it to various usages and conditions. Briefly, mice received intraperitoneal injections of 20 ug What is claimed is: ovalbumin (OVA, Grade V: Sigma-Aldrich, St. Louis, Mo.), 30 previously emulsified in 2.25 mg of alum (AlumImuject; 1. A compound which is a salt, hydrate, or polymorph of Pierce, Rockford, Ill.) in a total volume of 100 uI (sensitiza structural Formula (XII) tion phase). Injections occurred on days 0 and 14. Starting on day 28 onward (challenge phase), mice received daily intra 35 (XII) nasal instillation of OVA (25 mg/ml in phosphate-buffered eN saline), 15ul in each nostril without anesthesia. Instillations ( \, occurred for 6 days to evoke allergic nasal inflammation and S Nna congestion. Compounds were tested for the ability to prevent induction of nasal inflammation and congestion by intranasal 40 instillation 2.5 hours prior to OVA instillation. Instillations of compounds were performed using 10 ul (0.1% weight/vol oYoz. ume 1 mg/ml) in each nostril without anesthesia, in formu lation vehicle: either (a) unbuffered saline, pH approxi 45 wherein: mately 6.0, 0.2% volume/volume Tween-80 (Sigma X is chosen from CH and N: Aldrich, St. Louis, Mo.), or (b) 50 mM sodium acetate pH m and n are each an integer chosen from 1 and 2, and 5.0, 100 mM sodium chloride, 0.2% volume/volume Tween mn; 80. On day 4 (early phase) and day 7 (late phase) after starting R’ is chosen from lower amino, lower alkylamino, and OVA challenges, respiratory frequency (RF) was measured in 50 lower alkyl; conscious animals by single chamber restrained whole-body Y is chosen from a counterion and null; and plethysmography (WBP) Buxco Research Systems, Troy, Z is a chosen from a hydrate and null. N.Y.). Because mice are obligate nasal breathers, OVA 2. The compound as recited in claim 1, wherein: induced nasal inflammation and congestion results in 55 X is N: decreased breathing frequency. Compounds that block OVA m and n are each 2; and induced nasal inflammation and congestion prevent the R’ is lower alkyl. decrease in RF compared to positive control (instillation with 3. The compound as recited in claim 2, wherein the com formulation vehicle only prior to OVA challenge). The assay pound is a salt, hydrate, or polymorph of 8-(4-Methylpiper negative control measures baseline RF, where challenge is 60 azin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-alpyrazine. performed with phosphate-buffered saline lacking OVA. 4. The compound as recited in claim 1, wherein: After whole-body plethysmography on day 7, nasal airflow X is CH: impedance was measured as described (R, see Methods m and n are each 1; and section for Miyahara S. et al. above in the online supple 65 R’ is chosen from lower amino and lower alkylamino. mental material at the Journal of Allergy and Clinical Immu 5. The compound as recited in claim 4, wherein R is nology: www.jacionline.org), using a custom-designed ven lower alkylamino. US 8,859,550 B2 91 92 6. The compound as recited in claim 5, wherein the com pound is a salt, hydrate, or polymorph of N-methyl-1-(5- (XIV) (thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- eN amine. (S Nna\, 7. The compound as recited in claim 1, wherein: Y is a counterion, if the compound of Formula XII is a salt, 2 O Y is chosen from a counterion and null, if the compound of N TV 10 N1 Formula XII is a polymorph. H oYoz. 8. The compound as recited in claim 7, wherein Y is a counterion and the compound is a salt. 9. The salt as recited in claim 8, or a polymorph or hydrate or a polymorph or hydrate thereof, wherein: thereof, wherein Y is a counterion chosen from acetate, cit 15 Y is a counterion chosen from citrate, hydrochloride, rate, Sulfate, phosphate, hydrochloride, aspartate, mesylate, mesylate; and malate, tartrate, Stearate, and Succinate. 10. The salt as recited in claim 9, or a polymorph or hydrate Z is a chosen from a monohydrate, a hemihydrate, and thereof, wherein the counterion is chosen from tartrate, mesy null. late, citrate, and hydrochloride. 22. The salt as recited in claim 21, which is N-methyl-1- 11. The salt as recited in claim 10, wherein Zis chosen from (5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- a stoichiometric hydrate and a nonstoichiometric hydrate, or amine citrate, or a hydrate or polymorph thereof. a polymorph thereof. 23. The polymorph of the salt as recited in claim 22, which 12. The salt as recited in claim 10, wherein Zis chosen from is N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8- a monohydrate and a hemihydrate. 25 yl)azetidin-3-amine citrate Form 1. 13. The salt as recited in claim 10, having structural For 24. The salt as recited in claim 21, which is N-methyl-1- mula (XIII) (5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- amine hydrochloride, or a hydrate or polymorph thereof. 25. The polymorph of the salt as recited in claim 24, which (XIII) 30 is N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8- 7 - yl)azetidin-3-amine hydrochloride Form 1. S Naa 26. The salt as recited in claim 21, which is, N-methyl-1- (5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl)azetidin-3- 35 amine mesylate, or a hydrate or polymorph thereof. 27. The polymorph of the salt as recited in claim 26, which is N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8- yl)azetidin-3-amine mesylate Form 1.

40 28. The compound as recited in claim 1 which forms a or a polymorph or hydrate thereof, wherein: crystalline solid which has a solubility, in aqueous media at Y is a counterion chosen from tartrate and mesylate; and about pH 3.5 to about pH 5.0, of at least 2.5 mg/mL. Z is a chosen from a monohydrate, a hemihydrate, and 29. The compound as recited in claim 28, which has a null. solubility, in aqueous media at about pH 3.5 to about pH 5.0, 14. The salt as recited in claim 13, which is 8-(4-methylpip 45 of at least 5.0 mg/mL. erazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-apyrazine 30. The compound as recited in claim 29, which has a mesylate, or a hydrate or polymorph thereof. solubility, in aqueous media at about pH 3.5 to about pH 5.0, 15. The hydrate of the salt as recited in claim 14, which is of at least 10.0 mg/mL. 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5- apyrazine mesylate monohydrate, or a polymorph thereof. 50 31. The compound as recited in claim 30, which has a 16. The polymorph of the hydrate as recited in claim 15, solubility, in aqueous media at about pH 3.5 to about pH 5.0, which is 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetra of at least 20.0 mg/mL. Zolo 1.5-alpyrazine mesylate monohydrate Form 1. 32. The compound as recited in claim 31, which has a 17. The polymorph as recited in claim 13, which is 8-(4- solubility, in aqueous media at about pH 3.5 to about pH 5.0, methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo 1.5-a 55 of at least 30.0 mg/mL. pyrazine mesylate Form 3. 33. The compound as recited in claim 1, wherein the com 18. The salt as recited in claim 13, which is 8-(4-methylpip pound is a polymorph and Y is null. erazin-1-yl)-5-(thiophen-2-yl)tetraZolo 1.5-alpyrazine tar 34. The polymorph as recited in claim 33, which is a trate, or a hydrate or polymorph thereof. polymorph of 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl) 19. The polymorph of the salt as recited in claim 18, which 60 is 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo.1, tetrazolo 1.5-alpyrazine. 5-alpyrazine tartrate hemihydrate (Form 1). 35. The polymorph as recited in claim 33, which is a 20. The polymorph of the salt as recited in claim 18, which polymorph of N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a is 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo.1, pyrazin-8-yl)aZetidin-3-amine. 5-alpyrazine tartrate monohydrate (Form 2). 65 36. The compound 8-(4-Methylpiperazin-1-yl)-5- 21. The salt has recited in claim 10, having structural (thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Hemihy Formula (XIV) drate, having the structural formula (XV) US 8,859,550 B2 93 94 40. The compound 8-(4-Methylpiperazin-1-yl)-5- (XV) (thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate Monohy eN drate, having the structural formula (XVII) (S Na Z\, (XVII) 2 N r ( eN\, N-N 10 S NaNZ ON^ O Ho OH O /N

HO 0.5 H2O 15 OH O OH in crystalline form. in a crystalline form. 41. The crystalline form of the compound as recited in 37. The crystalline form of the compound as recited in claim 40, characterized in that said form has at least one of the claim 36, characterized in that said form has at least one of the following characteristics: following characteristics: a) an X-ray powder diffraction pattern with peaks at 5.3, a) an X-ray powder diffraction pattern with peaks at 10.2, 10.5, 15.1, 20.4, 21.2, 21.8 and 23.0 degrees two theta 13.4, 17.4, 18.1, 23.5 and 26.0 degrees two theta (+0.2 25 degree) (CuKO. J.-1.54059 A), or an X-ray powder dif (+0.2 degree) (CuKO. = 1.54059A), or an X-ray powder fraction pattern Substantially in accordance with that diffraction pattern substantially in accordance with that shown in FIG. 1; and shown in FIG.9; b) an FT-Raman spectrum with the bands at 1339, 1356, b) an FT-Raman spectrum with the bands at 1299, 1313, 1524 and 1589 cm oran FT-Raman spectrum substan 30 1354, 1422, 1494, 1523 and 1590 cm oran FT-Raman tially in accordance with that shown in FIG. 2. spectrum Substantially in accordance with that shown in 38. The compound 8-(4-Methylpiperazin-1-yl)-5- FIG 10. (thiophen-2-yl)tetrazolo 1.5-alpyrazine Tartrate Monohy drate, having the structural formula (XVI) 42. The compound 8-(4-Methylpiperazin-1-yl)-5- 35 (thiophen-2-yl)tetrazolo 1.5-alpyrazine Mesylate, having the structural formula (XVIII) (XVI) NeN ( \, (XVIII) S Nna 40 / - 2 N r / Yo, N- N N 45

OH O

50 in a crystalline form. ". OH HO 43. The crystalline form of the compound as recited in O OH claim 42, characterized in that said form has at least one of the following characteristics: in a crystalline form. 55 a) an X-ray powder diffraction pattern with peaks at 10.4, 39. The crystalline form of the compound as recited in 16.3, 17.2, 17.8, 22.8, 24.0 and 27.8 degrees two theta claim38, characterized in that said form has at least one of the (+0.2 degree) (CuKO. = 1.54059A), or an X-ray powder following characteristics: diffraction pattern substantially in accordance with that a) an X-ray powder diffraction pattern with peaks at 11.4. shown in FIG. 17; and 16.2, 17.1 19.0, 23.8 and 24.1 degrees two theta (+0.2 60 degree) (CuKO. J.-1.54059 A), or an X-ray powder dif b) an FT-Raman spectrum with the bands at 1346, 1423, fraction pattern Substantially in accordance with that 1493, 1524 and 1589 cm or an FT-Raman spectrum shown in FIG. 5; and substantially in accordance with that shown in FIG. 18. b) an FT-Raman spectrum with the bands at 1350, 1422, 65 44. The compound N-methyl-1-(5-(thiophen-2-yl)tetra 1494, 1522 and 1593 cm or an FT-Raman spectrum Zolo 1.5-alpyrazin-8-yl)aZetidin-3-amine Citrate, having the substantially in accordance with that shown in FIG. 6. structural formula (XIX) US 8,859,550 B2 95 96

(XIX) N (XXI) NeN Ne ( - 7 I N O O S N2 5 S Na Z N^ /N 2 2 OH

N VA N NAs 1. 1 10 N O OH O O in a crystalline form. HO OH 49. The crystalline form of the compound as recited in OH is claim 48, characterized in that said form has at least one of the following characteristics: in a crystalline form. a) an X-ray powder diffraction pattern with peaks at 11.6, 15.0, 17.7, 21.0, 24.3 and 25.2 degrees two theta (+0.2 45. The crystalline form of the compound as recited in degree) (CuKO. J.-1.54059 A), or an X-ray powder dif claim 44, characterized in that said form has at least one of the fraction pattern Substantially in accordance with that following characteristics: shown in FIG. 29; and b) an FTIR spectrum with the bands at 733, 781, 1038, a) an X-ray powder diffraction pattern with peaks at 9.4, 1140, 1199 and 1557 cm or an FTIR spectrum sub 12.4, 18.9, 19.9, 26.2 and 28.6 degrees two theta (+0.2 stantially in accordance with that shown in FIG. 30. degree) (CuKO. J.-1.54059 A), or an X-ray powder dif 25 50. A pharmaceutical composition comprising a com fraction pattern Substantially in accordance with that pound as recited in claim 1, together with a pharmaceutically shown in FIG. 20; and acceptable carrier. 51. The pharmaceutical composition as recited in claim 50, b) an FTIR spectrum with the bands at 731, 844, 1110, wherein the compound is chosen from a salt, polymorph or 1199, 1209, 1310, 1448 and 1564 cm or an FTIR 30 hydrate of 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tet spectrum Substantially in accordance with that shown in razolo 1.5-alpyrazine and a salt, polymorph or hydrate of FIG. 21. N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8-yl) 46. The compound N-methyl-1-(5-(thiophen-2-yl)tetra aZetidin-3-amine. Zolo 1.5-alpyrazin-8-yl)azetidin-3-amine Hydrochloride, 52. The pharmaceutical composition as recited in claim 51, 35 wherein the compound is a salt and Y is chosen from tartrate, having the structural formula (XX) mesylate, citrate, and hydrochloride. 53. The pharmaceutical composition as recited in claim 52, comprising a salt, or a polymorph thereof, chosen from N (XX) 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo1, Ne 40 5-alpyrazine tartrate hemihydrate; ( \, 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo1, S Naa 5-alpyrazine tartrate monohydrate; HCI 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl)tetrazolo1, 2 5-alpyrazine mesylate monohydrate; N Tys 45 N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8- N1 yl)aZetidin-3-amine citrate; H N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8- yl)azetidin-3-amine hydrochloride; and N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-alpyrazin-8- in a crystalline form. 50 yl)aZetidin-3-amine mesylate. 47. The crystalline form of the compound as recited in 54. The pharmaceutical composition as recited in claim 53, comprising 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl) claim 46, characterized in that said form has at least one of the tetrazolo 1.5-alpyrazine mesylate monohydrate or a poly following characteristics: morph thereof. a) an X-ray powder diffraction pattern with peaks at 6.0, 55 55. The pharmaceutical composition as recited in claim 54, 10.8, 12.7, 18.1, 20.2, 25.1 and 25.4 degrees two theta comprising 8-(4-methylpiperazin-1-yl)-5-(thiophen-2-yl) (+0.2 degree) (CuKO. = 1.54059A), or an X-ray powder tetrazolo 1.5-alpyrazine mesylate monohydrate Form 1. 56. The pharmaceutical composition as recited in claim 53, diffraction pattern substantially in accordance with that comprising N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a shown in FIG. 24; and 60 pyrazin-8-yl)azetidin-3-amine hydrochloride. b) an FTIR spectrum with the bands at 632,715,850, 1196, 57. The pharmaceutical composition as recited in claim 56, 1421, 1551 and 2689 cm or an FTIR spectrum sub comprising N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a stantially in accordance with that shown in FIG. 25. pyrazin-8-yl)azetidin-3-amine hydrochloride Form 1. 58. The pharmaceutical composition as recited in claim 53, 48. The compound N-methyl-1-(5-(thiophen-2-yl)tetra comprising N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a Zolo 1.5-alpyrazin-8-yl)aZetidin-3-amine Mesylate, having pyrazin-8-yl)aZetidin-3-amine mesylate or a polymorph the structural formula (XXI) thereof. US 8,859,550 B2 97 98 59. The pharmaceutical composition as recited in claim 58, comprising N-methyl-1-(5-(thiophen-2-yl)tetrazolo 1.5-a pyrazin-8-yl)aZetidin-3-amine mesylate Form 1. k k k k k