Crystalline forms of plasma kallikrein inhibitors

Abstract

The present invention discloses a pure crystalline form and an amorphous form of 1-benzyl-N-(4-carbamimidylbenzyl)-1H-pyrazole-4-carboxamide acetate, a pharmaceutical composition thereof and a preparation method thereof. #imgabs0#

Description

Technical Field

[0001] The present invention relates to crystalline forms of plasma kallikrein inhibitors, methods of preparing polymorphs of plasma kallikrein inhibitors substantially free of other polymorphs, and compositions comprising substantially pure polymorphs of plasma kallikrein inhibitors. Background Art

[0002] Thrombosis is essential to prevent blood loss and repair damaged blood vessels, a process known as hemostasis, but thrombosis can also be pathological when it occludes a vessel and deprives tissue of oxygen. Thrombotic occlusion of an artery, i.e., arterial thrombosis, most often occurs at sites of rupture or erosion of atherosclerotic plaques (V. Kou et al., Mt Sinai J Med (2006) Vol. 73: pp. 449-468). Specific occlusion of a coronary artery causes acute coronary syndromes, which include unstable angina and myocardial infarction (MI).

[0003] Fibrin clots in the blood can be generated by initiating either of two different pathways, the intrinsic pathway and the extrinsic pathway, which converge into a common coagulation pathway (RG Macfarlane, Nature (1964) Vol. 202: pp. 498-499; EU Davie et al., Science (1964) Vol. 145: pp. 1310-1312; K. Joseph et al., Adv Immunol (2005) Vol. 86: pp. 159-208). Experimental data indicate that although both individuals deficient in plasma kallikrein (PK) and individuals deficient in coagulation factor XII (FXII) lack a bleeding phenotype, their intrinsic pathway-mediated clot formation is severely impaired (OD Ratnoff et al., J Clin Invest (1955) Vol. 34: 602-613; RW Colman (2001) Hemostasis and Thrombosis: Basic Principles and Clinical Practice (RW Colman et al., eds., Lippincott Williams & Wilkins Publishers, Philadelphia, Pennsylvania, pp. 103-122; ED Rosen et al., Nature (1997) Vol. 390: 290-294; WE Hathaway et al., Blood (1965) Vol. 26: 521-532; AS Lawrie et al., Clin Lab Haematol (1998) Vol. 20: pp. 179-186; and SM Bates et al., Circulation (2005) Vol. 112: pp. 53-60). In the intrinsic pathway, a small amount of FXII is activated (FXIIa) by binding to the surface, and FXIIa in turn activates PK by proteolysis. Importantly, PK then produces additional FXIIa in a feedback loop, and FXIIa in turn activates factor XI (FXI) to FXIa and connects to the common pathway. Although the intrinsic pathway is initially activated by a small amount of FXIIa activating a small amount of PK, the extent of subsequent intrinsic pathway activation is controlled by the feedback activation of FXII by PK, thereby controlling downstream coagulation (WE Hathaway et al., Blood (1965) Vol. 26: pp. 521-532).

[0004] At present, the treatment of acute MI or ischemic stroke in hospital setting requires emergency measures to dissolve the occluded thrombus and allow reperfusion (restoration of blood flow). A common way of doing this is to treat patients with fibrinolytic agents, such as tissue plasminogen activator (t-PA) or streptokinase, which cause active plasmin to be produced from plasminogen. Plasmin cleaves the fibrin network of thrombus, dissolving the clot. Such fibrinolytic agents are the most commonly used reperfusion therapy worldwide. However, fibrinolysis is also associated with a high degree of rethrombosis, with subsequent reocclusion rates as high as 50% according to the following studies (F. Zijlstra et al., N Engl J Med (1993) Vol. 328: pp. 680-684; BR Brodie et al., Circulation (1994) Vol. 90: pp. 156-162; GW Stone et al., Circulation (1999) Vol. 99: pp. 1548-1554; H. Tamai et al., Am Heart J (2004) Vol. 147: E9; FW Verheugt et al., J Am Coll Cardiol (1996) Vol. 27: pp. 766-773).

[0005] There is clinical evidence that patients who have experienced an acute MI are in a hypercoagulable (procoagulant) state. Paradoxically, this hypercoagulable state is additionally exacerbated in patients who receive fibrinolytic therapy. As measured by thrombin-antithrombin III (TAT) levels, an increase in thrombin generation can be observed in patients receiving such treatment, with thrombin levels as high as 2-fold higher than the already high levels observed in patients receiving heparin alone (HM Hoffmeister et al., Circulation (1998) Vol. 98: pp. 2527-2533). It has been proposed that the increase in thrombin is caused by the activation of the intrinsic pathway mediated by plasmin. It is known that the activation of the intrinsic pathway system mediated by plasmin occurs in the blood (GA Ewald et al., Circulation (1995) Vol. 91: pp. 28-36), and it has been proposed that this occurs due to direct activation of FXII by plasmin.

[0006] The hypercoagulable state induced by fibrinolysis not only leads to an increased reocclusion rate, but may also be at least partially responsible for the inability to achieve complete fibrinolysis of the clot, which is a major drawback of fibrinolytic therapy (ECKeeley et al., Lancet (2003) Vol. 361: pp. 13-20). Another problem with fibrinolytic therapy is the 3-fold increase in the risk of intracranial hemorrhage (ICH) that accompanies it (V. Menon et al., Chest (2004) Vol. 126: pp. 549S-575S; Fibrinolytic Therapy Trialists' Collaboration Group, Lancet (1994) Vol. 343: pp. 311-322). Therefore, an adjuvant anticoagulant therapy that does not increase the risk of bleeding but inhibits the formation of new thrombin would be very beneficial.

[0007] Studies have found that treatment of wild-type mice with an irreversible inhibitor of FXII reduces vascular occlusion and ischemic cortical damage, and that inhibition of FXII protects against arterial thrombosis, such as that which occurs during acute MI or during thrombotic stroke (WO2006 / 066878). However, peptide drugs have many disadvantages, including limited use in acute studies due to their short half-life, the need for medical intervention for intravenous administration, and the development of anti-peptide antibodies in patients receiving treatment.

[0008] Plasma kallikrein has also been implicated in the following diseases: diabetic macular edema and retinopathy (A. Clermont et al., Diabetes (2011) Vol. 60: pp. 1590-1598; J.A. Hipps et al., Hypertension (2009) Vol. 53: pp. 175-181); hereditary angioedema with C1 inhibitor deficiency (A. Banerji et al., N Engl J Med (2017) Vol. 376: pp. 717-728; E. et al., N Engl J Med (2018) Vol. 379 (No. 4): pp. 352-362); acute liver injury (M. Li et al., Biochem Biophys Res Commun (2018) Vol. 504 (No. 4): pp. 857-864); inflammation and allergic reactions (L. Bender et al., Front Immunol (2017) Vol. 8: p. 1115); worsening of hemorrhagic transformation and cerebral edema after treatment with recombinant tPA (F. et al., Blood (2017) Vol. 129 (Issue 16): pp. 2280-2290; and chemical sensitization renal injury (H. Wang et al., J Immunotoxicol (2016) Vol. 13 (Issue 4): pp. 567-579).

[0009] The polymorphic behavior of a drug can play a key role in pharmacology. Polymorphs are different solid forms of the same molecule, and can have different physical properties due to the different arrangements of molecules in the crystal lattice. These different properties can affect drug parameters, such as storage stability, compressibility, density, hygroscopicity, dissolution and bioavailability. Usually a polymorph can be converted into another polymorph, and this occurs spontaneously in some cases. When a polymorph can be converted into different polymorphs with different densities or hygroscopicities (which can cause tablet expansion or crushing), storage stability is affected. A polymorphic form of a compound may be more soluble or dissolve at a higher rate than another polymorphic form, thereby improving its bioavailability. If a polymorph is converted into a form with lower bioavailability, the effectiveness of the dosage form may be reduced: if it is converted into a form with higher bioavailability, the toxicity of the drug may exceed the limit. In addition, the physical properties of the form may affect manufacturing: for example, a polymorph may be more likely to form a solvate, or may be difficult to filter and wash out impurities.

[0010] Suitable plasma kallikrein inhibitors have been developed (see, e.g., Sinha et al., WO2008 / 016883; US 8258170). However, the identification of crystalline forms of such compounds and their suitability for solid form formulations has not been reported. Since different crystalline forms may differ in solubility, density, stability, and bioavailability, the identification of such crystalline forms remains an unmet need. Summary of the invention

[0011] Provided herein are pure crystalline forms, amorphous forms, and methods for preparing each crystalline form of 1-benzyl-N-(4-carbamimidoylbenzyl)-1H-pyrazole-4-carboxamide acetate.

[0012] One aspect of the present invention is a crystalline form of 1-benzyl-N-(4-carbamimidobenzyl)-1H-pyrazole-4-carboxamide acetate ("Compound 1"), which may be Form I, Form II, Form III or Form IV, wherein the crystalline form is substantially free of any other polymorphic forms.

[0013] Another aspect of the invention is an amorphous form of Compound 1 ("Form V"), wherein the amorphous form is substantially free of any crystalline form.

[0014] Another aspect of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of a pure solid form of Compound 1 (Form I, Form II, Form III, Form IV or Form V) and a pharmaceutically acceptable carrier.

[0015] Another aspect of the present invention is a method for preparing Form I which is substantially free of any other forms.

[0016] Another aspect of the present invention is a method for preparing Form II which is substantially free of any other forms.

[0017] Another aspect of the present invention is a method for preparing Form III which is substantially free of any other crystalline forms.

[0018] Another aspect of the present invention is a method for preparing Form III which is substantially free of any other Form IV.

[0019] Another aspect of the present invention is a method for preparing Form V, which is substantially free of any crystalline form.

[0020] Another aspect of the invention is a method for treating a disease mediated by plasma kallikrein by administering a pharmaceutical composition of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is a powder X-ray diffraction (XRPD) pattern of Form I of Compound 1 prepared using Cu Ka radiation.

[0022] Figure 2 Shown is a powder X-ray diffraction (XRPD) pattern of Form II of Compound 1 prepared using Cu Ka radiation.

[0023] Figure 3 Shown is a powder X-ray diffraction (XRPD) pattern of Form III of Compound 1 prepared using Cu Ka radiation.

[0024] Figure 4 Shown is a powder X-ray diffraction (XRPD) pattern of Form IV of Compound 1 prepared using Cu Ka radiation.

[0025] Figure 5 Shown is a powder X-ray diffraction (XRPD) pattern of amorphous Form V of Compound 1 prepared using Cu Ka radiation.

[0026] Figure 6 Shown in 11×10 5Powder X-ray diffraction (XRPD) patterns of Form I and Form II before and after compression at a force of 100 N for about 25-28 hours. The first and third traces show the XRPD patterns of Form I and Form II before compression, respectively. Traces 2 and 4 show the XRPD patterns of Form I and Form II after compression, respectively.

[0027] Figure 7 Powder X-ray diffraction (XRPD) patterns of Form I and Form II before and after grinding are shown. The first and third traces show the XRPD patterns of Form I and Form II before grinding, respectively. Traces 2 and 4 show the XRPD patterns of Form I and Form II after grinding, respectively.

[0028] Figure 8 Shown is an overlay of DSC and TGA thermal images of Compound 1, Form I.

[0029] Fig. 9 Shown is an overlay of DSC and TGA thermal images of Compound 1, Form II.

[0030] Fig.10 Shown is an overlay of DSC and TGA thermal images of Compound 1, Form III.

[0031] Fig.11 Shown is an overlay of DSC and TGA thermal images of Compound 1, Form IV.

[0032] Fig.12 Overlay of DSC and TGA thermal images of amorphous Compound 1, Form V is shown. DETAILED DESCRIPTION

[0033] definition

[0034] Unless otherwise stated, the following terms used in the specification and claims have the meanings given below.

[0035] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon radical (i.e., C 1 -C 8 alkyl, alkylamino, alkylthio, alkylene, haloalkyl, etc. For each definition herein (e.g., alkyl, alkoxy, alkylamino, alkylthio, alkylene, haloalkyl), when no prefix is ​​included to indicate the number of main chain carbon atoms in the alkyl portion, the group or portion thereof will have 12 or fewer main chain carbon atoms.

[0036] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0037] The term "polymorphic form" refers to a crystalline or amorphous solid form of a compound. Polymorphic forms differ from each other in the orientation or arrangement of the molecules within the crystal structure. The differences in arrangement lead to different intermolecular forces, which can produce differences in melting point, solubility, hardness, stability, bioavailability, chemical reactivity, etc. Polymorphic forms of the same compound can be distinguished by these properties and by their X-ray diffraction patterns. We have now prepared four crystalline polymorphic forms and an amorphous form of Compound 1.

[0038] The term "amorphous" refers to a solid form with at most limited crystallinity as determined by XRPD. In an amorphous solid, the atoms exist in a disordered structure. In a crystalline material or crystalline region, the atoms have both short-range order and long-range order. In contrast, amorphous materials have only short-range order. The crystallinity of an active substance can be determined, for example, by means of dynamic differential calorimetry or X-ray diffraction methods (including XRPD).

[0039] The term "solid form" refers to any crystalline or amorphous form of 1-benzyl-N-(4-carbamimidobenzyl)-1H-pyrazole-4-carboxamide acetate, and mixtures thereof.

[0040] The terms "compound of formula I" and "Compound 1" refer to 1-benzyl-N-(4-carbamimidoylbenzyl)-1H-pyrazole-4-carboxamide acetate.

[0041] The term "Form I" as used herein refers to a pure anhydrous crystalline form of Compound 1. The XRPD diffraction pattern contains the following characteristic peaks: peaks at 10.0 degrees, 18.1 degrees, 18.6 degrees, 20.1 degrees and 23.9 degrees with a deviation of ±0.5 degrees, ±0.2 degrees or ±0.1 degrees at 2θ, wherein the XRPD is made using Cu Kα radiation. The complete diffraction pattern of Form I is shown in Figure 1 A "pure" crystalline form is substantially free of any other crystalline form.

[0042] The term "Form II" as used herein refers to a pure hydrated crystalline form of Compound 1. The XRPD diffraction pattern contains the following characteristic peaks: peaks at 4.2, 5.6, 8.4, 17.8 and 19.8 degrees with a deviation of ±0.5, ±0.2 or ±0.1 degrees 2θ, wherein the XRPD is made using Cu Kα radiation. The complete diffraction pattern of Form II is shown in FIG. Figure 2 shown.

[0043] The term "Form III" as used herein refers to another pure crystalline form of Compound 1. The XRPD diffraction pattern contains the following characteristic peaks: peaks at 7.7 degrees, 10.7 degrees, 20.1 degrees, 23.7 degrees and 24.3 degrees with a deviation of ±0.5 degrees, ±0.2 degrees or ±0.1 degrees at 2θ, wherein the XRPD is made using Cu Kα radiation. The complete diffraction pattern of Form III is shown in Figure 3 shown.

[0044] The term "Form IV" as used herein refers to another pure crystalline form of Compound 1. The XRPD diffraction pattern contains the following characteristic peaks: peaks at 18.5 degrees, 19.6 degrees and 23.7 degrees with a deviation of ±0.5 degrees, ±0.2 degrees or ±0.1 degrees 2θ, wherein the XRPD is made using Cu Kα radiation. The complete diffraction pattern of Form IV is shown in Figure 4 shown.

[0045] The term "Form V" as used herein refers to a pure amorphous form of Compound 1. The XRPD diffraction pattern contains few or no characteristic peaks. The complete diffraction pattern of Form V is shown in FIG. Figure 5 shown.

[0046] As used herein, the term "substantially free" refers to a solid form of Compound 1 that has at most a limited amount of different polymorphs. In some embodiments, a solid form of Compound 1 is substantially free of other polymorphs if it contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% by weight of other polymorphs. For example, if Form I consists of less than about 10%, less than about 5%, or less than about 1% by weight of Form II, Form III, Form IV, and / or Form V, Form I is substantially free of other polymorphs. The purity of such crystalline forms can be determined using XRPD, differential scanning calorimetry, thermogravimetric analysis and other methods known in the art.

[0047] As used herein, the term "subject" includes animals such as mammals, including but not limited to primates (including humans), cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like.

[0048] The term "effective amount" refers to an amount that provides a therapeutic benefit in the treatment, prevention or control of a pathological process mediated by plasma kallikrein or a significant symptom of a pathological process mediated by plasma kallikrein. The specific amount that is effective can be readily determined by an ordinary physician and can vary according to factors known in the art, such as the type of pathological process, the patient's medical history and age, the stage of the pathological process, etc. The inhibition of plasma kallikrein can be determined using known assays, such as, but not limited to, using an in vitro colorimetric or fluorometric substrate cleavage assay, or by measuring the clotting time of ex vivo whole blood or plasma (see, e.g., US 8258170).

[0049] Overview

[0050] Compound 1 is used as a plasma kallikrein (PK) inhibitor for the prevention and treatment of plasma kallikrein-dependent diseases or conditions, including coagulopathy (such as thrombosis) and other PK-dependent diseases and conditions. For example, these compounds inhibit the formation of thrombin through the intrinsic pathway, and thus reduce the risk of new pathogenic thrombosis (reocclusion), and when given as an adjunct therapy to a fibrinolytic regimen, also improve fibrinolysis-induced reperfusion. Compound 1 can also be used to treat other diseases and conditions mediated by plasma kallikrein, such as but not limited to diabetic macular edema, diabetic retinopathy, hereditary angioedema with C1 inhibitor deficiency, acute liver injury, inflammation and allergic reactions, hemorrhagic transformation and worsening of cerebral edema after treatment with recombinant tissue plasminogen activator (tPA), chemical sensitization renal injury, ischemic stroke, hemorrhagic stroke, hypertension and its vascular complications (including retinopathy and nephropathy), cerebral angioedema, pulmonary hypertension, inflammation, pain, acute myocardial infarction (MI), deep vein thrombosis (DVT), stroke or MI Complications of post-fibrinolytic therapy (e.g., treatment with tissue plasminogen activator, streptokinase), angina pectoris, angioedema, sepsis, arthritis, complications of extracorporeal circulation, capillary leak syndrome, inflammatory bowel disease, diabetes and its vascular complications (including retinopathy, diabetic macular edema, nephropathy and neuropathy), age-related macular degeneration, retinal vein occlusion, cerebral edema, ischemia-reperfusion injury, angiogenesis (e.g., angiogenesis in cancer), asthma, allergic reactions, and cerebrovascular complications of neurological disorders (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, CNS infections, and glioblastoma multiforme).

[0051] Plasma kallikrein (PK) is a serine protease present in plasma as an inactive zymogen precursor plasma kallikrein (prePK), which is activated by FXIIa through proteolysis. In a positive feedback loop, PK activates the zymogen FXII by proteolysis, resulting in the formation of additional FXIIa, further expanding its own activation. FXIIa also activates the zymogen FXI to activate FXIa, thereby starting the endogenous (contact) pathway of blood coagulation, resulting in the generation of thrombin and the cleavage of fibrinogen. Importantly, PK cleaves high molecular weight kininogen (HMWK) to produce bradykinin. Bradykinin can open the tight junctions between vascular endothelial cells by activating its receptors B1 and B2 present on the surface of endothelial cells, thereby causing fluid and plasma proteins to extravasate into the tissue, a situation referred to as increased vascular permeability. The tight junctions of the blood-brain barrier are destroyed, followed by leakage of plasma and proteins into the brain (edema), which is also associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis (MS), as well as CNS infections and brain tumors. For example, peritumoral brain edema leads to a poor prognosis for patients with glioblastoma multiforme (K. Schoenegger et al., Eur J Neurol. (2009) Vol. 16 (No. 7): pp. 874-878). Increased vascular permeability caused by bradykinin formation can lead to excessive fluid accumulation (edema) in many tissues and organs in various diseases, such as angioedema, cystoid macular edema, diabetic macular edema, macular edema after retinal vein occlusion, cerebral vascular edema after stroke or head trauma, and capillary leak syndrome. For example, studies have shown that compound 1, like the BK receptor antagonist hoe-140, reduces retinal vascular permeability in angiotensin II-treated rodents (JA Hipps et al., Hypertension (2009) Vol. 53: pp. 175-181). Studies have also shown that activation of prePK and contact systems, for example in patients treated with contaminated heparin, causes allergic reactions (TK Kishimoto et al., N. Engl. J. Med. (2008) Vol. 358: pp. 2457-2467).

[0052] The importance of BK in angioedema is further illustrated in hereditary angioedema, in which individuals have little or no functional C1 inhibitor, the major endogenous inhibitor of PK. High levels of bradykinin are produced in these individuals, leading to extravasation of fluid and protein from the plasma into the soft tissues, causing life-threatening edema. C1-inhibitor is also known to be involved in the pathogenesis of age-related macular degeneration (S. Ennis et al., Lancet (2008) Vol. 372: pp. 1828-1834) and ischemia-reperfusion injury after organ transplantation or myocardial infarction (D. Inderbitzin et al., Eur. Surg. Res. (2004) Vol. 36: pp. 142-147; G. Horstick et al., Circulation (2001) Vol. 104: pp. 3125-3131). Studies have shown that bradykinin and its receptors are involved in tumor angiogenesis (Y. Ikeda et al., Cancer Res (2004) Vol. 64: pp. 5178-5185), pulmonary hypertension (L. Taraseviciene-Stewart et al., Peptides (2005) Vol. 26: pp. 1292-1300) and asthma (PJ Barnes, "Recent Progress on Kinins" (1992) AAS 38 / III, Birkhauser Verlag, Basel).

[0053] In patients with angioedema, small polypeptide PK inhibitors (DX-88, ecallantide) relieve edema in patients with hereditary angioedema (A. Williams et al., Transfus. Apher. Sci. (2003) Vol. 29: pp. 255-258; L. Schneider et al., J Allergy Clin Immunol (2007) Vol. 120 (No. 2): pp. 416-422; JH Levy et al., Expert Opin. Invest. Drugs (2006) Vol. 15: pp. 1077-1090). Icatibant, a bradykinin B2 receptor antagonist, is also effective in treating hereditary angioedema (K. Bork et al., J. Allergy Clin. Immunol. (2007) Vol. 119: pp. 1497-1503). PK generates bradykinin, so inhibiting PK can inhibit the production of bradykinin.

[0054] In thrombosis caused by fibrinolytic therapy (e.g., tissue plasminogen activator, streptokinase), higher levels of PK are present in patients receiving fibrinolysis (HM Hoffmeister et al., J. Cardiovasc. Pharmacol. (1998) Vol. 31: pp. 764-772). Studies have shown that plasmin-mediated activation of the intrinsic pathway occurs in plasma and blood and is significantly attenuated in plasma from individuals lacking any of the intrinsic pathway components (GA Ewald et al., Circulation (1995) Vol. 91: pp. 28-36). Individuals with acute MI were found to have elevated levels of activated PK and thrombin (HM Hoffmeister et al., Circulation (1998) Vol. 98: pp. 2527-2533).

[0055] Icallantide reduces brain edema, infarct volume and neurological deficits in animal models of ischemic stroke (C. Storini et al., J. Pharm. Exp. Ther. (2006) Vol. 318: pp. 849-854). C1-INH reduces infarct size in a mouse model of middle cerebral artery occlusion (MG De Simoni et al., Am. J. Pathol. (2004) Vol. 164: pp. 1857-1863; N. Akita et al., Neurosurg. (2003) Vol. 52: pp. 395-400). Studies have shown that compound 1 reduces infarct volume and cerebral vascular edema in a rat model of ischemic stroke, and reduces the expansion of intracerebral hemorrhage in a model of hemorrhagic stroke (WO2009 / 0971). B2 receptor antagonists were found to reduce infarct volume, brain swelling and neutrophil accumulation, and have neuroprotective effects in animal models of ischemic stroke (S. Zausinger et al., Acta Neurochir. Suppl. (2003) Vol. 86: pp. 205-207; DB Lumenta et al., Brain Res. (2006) Vol. 1069: pp. 227-234; L. Ding-Zhou et al., Br. J. Pharmacol. (2003) Vol. 139: pp. 1539-1547).

[0056] Studies have found that prePK levels are higher in diabetic patients, especially those with proliferative retinopathy, and correlate with fructosamine levels (B.-B. Gao et al., Nature Med. (2007) Vol. 13: pp. 181-188; K. Kedzierska et al., Archives Med. Res. (2005) Vol. 36: pp. 539-543). PrePK levels have also been found to be elevated in diabetic patients, with the highest levels in patients with sensorimotor neuropathy (M. Christie et al., Thromb. Haemostas. (1984) Vol. 52: pp. 221-223). PrePK levels are elevated in diabetic patients, correlate with elevated blood pressure, are independently associated with albumin excretion rate, and are elevated in diabetic patients with macroalbuminuria, suggesting that prePK may be a marker for progressive renal disease (AA Jaffa et al., Diabetes (2003) Vol. 52: pp. 1215-1221). B1 receptor antagonists have been found to reduce increased vascular permeability in rats with streptozotocin-induced diabetes, as well as leakage of plasma to various organs, including the skin and retina (SR Lawson et al., European Pharmacopoeia (2005) Vol. 514: pp. 69-78; SR Lawson et al., Regul Pept. (2005) Vol. 124: pp. 221-224). B1 receptor antagonists can also prevent hyperglycemia and renal dysfunction in streptozotocin-treated mice (A. Zuccollo et al., Can. J. Physiol. Pharmacol. (1996) Vol. 74: pp. 586-589).

[0057] One aspect of the present invention is a crystalline form of 1-benzyl-N-(4-amidinobenzyl)-1H-pyrazole-4-carboxamide acetate ("Compound 1") that is substantially free of other polymorphic forms of 1-benzyl-N-(4-amidinobenzyl)-1H-pyrazole-4-carboxamide acetate. One embodiment of the present invention is a crystalline form having less than 10% by weight of any other polymorphic form. One embodiment of the present invention is a crystalline form having less than 5% by weight of any other polymorphic form. One embodiment of the present invention is a crystalline form having less than 1% by weight of any other polymorphic form.

[0058] Another aspect of the present invention is an amorphous form of Compound 1 that is substantially free of other polymorphic forms of Compound 1.

[0059] Another aspect of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a solid form of Compound 1 that is substantially free of any other solid form of Compound 1.

[0060] Another aspect of the invention is a method for preparing a solid form of Compound 1 that is substantially free of any other solid form of Compound 1.

[0061] Preparation of compounds

[0062] Compounds of formula I are prepared by the methods described herein:

[0063]

[0064] The process of the present invention has been optimized in terms of yield and purity to a sufficient degree for commercialization. 1-Benzyl-1H-pyrazole-4-carboxylic acid ("Compound 4") is commercially available or can be prepared from commercially available precursor compounds by methods known in the art. For example, 1-benzyl-1H-pyrazole-4-carboxylic acid can be prepared by contacting ethyl 1H-pyrazole-4-carboxylate with an appropriately substituted benzyl halide (e.g., benzyl bromide) in an aprotic solvent in the presence of a strong base and then hydrolyzing the ester. For example, K 2 CO 3 1H-pyrazole-3-carboxylic acid ethyl ester is treated with a solution of 1H-pyrazole-3-carboxylic acid in acetone and then treated with benzyl bromide to obtain compound 1-benzyl-1H-pyrazole-3-carboxylic acid ethyl ester. The ethyl ester is then treated with, for example, a solution of KOH in methanol to obtain the free acid (Compound 4).

[0065] A. Method Step 1:

[0066] (A) 1-Propanephosphonic acid cyclic anhydride can be used and triethylamine (Et 3 N) Compound 4 is coupled with 4-aminomethyl-benzonitrile in an aprotic solvent to produce 1-benzyl-N-(4-cyanobenzyl)-1H-pyrazole-4-carboxamide ("Compound 3").

[0067] In one embodiment of the method, Provided as a 50% ethyl acetate solution. Compound 4 and 4-aminomethyl-benzonitrile can be provided in approximately equimolar amounts, or one of the reactants can be provided in an excess of about 0.2 equivalents to about 5 equivalents relative to the other reactant. In one embodiment of the method, the ratio of compound 4 to 4-aminomethyl-benzonitrile is about 0.2 to about 5, about 0.5 to about 2, about 0.9 to about 1.2, or about 1.0. A range of ratios may also be provided. In an embodiment of the method, The ratio of Et to Compound 4 can be from about 0.5 to about 5, from about 0.8 to about 4, from about 1.0 to about 3, from about 1.2 to about 2.0, and from about 1.2 to about 1.8. Triethylamine can also be provided in a range of ratios to Compound 4. In an embodiment of the method, Et 3 The ratio of N to compound 4 is from about 0.5 to about 10, from about 1 to about 8, from about 2 to about 5, and from about 3 to about 5.

[0068] In some embodiments of the method, an aprotic solvent is used, such as, but not limited to, dichloromethane (DCM), tetrahydrofuran (THF), methyl ethyl ketone (MEK), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), methyl tert-butyl ether (MTBE), and mixtures thereof. In one embodiment of the method, the aprotic solvent is DCM.

[0069] The coupling of compound 4 with 4-(aminomethyl)benzonitrile is typically carried out at a temperature ranging from about 0°C to about 100°C. In some embodiments, the coupling reaction temperature is from about 15°C to about the reflux temperature of the selected aprotic solvent. In embodiments of the method, the reaction temperature is from about 20°C to about 80°C. In some embodiments of the method, the reaction temperature is from about 20°C to about 30°C.

[0070] The above-mentioned coupling reaction is usually carried out in a suitable time period so that the reaction is substantially completed, and the time period can vary with the selected aprotic solvent and the reaction temperature. In some embodiments of the method, the reaction time is about 30 minutes to about 48 hours. In some embodiments of the method, the reaction time is about 1 hour to about 24 hours. In other embodiments of the method, the reaction time is about 4 hours to about 12 hours. In other embodiments of the method, the reaction time is about 6 hours to about 10 hours. In some embodiments of the method, the reaction time is about 8 hours.

[0071] In some embodiments of the method, the reaction is carried out under an inert atmosphere or anhydrous conditions. In some embodiments of the method, the reaction is carried out under nitrogen.

[0072] (B) The product of the coupling reaction, i.e., compound 3, is then purified by extraction. Typically, (i) the aprotic solvent containing compound 3 is combined with water, (ii) mixed thoroughly (e.g., by stirring or shaking), (iii) the organic layer and the aqueous layer are separated, (iv) the aqueous layer is removed, and (v) the organic layer is dried to remove water. These steps may be repeated one, two, three or more times, either together or separately. In addition, the water may also contain salts, such as NaCl, NaHCO 3 Etc. The aqueous layer may also be extracted with an organic solvent, such as DCM, and the organic solvent may be combined with the other organic layers obtained. The organic layer may then be dried with a suitable drying agent such as sodium sulfate.

[0073] In some embodiments of the method, a DCM solution of compound 3 is stirred with water, the layers are separated, and then 10% NaHCO is added. 3 The aqueous solution was stirred, separated, and then saturated NaCl aqueous solution was stirred, separated, and then Na 2 SO 4 dry.

[0074] In some embodiments of the method, the dried organic layer is then concentrated under reduced pressure, dissolved in acetone, washed with water, filtered, dried by suction, and dried in vacuo (or dried under reduced pressure) to obtain purified compound 3. The drying step can be performed at an elevated temperature, for example, at a temperature above about 25°C, above about 30°C, above about 35°C, above about 40°C, above about 45°C, above about 50°C, above about 55°C, and above about 60°C. The drying temperature is generally below the melting temperature of compound 3, and can be below about 150°C, below about 120°C, below about 100°C, below about 90°C, below about 80°C, below about 75°C, below about 70°C, and below about 65°C.

[0075] B. Method Step 2:

[0076] (A) Compound 3 is then reacted with hydroxylamine (NH 2 OH) or a salt thereof in a suitable solvent to obtain a solution of compound 2. In an embodiment of the method, hydroxylamine is hydroxylamine hydrochloride. Hydroxylamine or a hydroxylamine salt is added to the reaction in a ratio of about 10 to about 0.5 to compound 3. In some embodiments of the method, NH 2 The ratio of OH or salt to compound 3 is about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1. In some embodiments of the method, the ratio is at least about 0.5, about 1, about 2, or about 3.

[0077] In some embodiments of the method, the weak base used when converting compound 3 to compound 2 is triethylamine or diisopropylamine. The weak base is generally added to the reaction mixture in a ratio of about 10 to about 0.5 to compound 3. In some embodiments of the method, the ratio of weak base to compound 3 is about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1. In some embodiments of the method, the ratio is at least about 0.5, about 1, about 2, or about 3.

[0078] In some embodiments of this method (conversion of compound 3 to compound 2), the solvent is ethanol, isopropanol, methanol, DCM, EtOAc, or a mixture thereof.

[0079] In some embodiments of the method for converting compound 3 to compound 2, the reaction is carried out at an elevated temperature, for example, at a temperature above about 25° C., above about 30° C., above about 35° C., above about 40° C., above about 45° C., above about 50° C., above about 55° C., above about 60° C., above about 65° C., and above about 70° C. The reaction temperature is generally at or below the reflux temperature of the selected solvent, and may be below about 120° C., below about 100° C., below about 90° C., below about 80° C., below about 75° C., below about 70° C., and below about 65° C.

[0080] In some embodiments of the method that compound 3 is converted into compound 2, the reaction time is generally the time length required for the reaction to be substantially completed, and the time length can vary with the selected specific reactant, aprotic solvent and reaction temperature. In some embodiments of the method, the reaction time is about 30 minutes to about 48 hours. In embodiments of the method, the reaction time is about 1 hour to about 24 hours. In embodiments of the method, the reaction time is about 4 hours to about 12 hours. In embodiments of the method, the reaction time is about 6 hours to about 10 hours. In some embodiments of the method, the reaction time is about 7 hours.

[0081] (B) then (i) concentrate the compound 2 solution, (ii) precipitate the compound by adding water, (iii) filter the solid, (iv) wash and (v) dry to obtain purified compound 2. In some embodiments of the method, (i) concentration is achieved by heating the solution containing compound 2, by reducing the pressure, or both. In some embodiments of the method, the solution is concentrated to a volume of about 20% of the volume of the reaction mixture by heating under reduced pressure. In some embodiments of the method, the solid is dried by suction filtration, reduced pressure drying, high temperature drying, or a combination thereof. In some embodiments of the method, the solid is first dried by suction filtration and then by reduced pressure drying at high temperature to obtain purified compound 2.

[0082] C. Method Step 3:

[0083] (A) Compound 2 is then treated with reducing conditions in a protic solvent at elevated temperature to obtain a crude compound 1. In some embodiments of the method, the reducing conditions include catalytic hydrogenation. In some embodiments of the method, the catalytic hydrogenation uses Raney nickel and hydrogen. In some embodiments of the method, the protic solvent is acetic acid. In some embodiments, the method can provide compound 1 as an acetate.

[0084] In some embodiments of the method for converting Compound 2 to Compound 1, a high reaction temperature of above about 30° C., above about 35° C., above about 40° C., above about 45° C., above about 50° C., above about 55° C., or above about 60° C. is used. The reaction temperature is typically equal to or below the reflux temperature of the solvent and is below about 120° C., below about 100° C., below about 90° C., below about 80° C., below about 75° C., below about 70° C., below about 65° C., below about 60° C., or below about 55° C. In some embodiments of the method, the reaction temperature is from about 50° C. to about 55° C.

[0085] In some embodiments of the method for converting compound 2 to compound 1, the catalytic hydrogenation uses a metal catalyst. In some embodiments, the metal catalyst includes nickel. In other embodiments, the metal catalyst includes Raney nickel.

[0086] The amount of catalyst used when reducing compound 2 to compound 1 can vary with the selected catalyst and other reaction conditions. In some embodiments of the method, the amount of Raney nickel used is (expressed in mol% based on the amount of compound 2) at least about 1 mol%, at least about 5 mol%, at least about 10 mol%, at least about 15 mol%, at least about 20 mol%, at least about 25 mol%, at least about 30 mol%, at least about 35 mol%, at least about 40 mol%, at least about 45 mol%, at least about 50 mol%, or at least about 60 mol%.

[0087] Similarly, the amount of hydrogen used (expressed as kg pressure / cm 3 The amount of hydrogen (catalyst) will also vary with the amount of catalyst selected and other reaction conditions. In some embodiments of the method, the amount of hydrogen is at least about 1 kg / cm 3 , at least about 2kg / cm 3 , at least about 3kg / cm 3 , at least about 4kg / cm 3 , at least about 5kg / cm 3 , at least about 6kg / cm 3 , at least about 7kg / cm 3 , at least about 8kg / cm 3 , at least about 9kg / cm 3 , at least about 10kg / cm 3 , at least about 11kg / cm 3 , at least about 12kg / cm 3 , at least about 15kg / cm 3 , at least about 20kg / cm 3 or at least about 25kg / cm 3In some embodiments, the reaction uses about 20 mol% Raney nickel and about 10 kg / cm 3 H 2 .

[0088] The reaction time is generally the time length required for the reduction to be completed substantially, and this time length can change with selected specific conditions and reaction temperature. In some embodiments, the reaction time is at least about 30 minutes, at least about 1 hour, at least about 4 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours or at least about 24 hours. In other embodiments, the reaction time is less than about 48 hours, less than about 40 hours, less than about 36 hours, less than about 30 hours, less than about 24 hours, less than about 18 hours or less than about 14 hours. In some embodiments, the reaction time is about 12 hours.

[0089] (B) (i) filtering the crude product of the reaction mixture of compound 1, (ii) washing the residue with a first solvent, and then (iii) concentrating the reaction mixture to about 10-20% of the volume of the reaction mixture, and (iv) placing the reaction mixture in a second solvent in which the compound is less soluble, (v) filtering and (vi) drying to obtain a semi-purified product. The first solvent (step ii) can be a lower alkyl alcohol, dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In some embodiments, the first solvent is methanol or ethanol. In some embodiments of the method, the second solvent (step iv) is ethyl acetate.

[0090] The filtration process (step i and step v) may include suction filtration and may also include washing the solid with an additional second solvent. In an embodiment of the method, the drying process (step vi) may include suction filtration drying, reduced pressure drying, high temperature drying or a combination thereof. In other embodiments of the method, the drying process includes suction filtration drying, followed by reduced pressure drying at a temperature of at least 35°C. In some embodiments, the reduced pressure is less than 600mmHg, less than 500mmHg, less than 400mmHg, less than 300mmHg or less than 200mmHg. In some embodiments, the drying temperature is at least about 40°C, at least about 45°C or at least about 50°C. Those skilled in the art will appreciate that the drying temperature is lower than the melting point or decomposition temperature of compound 1. In some embodiments, the drying temperature is less than about 120°C, less than about 110°C, less than about 100°C, less than about 90°C, less than about 80°C, less than about 70°C or less than about 65°C.

[0091] (C) At this point, the semi-purified product (Compound 1) may still contain an unacceptable amount of nickel (or other catalyst metals). To further purify the product, the dried solid is (i) treated with water, (ii) heated and stirred at an elevated temperature to form a slurry, (iii) cooled and (iv) filtered, (v) dried for the first time, (vi) dissolved in a mixture of ethanol and acetic acid, (vii) heated to a holding temperature, (viii) cooled, (ix) filtered, and (x) dried for the second time to obtain a nickel-depleted product. In some embodiments of the method, the elevated temperature of step (ii) is at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C, at least about 55°C, at least about 60°C, or at least about 65°C. In some embodiments of the method, the elevated temperature is less than about 100°C, less than about 90°C, less than about 80°C, less than about 70°C, or less than about 65°C. In some embodiments of the method, the elevated temperature is about 55°C. In some embodiments of the method, the filtration process of step (iv) includes suction filtration and washing with water. In some embodiments of the method, the drying step of step (v) comprises drying by suction, followed by drying under reduced pressure at a temperature of at least 35°C. In some embodiments of the method, the reduced pressure is less than 600 mmHg, less than 500 mmHg, less than 400 mmHg, less than 300 mmHg, or less than 200 mmHg. In some embodiments of the method, the drying temperature of step (v) is at least about 40°C, at least about 45°C, or at least about 50°C. In embodiments of the method, the drying temperature is less than about 120°C, less than about 110°C, less than about 100°C, less than about 90°C, less than about 80°C, less than about 70°C, or less than about 65°C. In some embodiments of the method, the drying temperature of step (v) is about 45°C.

[0092] In step (vi), the ratio of ethanol to acetic acid may be about 1:20 to about 20: 1. In some embodiments of the method, the ratio is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1, v / v ethanol:acetic acid.

[0093] In some embodiments of the method, the holding temperature of step (vii) is at least about 30° C., at least about 35° C., at least about 40° C., at least about 45° C., at least about 50° C., at least about 55° C., at least about 60° C., or at least about 65° C. In some embodiments of the method, the holding temperature is about the reflux temperature of the solvent mixture of ethanol and acetic acid, or a temperature of less than about 80° C., less than about 75° C., less than about 70° C., less than about 65° C., or less than about 60° C. In some embodiments of the method, the holding temperature is about the reflux temperature of the solvent mixture.

[0094] In some embodiments of the method, the mixture is kept at a holding temperature for at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, at least about 120 minutes, at least about 150 minutes, at least about 240 minutes, or at least about a 3-hour time period. In some embodiments of the method, the time period is no more than about 5 hours, no more than about 4 hours, no more than about 3 hours, no more than about 2 hours, no more than about 1 hour, or no more than about 30 minutes. In some embodiments of the method, the time period is about 1 hour.

[0095] The cooling described in step (viii) is usually carried out in a time period of at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour or at least about 2 hours. The final temperature of step viii is less than about 35°C, less than about 30°C, less than about 25°C, less than about 20°C, less than about 15°C, less than about 10°C or less than about 5°C. In some embodiments of the method, the final temperature of step (viii) is about ambient temperature. In some embodiments, step (viii) also includes stirring the mixture.

[0096] The filtration of step (ix) may also include washing with a lower alkyl alcohol. In some embodiments, the filtered solid is washed with ethanol. The drying step of step (x) may include suction drying, reduced pressure drying, high temperature drying or a combination thereof. In an embodiment of the method, the drying method of step (x) includes suction drying, followed by reduced pressure drying at a temperature of at least 35°C. In an embodiment of the method, the reduced pressure is less than 600mmHg, less than 500mmHg, less than 400mmHg, less than 300mmHg or less than 200mmHg. In some embodiments of the method, the drying temperature of step (x) is at least about 40°C, at least about 45°C or at least about 50°C. As described above, the drying temperature used will be lower than the melting point of compound 1 and lower than the decomposition temperature of compound 1. In some embodiments of the method, the drying temperature is less than about 120°C, less than about 110°C, less than about 100°C, less than about 90°C, less than about 80°C, less than about 70°C or less than about 65°C. In some embodiments of the method, the drying temperature of step (x) is about 45°C. In some embodiments of the method, steps (vi)-(x) are repeated 1, 2 or 3 times. In embodiments of the method, steps (vi)-(x) are repeated 1 time.

[0097] Crystal form

[0098] One aspect of the present invention is a crystalline form of 1-benzyl-N-(4-amidinobenzyl)-1H-pyrazole-4-carboxamide acetate ("Compound 1") that is substantially free of other polymorphic forms of 1-benzyl-N-(4-amidinobenzyl)-1H-pyrazole-4-carboxamide acetate. One embodiment of the present invention is a crystalline form having less than 10% by weight of any other polymorphic form. One embodiment of the present invention is a crystalline form having less than 5% by weight of any other polymorphic form. One embodiment of the present invention is a crystalline form having less than 1% by weight of any other polymorphic form.

[0099] A. Crystal Form I:

[0100] (A) The nickel-depleted product of process step 3 is further purified by (i) contacting the compound with a first solvent, (ii) raising the mixture to a first elevated temperature, (iii) adding a second solvent, (iv) cooling the resulting mixture to a crystallization temperature, (v) stirring the mixture, (vi) filtering the solid, and (vii) drying the solid to provide Compound 1 as pure anhydrous crystalline Form I.

[0101] In some embodiments, the first solvent of step (i) is methanol, ethanol, 1-propanol or 2-propanol or a mixture thereof. In some embodiments, the lower alkyl alcohol is methanol.

[0102] In some embodiments, the first elevated temperature of step (ii) is at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C, at least about 55°C, at least about 60°C, or at least about 65°C. The elevated temperature will be no greater than the reflux temperature of the first solvent, or no more than about 80°C, no more than about 75°C, no more than about 70°C, no more than about 60°C, no more than about 55°C, no more than about 50°C, no more than about 45°C, no more than about 40°C, or no more than about 35°C. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, step (ii) further comprises slowly cooling the solution to a second elevated temperature. In some embodiments, the second elevated temperature is about 5°C, about 10°C, about 15°C, or about 20°C lower than the first elevated temperature. The second elevated temperature is about 5° C., about 10° C., about 15° C., about 20° C., or above 20° C. In some embodiments, step (ii) further comprises filtering the solution.

[0103] In some embodiments, the second solvent of step (iii) is MTBE or THF. In some embodiments, the second solvent is MTBE. In some embodiments, the first solvent and the second solvent are anhydrous. In some embodiments, the second solvent is slowly added in the extended time period of at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes, at least about 150 minutes, at least about 180 minutes or at least about 240 minutes. The extended time period is less than about 24 hours, less than about 18 hours, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours or less than about 1 hour. In some embodiments, the extended time period is about 2 hours. The ratio of the first solvent to the second solvent can vary between about 1:20 to about 20:1v / v. In some embodiments, the ratio of MeOH to MTBE is about 5:1, about 4:1, about 3:1, about 2.7:1, about 2.5:1, about 2.3:1, about 2:1, about 1.5:1, about 1.3:1, about 1.2:1, about 1:1, about 1:1.5, about 1:2, about 1:3, or about 1:4.

[0104] In some embodiments, step (iii) further comprises adding seed crystals.

[0105] The crystallization temperature of step (iv) is no more than about 35°C, less than about 30°C, less than about 25°C, less than about 20°C, less than about 15°C, less than about 10°C or less than about 5°C. In some embodiments, the crystallization temperature is about 25°C. It will be appreciated by those skilled in the art that cooling will generally occur over an extended period of time. In some embodiments, the cooling time of step (iv) is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes, at least about 150 minutes or at least about 180 minutes. In some embodiments, the cooling time is about 45 minutes to about 90 minutes.

[0106] In some embodiments of the present invention, step (v) also comprises adding the second solvent of additional amount in the extended time period.In some embodiments, the extended time period of step (v) is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes or at least about 150 minutes.In other embodiments, the extended time period is less than about 24 hours, less than about 18 hours, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours or less than about 1 hour.

[0107] The filtering step of step (vi) may also include washing the solid with an additional amount of the second solvent. The drying step of step (vii) may include suction drying, reduced pressure drying, high temperature drying or a combination thereof. In some embodiments, the drying invention of step (x) includes suction drying, followed by reduced pressure drying at a temperature of at least 35°C. In some embodiments, the reduced pressure is less than 600mmHg, less than 500mmHg, less than 400mmHg, less than 300mmHg or less than 200mmHg. In some embodiments, the drying temperature of step (vii) is at least about 40°C, at least about 45°C or at least about 50°C. As described above, the drying temperature is lower than the melting point of compound 1 and lower than the decomposition temperature of compound 1. In some embodiments, the drying temperature is lower than about 120°C, lower than about 110°C, lower than about 100°C, lower than about 90°C, lower than about 80°C, lower than about 70°C or lower than about 65°C. In some embodiments, the drying temperature of step vii is about 45°C.

[0108] One aspect of the invention is a crystalline form of Compound 1, which is an anhydrous crystalline form and is characterized by having a melting point of about 253°C and an aqueous solubility of about 8.3 mg / mL at 25°C ("Form I"). One embodiment of the invention is a crystalline form of Compound 1, which has an XRPD pattern including peaks at 10.0 degrees, 18.1 degrees, 18.6 degrees, 20.1 degrees, and 23.9 degrees with a deviation of ±0.5 degrees, ±0.2 degrees, or ±0.1 degrees of 2θ, wherein the XRPD pattern is made using Cu Ka radiation. One embodiment of the invention is a crystalline form of Compound 1, wherein the XRPD pattern also includes a peak at 20.5 degrees with a deviation of ±0.5 degrees, ±0.2 degrees, or ±0.1 degrees of 2θ. One embodiment of the invention is a crystalline form of Compound 1, which has a substantially similar Figure 1 XRPD pattern of the spectrum.

[0109] One aspect of the invention is a method for preparing Form I, comprising contacting a solid form of Compound 1 with a solvent selected from methanol, isopropanol, tetrahydrofuran, 2-methyl-tetrahydrofuran, anhydrous acetone, dichloromethane, diethyl ether, 3-methyl-1-butanol and nitromethane or mixtures thereof for a period of time sufficient to produce Form I. One embodiment of the invention is the method, wherein the solvent is methanol.

[0110] B. Crystal form II:

[0111] In the following method, any solid form of compound 1 is converted into Form II. Compound 1 (i) is contacted with a mixture of an organic solvent and water, (ii) heated and maintained for a first period of time, (iii) a second solvent is added, (iv) the mixture is cooled and maintained for a second period of time to crystallize, and (v) the resulting solid is separated to obtain a pure Form II compound 1. In some embodiments, the organic solvent of step (i) is methanol, isopropanol, THF or acetone. In some embodiments, the organic solvent is methanol. In some embodiments, the mixture of organic solvent and water comprises at least about 1% water, at least about 2% water, at least about 3% water, at least about 4% water or at least about 5% water. In some embodiments, the mixture of organic solvent and water comprises at least about 10% water, at least about 15% water, at least about 20% water, at least about 25% water, at least about 30% water, at least about 35% water, at least about 40% water, at least about 45% water or at least about 50% water. In some embodiments, the mixture of organic solvent and water comprises no more than about 45% water, no more than about 40% water, no more than about 35% water, no more than about 30% water, no more than about 25% water, no more than about 20% water, no more than about 15% water, or no more than about 10% water.

[0112] The elevated temperature of step (ii) is at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C, at least about 55°C, at least about 60°C, or at least about 65°C. The elevated temperature will be no greater than the reflux temperature of the solvent mixture, or no more than about 80°C, no more than about 75°C, no more than about 70°C, no more than about 60°C, no more than about 55°C, no more than about 50°C, no more than about 45°C, no more than about 40°C, or no more than about 35°C. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, step (ii) further comprises filtering the solution. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, the time period may be about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 120 minutes. In some embodiments, the time period will be less than about 180 minutes, less than about 120 minutes, less than about 60 minutes, less than about 45 minutes, or less than about 30 minutes.

[0113] The second solvent of step (iii) is MTBE or THF. In some embodiments, the second solvent is MTBE. Step (iii) may optionally include adding seed crystals of Form II before or after the second solvent is added.

[0114] The crystallization temperature of step (iv) is no more than about 35° C., less than about 30° C., less than about 25° C., less than about 20° C., less than about 15° C., less than about 10° C., or less than about 5° C. In some embodiments, the crystallization temperature is about 25° C.

[0115] The cooling of step (iv) usually occurs in the extended time period.In some embodiments, the cooling time of step (iv) is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes, at least about 150 minutes or at least about 180 minutes.In some embodiments, the cooling time is about 45 minutes to about 90 minutes.In some embodiments, the crystallization / holding time period of step (iv) is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes or at least about 150 minutes.In some embodiments, the time period is less than about 24 hours, less than about 18 hours, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours or less than about 1 hour.

[0116] The separation step of step (v) is substantially similar to steps (vi) and (vii) of the Form I process.

[0117] One aspect of the present invention is a crystalline form of Compound 1, which is a hydrated crystalline form and is characterized by having a melting point of about 253°C and an aqueous solubility of about 5.5 mg / mL at 25°C ("Form II"). One embodiment of the present invention is a crystalline form of Compound 1, which has an XRPD pattern including peaks at 2θ of 4.2 degrees, 5.6 degrees, 8.4 degrees, 17.8 degrees, and 19.8 degrees with a deviation of ±0.5 degrees, ±0.2 degrees, or ±0.1 degrees, wherein the XRPD pattern is made using Cu Kα radiation. One embodiment of the present invention is a crystalline form of Compound 1, wherein the XRPD pattern also includes a peak at 2θ of 12.7 degrees with a deviation of ±0.5 degrees, ±0.2 degrees, or ±0.1 degrees. One embodiment of the present invention is a crystalline form of Compound 1, which has a substantially similar Figure 2 XRPD pattern of the spectrum.

[0118] One aspect of the present invention is a method for preparing Form II, the method comprising contacting a solid form of Compound 1 with a mixture of water and a solvent for a period of time sufficient to produce Form II, the solvent being selected from isopropanol, tetrahydrofuran, acetone and ethyl acetate or a combination thereof. In one embodiment, the solvent is ethyl acetate. In another embodiment, the solvent is methanol. In another embodiment, the method further comprises adding MIBK to the solvent.

[0119] C. Crystalline Form III:

[0120] (A) Compound 1 in any solid form is converted to Form III in the following method. Compound 1 is (i) contacted with an organic solvent, (ii) heated and maintained for a first period of time, (iii) a second solvent is added, (iv) the mixture is cooled and maintained for a second period of time to crystallize, and (v) the resulting solid is isolated to obtain Compound 1 in pure Form III.

[0121] In some embodiments, the organic solvent of step (i) is MeOH. In some embodiments, the organic solvent contains less than about 10% water. In some embodiments, the organic solvent contains no more than about 10% water, no more than about 8% water, no more than about 5% water, no more than about 2% water, no more than about 1% water, or no more than about 0.1% water.

[0122] The elevated temperature of step (ii) is at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C, at least about 55°C, at least about 60°C, or at least about 65°C. The elevated temperature will be no greater than the reflux temperature of the solvent mixture, or no more than about 80°C, no more than about 75°C, no more than about 70°C, no more than about 60°C, no more than about 55°C, no more than about 50°C, no more than about 45°C, no more than about 40°C, or no more than about 35°C. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, step (ii) further comprises filtering the solution. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, the time period may be about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 120 minutes. In some embodiments, the time period will be less than about 180 minutes, less than about 120 minutes, less than about 60 minutes, less than about 45 minutes, or less than about 30 minutes.

[0123] In some embodiments, the second solvent of step (iii) is acetonitrile (CH 3 CN). The ratio of acetonitrile to MeOH solution is at least about 1:1, at least about 1:2, at least about 1:3, at least about 1:4, at least about 1:5, at least about 1:7, at least about 1:10, at least about 1:20, at least about 1:30, at least about 1:40 or at least about 1:50. Step (iii) may optionally include adding seed crystals of Form III before or after the addition of the second solvent.

[0124] The crystallization temperature of step (iv) is no more than about 35°C, less than about 30°C, less than about 25°C, less than about 20°C, less than about 15°C, less than about 10°C or less than about 5°C. In some embodiments, the crystallization temperature is about 25°C. Cooling occurs over an extended period of time. In some embodiments, the cooling time of step (iv) is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes, at least about 150 minutes or at least about 180 minutes. In some embodiments, the cooling time is about 45 minutes to about 90 minutes. In some embodiments, the crystallization / holding time period of step iv is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes or at least about 150 minutes. In some embodiments, the time period is less than about 24 hours, less than about 18 hours, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour.

[0125] The separation step of step (v) is substantially similar to steps (vi) and (vii) of the Form I process.

[0126] (B) Form III can also be prepared by other methods. Compound 1 (i) is contacted with an organic solvent, (ii) heated and maintained for a first period of time, (iii) the solvent is quickly removed under reduced pressure, and (iv) the resulting solid is separated to obtain Compound 1 as pure Form III. In some embodiments, the organic solvent of step (i) is MeOH or a mixture of MeOH and DCM. In some embodiments, the organic solvent is MeOH. In some embodiments, the organic solvent is a mixture of MeOH and DCM. In some embodiments, the ratio of MeOH to DCM is about 1: 1. In some embodiments, the organic solvent contains less than about 10% water. In some embodiments, the organic solvent contains no more than about 10% water, no more than about 8% water, no more than about 5% water, no more than about 2% water, no more than about 1% water, or no more than about 0.1% water.

[0127] The elevated temperature of step (ii) is at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C, at least about 55°C, at least about 60°C, or at least about 65°C. The elevated temperature will be no greater than the reflux temperature of the solvent mixture, or no more than about 80°C, no more than about 75°C, no more than about 70°C, no more than about 60°C, no more than about 55°C, no more than about 50°C, no more than about 45°C, no more than about 40°C, or no more than about 35°C. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step ii further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, step (ii) further comprises filtering the solution. In some embodiments, the elevated temperature is about 55°C. In some embodiments, the time period may be about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 120 minutes. In some embodiments, the period of time will be less than about 180 minutes, less than about 120 minutes, less than about 60 minutes, less than about 45 minutes, or less than about 30 minutes.

[0128] The solvent removal of step (iii) is carried out quickly under reduced pressure. The pressure may be less than about 700 mmHg, less than about 600 mmHg, less than about 500 mmHg, less than about 400 mmHg, less than about 350 mmHg, less than about 300 mmHg, less than about 250 mmHg, less than about 200 mmHg, less than about 150 mmHg, less than about 100 mmHg or less than about 50 mmHg. The rapid solvent removal is carried out in less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, less than about 1 minute.

[0129] The separation step of step (iv) is substantially similar to steps (vi) and (vii) of the Form I process.

[0130] One aspect of the present invention is a crystalline form of Compound 1, which is a non-anhydrous crystalline form and is characterized by having a melting point of about 249°C and an aqueous solubility of about 7.2 mg / mL at 25°C ("Form III"). One embodiment of the present invention is a crystalline form of Compound 1, which has an XRPD pattern including peaks at 7.7 degrees, 10.7 degrees, 20.1 degrees, 23.7 degrees and 24.3 degrees with a deviation of ±0.5 degrees, ±0.2 degrees or ±0.1 degrees at 2θ, wherein the XRPD pattern is made using Cu Ka radiation. One embodiment of the present invention is a crystalline form of Compound 1, wherein the XRPD pattern also includes peaks at 18.5 degrees and 26.5 degrees with a deviation of ±0.5 degrees, ±0.2 degrees or ±0.1 degrees at 2θ. One embodiment of the present invention is a crystalline form of Compound 1, which has a substantially similar Figure 3 XRPD pattern of the spectrum.

[0131] One aspect of the invention is a method for preparing Form III, the method comprising contacting any solid form of Compound 1 with a solvent comprising acetonitrile for a period of time sufficient to produce Form III. One embodiment of the invention is the method, wherein the solvent comprises less than about 10% water. One embodiment of the invention is the method, wherein the solvent comprises less than about 5% water. One embodiment of the invention is the method, wherein the solvent comprises less than about 1% water.

[0132] D. Crystalline Form IV:

[0133] (A) Compound 1 in any solid form is converted to Form IV in the following method. Compound 1 is (i) contacted with an organic solvent, (ii) heated and maintained for a first period of time, (iii) a second solvent is added, (iv) the mixture is cooled and maintained for a second period of time to crystallize, and (v) the resulting solid is isolated to obtain Compound 1 as pure Form IV.

[0134] In some embodiments, the organic solvent of step (i) is MeOH. In some embodiments, the organic solvent contains less than about 5% water, no more than about 2%, no more than about 1%, or no more than about 0.1% water.

[0135] The elevated temperature of step (ii) is at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C, at least about 55°C, at least about 60°C, or at least about 65°C. The elevated temperature will be no greater than the reflux temperature of the solvent mixture, or no more than about 80°C, no more than about 75°C, no more than about 70°C, no more than about 60°C, no more than about 55°C, no more than about 50°C, no more than about 45°C, no more than about 40°C, or no more than about 35°C. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, step (ii) further comprises filtering the solution. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, the time period may be about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 120 minutes. In some embodiments, the time period will be less than about 180 minutes, less than about 120 minutes, less than about 60 minutes, less than about 45 minutes, or less than about 30 minutes.

[0136] The second solvent of step (iii) is ethyl acetate (EtOAc), isopropyl acetate, MEK or MIBK. In some embodiments, the second solvent is ethyl acetate (EtOAc), isopropyl acetate, MEK or MIBK. In some embodiments, the second solvent is EtOAc. In some embodiments, the second solvent is MEK. In some embodiments, the second solvent is MIBK.

[0137] The ratio of the second solvent (of step (i)) to the MeOH solution is at least 0.1:1, at least about 0.2:1, at least about 0.3:1, at least about 0.4:1, at least about 0.5:1, at least about 1:1, at least about 1:2, at least about 1:3, at least about 1:4, at least about 1:5, at least about 1:7, at least about 1:8, at least about 1:9, at least about 1:10, at least about 1:20, at least about 1:30, at least about 1:40, or at least about 1:50.

[0138] Step (iii) may optionally include adding seed crystals of Form IV before or after the addition of the second solvent.

[0139] The crystallization temperature of step (iv) is no more than about 35°C, less than about 30°C, less than about 25°C, less than about 20°C, less than about 15°C, less than about 10°C or less than about 5°C. In some embodiments, the crystallization temperature is about 25°C. Cooling occurs over an extended period of time. In some embodiments, the cooling time of step (iv) is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes, at least about 150 minutes or at least about 180 minutes. In some embodiments, the cooling time is about 45 minutes to about 90 minutes. In some embodiments, the crystallization / holding time period of step (iv) is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes or at least about 150 minutes. The time period is less than about 72 hours, less than about 48 hours, less than about 24 hours, less than about 18 hours, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours or less than about 1 hour.

[0140] The separation step of step (v) is substantially similar to steps (vi) and (vii) of the Form I process.

[0141] One aspect of the invention is a crystalline form of Compound 1, characterized in that it has a melting point of about 251°C ("Form IV"). One embodiment of the invention is a crystalline form of Compound 1, having an XRPD pattern including peaks at 18.5 degrees, 19.6 degrees, and 23.7 degrees with a deviation of ±0.5 degrees, ±0.2 degrees, or ±0.1 degrees 2θ, wherein the XRPD pattern is made using Cu Ka radiation. One embodiment of the invention is a crystalline form of Compound 1, wherein the XRPD pattern also includes a peak at 24.6 degrees with a deviation of ±0.5 degrees, ±0.2 degrees, or ±0.1 degrees 2θ. One embodiment of the invention is a crystalline form of Compound 1, having a substantially similar Figure 4 XRPD pattern of the spectrum.

[0142] E. Form V—Amorphous:

[0143] In the following method, any solid form of Compound 1 is converted to Form V. Compound 1 is (i) contacted with an aqueous solvent, (ii) heated and maintained for a first period of time, (iii) the solution is frozen and lyophilized, and (iv) the resulting solid is isolated to provide Compound 1 as pure amorphous Form V.

[0144] In some embodiments, the aqueous solvent of step (i) is water, deionized water or water for injection. In one embodiment of the present invention, the aqueous solvent is water for injection. In one embodiment of the present invention, the aqueous solvent is deionized water.

[0145] The elevated temperature of step (ii) is at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, at least about 50°C, at least about 55°C, at least about 60°C, or at least about 65°C. The elevated temperature will be no greater than the reflux temperature of the solvent mixture, or no more than about 80°C, no more than about 75°C, no more than about 70°C, no more than about 60°C, no more than about 55°C, no more than about 50°C, no more than about 45°C, no more than about 40°C, or no more than about 35°C. In one embodiment of the invention, the elevated temperature is about 45°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, step (ii) further comprises filtering the solution. In some embodiments, the elevated temperature is about 55°C. In some embodiments, step (ii) further comprises maintaining the mixture at or near the elevated temperature until compound 1 is completely dissolved and a clear solution is formed. In some embodiments, the time period may be about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 120 minutes. In some embodiments, the time period will be less than about 180 minutes, less than about 120 minutes, less than about 60 minutes, less than about 45 minutes, or less than about 30 minutes.

[0146] The freeze drying step (iii) occurs over an extended period of time. In some embodiments, the freeze drying time of step (iii) is at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 75 minutes, at least about 90 minutes, at least about 105 minutes, at least about 120 minutes, at least about 150 minutes, at least about 180 minutes, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, or at least about 10 hours. The time period is less than about 24 hours, less than about 18 hours, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour.

[0147] The isolation step of step (iv) generally entails simply collecting the amorphous solid from the lyophilization apparatus.

[0148] One aspect of the invention is an amorphous form of Compound 1 characterized by having a melting point of about 240°C and an aqueous solubility of about 7.1 mg / mL at 25°C ("Form V"). One embodiment of the invention is an amorphous form of Compound 1 having a substantially similar Figure 5 XRPD pattern of the spectrum.

[0149] preparation

[0150] Compound 1 is formulated and administered according to methods known in the art. Pharmaceutical compositions containing active ingredients may be in a form suitable for oral use, for example, tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self-emulsifiers, hard or soft capsules, syrups, elixirs, solutions, oral patches, oral gels, chewing gums, chewing tablets, effervescent powders and effervescent tablets as described in US 2002-0012680. Compositions for oral use may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more agents selected from sweeteners, flavoring agents, coloring agents, antioxidants and preservatives to provide pharmaceutically aesthetic and palatable preparations. Tablets contain active ingredients mixed with nontoxic pharmaceutically acceptable excipients suitable for preparing tablets. These excipients can be, for example, inert diluents, such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants, such as corn starch or alginic acid; binders, such as PVP, cellulose, PEG, starch, gelatin or gum arabic; and lubricants, such as magnesium stearate, stearic acid or talc. Tablets can be uncoated, or can be enterically coated or otherwise coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer sustained effect. For example, sustained-release materials such as monostearate or distearate can be used. Tablets can also be coated by the techniques described in US 4256108, US 4166452 and US 4265874 to form osmotic therapeutic tablets for controlled release.

[0151] Formulations for oral use may also be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil. In addition, emulsions may be prepared from water-immiscible ingredients such as oils and stabilized by surfactants such as mono- and di-glycerides, PEG esters, and the like.

[0152] Aqueous suspensions contain the active material in admixture with excipients suitable for the preparation of aqueous suspensions. Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersants or wetting agents may be naturally occurring phosphatides such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long chain fatty alcohols, such as heptadecaethyleneoxyhexadecanoate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0153] Oily suspensions can be prepared by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil), or by suspending in a mineral oil such as liquid paraffin. Oily suspensions can contain thickeners, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners such as those described above, and flavorings can be added to provide a palatable oral preparation. These compositions can be preserved by adding an antioxidant such as ascorbic acid.

[0154] Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide the active ingredient mixed with a dispersant or wetting agent, a suspending agent and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those formulations already mentioned above. Additional excipients, such as sweeteners, flavoring agents and coloring agents may also be present.

[0155] The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin or a mixture of these liquid paraffins. Suitable emulsifiers may be naturally occurring gums, such as gum arabic or tragacanth; naturally occurring phospholipids, such as soybean, lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of the partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweeteners and flavorings.

[0156] Syrups and elixirs can be formulated with sweeteners, such as glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain demulcents, preservatives and flavoring and coloring agents. Oral solutions can be prepared in combination with, for example, cyclodextrins, PEG and surfactants.

[0157] The pharmaceutical composition can be in the form of a sterile injection aqueous or oily suspension. The suspension can be prepared according to known techniques using those suitable dispersants or wetting agents and suspending agents mentioned above. The sterile injection preparation can also be a sterile injection solution or suspension in a non-toxic parenteral acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable solvents and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injections.

[0158] Compound 1 can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient, which is solid at room temperature but liquid at rectal temperature, and will therefore dissolve in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycol. In addition, these compounds can be administered by solution or ointment via ocular delivery. Further, transdermal delivery of the target compound can be accomplished by iontophoresis patches and the like. For topical application, creams, ointments, gels, solutions or suspensions containing the compounds of the invention are used. As used herein, topical application also means the use of mouthwashes and gargles.

[0159] Compound 1 can be formulated for deposition into a medical device, which can include any of a variety of conventional grafts, stents, including stent grafts, catheters, balloons, baskets, or other devices that can be deployed or permanently implanted in a body cavity. As a specific example, there is a need for devices and methods that can deliver the compounds of the invention to areas of the body that have been treated by interventional techniques.

[0160] Compound 1 can be deposited in a medical device such as a stent and delivered to a treatment site for treating a part of the body. Stents have been used as delivery vehicles for therapeutic agents (i.e., drugs). Intravascular stents are usually permanently implanted in coronary or peripheral blood vessels. Stent designs include stent designs of US 4733655 (Palmaz), US 4800882 (Gianturco), and US 4886062 (Wiktor). Such designs include metal stents and polymer stents, as well as self-expanding stents and balloon-expandable stents. Stents can also be used to deliver drugs at sites in contact with the vascular system, such as those disclosed in US 5102417 (Palmaz) and WO 91 / 12779 (Medtronic, Inc.) and WO 90 / 13332 (Cedars-Sanai Medical Center), US 5419760 (Narciso, Jr.) and US 5429634 (Narciso, Jr.).

[0161] The term "deposited" refers to coating, adsorbing, placing or otherwise incorporating an inhibitor into a device by methods known in the art. For example, the inhibitor may be embedded in a medical device and released from it ("matrix type"), or surrounded by and released from a polymer material that coats or covers the medical device ("reservoir type"). In the latter example, the inhibitor may be entrapped in a polymer material or coupled to a polymer material using one or more techniques known in the art for generating such materials. In other formulations, the inhibitor may be attached to the surface of the medical device by a detachable bond without the need for a coating and released over time, the inhibitor may be removed by an active mechanical or chemical process, or may be in a permanently fixed form so that the inhibitor is present at the implant site.

[0162] The polymer may be a biostable or bioabsorbable polymer, depending on the desired release rate or degree of polymer stability desired. Bioabsorbable polymers that may be used include, but are not limited to, poly(l-lactic acid), polycaprolactone, polyglycolide (PGA), poly(lactide-co-glycolide) (PLLA / PGA), poly(hydroxybutyrate), poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoesters, polyanhydrides, poly(glycolic acid), poly(d-lactic acid), poly(l-lactic acid), poly(d,l-lactic acid), poly(d,l-lactide) (PLA), poly(l-lactide) (PLLA), poly(glycolic acid-co-trimethylene carbonate) (PGA / PTMC), polyethylene oxide ( Suitable bioresorbable copolymers include, for example, PEO, polydioxanone (PDS), polyphosphates, polyphosphate urethanes, poly(amino acids), cyanoacrylates, poly(trimethylene carbonate), poly(imino carbonate), copoly(ether-esters) (e.g., PEO / PLA), polyalkylene oxalates, polyphosphazenes, and biomolecules (such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid, poly-ε-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, cross-linked or amphiphilic block copolymers of hydrogels), and other suitable bioresorbable copolymers known in the art. In addition, biostable polymers with relatively low chronic tissue response can be used, such as polyurethanes, silicones, and polyesters, and other polymers can also be used if they can be dissolved and cured or polymerized on the medical device, such as polyolefins, polyisobutylene, and ethylene-alpha olefin copolymers; acrylic polymers and copolymers, vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl pyrrolidone; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketone; polyvinyl aromatic compounds, such as polystyrene, polyvinyl esters, such as polyvinyl acetate; vinyl monomers with a molecular weight of 1000 Å and a molecular weight of 1000 Å. and copolymers with olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins and ethylene-vinyl acetate copolymers; pyran copolymers; polyhydroxypropylmethacrylamide-phenol; polyhydroxyethyl-aspartamide-phenol; polyethylene oxide-polylysine substituted with palmitoyl residues; polyamides, such as nylon 66 and polycaprolactam; alkyd resins, polycarbonates; polyoxymethylene; polyimides; polyethers; epoxy resins, polyurethanes; rayon; rayon-triacetate; cellulose, cellulose acetate, cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; and carboxymethyl cellulose.

[0163] Polymers and semipermeable polymer matrices can form molded articles, such as valves, stents, tubes, prostheses, etc. Typically, polymers are applied to the surface of an implantable device by spin coating, dipping or spraying. Other methods known in the art may also be used for this purpose. Spray coating methods include conventional methods and micro-deposition techniques utilizing inkjet dispensers. In addition, light patterning or 3D printing can be used to deposit polymers on an implantable device so that the polymer is only located at a specific portion of the device. The coating of the device provides a uniform layer around the device, which allows for improved diffusion of various analytes through the device coating.

[0164] Compound 1 can be formulated to be released from the polymer coating into the environment in which the medical device is placed. For example, at least one of several well-known techniques involving polymer carriers or layers is used to control elution, releasing the compound in a controlled manner over an extended time frame (e.g., months). Some of these techniques have been previously described in US2004 / 0243225, the entire disclosure of which is incorporated herein in its entirety.

[0165] In addition, as described in, for example, US 6770729, which is incorporated herein as a whole, the reagents and reaction conditions of the polymer composition can be manipulated, so that the release of the inhibitor from the polymer coating can be controlled. For example, the diffusion coefficient of one or more polymer coatings can be adjusted to control the release of the inhibitor from the polymer coating. In a variation of this theme, the diffusion coefficient of one or more polymer coatings can be controlled to adjust the ability of the analyte (for example, the analyte that promotes the decomposition or hydrolysis of some parts of the polymer) present in the environment where the medical device is placed to approach one or more components in the polymer composition (and for example, so as to adjust the release of the inhibitor from the polymer coating). Another embodiment of the method includes a device with multiple polymer coatings, each coating having multiple diffusion coefficients. In such embodiments of the method, the release of the inhibitor from the polymer coating can be adjusted by the multiple polymer coatings.

[0166] The release of the inhibitor from the polymer coating can be controlled by adjusting one or more properties of the polymer composition, such as the presence of one or more endogenous compounds or exogenous compounds, or alternatively, by adjusting the pH of the polymer composition. For example, certain polymer compositions can be designed to release the inhibitor in response to a decrease in the pH of the polymer composition. Alternatively, certain polymer compositions can be designed to release the inhibitor in response to the presence of hydrogen peroxide.

[0167] One aspect of the present invention is a pharmaceutical composition for treating a plasma kallikrein-dependent disease or condition, the pharmaceutical composition comprising a therapeutically effective amount of a solid form of Compound 1 and a pharmaceutically acceptable carrier, wherein the solid form of Compound 1 is Form I, Form II, Form III, Form IV, or Form V, and is substantially free of any other polymorphic form or amorphous form of Compound 1. One embodiment of the present invention is a pharmaceutical composition, wherein the solid form is Form I. One embodiment of the present invention is a pharmaceutical composition, wherein the solid form is Form II. One embodiment of the present invention is a pharmaceutical composition, wherein the solid form is Form III. One embodiment of the present invention is a pharmaceutical composition, wherein the solid form is Form IV. One embodiment of the present invention is a pharmaceutical composition, wherein the solid form is Form V.

[0168] One aspect of the present invention is the use of a solid form of Compound 1 for the preparation of a medicament for treating a plasma kallikrein-dependent disease or condition, wherein the solid form of Compound 1 is substantially free of any other polymorphic or amorphous form of Compound 1. Another aspect of the present invention is the use of a solid form of Compound 1 for the preparation of a medicament for treating a plasma kallikrein-dependent disease or condition, wherein the solid form of Compound 1 is substantially free of any other polymorphic or amorphous form of Compound 1.

[0169] Application

[0170] The pharmaceutical compositions of the present invention are administered in a therapeutically effective amount by any of the modes of administration suitable for agents with similar uses. The actual amount of Compound 1 administered will depend on many factors, such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors.

[0171] One aspect of the invention is a method for treating a plasma kallikrein-dependent disease or disorder by administering to a subject in need thereof an effective amount of a pharmaceutical composition of the invention. One embodiment of the invention is the method, wherein the plasma kallikrein-dependent disease or disorder is selected from diabetic macular edema, diabetic retinopathy, hereditary angioedema with C1 inhibitor deficiency, acute liver injury, inflammation, allergic reaction, chemical sensitization kidney injury, ischemic stroke, hemorrhagic stroke, hypertension, vascular complications of hypertension, retinopathy, nephropathy, cerebral angioedema, pulmonary hypertension, inflammation, pain, acute myocardial infarction, deep vein thrombosis, complications of fibrinolytic therapy, angina pectoris, angioedema, sepsis, arthritis, complications of extracorporeal circulation, capillary leak syndrome, inflammatory bowel disease, diabetes, diabetic retinopathy, diabetic macular edema, diabetic nephropathy, diabetic neuropathy, age-related macular degeneration, retinal vein occlusion, cerebral edema, ischemia-reperfusion injury, cancer-related angiogenesis, asthma, allergic reaction and cerebrovascular complications of Alzheimer's disease, Parkinson's disease, multiple sclerosis, central nervous system infection and glioblastoma multiforme.

[0172] Example

[0173] The following examples are provided as illustrations and are not intended to limit the claimed invention. In the following examples, unless otherwise stated, concentration under reduced pressure was performed at 500 mmHg-600 mmHg. The following abbreviations are used: DCM = dichloromethane; MeOH = methanol; EtOH = ethanol; AcOH = acetic acid; EtOAc and AcOEt = ethyl acetate; 1-Propanephosphonic anhydride in EtOAc; MTBE = methyl tert-butyl ether; MEK = methyl ethyl ketone (2-butanone); MIBK = methyl isobutyl ketone (4-methyl-2-pentanone).

[0174] The RT-PCR system was constructed under ambient conditions (about 20 °C) using Cu Kα radiation (45 kV, 40 mA), a θ-θ goniometer, a focusing mirror, a divergence slit (1 / 2”), Soller slits (4 mm) at the incident and divergence beams, a transmission foil stage ( X-ray powder diffraction patterns were collected on a PANalytical X'Pert PRO X-ray diffractometer (Malvern Panalytical, Malvern, UK) with a polyimide (polyimide, 12.7 μm thick film) and a PIXcel detector. The software used for data collection was X'Pert Data Collector version 2.2f, and the data were presented using X'Pert Data Viewer version 1.2d. The data collection range was 2.994°-35° 2θ, and the continuous scan speed was 0.202004° s -1 .

[0175] Differential scanning calorimetry data were collected on a PerkinElmer Pyris 6000DSC equipped with a 45-position sample holder. Certified indium was used to verify the energy and temperature calibration of the instrument. In the following examples, a predetermined amount of sample (0.5 mg-3.0 mg) was placed in a pinhole aluminum pan and heated from 30°C to 350°C at a rate of 20°C / min, or adjusted according to experimental requirements. The sample was purged with dry nitrogen at a rate of 20 mL / min. Instrument control, data acquisition, and analysis were performed using Pyris software v11.1.1 Revision H.

[0176] Thermogravimetric analysis data were collected on a PerkinElmer Pyris 1TGA equipped with a 20-position autosampler. The temperature of the instrument was calibrated using certified weights and certified aluminum nickel alloys and Parker alloys (Perkalloy). In the following examples, unless otherwise stated, a predetermined amount of sample (about 5 mg) was loaded into a pre-peeled aluminum crucible and heated from ambient temperature to 400°C at a rate of 20°C / min. The sample was purged with nitrogen at a rate of 20 mL / min. Instrument control, data acquisition, and analysis were performed using Pyris software v11.1.1 Revision H.

[0177] Example 1: Synthesis of Compound of Formula IV

[0178] Compounds of formula IV are purchased from commercial sources or prepared by the methods described below or by other methods known in the art.

[0179]

[0180] At 20°C-25°C, 1H-pyrazole-4-carboxylic acid ethyl ester (23.5 g, 1 equivalent) and acetone (587 mL) were stirred under N 2 atmosphere into a round-bottom flask, and the mixture was stirred for 10 minutes, and then K 2 CO 3(70.4 g, 3 eq.). The reaction mass was cooled to 0°C-5°C and benzyl bromide (28.66 g, 1.1 eq.) was added very slowly at 0°C-5°C over 15 minutes. The reaction mixture was raised to 20°C-25°C and then heated to 50°C-60°C and maintained at this temperature for 3 hours. After completion of the reaction (monitored by HPLC), the reaction mixture was concentrated under reduced pressure at 45°C-50°C, quenched with 10% NaOH, and extracted with DCM (117 mL). The aqueous layer was separated, back-extracted with DCM (117 mL), and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure at 45°C-50°C. Petroleum ether or n-heptane (117 mL) was added to the concentrate, stirred for 1 hour, then filtered and dried under reduced pressure at 40°C-45°C for 12 hours to obtain 1-benzyl-1H-pyrazole-4-carboxylic acid ethyl ester (32.5 g).

[0181] Ethyl 1-benzyl-1H-pyrazole-4-carboxylate (30 g) and methanol (300 mL) were added to a 3 L round bottom flask and the resulting solution was stirred at 24°C for 10 minutes. KOH (14.6 g, 2 eq.) was then added and the mixture was heated to 65°C-70°C for 4 hours. After the reaction was complete (as determined by HPLC), the reaction mixture was concentrated to 40 mL-60 mL under reduced pressure at 45°C-50°C. The resulting residue was dissolved in water (300 mL) and extracted with DCM (2×150 mL). The aqueous layer was separated and acidified to pH 2 with 6N HCl. The precipitated solid was filtered, washed with water (30 mL), and dried under reduced pressure at 45°C-50°C for 12 hours to give 1-benzyl-1H-pyrazole-4-carboxylic acid (19.5 g) as a light brown solid with a purity of 99.1% as determined by HPLC. 1 H NMR (400 MHz, DMSO-d6): δ 5.36 (s, 2H), 7.26-7.37 (m, 5H), 7.83 (s, 1H), 8.38 (s, 1H), 12.33 (broad peak S, 1H).

[0182] Example 2: Synthesis of Compound III

[0183] The compound of formula III is prepared as follows:

[0184]

[0185] DCM (285 mL) and 4-aminomethyl-benzonitrile hydrochloride (19.2 g, 1.2 eq) were added to a 3 L round bottom flask, and the mixture was cooled to 0°C. Triethylamine (39.4 g, 3 eq) was added at 0°C, and the resulting mixture was stirred for 30 minutes. Next, 1-benzyl-1H-pyrazole-4-carboxylic acid (19 g, 1 eq) was added at 0°C-5°C, and the temperature was raised to 20°C-25°C. A 50% 1-propanephosphonic anhydride solution in ethyl acetate ( Spectrochem, 72 mL, 1.28 eq) and stirred at 20°C-25°C for 3 hours. After the reaction was complete, water (95 mL) was added and stirred for 10-15 minutes, and then the organic layer was separated. The aqueous layer was extracted again with DCM (95 mL), and the organic layers were combined and washed with water (95 mL). The organic layer was washed with Na 2 SO 4 The mixture was dried and concentrated under reduced pressure to about 40 mL. Acetone (95 mL) was then added and the mixture was co-distilled until only 20 mL-30 mL remained in the reaction tank. Water (285 mL) was then added and stirred at 20° C.-25° C. for 1 hour. The resulting solid was filtered, washed with acetone: water (1:3 v / v, 10 mL), then filtered and dried under reduced pressure at 45° C.-50° C. for 12 hours to obtain 1-benzyl-N-(4-cyanobenzyl)-1H-pyrazole-4-carboxamide (26.9 g, 94%) as a light brown solid with a purity of 98.51% as measured by HPLC. 1 H NMR (400MHz, DMSO-d6): δ4.49(d,J=5.9Hz,2H),5.37(s,2H),7.27-7.38(m,5H),7.48(d ,J=8.1Hz,2H),7.79(d,J=8.1Hz,2H),7.95(s,1H),8.31(s,1H),8.77(t,J=5.9Hz,1H).

[0186] Example 3: Synthesis of Compounds of Formula II

[0187] The compound of formula II is prepared as follows:

[0188]

[0189] Ethanol (250 mL) and 1-benzyl-N-(4-cyanobenzyl)-1H-pyrazole-4-carboxamide (25 g) were added to a 1 L round bottom flask at 20°C-25°C under a nitrogen atmosphere. Hydroxylamine hydrochloride (16.3 g, 3 equivalents) and triethylamine (24.64 g, 3 equivalents) were added to the reaction mixture at 20°C-25°C. The mixture was then heated to 60°C-65°C and maintained at this temperature for 7 hours. After the reaction was complete, the mixture was concentrated to about 30 mL-50 mL under reduced pressure at 45°C-50°C. Water (250 mL) was then added and stirred at ambient temperature for 30 minutes. The obtained solid was filtered, washed with water (125 mL), filtered to dryness, and then further dried under reduced pressure at 45°C-50°C for 12 hours to obtain 1-benzyl-N-(4-(N-hydroxycarbamimidyl)benzyl)-1H-pyrazole-4-carboxamide (25.5 g, 92.3 yield) as a light yellow solid with a purity of 95.41% as measured by HPLC. 1 H NMR (400 MHz, DMSO-d6): δ 4.40 (d, J = 5.9 Hz, 2H), 5.34 (s, 2H), 5.76 (broad peak s, 2H), 7.25-7.37 (m, 7H), 7.61 (d, J = 8.2 Hz, 2H), 7.91 (s, 1H), 8.27 (s, 1H), 8.63 (t, J = 5.9 Hz, 1H), 9.57 (broad peak s, 1H).

[0190] Example 4: Synthesis of compounds of formula I

[0191] The compound of formula I is prepared as follows:

[0192]

[0193] Acetic acid (2100 g) and 1-benzyl-N-(4-(N-hydroxycarbamimidoyl)benzyl)-1H-pyrazole-4-carboxamide (200 g) were stirred in a hydrogenator at 25°C for 10 to 15 minutes.

[0194] Raney nickel (40 g) and water (1 volume) were slurried in a flask and then allowed to settle for 5 minutes. The water was decanted and another volume of water was added, slurried, allowed to settle for 5 minutes and decanted. Acetic acid (1 volume) was added and the mixture was slurried for 10 minutes and then allowed to settle for 5 minutes and decanted. Raney nickel was then added to the hydrogenator along with acetic acid (1 volume). The reaction mixture was heated to 60°C and hydrogen (10 Kg pressure) was applied for 30 minutes. The resulting mixture was cooled to ambient temperature and the resulting solid was heated to 40°C. The mixture was stirred at 25 ° C for 1 hour, then filtered under suction, washed with EtOAc (400g), and dried under suction for 2 hours. The product was further dried under reduced pressure (<300mmHg) at 25 ° C for 2 hours, and then dried under reduced pressure (<300mmHg) at 45 ° C for 12 hours to obtain 1-benzyl-N-(4-amidinobenzyl)-1H-pyrazole-4-carboxamide acetate (220g) as a crude product.

[0195] Example 5: Purification

[0196] The dried product (219 g) was cooled to 25°C and added to a round-bottomed flask with water (2190 mL) and stirred for 10 minutes to form a slurry. The slurry was heated to 55°C in 20 minutes, stirred at this temperature for one hour, cooled to 25°C in 20 minutes with stirring, and stirred for another 30 minutes at 25°C. The solid was filtered, washed with water (220 mL), and dried by suction for 2 hours. The product was dried again under reduced pressure (<300 mmHg) at 45°C for 12 hours, then cooled to 25°C and added to a round-bottomed flask. Anhydrous ethanol (1250 g) and acetic acid (183 g) were added thereto, and the mixture was heated to reflux temperature (75°C) in 30 minutes, and kept under reflux for 30 minutes. The mixture was then slowly cooled to 25°C in 30 minutes, stirred at 25°C for 45 minutes, filtered and washed with EtOH, and then dried by suction at 25°C for 2 hours. The product was then dried at 45°C under reduced pressure (<300 mmHg) for 10 hours.

[0197] The dried product (143 g) was cooled to 25°C and added to a round bottom flask with anhydrous ethanol (1027 g) and acetic acid (150 g), and the mixture was heated to reflux temperature (75°C) within 30 minutes and kept at reflux for 1 hour. The mixture was then slowly cooled to 25°C within 45 minutes, stirred at 25°C for 45 minutes, filtered and washed with EtOH, and then dried by suction at 25°C for 2 hours. The product was then dried at 45°C under reduced pressure (<300 mmHg) for 12 hours to obtain purified 1-benzyl-N-(4-carboxamidinobenzyl)-1H-pyrazole-4-carboxamide acetate (126 g, 56% yield, purity of 99.6% as determined by HPLC) containing less than 30 ppm nickel.

[0198] Example 6: Pure Crystal Form 1

[0199] A mixture of crude 1-benzyl-N-(4-carbamimidoylbenzyl)-1H-pyrazole-4-carboxamide acetate (Compound 1, 10 g) in MeOH (450 mL) was added to a 2L round-bottom flask, and the mixture was heated to 50°C-55°C to obtain a clear solution. The solution was maintained at 50°C-55°C for 30 minutes, filtered, and added to a reactor at 50°C-55°C. MTBE (450 mL) was slowly added at 50°C-55°C, and the mixture was cooled to 25°C within 1 hour. A white suspension was observed after cooling. MTBE (450 mL) was slowly added at 20°C-25°C, and the resulting mixture was stirred for 16 hours, filtered, and washed with MTBE (10 mL). The product was dried under reduced pressure at 50°C-55°C for 24 hours to give pure 1-benzyl-N-(4-carbamimidoylbenzyl)-1H-pyrazole-4-carboxamide acetate (8.25 g, 82.5% yield) as an anhydrous crystalline polymorph ("Form 1") of an off-white solid. The product purity was >99% as determined by HPLC and contained less than 14.5 ppm nickel. 1 H NMR (300 MHz, DMSO-d6): δ1.71 (s, 3H), 4.47 (d, J = 5.4 Hz, 2H), 5.36 (s, 2H), 7.26-7.37 (m, 5H), 7.46 (d, J = 7.8 Hz, 2H), 7.74 (d, J = 7.8 Hz, 2H), 7.92 (s, 1H), 8.29 (s, 1H), 8.77 (broad peak s, 1H), 10.34 (broad peak s, 3H). 13 C NMR (75 MHz, DMSO-d6): δ 24.7, 41.7, 55.0, 118.4, 127.4 (2C), 127.5 (2C), 127.8 (2C), 128.2 (2C), 128.6 (2C), 131.6, 136.8, 145.2, 161.8, 165.7, 176.5. The X-ray powder diffraction (XRPD) pattern includes peaks at 10.0, 18.1, 18.6, 20.1 and 23.9 degrees with a deviation of ±0.5, ±0.2 or ±0.1 degrees 2θ (Cu Kα radiation), which is consistent with Figure 1 Thermal examination showed a simple thermal curve with only a major melt endotherm at 251°C on the DSC thermogram and a single weight decrease ( Figure 2 ).

[0200] Example 7: Pure Crystal Form 2

[0201] (A) crystallization : Compound 1 (53 mg) was weighed into a vial and dissolved in 3:1 MeOH:H at 55 °C. 2O (1 mL, 20 vol) and clarified at 50 °C into a crystallization tube.

[0202] MTBE (1 mL) was added to the solution and maintained at 50°C for about 10 minutes. Within 3 minutes of the addition of MTBE, the solution formed a suspension. The mixture was cooled to 25°C in about 1 hour and equilibrated for another 1 hour, but remained a suspension. The solid was separated by filtration and dried in vacuo at 50°C for about 16 hours to give an off-white solid with a recovery of 62.3%. The X-ray powder diffraction (XRPD) pattern included peaks at 4.2, 5.6, 8.4, 17.8 and 19.8 degrees with deviations of ±0.5, ±0.2 or ±0.1 degrees 2θ (Cu Kα radiation), which was consistent with Figure 2 The XRPD pattern is consistent with that of

[0203] (B) Seed crystallization : Weigh compound 1 (1.008 g) into a container and add 3:1 MeOH:H 2 O (22 mL, 22 vol). The mixture was heated to 55°C to achieve dissolution. The solution was clarified and added to a container at 50°C. Form II seeds (approximately 10 mg) were added to the solution and their persistence was observed.

[0204] MTBE (22 mL, 22 volumes) was added to the vessel and the mixture was cooled to 25°C over approximately 1 hour. A fine off-white suspension was observed to mature as it cooled. The mixture was re-equilibrated for 45 minutes and additional MTBE (22 mL, 22 volumes) was added. The suspension was re-equilibrated at 25°C for 16.5 hours. The solid was isolated by filtration and dried under vacuum at 50°C for approximately 23.5 hours to give a free-flowing white powder solid in 73.5% yield. The X-ray powder diffraction (XRPD) pattern included peaks at 4.2, 5.6, 8.4, 17.8 and 19.8 degrees with deviations of ±0.5, ±0.2 or ±0.1 degrees 2θ (Cu Kα radiation), consistent with Figure 2 The XRPD pattern of Fig. 9 ) shows a typical DSC thermogram of Form II with a prominent endotherm at 158° C. followed by a major melt endotherm at 254° C. The TGA thermogram shows a weight loss of 4.067 wt % from 40° C. to 105° C., indicating a near stoichiometric loss of water prior to the acetate weight loss event.

[0205] Example 8: Pure Crystal Form 3

[0206] (A) Via CH 3 CN preparation: Weigh compound 1 (25 mg) into a crystallization tube, and add acetonitrile (MeCN) in 0.1 mL (4 volumes) aliquots to 0.5 mL (20 volumes), and record the observations immediately. Stir the mixture with a magnetic stirring bar and heat to 50°C, and record the observations after 4 hours. If there is no obvious dissolution, double the solvent volume to 1 mL (40 volumes) and record the observations immediately. Cool the mixture to 25°C and balance for 65 hours. Record the observations and separate half of the mixture. Heat the mixture to 50°C and balance for 6 hours, then separate the remainder.

[0207] The solid was dried under vacuum at 50°C for 17 hours and then analyzed by XRPD detection. The X-ray powder diffraction (XRPD) pattern included peaks at 7.7 degrees, 10.7 degrees, 20.1 degrees, 23.7 degrees and 24.3 degrees, which were consistent with Figure 3 The XRPD pattern of Form III is consistent. Fig.10 ) shows a single endothermic peak at 249° C. and a small shoulder at 234° C. in the DSC thermogram. Only the disproportionation weight reduction of acetate due to TGA is present in the melt.

[0208] (B) Prepared via MeOH : Compound 1 (1.050 g) was weighed and added to a flask, and MeOH (50 mL, 50 volumes) was added. The mixture was heated to 50 ° C and maintained for 20 minutes. Only a portion was dissolved, so it was further heated to 60 ° C and additional MeOH (10 mL, 60 volumes in total) was applied to achieve dissolution. The solution was clarified and concentrated to dryness in vacuo, and then further vacuum dried at 50 ° C for 16.5 hours to regain a pink, large volume, low density solid with a recovery rate of 76.1%.

[0209] Example 9: Pure Crystal Form 4

[0210] (A) Prepared from Form 1 Compound 1 (211 mg) was weighed into a vial and MeOH (8.44 mL, 40 volumes) was added. The mixture was heated to 50°C and maintained for 1 hour. Only a portion dissolved, so further heating was performed to achieve dissolution. The solution was clarified and added to a crystallization tube at 50°C in 1 mL aliquots. EtOAc was added to 1 mL in 0.1 mL aliquots. The mixture was cooled to 20°C over 2 hours and equilibrated at 20°C for another 19.25 hours.

[0211] The solid was dried under vacuum at 50°C for about 22.5 hours. The X-ray powder diffraction (XRPD) pattern included peaks at 18.5, 19.6 and 23.7 degrees with deviations of ±0.5, ±0.2 or ±0.1 degrees 2θ, and Figure 4The thermal examination of Form IV shows a simple thermal curve with only a main melt endotherm at 253°C on the DSC thermogram ( Fig.11 ), and a smaller weight drop of about 0.40 wt% from 100°C to 150°C before acetate loss.

[0212] (B) Prepared from amorphous : Amorphous compound 1 (4×50 mg) and Form 4 (4×2 mg) were weighed into 4 crystallization tubes, and 4 solvents (THF, EtOAc, MIBK and MEK: 2 mL, 40 volumes) were added. The mixture was stirred at 50°C for about 21 hours by a magnetic stirring bar and then cooled to 25°C. The suspension was equilibrated at 25°C for about 7.5 hours and then heated to 50°C. The suspension was equilibrated at 50°C for about 16 hours, then cooled to 25°C, and the solid was separated by filtration.

[0213] The solid was dried under vacuum at 50°C for about 72 hours. The solid recovered from THF or EtOAc was confirmed to be Form 1 by XRPD, while the solid recovered from MIBK or MEK was shown to be Form 4.

[0214] Example 10: Pure Amorphous Form 5

[0215] Compound 1 (495 mg) was weighed and added to a flask containing deionized water (250 mL) and stirred at 45°C to achieve dissolution. The clarified solution was added to a 3L flask, frozen and lyophilized within about 9 hours. A white bulk solid was isolated with a recovery of about 83.6%. XRPD detection showed that the solid was mainly amorphous, but there were traces of Form II.

[0216] The above process was repeated and the solid was freeze-dried in about 6.25 hours to obtain a white bulk solid with a recovery rate of 70.5%. XRPD detection showed that the material was mainly amorphous ( Figure 5 ). Thermal Detection( Fig.12 ) shows a minor endothermic event at about 112°C similar to Form II, and major endotherms at about 229°C and 240°C with slightly reduced onsets and peaks compared to Form II. A weight loss of 1.300 wt% was observed from 40°C to 80°C, which is primarily residual surface moisture. A second significant weight loss of 15.837 wt% from 170°C to 240°C corresponds to the melt, indicating acetate loss (15.01 wt%), and may also indicate trapped water loss or a minor degradation event leading to decomposition starting at 250°C.

[0217] Example 11: Compression and grinding stability

[0218] The preparation method of solid dosage forms may require high compression and / or grinding, which may result in changes in the crystal structure of some compounds. 5 The samples were compressed under N pressure for about 25-28 hours to evaluate the effect of compression on the crystalline stability of these crystalline forms.

[0219] XRPD examination showed a slight decrease in diffraction resolution of Form I. Examination of Form II showed a significant change in the diffraction pattern, becoming a pattern with reduced crystallinity, such as Figure 6 Thermal examination of Form I shows no change in the TGA thermogram after compression. The intensity and temperature of the melt endotherm in the DSC thermogram decrease after compression. Thermal examination of Form II shows a slight increase in water content in the TGA thermogram, however, this may be due to aging of the sample after isolation rather than compression. The DSC thermogram shows a decrease in intensity but an increase in the temperature of the main melt endotherm, and a significant endotherm is added at about 150°C after compression.

[0220] The grinding operation was performed on Form I and Form II samples (about 100 mg each) to evaluate the effect on solid form and microscopic observation. The generation of amorphous content was noted and the effect on the water content of the batch was examined, especially for the Form II hydrate.

[0221] The data sets after grinding for both samples clearly show no thermal or crystallographic changes induced. Form II shows minor changes in reflection resolution and intensity, but neither sample indicates amorphous content either thermally or in XRPD compared to the input, as shown in Table 1. Figure 7 The most notable change is that for the compressed material, the water content of Form II increased from 2.76 wt% to 3.93 wt% as produced, consistent with the expected hydrate.

[0222] Microscopic examination was performed in which both samples were compared to their original state. After grinding, both samples dispersed very well under silicone oil. Form II retained more particles of 5-10 μm size, but most of the solids were <5 μm. Form I was broken down into very fine and evenly dispersed solids, mainly particles <5 μm (mostly 1 μm-2 μm), which was expected considering the relatively small particle sizes observed in the input batch. In summary, Form I showed better stability under artificial pressure than hydrated Form II.

Claims

1. Crystalline Form I of 1-benzyl-N-(4-carbamimidoylbenzyl)-1H-pyrazole-4-carboxamide acetate, characterized in that: The X-ray powder diffraction (XRPD) pattern of the crystalline form includes peaks at 2θ of 10.0 degrees, 18.1 degrees, 18.6 degrees, 20.1 degrees and 23.9 degrees with a deviation of ±0.2 degrees, wherein the XRPD is made using Cu Kα radiation.

2. The crystal form according to claim 1, characterized in that The XRPD pattern also includes a peak at 20.5 degrees 2θ with a deviation of ±0.2 degrees.

3. The crystal form according to claim 1, characterized in that The XRPD pattern is consistent with Figure 1.

4. The crystal form according to claim 3, characterized in that The crystalline form has a melting point of about 253°C.

5. The crystal form according to claim 3, characterized in that: The crystalline form has a water solubility of about 8.3 mg / mL at 25°C.

6. The crystalline form according to any one of claims 1 to 5, characterized in that The crystalline form has less than 10% by weight of any other polymorphic form.

7. The crystal form according to claim 6, characterized in that: The crystalline form has less than 5% by weight of any other polymorphic form.

8. The crystal form according to claim 6, characterized in that: The crystalline form has less than 1% by weight of any other polymorphic form.

9. A pharmaceutical composition comprising the crystalline form according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

10. Use of the crystalline form according to any one of claims 1 to 5 in the preparation of a medicament for treating a disease or condition selected from the group consisting of diabetic retinopathy, hereditary angioedema with C1 inhibitor deficiency, allergic reaction, ischemic stroke, hemorrhagic stroke, inflammation, pain, acute myocardial infarction, deep vein thrombosis, angina pectoris, angioedema, sepsis, arthritis, inflammatory bowel disease, diabetes, age-related macular degeneration, retinal vein occlusion, and cerebral edema.

11. Use of the crystalline form according to any one of claims 1 to 5 in preparing a medicament for treating a disease or condition, characterized in that: The disease or condition is diabetic macular edema.

12. The crystalline form according to any one of claims 1 to 5, characterized in that The XRPD peaks were within ±0.1 degrees 2Θ of the peak positions.

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