Process-scale synthesis of plasma kallikrein inhibitors

Abstract

Methods for preparing Compound (I) and salts thereof are disclosed. The methods for preparing Compound (I) are suitable for use on a process scale. [Formula 1] TIFF2022543419000034.tif46164

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 883,396, filed August 6, 2019. [Background technology]

[0002] Serine proteases constitute the largest and most extensively studied group of proteolytic enzymes. Their important roles in physiological processes span a variety of areas, such as blood clotting, fibrinolysis, complement activation, regeneration, degradation, and release of physiologically active peptides. Many of these important processes begin with the cleavage of a single or a small number of peptide bonds in a precursor protein or peptide. A defined series of proteolytic reactions or cascades are involved in blood clotting, fibrinolysis, and complement activation. The biological signals to initiate these cascades can also be controlled and amplified. Similarly, controlled proteolysis can stall or inactivate a protein or peptide by cleavage of a single bond.

[0003] Kallikreins are a subgroup of serine proteases. In humans, plasma kallikrein (KLKB1) has no known homologues, but tissue kallikrein-related peptidases (KLKs) encode a family of 15 closely related serine proteases. Plasma kallikreins are involved in several pathways related to the intrinsic pathway of coagulation, inflammation, and the complement system.

[0004] Coagulation is the process by which blood forms clots, for example to stop bleeding. The physiology of coagulation is somewhat complex in that it involves two separate early pathways that converge into a final common pathway leading to clot formation. In the final common pathway, prothrombin is converted to thrombin, which in turn converts fibrinogen to fibrin, the main building block of the cross-linked fibrin polymer that forms the hemostatic plug. Of the two early pathways upstream of the final common pathway, one is known as the contact activation or intrinsic pathway, and the other is known as the tissue factor or extrinsic pathway.

[0005] The intrinsic pathway begins with the formation of a primary complex on collagen by high molecular weight kininogen (HMWK), prekallikrein, and FXII (factor XII; Hageman factor). Prekallikrein is converted to kallikrein, and FXII is activated to FXIIa. FXIIa then converts factor XI (FXI) to FXIa, which in turn activates factor IX (FIX), which forms a "tenase" complex with its cofactor FVIIIa, which activates factor X (FX) to FXa. It is FXa that is involved in the conversion of prothrombin to thrombin in the final common pathway.

[0006] Prekallikrein, the inactive precursor of plasma kallikrein, is synthesized in the liver and circulates in plasma bound to HMWK or as a free zymogen. Prekallikrein is cleaved by activated factor XII (FXIIa) to release activated plasma kallikrein (PK). Activated plasma kallikrein exhibits endopeptidase activity at the peptide bonds after arginine (preferred) and lysine. PK then generates additional FXIIa in a feedback loop, which in turn activates factor XI (FXI) to FXIa, leading to a common pathway. The initial activation of the intrinsic pathway is due to a small amount of FXIIa activating a small amount of PK, but it is the subsequent feedback activation of FXII by PK that controls the degree of activation of the intrinsic pathway and thus downstream coagulation. Non-patent document 1.

[0007] Active plasma kallikrein also cleaves HMWK to release the potent vasodilatory peptide bradykinin. It can also cleave several inactive precursor proteins to generate active products such as plasmin (from plasminogen) and urokinase (from pro-urokinase). Plasmin, a regulator of blood clotting, proteolytically cleaves fibrin into fibrin degradation products, which inhibit excessive fibrin formation.

[0008] Patients who have suffered an acute myocardial infarction (MI) show clinical evidence of being in a hypercoagulable (procoagulant) state. This hypercoagulable state is paradoxically exacerbated in patients receiving fibrinolytic therapy. In such treated patients, an increase in thrombin generation, as measured by thrombin-antithrombin III (TAT) levels, is observed compared to the already high levels observed in patients receiving heparin alone. (2003) It has been proposed that the increase in thrombin is due to plasmin-mediated activation of the intrinsic pathway through direct activation of FXII by plasmin.

[0009] The fibrinolysis-induced hypercoagulable state is a major drawback of fibrinolytic therapy, which not only leads to an increased reocclusion rate but also, perhaps at least in part, is the reason why complete fibrinolysis of the clot (thrombus) cannot be achieved (Non-Patent Document 3). Another problem with fibrinolytic therapy is the associated increased risk of intracranial hemorrhage (Non-Patent Document 4). Therefore, an adjunctive anticoagulant therapy that does not increase the risk of hemorrhage but inhibits the generation of new thrombin would be highly beneficial.

[0010] Plasma kallikrein inhibitors also have therapeutic potential for treating hereditary angioedema (HAE). HAE is a severe and sometimes life-threatening rare genetic disease caused by mutations in the C1-esterase inhibitor (C1INH) gene located on chromosome 11q. HAE is inherited as an autosomal dominant condition, but one-quarter of diagnosed cases result from de novo mutations. HAE is classified as a rare disease in Europe, with an estimated prevalence of 1 in 50,000. Individuals with HAE experience recurrent acute attacks of painful subcutaneous or submucosal edema of the face, larynx, gastrointestinal tract, limbs or genitals, which may last up to 5 days if untreated. Attacks vary in frequency, severity and location and can be life-threatening. Laryngeal attacks, which can be asphyxiating, pose the greatest risk. Abdominal attacks are particularly painful and often lead to exploratory procedures or unnecessary surgery. Facial and peripheral attacks are disfiguring and debilitating.

[0011] There are several subtypes of HAE. Type I HAE is defined by C1INH gene mutations that produce low levels of C1-inhibitor, while type II HAE is defined by mutations that produce normal levels of ineffective C1 protein. Type III HAE has a different cause and is caused by mutations in the F12 gene that encodes a serine protease known as factor XII. Diagnostic criteria for identifying subtypes of HAE and for distinguishing HAE from other angioedemas can be found in Non-Patent Document 5, which is incorporated herein by reference.

[0012] Current treatments for HAE are classified into two main types. Older non-specific treatments, including androgens and antifibrinolytic drugs, are associated with significant side effects, especially in women. Newer treatments are based on the understanding of the molecular pathology of the disease, namely that C1INH is the most important inhibitor of kallikrein in human plasma and that C1INH deficiency leads to unhindered activation of the kallikrein-bradykinin cascade, with bradykinin being the most important mediator of the local increase in vascular permeability that characterizes attacks. All currently available targeted therapies are administered by intravenous or subcutaneous injection. There are currently no targeted oral long-term treatments specific for HAE.

[0013] Therefore, there is a need to develop inhibitors of PK that can change the fibrinolysis / thrombosis situation towards dissolving occluding clots, thereby promoting reperfusion and also attenuating the hypercoagulable state, thus preventing clots from reforming and reoccluding the vessel. In particular, the creation of plasma kallikrein inhibitors that are specific and can be formulated for in vivo use could lead to a new class of therapeutic agents. Therefore, what is needed are improved methods for preparing and formulating plasma kallikrein inhibitors, especially on a process scale. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Hathaway, WE, et al. (1965) Blood 26:521-32 [Non-Patent Document 2] Hoffmeister,HMet al.(1998)Circulation 98:2527-33 [Non-Patent Document 3] Keeley, EC et al. (2003) Lancet 361:13-20 [Non-Patent Document 4] Menon, V. et al. (2004)(Chest 126:549S-575S;Fibrinolytic Therapy Trialists' Collaborative Group(1994)Lancet 343:311-22 [Non-Patent Document 5] Ann Allergy Asthma lmmunol 2008;100(Suppl2):S30-S40 and J Allergy Clin lmmunol 2004;114:629-37 Summary of the Invention [Means for solving the problem]

[0015] One aspect of the invention relates to a synthesis of Compound I or a salt thereof that can be carried out on a process scale (eg, to produce about 100 kilograms of Compound I or a salt thereof). [ka]

[0016] In certain embodiments, the present invention provides a method for producing a compound comprising the steps of combining Compound C, or a salt thereof, and Compound F, or a salt thereof, under conditions sufficient to produce Compound D, or a salt thereof; Compound C is [ka] and compound F is represented by [ka] and compound D is represented by [ka] The present invention provides a method, represented by

[0017] In certain embodiments, the method comprises: (a) reacting Compound B, or a salt thereof, with [ka] to produce a first reaction mixture; and thereafter, (b) combining the first reaction mixture with a reducing agent under conditions sufficient to produce compound C, or a salt thereof; Compound B is [ka] It is represented by:

[0018] In certain embodiments, the method further comprises: (a) combining Compound A and a second acid under conditions sufficient to produce Compound B, or a salt thereof; Compound A is [ka] It is represented by:

[0019] In certain embodiments, the method further includes (c) combining compound D and a deprotection reagent to form a second reaction mixture, and then (d) exposing the second reaction mixture to conditions sufficient to form compound I as a free base.

[0020] In certain embodiments, the method further comprises e) providing a first free base mixture of compound I as a free base in a fourth organic solvent; f) mixing the free base mixture with a first reagent solution comprising a fourth acid and a fifth organic solvent under conditions sufficient to produce a third reaction mixture comprising a salt of compound I; and g) crystallizing the salt of compound I from the mixture comprising the salt of compound I. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention relates to syntheses of Compound I that can be carried out on a process scale (eg, to produce about 100 kilograms of Compound I).

[0022] Method for Producing Compound I In certain embodiments, the present invention provides a method for producing a compound comprising the steps of combining compound C, or a salt thereof, and compound F, or a salt thereof, under conditions sufficient to produce compound D, or a salt thereof; Compound C is [ka] and compound F is represented by [ka] and compound D is represented by [ka] The present invention relates to a method represented by the following formula:

[0023] In certain embodiments, the conditions sufficient to produce compound D include an amide coupling reagent and a first base.

[0024] In certain embodiments, the amide coupling reagent is propylphosphonic anhydride (T3P), N,N'-di(isopropyl)carbodiimide, N,N'-di(cyclohexyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, or ethyl 2-cyano-2-(hydroxyimino)acetate, preferably, the amide coupling reagent is propylphosphonic anhydride (T3P).

[0025] In certain embodiments, the first base is a first organic base. Exemplary organic bases include amine bases and alkoxide bases. In certain embodiments, the first base is triethylamine, pyridine, diisopropylethylamine, diisopropylmethylamine, imidazole, pyrimidine, N-methylmorpholine, quinuclidine, or 1,4-diazabicyclo[2.2.2]octane (DABCO). In a preferred embodiment, the first base is pyridine.

[0026] In certain embodiments, the conditions sufficient to produce compound D further include a first solvent. The first solvent may be a polar aprotic solvent such as dichloromethane, tetrahydrofuran, acetone, acetonitrile, or ethyl acetate. In a preferred embodiment, the first solvent is ethyl acetate.

[0027] In certain embodiments, compound C is present as an acid salt, The method further comprises the step of combining the acid salt of Compound C with a second water-soluble base, thereby producing a free base of Compound C; The step of mixing the acid salt of compound C with the second water-soluble base is performed prior to mixing compound C and compound F.

[0028] In certain embodiments, "acid salt" refers to a salt formed in the presence of a Bronsted acid. For example, an amine such as R-NH2 is readily protonated by a Bronsted acid HX to give R-NH3 + X - Therefore, R-NH3 + X - is the acid salt of R-NH2. Common Bronsted acids that can cause the formation of acid salts include hydrochloric acid, hydrobromic acid, hydroiodic acid, and oxalic acid. When they contact an amine, such Bronsted acids will generate the hydrochloride, hydrobromide, hydroiodide, or oxalate salts, respectively.

[0029] In certain embodiments, the acid salt of compound C is a hydrochloride, a hydrobromide, a hydroiodide, or an oxalate. Preferably, the acid salt of compound C is an oxalate.

[0030] In certain embodiments, the second water-soluble base comprises potassium hydroxide, sodium hydroxide, ammonium hydroxide, potassium bicarbonate, sodium bicarbonate, potassium carbonate, or sodium carbonate. In a preferred embodiment, the second water-soluble base comprises potassium hydroxide.

[0031] In certain embodiments, the method includes: (a) reacting Compound B, or a salt thereof, with [ka] to produce a first reaction mixture; and thereafter, (b) combining the first reaction mixture with a reducing agent under conditions sufficient to produce compound C, or a salt thereof; Compound B is [ka] It is represented by:

[0032] In certain embodiments, the reducing agent is LiAlH4 or NaBH4, preferably NaBH4.

[0033] In certain embodiments, the conditions sufficient to produce compound C further include a second solvent.

[0034] In certain embodiments, the second solvent is a second polar protic solvent. Exemplary polar protic solvents include methanol, ethanol, and isopropanol. In certain embodiments, the second solvent is methanol.

[0035] In certain embodiments, the method further comprises contacting compound C with a first acid to produce an acid salt of compound C.

[0036] In certain such embodiments, the first acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, or oxalic acid, and the acid salt of Compound C is a hydrochloride, hydrobromide, hydroiodide, or oxalate. In a preferred embodiment, the first acid is oxalic acid, and the acid salt of Compound C is an oxalate.

[0037] In certain embodiments, compound B is present as an acid salt, The method further comprises the step of combining the salt of Compound B with a third organic base, thereby producing the free base of Compound B; The step of mixing the salt of compound B with a third organic base provides a compound B and [ka] This is done before mixing.

[0038] In certain such embodiments, the acid salt of compound B is a hydrochloride, a hydrobromide, or a hydroiodide salt. Preferably, the acid salt of compound B is a hydrochloride salt.

[0039] In certain embodiments, the third organic base comprises sodium methoxide.

[0040] In certain embodiments, the method further comprises: (a) combining Compound A and a second acid under conditions sufficient to produce Compound B, or a salt thereof; Compound A is [ka] It is represented by:

[0041] In certain embodiments, the second acid is hydrochloric acid, hydrobromic acid, or hydroiodic acid, preferably hydrochloric acid.

[0042] In certain embodiments, the conditions sufficient to produce compound B include a third polar protic solvent. Exemplary polar protic solvents include methanol, ethanol, and isopropanol. In a preferred embodiment, the third polar protic solvent is isopropanol.

[0043] In certain embodiments, compound B is prepared as an acid salt, such as a hydrochloride, hydrobromide, or hydroiodide salt. In a preferred embodiment, the acid salt of compound B is a hydrochloride salt.

[0044] In certain embodiments, the method further comprises: (c) combining compound D and a deprotection reagent to form a second reaction mixture; and then (d) exposing the second reaction mixture to conditions sufficient to form compound I as a free base; Compound I is [ka] It is represented by:

[0045] In certain embodiments, the deprotection reagent is a third acid. Exemplary acids include hydrochloric acid, hydrobromic acid, and hydroiodic acid. In a preferred embodiment, the third acid is hydrochloric acid.

[0046] In certain embodiments, the conditions sufficient to produce compound I as a free base include a fourth base. In certain embodiments, the fourth base is aqueous ammonia.

[0047] In certain embodiments, the method comprises: e) providing a first free base mixture of Compound I as a free base in a fourth organic solvent; f) combining the free base mixture with a first reagent solution comprising a fourth acid and a fifth organic solvent under conditions sufficient to produce a third reaction mixture comprising a salt of Compound I; g) crystallizing a salt of Compound I from a mixture containing the salt of Compound I; Further includes:

[0048] In certain embodiments, the crystalline salt is a hydrochloride salt, for example a bis(hydrochloride) salt.

[0049] In certain embodiments, the fourth organic solvent comprises a fourth polar aprotic solvent. Exemplary polar aprotic solvents include acetonitrile, N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulfoxide (DMSO), diethyl ether, ethyl acetate, isopropyl acetate, methyl ethyl ketone, methyl tert-butyl ether (MTBE), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran, dichloromethane, and acetone. In certain embodiments, the fourth polar aprotic solvent is methyl tert-butyl ether.

[0050] In certain embodiments, the fourth organic solvent further comprises a fourth non-polar solvent. Non-polar solvents include, for example, benzene, heptane, hexane, and toluene. In certain embodiments, the non-polar solvent is toluene.

[0051] In certain embodiments, the fourth acid is hydrochloric acid.

[0052] In certain embodiments, the fifth organic solvent is a fifth polar protic solvent, such as ethanol, methanol, 2-propanol, 1-butanol, water, or any combination thereof. Preferably, the fifth polar protic solvent is methanol.

[0053] In certain embodiments, compound C is used in an amount of at least 1 kg, at least 5 kg, at least 10 kg, at least 15 kg, at least 20 kg, at least 25 kg, at least 30 kg, at least 35 kg, at least 40 kg, at least 45 kg, at least 50 kg, at least 55 kg, or at least 60 kg. In yet other embodiments, compound C is used in an amount of at least 50 kg.

[0054] In certain embodiments, the methods of the invention produce Compound I or a salt thereof on a scale of at least 1 kg, at least 5 kg, at least 10 kg, at least 15 kg, at least 20 kg, at least 25 kg, at least 30 kg, at least 35 kg, at least 40 kg, at least 45 kg, at least 50 kg, at least 55 kg, at least 60 kg, at least 65 kg, at least 70 kg, at least 75 kg, at least 80 kg, at least 85 kg, at least 90 kg, at least 95 kg, or at least 100 kg.

[0055] Pharmaceutical Compositions Compound I, synthesized by the methods described herein, may be formulated as a pharmaceutical composition. Such a pharmaceutical composition comprises Compound I and a pharma- ceutically acceptable carrier.

[0056] The terms "carrier" and "pharmaceutical acceptable carrier" as used herein refer to both diluents, adjuvants, excipients, or vehicles in which a compound is administered or formulated for administration. Non-limiting examples of such pharmaceutical acceptable carriers include liquids such as water, saline, and oils, as well as solids such as gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating, flavoring, and coloring agents may be used. Other examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein in its entirety by reference.

[0057] In certain embodiments, the pharmaceutical composition of the present invention further comprises at least one additional pharmacologic active agent other than compound I. The at least one additional pharmacologic active agent may be an agent useful for treating a disease or condition characterized by abnormal plasma kallikrein activity.For example, the at least one additional pharmacologic active agent may be an anticoagulant, an antiplatelet agent, or a thrombolytic agent.

[0058] Anticoagulants prevent the clotting of blood components, thereby preventing clot formation, for example, in atrial fibrillation. Anticoagulants include, but are not limited to, heparin, warfarin, coumadin, dicoumarol, phenprocoumon, acenocoumarol, ethyl biscoumaacetate, hirudin, bivalartin, direct thrombin inhibitors, and indandione derivatives.

[0059] Antiplatelet drugs inhibit platelet aggregation and are often used to prevent thromboembolic stroke in patients who have experienced transient ischemic attack, stroke, or atrial fibrillation. Antiplatelet drugs include, but are not limited to, aspirin, thienopyridine derivatives such as ticlopodine and clopidogrel, dipyridamole, and sulfinpyrazone, and RGD mimetics.

[0060] Thrombolytic agents dissolve blood clots that cause thromboembolic events such as stroke, myocardial infarction, and pulmonary thromboembolism. Thrombolytic agents include, but are not limited to, plasminogen, a2-antiplasmin, streptokinase, antistreplase, TNK, tissue plasminogen activator (tPA), and urokinase. Tissue plasminogen activators include native tPA and recombinant tPA, as well as modified forms of tPA that retain the enzymatic or fibrinolytic activity of native tPA.

[0061] The pharmaceutical compositions of the present invention can be prepared by mixing Compound I with a pharma- ceutically acceptable carrier and, optionally, one or more additional pharma- ceutical active agents.

[0062] In certain embodiments, the invention provides pharmaceutical compositions formulated for the prophylactic or therapeutic treatment of diseases or conditions characterized by abnormal plasma kallikrein activity.

[0063] Treatment method The present invention provides a method for producing compounds that inhibit thrombin generation via the intrinsic pathway, thereby reducing the risk of pathological new thrombus formation (vascular occlusion or reocclusion) and also improve fibrinolysis-induced reperfusion when given as an adjunct treatment with a fibrinolytic regimen. Diseases and conditions that can be treated using the compounds of the present invention include, but are not limited to, stroke, inflammation, reperfusion injury, acute myocardial infarction, deep vein thrombosis, post-fibrinolytic treatment conditions, angina, edema, angioedema, hereditary angioedema, sepsis, arthritis, hemorrhage, blood loss during cardiopulmonary bypass, inflammatory bowel disease, diabetes mellitus, retinopathy, diabetic retinopathy, diabetic macular edema, diabetic macular degeneration, age-related macular edema, age-related macular degeneration, proliferative retinopathy, neuropathy, hypertension, cerebral edema, increased albumin excretion, macroalbuminuria, and nephropathy.

[0064] For example, in patients with angioedema conditions, the small polypeptide PK inhibitor DX-88 (ecallantide) reduces edema in patients with hereditary angioedema (HAE). Williams, A. et al. (2003) Transfus. Apher. Sci. 29:255-8; Schneider, L. et al. (2007) J Allergy Clin Immunol. 120:416-22; and Levy, J Het al. (2006) Expert Opin. Invest. Drugs 15:1077-90. Icatibant, a bradykinin B2 receptor antagonist, is also effective in treating HAE. Bork, K. et al. (2007) J. Allergy Clin. Immunol. 119:1497-1503. Because plasma kallikrein generates bradykinin, inhibition of plasma kallikrein is expected to inhibit bradykinin production.

[0065] For example, in coagulation resulting from fibrinolytic treatment (e.g., treatment with tissue plasminogen activator or streptokinase), higher levels of plasma kallikrein are seen in patients undergoing fibrinolysis. Hoffmeister, HMet al. (1998) J. Cardiovasc. Pharmacol. 31:764-72. Plasmin-mediated activation of the intrinsic pathway has been shown to occur in plasma and blood and was significantly attenuated in the plasma of individuals deficient in any of the intrinsic pathway components. Ewald, GA et al. (1995) Circulation 91:28-36.

[0066] Individuals who have had an acute MI have been found to have elevated levels of activated plasma kallikrein and thrombin. Hoffmeister, HM, et al. (1998) Circulation 98:2527-33.

[0067] DX-88 reduced brain edema, infarct volume, and neurological abnormalities in an animal model of ischemic stroke. Storini, C. et al. (2006) J. Pharm. Exp. Ther. 318:849-854. C1-inhibitor reduced infarct size in a mouse model of middle cerebral artery occlusion (MCAO). De Simoni, MGet al. (2004) Am. J. Pathol. 164:1857-1863; and Akita, N. et al. (2003) Neurosurgery 52:395-400). B2 receptor antagonists were found to reduce infarct volume, brain swelling, and neutrophil accumulation in an MCAO animal model and were neuroprotective. Zausinger, S. et al. (2003) Acta Neurochir. Suppl. 86:205-7; Lumenta, DBet al. (2006) Brain Res. 1069: 227-34; Ding-Zhou, L. et al. (2003) Br. J Pharmacol. 139: 1539-47.

[0068] With regard to blood loss during cardiopulmonary bypass (CPB), the kallikrein-kinin (i.e., contact) system has been found to be activated during CABG. Wachtfogel, YT (1989) Blood 73:468. Activation of the contact system during CPB increases plasma bradykinin up to 20-fold. Cugno, M. et al. (2006) Chest 120:1776-82; and Campbell, DJ et al. (2001) Am. J. Physiol. Reg. Integr. Comp. Physiol. 281:1059-70.

[0069] The plasma kallikrein inhibitors P8720 and PKSI-527 have also been shown to reduce joint swelling in a rat model of arthritis. De La Cadena, RA et al. (1995) FASEB J. 9:446-52; Fujimori, Y. (1993) Agents Action 39:42-8. Inflammation in animal models of arthritis has also been shown to be accompanied by activation of the contact system. Blais, C. Jr. et al. (1997) Arthritis Rheum. 40:1327-33.

[0070] Furthermore, the plasma kallikrein inhibitor P8720 was found to reduce inflammation in acute and chronic rat models of inflammatory bowel disease (IBD). Stadnicki, A. et al. (1998) FASEB J. 12:325-33; Stadnicki, A. et al. (1996) Dig. Dis. Sci. 41:912-20; and De La Cadena, RA, et al. (1995) FASEB J. 9:446-52. The contact system is activated during acute and chronic small intestinal inflammation. Sartor, RB et al. (1996) Gastroenterology 110:1467-81. It was found that B2 receptor antagonists, antibodies against high molecular weight kininogen, or reduction of kininogen levels reduced clinical pathology in animal models of IBD. Ibid; Arai, Y. et al. (1999) Dig. Dis. Sci. 44:845-51; and Keith, JC et al. (2005) Arthritis Res. Therapy 7: R769-76.

[0071] Hd-Pro-Phe-Arg-chloromethylketone (CMK), an inhibitor of PK and FXII, as well as a physiological inhibitor (C1-inhibitor), was found to reduce vascular permeability in multiple organs and reduce the lesions of lipopolysaccharide (LPS) or bacteria-induced sepsis in animals. Liu, D. et al. (2005) Blood 105:2350-5; Persson, K. et al. (2000) J. Exp. Med. 192:1415-24. Clinical improvement was observed in septic patients treated with C1-inhibitor. Zeerleder, S. et al. (2003) Clin. Diagnost. Lab. Immunol. 10:529-35; Caliezi, C., et al. (2002) Crit. Care Med. 30:1722-8; and Marx, G. et al. (1999) Intensive Care Med. 25:1017-20. Life-threatening cases of sepsis have been shown to have a higher degree of contact activation. Martinez-Brotons, F. et al. (1987) Thromb. Haemost. 58:709-713; and Kalter, ES et al. (1985) J. Infect. Dis. 151:1019-27.

[0072] PrePK levels have also been found to be higher in diabetic patients, especially those with proliferative retinopathy, and correlate with fructosamine levels. Gao, B.-B., et al. (2007) Nature Med. 13:181-8; and Kedzierska, K. et al. (2005) Archives Med. Res. 36:539-43. PrePK has also been found to be highest in patients with sensorimotor neuropathy. Christie, M. et al. (1984) Thromb. Haemostas. (Stuttgart) 52:221-3. PrePK levels are elevated in diabetic patients and associated with increased blood pressure. PrePK levels independently correlate with albumin excretion rate and are elevated in diabetic patients with macroalbuminuria, suggesting that prePK may be a marker for progressive renal damage. Jaffa, AA et al. (2003) Diabetes 52:1215-21. B1 receptor antagonists have been found to reduce plasma leakage in rats treated with streptozotocin. Lawson, SR et al. (2005) Eur. J. Pharmacol. 514:69-78. B1 receptor antagonists can also prevent streptozotocin-treated mice from developing hyperglycemia and renal insufficiency. Zuccollo, A. et al. (1996) Can. J. Physiol. Pharmacol. 74:586-9.

[0073] Compound I may be used as a medicine.

[0074] For example, compound I may be used in a method for treating or preventing a disease or condition characterized by abnormal plasma kallikrein activity. The method includes administering a therapeutically effective amount of compound I to a subject in need thereof, thereby treating or preventing a disease or condition characterized by abnormal plasma kallikrein activity. By reducing the subject's plasma kallikrein activity, the disease or condition characterized by abnormal plasma kallikrein activity is treated.

[0075] The terms "treat", "treating", and "treatment" as used herein mean to prevent, stop or slow the progression of, or eliminate a disease or condition in a subject. In some embodiments, "treat", "treating", and "treatment" mean to stop or slow the progression of, or eliminate a disease or condition in a subject. In some embodiments, "treat", "treating", and "treatment" mean to reduce at least one of the desired symptoms of a disease or condition in a subject.

[0076] The term "effective amount," as used herein, refers to an amount sufficient to produce a desired biological effect.

[0077] The term "therapeutically effective amount," as used herein, refers to an amount sufficient to produce a desired therapeutic effect.

[0078] The term "inhibit" as used herein means to reduce by an objectively measurable amount or extent. In various embodiments, "inhibit" means to reduce by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 percent compared to a relevant control. In one embodiment, "inhibit" means to reduce by 100 percent, i.e., to stop or eliminate.

[0079] The term "subject" as used herein refers to a mammal. In various embodiments, the subject is a mouse, rat, rabbit, cat, dog, pig, sheep, horse, cow, or non-human primate. In one embodiment, the subject is a human.

[0080] Alternatively, in certain embodiments, Compound I may be used for the treatment of diseases or conditions characterized by abnormal plasma kallikrein activity.

[0081] Alternatively, in certain embodiments, Compound I may be used for the manufacture of a medicament for use in the treatment of a disease or condition characterized by abnormal plasma kallikrein activity.

[0082] As used herein, a "disease or condition characterized by abnormal plasma kallikrein activity" refers to any disease or condition in which it is desirable to reduce plasma kallikrein activity. For example, it may be desirable to reduce plasma kallikrein activity in situations of inappropriate activation or overactivation of kallikrein. As another example, it may be desirable to reduce plasma kallikrein activity in situations of hypercoagulable states. As another example, it may be desirable to reduce plasma kallikrein activity in situations of tissue ischemia associated with the presence or formation of a thrombus.

[0083] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is selected from the group consisting of stroke, inflammation, reperfusion injury, acute myocardial infarction, deep vein thrombosis, post-fibrinolytic treatment conditions, angina, edema, angioedema, hereditary angioedema, sepsis, arthritis, hemorrhage, blood loss during cardiopulmonary bypass, inflammatory bowel disease, diabetes mellitus, retinopathy, diabetic retinopathy, diabetic macular edema, diabetic macular degeneration, age-related macular edema, age-related macular degeneration, proliferative retinopathy, neuropathy, hypertension, cerebral edema, increased albumin excretion, macroalbuminuria, and nephropathy.

[0084] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is angioedema.

[0085] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is acquired angioedema or hereditary angioedema (HAE).

[0086] Acquired angioedema (AAE) (Caldwell JR, et al. Clin Immunol Immunopathol. 1972;1:39- 52) is characterized in several ways, including angioedema symptoms involving acquired deficiency of C1 inhibitor (C1-INH), overactivation of the classical pathway of human complement, and bradykinin released by inappropriate activation of the contact kinin system. AAE may exist in two forms: type 1 AAE (usually associated with another disease) and type II AAE, which is usually associated with an autoimmune disease. AAE may be caused by several factors, including, but not limited to, autoimmune disease (e.g., production of anti-C1INH antibodies) or by acquired mutations in C1 INH. Additionally, Compound I may be used to treat side effects of angiotensin-converting enzyme (ACE) inhibitor treatment. ACE inhibitors block a pathway important in the breakdown of bradykinin. Inhibiting kallikrein production through the use of Compound I reduces the production of bradykinin.

[0087] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is hereditary angioedema (HAE). In certain embodiments, the hereditary angioedema is type I hereditary angioedema. Alternatively, the hereditary angioedema may be type II hereditary angioedema. Alternatively, the hereditary angioedema may be type III hereditary angioedema.

[0088] In certain embodiments, Compound I is used for the prophylactic treatment of HAE. In other embodiments, Compound I is used for the acute treatment of HAE.

[0089] In certain embodiments, Compound I is used to prevent or treat angioedema attacks in subjects with HAE. In certain embodiments, Compound I is used as a prophylactic treatment to reduce the frequency of angioedema attacks in subjects with HAE. In other embodiments, Compound I is used to treat acute angioedema attacks in subjects with HAE.

[0090] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is stroke.

[0091] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is reperfusion injury.

[0092] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is acute myocardial infarction.

[0093] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is bleeding.

[0094] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is blood loss during cardiopulmonary bypass.

[0095] In certain embodiments, the disease or condition characterized by abnormal plasma kallikrein activity is selected from the group consisting of retinopathy, diabetic retinopathy, diabetic macular edema, diabetic macular degeneration, age-related macular edema, age-related macular degeneration, and proliferative retinopathy.

[0096] Formulations, Routes of Administration, and Dosing Compound I, synthesized as described herein, can be formulated as a pharmaceutical composition and administered to a mammalian host, such as a human patient, in a variety of forms compatible with the selected route of administration, for example, orally or parenterally by intravenous, intraperitoneal, intramuscular, topical, or subcutaneous routes. Additional routes of administration are also contemplated by the present invention.

[0097] Thus, compound I (also referred to herein as "active compound") may be administered systemically, for example, orally, in combination with a pharma- ceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or mixed directly with the food of the patient's diet. For therapeutic oral administration, the active compound may be mixed with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, vary and may conveniently be between about 2% and about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level is obtained.

[0098] Tablets, troches, pills, capsules, etc. may also contain the following diluents and carriers: binders, such as gum tragacanth, gum arabic, corn starch, or gelatin; excipients, such as calcium hydrogen phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, fructose, lactose, or aspartame, or flavorings, such as peppermint, mung bean oil, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain a liquid carrier, such as vegetable oil or polyethylene glycol, in addition to the above types of materials. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetener, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavoring.Of course, any material used in preparing any unit dosage form must be pharma-ceutically acceptable and substantially non-toxic in the amounts employed.Furthermore, the active compound may be incorporated into sustained-release preparations and devices.

[0099] The active compound may also be administered intravenously or intraperitoneally by infusion or injection.The solution of the active compound may be prepared in water or a physiologically acceptable aqueous solution, optionally mixed with a non-toxic surfactant.Dispersions may also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, as well as in oil.Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0100] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions containing the active compound, optionally encapsulated in liposomes, or sterile powders suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion medium, including, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Prevention of microbial activity may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0101] Sterile injectable solution is prepared by mixing the active compound in the required amount in a suitable solvent with various other components as listed above as necessary, followed by filtration sterilization.In the case of sterile powder for preparing sterile injectable solution, the preparation method can include vacuum drying and freeze-drying technology, which produces a powder of active compound and any additional desired components present in the previously sterile-filtered solution.

[0102] For topical administration, the compounds I may be utilized in pure form, i.e., when they are formulated as liquids, although it is generally desirable to administer them to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be a solid or liquid.

[0103] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, alcohol or glycol or water-alcohol / glycol blends in which Compound I can be dissolved or dispersed at effective levels, optionally with non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents may be added to optimize the properties for a given use. The resulting liquid compositions may be applied from absorbent pads, used in impregnated bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprays.

[0104] Thickening agents, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials may also be utilized with liquid carriers to produce spreadable pastes, gels, ointments, soaps, and the like for application directly to the user's skin.

[0105] Examples of useful dermatological compositions that can be used to deliver Compound I to the skin are known in the art, see, e.g., Jacquet et al. (U.S. Pat. No. 4,608,392, incorporated herein by reference), Geria (U.S. Pat. No. 4,992,478, incorporated herein by reference), Smith et al. (U.S. Pat. No. 4,559,157, incorporated herein by reference), and Wortzman (U.S. Pat. No. 4,820,508, incorporated herein by reference).

[0106] Useful dosages of Compound I can be determined, at least initially, by comparing the in vitro activity with the in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known in the art; see, for example, U.S. Patent No. 4,938,949, incorporated herein by reference.

[0107] The amount of Compound I required for use in treatment will vary with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and is ultimately at the discretion of the attending physician or clinician.

[0108] In general, however, suitable dosages will be in the range of about 0.5 to about 100 mg / kg of recipient body weight per day, e.g., about 3 to about 90 mg / kg of body weight per day, about 6 to about 75 mg per kilogram of body weight per day, about 10 to about 60 mg / kg of body weight per day, or about 15 to about 50 mg / kg of body weight per day.

[0109] Compound I is conveniently administered in an amount per unit dosage form, for example, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, and other such unit dosage amounts within the unit dosage ranges set forth below, for example, 5 to 1000 mg, 10 to 750 mg, 50 to 500 mg, 75 mg to 350 mg, 75 mg to 300 mg, 75 mg to 250 mg. g, 75mg to 200mg, 75mg to 175mg, 75mg to 150mg, 75mg to 125mg, 100mg to 750mg, 100mg to 500mg, 100mg to 350mg, 100mg to 300mg, 100mg to 250mg, 100mg to 200mg, 100mg to 175mg, 100mg to 150mg, 100mg to 125mg, 125mg to 350mg, 125mg to 300mg, 125mg to 250mg, 125mg to 200mg, 125mg to 175mg, 125mg to 150mg of active compound. In one embodiment, the present invention provides a composition comprising Compound I formulated in such a unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, e.g., two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into several loosely spaced separate administrations.

[0110] Compound I may also be administered in combination with other therapeutic agents, for example, other agents useful for treating or preventing ischemia, blood loss, or reperfusion injury.

[0111] Other delivery systems can include sequential sustained release, delayed release, or sustained release delivery systems as known in the art. Such systems can avoid repeated administration of active compounds, increasing convenience for subjects and physicians. Many types of release delivery systems are available and known to those skilled in the art. In some cases, it may be desirable to use long-term sustained release implants. Long-term release, as used herein, means that the delivery system or implant is constructed and arranged to deliver therapeutic levels of active compounds for at least 30 days, preferably 60 days.

[0112] In certain embodiments, Compound I is formulated for intraocular administration, eg, direct injection or insertion in or in association with an intraocular medical device.

[0113] Compound I may be formulated for deposition on a medical device, which may include any of a variety of conventional grafts, stents, including stent grafts, catheters, balloons, baskets, or other devices that may be placed or permanently implanted in a body lumen. As a specific example, it would be desirable to have devices and methods that can deliver Compound I to areas of the body that are treated by interventional techniques.

[0114] In an exemplary embodiment, Compound I may be deposited within a medical device, such as a stent, and delivered to a treatment site for treatment of a body part.

[0115] Stents have been used as delivery vehicles for therapeutic agents (i.e., drugs). Intravascular stents are generally permanently implanted in coronary or peripheral vessels. Stent designs include those in U.S. Pat. No. 4,733,655 (Palmaz), U.S. Pat. No. 4,800,882 (Gianturco), or U.S. Pat. No. 4,886,062 (Wiktor). Such designs include both metallic and polymeric stents, as well as self-expanding and balloon-expandable stents. Stents may also be used to deliver drugs to a site of contact with the vasculature, as disclosed, for example, in U.S. Pat. No. 5,102,417 (Palmaz), U.S. Pat. No. 5,419,760 (Narciso, Jr.), U.S. Pat. No. 5,429,634 (Narciso, Jr.), and International Patent Applications WO 91 / 12779 (Medtronic, Inc.) and WO 90 / 13332 (Cedars-Sanai Medical Center).

[0116] The term "deposited" means that the active compound is coated, adsorbed, placed, or otherwise incorporated into the device by methods known in the art. For example, the compound is embedded in and released from a polymeric material that coats or spreads on the medical device ("matrix type") or is surrounded by and released through a polymeric material ("reservoir type"). In the latter example, the compound may be encapsulated within or bound to the polymeric material using one or more techniques for producing such materials known in the art. In other formulations, the compound may be linked to the surface of the medical device without the need for a coating, for example by a detachable bond, and may be released over time or removed by an effective mechanical or chemical process. In other formulations, the compound may be in a permanently fixed form that provides the compound at the insertion site.

[0117] In certain embodiments, the active compound may be mixed with the polymer composition during the formation of a biocompatible coating for a medical device such as a stent. The coatings produced from such components are generally homogeneous and are useful for coating many devices designed for implantation.

[0118] The polymer may be either a biostable or bioabsorbable polymer depending on the desired release rate or degree of polymer stability desired, although bioabsorbable polymers are often preferred for this embodiment because, unlike biostable polymers, they do not persist long after implantation to cause any long-term local adverse reactions. Potentially usable bioabsorbable polymers include 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 (PEO), polydioxanone (PDS), polyphosphate esters, polyphosphates. These include, but are not limited to, ester urethanes, poly(amino acids), cyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonates), copoly(ether-esters) (e.g., PEO / PLA), polyalkylene oxalates, polyphosphazenes, and biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid, polyepsilon caprolactone, polyhydroxybutyrate, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, crosslinked hydrogels or amphiphilic block copolymers, and other suitable bioabsorbable polymers known in the art.Also, biostable polymers with relatively low long-term tissue reactions, such as polyurethanes, silicones, and polyesters, can be used and can be dissolved and cured or polymerized onto the medical device. Other polymers, e.g., polyolefins, polyisobutylene, and ethylene-alpha-olefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers, e.g., polyvinyl chloride; polyvinylpyrrolidone; polyvinyl ethers, e.g., polyvinyl methyl ether; polyvinylidene halides, e.g., polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketone; polyvinyl aromatics, e.g., polystyrene, polyvinyl esters, e.g., polyvinyl acetate; copolymers of vinyl monomers and olefins with each other, e.g., ethylene glycol; Other examples that may be used include ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; pyran copolymers; polyhydroxypropyl methacrylamide 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; nitrocellulose; cellulose propionate; cellulose ethers; and carboxymethylcellulose.

[0119] The polymers and semipermeable polymer matrices may be formed into shaped articles such as valves, stents, tubes, prostheses, and the like.

[0120] In certain embodiments of the invention, Compound I is bound to a polymer or semipermeable polymer matrix that is formed into a stent or stent-graft device.

[0121] Generally, the polymer is applied to the surface of the implantable device by spin coating, dipping, or spraying. Additional methods known in the art can also be utilized for this purpose. Spraying methods include conventional methods as well as microdeposition techniques using inkjet-type dispensers. Additionally, the polymer can be deposited on the implantable device using photopatterning, which places the polymer only on specific parts of the device. The coating of the device results in an even layer around the device, which allows for improved diffusion of various analytes through the device coating.

[0122] In certain embodiments of the present invention, active compound is formulated for release from polymer coating to the environment where medical device is placed.Preferably, compound is released in a controlled manner over an extended time frame (e.g., months) using at least one of several well-known techniques involving polymer carriers or layers that control elution.Some of these techniques are described in US Patent Application No. 2004 / 0243225A1, the entire disclosure of which is fully incorporated herein.

[0123] Additionally, the reagents and reaction conditions of the polymer composition can be manipulated to controllably release the active compound from the polymer coating, for example, as described in U.S. Pat. No. 6,770,729, which is incorporated herein in its entirety. For example, the diffusion coefficient of one or more polymer coatings can be adjusted to control the release of the compound from the polymer coating. In a variation of the present subject matter, the diffusion coefficient of one or more polymer coatings can be controlled to control the ability of an analyte present in the environment in which the medical device is placed (e.g., an analyte that promotes the degradation or hydrolysis of a portion of the polymer) to reach one or more components within the polymer composition (e.g., thereby controlling the release of the compound from the polymer coating). Yet another embodiment of the present invention includes a device having multiple polymer coatings, each having multiple diffusion coefficients. In such an embodiment of the present invention, the release of the active compound from the polymer coating can be controlled by the multiple polymer coatings.

[0124] In yet another embodiment of the invention, the release of the active compound from the polymer coating is controlled by the presence of one or more endogenous or exogenous compounds or by adjusting one or more properties of the polymer composition, such as the pH of the polymer composition. For example, a particular polymer composition can be designed to release a compound in response to a decrease in the pH of the polymer composition.

[0125] kit The invention also provides a kit comprising Compound I, at least one other therapeutic agent, packaging material, and instructions for administering Compound I and the other therapeutic agent(s) to a mammal to treat or prevent a disease or condition characterized by abnormal kallikrein activity in the mammal. In one embodiment, the mammal is a human. EXAMPLES

[0126] Example 1: Synthesis Protocol for Compound A The following scheme and accompanying steps illustrate a protocol for synthesizing compound A. [ka]

[0127] Step 1: (R,E)-N-(3-cyanobenzylidene)-2-methylpropane-2-sulfinamide (compound 03) [ka]

[0128] 3-Cyanobenzaldehyde (compound 01; 53.57 kg, 408.42 mol, 0.9 equiv.) was added to a stirred solution of (R)-(+)-2-methylpropane-2-sulfinamide (compound 02; 55.0 kg, 453.8 mol, 1.0 equiv.) in dichloromethane (DCM) (550.0 L, 10.0 vol) at room temperature. KHSO4 (46.2 kg, 340.35 mol, 0.75 equiv.) was added at room temperature and the reaction mixture was stirred at this temperature for 6 h. The progress of the reaction was monitored by HPLC analysis. The reaction mixture was quenched with water (220.0 L) and stirred for 30 min. The DCM layer was separated and the aqueous layer was extracted again with DCM (110.0 L). The combined organic extracts were washed with sodium metabisulfite (17.24 kg in DMW (165.0 L)) for 2 h. The aqueous layer was extracted with DCM (55.0 L). The combined organics were dried over Na2SO4 (13.75 kg) and filtered. The filtrate was concentrated under reduced pressure at 40° C. Add 110.0 L of n-heptane for crystallization, stir at 10° C. for 2 h, filter, and wash with 27.5 L of n-heptane. The solid was dried in a vacuum tray dryer at 35° C. to give (R,E)-N-(3-cyanobenzylidene)-2-methylpropane-2-sulfinamide (compound 03; 86.23 kg, 81.1%) as an off-white solid. 1HNMR(300MHz,DMSO-d6)δ8.63(s,1H),8.42(d,J=1.7Hz,1H),8.28(dt,J=7.9,1.4Hz,1H),8.07 (dt,J=7.8,1.4Hz,1H),7.76(t,J=7.8Hz,1H),1.21(s,9H);MS(ES+)235.2(M+1),257.2(M+Na).

[0129] Step 2: N-(5-bromo-2-fluorophenyl)-1,1,1-trimethyl-N-(trimethylsilyl)silanamine: [ka]

[0130] Trimethylsilyl trifluoromethanesulfonate (55.1 kg, 249.98 mol) was added to a stirred solution of 5-bromo-2-fluoroaniline (compound 04; 19.0 kg, 99.99 mol) in triethylamine (95.0 L) at room temperature under nitrogen. The reaction mixture was heated to reflux under nitrogen and stirred for 8-14 hours. The progress of the reaction was monitored. 1 The reaction mixture was cooled to room temperature under nitrogen and the layers were separated. The lower layer was collected in a separate HDPE drum under nitrogen. The upper layer was concentrated under reduced pressure at 70° C. to 80° C. The residue was purified by HVD to give the product N-(5-bromo-2-fluorophenyl)-1,1,1-trimethyl-N-(trimethylsilyl)silanamine (compound 05) as a pale yellow oil (29.09 kg, 86.99%).

[0131] Step 3: (3-(bis(trimethylsilyl)amino)-4-fluorophenyl)magnesium bromide: [ka]

[0132] A suspension of oven-dried Mg turnings (4.5 kg, 185.15 mol) and iodine (50.50 g, 0.0013 equiv.) in THF (15 L) was stirred at room temperature under nitrogen for 10 min. N-(5-bromo-2-fluorophenyl)-1,1,1-trimethyl-N-(trimethylsilyl)silanamine (compound 05) (1.5 kg, 4.49 mol) was added slowly to the above suspension at room temperature under nitrogen. An exothermic reaction was observed at the beginning of the reaction. THF (100.0 L) was added to the reaction under nitrogen. The remaining compound 05 (48.5 kg, 145.04 mol) was added to the reaction mixture at such a rate as to maintain the reaction temperature below 45° C. over a period of 5 to 7 hours. The reaction mixture was stirred at room temperature under nitrogen for 10 hours. The reaction mixture was used directly in the next step. Qty: 150.0 L

[0133] Step 4: (R)-N-((R)-(3-amino-4-fluorophenyl)(3-cyanophenyl)methyl)-2-methylpropane-2-sulfinamide (compound A): [ka]

[0134] A stirred solution of (R,E)-N-(3-cyanobenzylidene)-2-methylpropane-2-sulfinamide (compound 03) (62.0 kg, 264.6 mol) in toluene (1240.0 L) was cooled to -60 to -50°C. Grignard reagent solution (3-(bis(trimethylsilyl)amino)-4-fluorophenyl)magnesium bromide (compound 06) (450.0 L) was added under nitrogen over a period of 2 hours to maintain the reaction temperature between -60 to -35°C. The reaction mixture was stirred under nitrogen at -60 to -35°C for 1 hour before being quenched with aqueous KHSO4 (99.2 kg KHSO4 in 744 L DM W) at -60 to 15°C and the mixture was stirred at room temperature for 1 hour before the layers were separated. The lower aqueous layer was removed and back extracted with toluene (341.0 L). The combined organic layers were washed with brine (105.4 kg NaCl in 527.0 L DM W). The organic layer was dried over Na2SO4 (43.4 kg), filtered, and the filtrate was concentrated under reduced pressure at 65° C. to give crude compound A (99.0 kg) as a sticky reddish crude.

[0135] The sticky reddish crude compound A (99.1 kg) was dissolved in toluene (893.0 L) at 40-45° C. The solution was slowly cooled to room temperature. The solution was vigorously stirred and n-heptane (347.2 L) was added at room temperature. The mixture was stirred at 15-20° C. for 5 hours and the precipitated solid was collected by filtration, washed with a mixture of 20 parts toluene to 80 parts n-heptane (97.0 L) and dried at room temperature for 24 hours to give the product as a light brown solid (40.36 kg, 44.16%). mp 100.6° C.

[0136] Step 5: Recrystallization of (R)-N-((R)-(3-amino-4-fluorophenyl)(3-cyanophenyl)methyl)-2-methylpropane-2-sulfinamide (Compound A): [ka]

[0137] (R)-N-((R)-(3-amino-4-fluorophenyl)(3-cyanophenyl)methyl)-2-methylpropane-2-sulfinamide (compound A) (81.0 kg) was dissolved in toluene (567.0 L) at 40-50 °C. The reaction mixture was cooled to room temperature and then filtered through a hyflow bed and washed with toluene (162.0 L). The solution was charged into a reactor. The solution was vigorously stirred and n-heptane (324.0 L) was added at room temperature. The mixture was stirred at 15-20° C. for 5 hours, and the precipitated solid was collected by filtration, washed with a mixture of 10 parts toluene to 90 parts n-heptane (81.0 L), and dried at room temperature for 24 hours to give the product, (R)-N-((R)-(3-amino-4-fluorophenyl)(3-cyanophenyl)methyl)-2-methylpropane-2-sulfinamide (Compound A) as an off-white solid (67.96 kg, 83.9%). 1 HNMR(300MHz,DMSO-d6)δ7.82(d,J=1.7Hz,1H),7.70(ddt,J=8.1,5.2,1.4Hz,2H),7.54(t,J=7.7Hz,1H),6.93(dd,J=11.4,8.3Hz,1H),6.73(dd,J =8.8,2.3Hz,1H),6.57(ddd,J=8.4,4.4,2.2Hz,1H),5.99(d,J=6.0Hz,1H),5.48(d,J=6.0Hz,1H),5.14(s,2H),1.13(s,9H);MS(ES+)346.3(M+1).

[0138] Example 2: Synthesis protocol for compound I·2HCl The following scheme and accompanying steps illustrate a protocol for synthesizing the crystalline bis(hydrochloride) salt of Compound I. [ka]

[0139] Step 0: Preparation of Compound B HCl (100 kg scale) Compound A (151 kg) and 2-propanol (501 L) were charged to the reactor and stirred at 35±5°C until a clear solution was obtained. After temperature adjustment to 30-35°C, concentrated hydrochloric acid (37% aqueous solution, 64 kg, 1.5 equiv.) was transferred to the reactor over at least 10 minutes to maintain the reactor temperature at ≦35°C. The temperature of the reactor contents was adjusted to 25±5°C and after 6±1 hours of stirring the contents were cooled to 10±5°C and an IPC sample was collected for conversion analysis by IPC HPLC. When conversion to the reaction product, compound B HCl, was <99.5%, the reaction mixture was brought to 25±5°C and stirred for an additional 3±1 hours before being cooled again to 10±5°C and a new IPC sample was collected for analysis.

[0140] The reactor contents were aged at 10±5°C for 20-35 hours. The product slurry was transferred to a centrifuge and the product was isolated by centrifugation and washed with 2-propanol. After dry spinning the product, compound B HCl (wet) was drained from the centrifuge. The product was dried under vacuum at ≦30°C for ≥3 hours and ≦40°C for ≥2 hours. Yield (approximately 80%). 1 HNMR(300MHz,DMSO-d6)δ9.28(s,3H),8.04(s,1H),7.88-7.79(m,2H),7.63(t,J= 7.8Hz,1H),7.10-6.98(m,1H),6.76(d,J=10.3Hz,2H),5.58(s,1H),5.32(s,2H).

[0141] Step 1: Preparation of Compound C Oxalate (100 kg scale) Compound B HCl (94 kg) and methanol (412 L) were charged to the reactor and the temperature of the contents was adjusted to -10±5°C. 30% sodium methoxide in methanol (46 kg, 0.76 equiv.) was charged to maintain the internal reactor temperature at -10±5°C. The reaction mixture was stirred for 60 min or more, the pH was checked, and further adjusted to pH=7-8, if necessary, using optional portions of 30% sodium methoxide in methanol (2.6 kg, 0.04 equiv.). The reaction mixture was cooled to -22±4°C and pre-weighed cyclopropanecarboxaldehyde (23.4 kg, 0.99 equiv.) was charged in five portions to maintain the internal reactor temperature ≦-10°C. The reactor contents were then stirred at a temperature of -20±5°C for 1.5 h or more.

[0142] Sodium borohydride (4.5 kg, 0.35 equiv.) was charged simultaneously in one portion to maintain a reactor temperature of -20±5°C. The reactor contents were then stirred at a temperature of -20±5°C for ≥30 min and IPC assayed for conversion to compound C. If necessary, any portion of sodium borohydride (0.32 kg, 0.025 equiv.) was charged, followed by stirring of the reactor contents at -20±5°C for ≥30 min. Once the reaction was complete (≥95.0% conversion to compound C), methanol was distilled under reduced pressure at a jacket temperature ≤50°C to volumetric basis (approximately 213 L). Extraction with toluene (339 L) and process water (121 L) was performed at an internal temperature of 30±5°C, followed by separation for ≥15 min. The lower aqueous phase was discarded. 10% sodium chloride (99 kg) and process water (121 L) were premixed and then charged to the reactor at 30±5° C., followed by separation over 15 min. The lower aqueous phase was discarded. The process by vacuum distillation of toluene, jacket temperature≦50° C., was continued up to the specified volumetric standard (approximately 221 L).

[0143] Oxalic acid dihydrate (42 kg, 1 equiv.) and 2-propanol (254 L) were charged to the reactor and the content temperature was adjusted to 30 ± 5 °C. The content was then stirred for ≥ 30 min until a clear solution was obtained. The concentrated toluene phase from the above step was then transferred, the reactor temperature was adjusted to 30 ± 5 °C, stirred for ≥ 30 min, and checked for crystallization. The reactor content was cooled to 20 ± 5 °C and stirred for ≥ 6 h. The precipitated product was transferred to a centrifuge and washed with premixed toluene (59 L) and 2-propanol (62 L). After dry spinning the product, the compound C oxalate (wet) was discharged from the centrifuge. Recrystallization was carried out by charging the entire discharged batch of compound C oxalate (wet) to the reactor, followed by charging preheated (80-83 °C) 2-propanol (516 L) and toluene (348 L). The reactor contents were rapidly heated to 79±3° C. and stirred until a clear solution was obtained. Process water (6.1 L, 1 equiv.) was charged to the reactor contents and the contents were cooled to 20±3° C. and stirred for at least 12 hours until crystallization occurred. The product was isolated by centrifugation and washed with premixed isopropanol (62 L) and toluene (59 L). The product was drained, an IPC sample was taken, and a second recrystallization was performed using the same procedure as previously described and an IPC sample was taken (compound B≦0.05%, RRT 1.39≦1.04%). The product was dried in a cone dryer first at ≦30° C. for ≥3 hours and then at ≦40° C. for ≥3 hours. The dried material was drained to give compound C oxalate (yield approx. 77%). 1 HNMR (300MHz, deuterium oxide) δ7.77(d,J=3.3Hz,2H),7.73(s,1H),7.58(t,J=7.8Hz,1H),7.11(dd,J=10.9,8.4Hz,1H),6.98(dd,J=8.2,2.3Hz,1H),6 .86(dt,J=7.0,3.2Hz,1H),5.51(s,1H),2.88(d,J=7.4Hz,2H),1.03(tq, J=7.9,3.9,3.0Hz,1H),0.63(d,J=7.7Hz,2H),0.24(p,J=5.5,5.1Hz,2H).

[0144] Step 2: Preparation of Compound D (100 kg scale; 500 kg of toluene solution, 20 w / w% Compound D) A premix of KOH solution was prepared by charging water (141 L) and potassium hydroxide (24 kg, 85%, 2.1 equiv.). The mixture was stirred at 20±5° C. until a clear solution was obtained. Compound C oxalate (67 kg), 2-propanol (10 L) and toluene (292 L) were charged to the reactor and the reactor contents were stirred at 20±5° C. for 15 min or more. The prepared potassium hydroxide solution was transferred to another reactor at a temperature of ≦50° C. The reaction mixture was then heated to 50±5° C. and stirred until a clear solution was obtained. After checking the pH (≧13) of the lower aqueous phase, the reactor contents were allowed to separate for 10 min or more and the lower aqueous phase was discarded.

[0145] The remaining toluene phase was washed with process water (156 L) at 50±5°C and allowed to separate for over 15 minutes, followed by removal of the lower aqueous phase. The procedure was repeated and an IPC sample of the organic phase was taken (RRT 0.85, NMT 0.10%). The toluene phase was distilled under reduced pressure with a jacket temperature ≦70°C to a specified volume (173 L). Toluene (192 L) was charged and the distillation was repeated using the same procedure.

[0146] A separate reactor was charged with compound F (73.5 kg, 1.1 equiv. as 100%) through the manhole, the reactor was stirred at 20±5° C. for not less than 15 minutes, and the first portion of pyridine (13.7 kg, 1.0 equiv.) was charged. The contents were then stirred until a clear solution was obtained.

[0147] T3P in EtOAc, 50% (148.7 kg, 50%, 1.35 equiv.) was charged to the above solution at a temperature ≦3° C. The reactor contents were heated to 20±5° C. and the mixture was stirred for 1.5-2.5 hours before charging a second portion of pyridine (6.9 kg, 0.5 equiv.). After stirring the contents for 2-4 hours at 20±5° C., an IPC sample of the reaction mixture (including both the aqueous and organic phases) was taken for conversion analysis (compound D≧99.6%). Process water (265 L) and a third portion of pyridine (20.6 kg) were charged to the reactor. The reaction mixture was stirred at 35-40° C. for 15 minutes, then allowed to separate for 20 minutes or more, and the lower aqueous phase was discarded. The organic phase was washed with process water (265 L) and stirred at 35-40° C. for 15 minutes or more, then allowed to separate for 20 minutes or more, followed by removal of the lower aqueous phase. The procedure was repeated as necessary. A premixed solution of aqueous sodium bicarbonate (5%), water (487 L) and NaHCO3 (24.8 kg, 1.7 equiv.) was then transferred and the contents were stirred at 38±3°C for 20 min or more, allowed to separate for 20 min or more and the lower aqueous phase was discarded. The process continued in the reactor at 35-40°C by washing the contents with process water (266 L) followed by separation and removal of the lower aqueous phase.

[0148] A vacuum distillation of the reactor contents was performed in the reactor to a volume of ≦284 L at a Tm<70° C. Toluene (approximately 280 L) was charged to the specified target volume (approximately 563 L). The product (compound D) solution was cooled to 4±5° C. and sampled and discharged into a nitrogen flushed barrel before being stored in a cooling vessel at 4±5° C. 1 HNMR(300MHz,DMSO-d6)δ10.56(s,1H),7.89(t,J=1.6Hz,1H),7.74(d,J=7 .7Hz,1H),7.69-7.61(m,2H),7.58(s,1H),7.54-7.31(m,7H),7.22(dd,J=1 0.3,8.5Hz,1H),4.93(s,1H),4.19(d,J=6.2Hz,2H),2.25(s,2H),1.37(s,9 H),1.26(s,1H),1.00-0.80(m,1H),0.48-0.30(m,2H),0.10--0.03(m,2H); 19FNMR(282MHz,DMSO)a-60.62,-123.00;MS(ES+)663.5(M+1).

[0149] Step 3: Preparation of Compound I 2×HCl (100 kg scale) The reactor was charged with compound D (119.9 kg, as 614.7 kg of 19.5% toluene solution) and the solution was vacuum distilled to a volume content of 283 L at a jacket temperature ≦70° C. for removal of toluene. Solvent exchange was performed by charging 2-propanol (807 L) to the reactor followed by vacuum distillation to a volume content of 283 L at Tm<70° C. The procedure was repeated once and the distilled solution was then diluted with 2-propanol (306 L) and water (542 L) and the temperature was adjusted to 20±5° C. 37% hydrochloric acid (115.2 kg; 6.46 equiv.) was charged to the reactor to maintain the internal reactor temperature ≦45° C. The reaction mixture was then stirred at 40-47° C. for 5-12 h, after which IPC samples were taken for conversion control (compound I≧98%). The reactor contents were washed with toluene (420 L) and stirred at 35-40°C for at least 15 minutes, followed by separation of the product phase (lower aqueous phase) at 35-40°C for at least 15 minutes. The organic waste phase (upper phase) was discarded. This procedure was repeated twice (first with 540 L toluene and then with 633 L toluene). Toluene (662 L) was charged to the aqueous product phase, 25% aqueous ammonia (137 kg, 11.2 equiv.) was charged to the reactor via glass at a temperature ≦40°C, the basic mixture was stirred at 35-40°C for at least 30 minutes, the pH was checked (pH ≧10), followed by stopping of stirring to allow separation of the lower aqueous phase and the upper organic phase containing the product Compound I free base. The lower aqueous phase was discarded. The organic product phase was washed with process water (614 L) at a temperature of 35-40°C, and the aqueous phase was discarded. This procedure was repeated once. The toluene phase was distilled in the reactor under reduced pressure with a jacket temperature ≦70° C. to a specific volume (314 L). A solvent exchange to MTBE (627 L) was performed under reduced pressure distillation at Tm<70° C. The procedure was repeated once and the remaining contents in the reactor were adjusted by further addition or further distillation of MTBE to obtain a controlled specific volume (max 1372 L, min 1262 L) at 20±5° C. The contents were then cooled to −7±3° C.

[0150] Aqueous 37% hydrochloric acid (38.1 kg, 32.3 L, 2.14 equiv.) was charged to a clean, empty crystallization vessel, methanol (228.9 kg, 39.5 equiv.) was added as a rinse, and the contents were cooled to -7 ± 3 °C. The MTBE phase from the preceding step was filtered into the crystallization vessel through a polishing filter at a temperature of -5 ± 5 °C. After rinsing with MTBE, pre-weighed compound I 2 × HCl seeds (1.39 kg, 0.012 equiv.) were charged through the manhole. The vessel contents were heated to 30-33 °C, and the stirring speed was set to 25-50 rpm. After confirmation of crystallization, the slurry was stirred for an additional 3 to 4 hours. The product slurry was transferred to a centrifuge, isolated by centrifugation, and the product was washed with MTBE (585 L). After dry spinning the product, compound I 2×HCl (wet) was discharged from the centrifuge and the product was dried in a cone dryer under vacuum at ≦40° C. Typical yield=74-86%.

[0151] 1 The H NMR (300 MHz, DMSO-d6) data is shown in the table below. [Table 1]

[0152] 19 F NMR (282 MHz, DMSO-d6) data is shown in the table below. [Table 2]

[0153] Compound I has two basic sites: the conjugate acid of the primary amine is calculated to have a pKa value of 8.89, and the conjugate acid of the secondary amine is calculated to have a pKa value of 7.86.

[0154] Example 3: Compound Assay Compound I was assayed in an in vitro biochemical assay to determine the inhibition of human plasma kallikrein activity. The experimental protocol and assay results can be found in WO2015 / 134998 and US Patent Application Publication No. 2017 / 0073314A1 (both of which are incorporated by reference). The results of this biochemical assay show that Compound I is a potent inhibitor of human plasma kallikrein activity.

[0155] Incorporation by Reference All U.S. patents and U.S. and PCT published patent applications referred to herein are hereby incorporated by reference in their entirety as if each individual patent or published application was specifically and individually indicated to be incorporated by reference. In the case of conflict, the present application, including any definitions herein, will control.

[0156] Equivalent While specific embodiments of the subject invention have been described, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the appended claims. The full scope of the invention should be determined by reference to the specification, along with the claims and such variations, along with their full scope of equivalents. Finally, preferred embodiments of the present invention are described in sections. [Embodiment 1] mixing compound C, or a salt thereof, and compound F, or a salt thereof, under conditions sufficient to produce compound D, or a salt thereof; Compound C is [ka] and compound F is represented by [ka] and compound D is represented by [ka] The method is represented by [Embodiment 2] 2. The method of embodiment 1, wherein the conditions sufficient to produce compound D comprise an amide coupling reagent and a first base. [Embodiment 3] 3. The method of embodiment 2, wherein the amide coupling reagent is propylphosphonic anhydride (T3P), N,N'-di(isopropyl)carbodiimide, N,N'-di(cyclohexyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, or ethyl 2-cyano-2-(hydroxyimino)acetate. [Embodiment 4] 4. The method of embodiment 3, wherein the amide coupling reagent is propylphosphonic anhydride (T3P). [Embodiment 5] 5. The method according to any one of embodiments 2 to 4, wherein the first base is a first organic base. [Embodiment 6] 6. The method of any one of embodiments 2 to 5, wherein the first base is triethylamine, pyridine, diisopropylethylamine, diisopropylmethylamine, imidazole, pyrimidine, N-methylmorpholine, quinuclidine, or 1,4-diazabicyclo[2.2.2]octane (DABCO). [Embodiment 7] 7. The method according to any one of embodiments 2 to 6, wherein the first base is pyridine. [Embodiment 8]

[0023] 8. The method of any one of embodiments 1-7, wherein the conditions sufficient to produce compound D further comprise a first solvent. [Embodiment 9] 9. The method of embodiment 8, wherein the first solvent is a polar aprotic solvent. [Embodiment 10] 10. The method of embodiment 9, wherein the first solvent is dichloromethane, tetrahydrofuran, acetone, acetonitrile, or ethyl acetate. [Embodiment 11] 11. The method of embodiment 9 or 10, wherein the first solvent is ethyl acetate. [Embodiment 12] Compound C exists as an acid salt, The method further comprises the step of combining the acid salt of compound C with a second water-soluble base, thereby producing a free base of compound C; 12. The method of any of the preceding embodiments, wherein the step of combining the acid salt of compound C with a second water-soluble base is performed prior to combining compound C and compound F. [Embodiment 13] The method of embodiment 12, wherein the acid salt of compound C is a hydrochloride, a hydrobromide, a hydroiodide, or an oxalate. [Embodiment 14] The method of embodiment 13, wherein the acid salt of compound C is an oxalate salt. [Embodiment 15] 15. The method of any one of embodiments 12-14, wherein the second water-soluble base comprises potassium hydroxide, sodium hydroxide, ammonium hydroxide, potassium bicarbonate, sodium bicarbonate, potassium carbonate, or sodium carbonate. [Embodiment 16] 16. The method of any one of embodiments 12 to 15, wherein the second water-soluble base comprises potassium hydroxide. [Embodiment 17] (a) compound B, or a salt thereof, and

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Claims

1. mixing Compound C, or a salt thereof, with Compound F, or a salt thereof, under conditions sufficient to produce Compound D, or a salt thereof; Compound C is 【Chemistry 1】 is represented by Compound F is 【Chemistry 2】 is represented by Compound D is 【Chemistry 3】 is represented by The conditions sufficient to produce compound D include an amide coupling reagent and a first base; the amide coupling reagent is propylphosphonic anhydride (T3P), N,N'-di(isopropyl)carbodiimide, N,N'-di(cyclohexyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, or ethyl 2-cyano-2-(hydroxyimino)acetate; the first base is triethylamine, pyridine, diisopropylethylamine, diisopropylmethylamine, imidazole, pyrimidine, N-methylmorpholine, quinuclidine, or 1,4-diazabicyclo[2.2.2]octane (DABCO); The method wherein compound C is used in an amount of at least 1 kg.

2. 2. The method of claim 1, wherein the amide coupling reagent is propylphosphonic anhydride (T3P).

3. 3. The method of claim 1 or 2, wherein the first base is pyridine.

4. 4. The method of any one of claims 1 to 3, wherein the conditions sufficient to produce compound D further comprise a first solvent.

5. The method of claim 4 , wherein the first solvent is a polar aprotic solvent.

6. 6. The method of claim 5, wherein the first solvent is dichloromethane, tetrahydrofuran, acetone, acetonitrile, or ethyl acetate.

7. 7. The method of claim 5 or 6, wherein the first solvent is ethyl acetate.

8. Compound C exists as an acid salt; The method further comprises the step of combining the acid salt of Compound C with a second water-soluble base, thereby producing a free base of Compound C; 8. The method of claim 1, wherein the step of mixing the acid salt of compound C with a second water-soluble base is performed before mixing compound C with compound F.

9. 9. The method of claim 8, wherein the acid salt of compound C is a hydrochloride, a hydrobromide, a hydroiodide, or an oxalate.

10. 10. The method of claim 9, wherein the acid salt of compound C is an oxalate salt.

11. 11. The method of any one of claims 8 to 10, wherein the second water-soluble base comprises potassium hydroxide, sodium hydroxide, ammonium hydroxide, potassium bicarbonate, sodium bicarbonate, potassium carbonate, or sodium carbonate.

12. The method of any one of claims 8 to 11, wherein the second water-soluble base comprises potassium hydroxide.

13. (a) compound B or a salt thereof; 【Chemistry 4】 to produce a first reaction mixture, and thereafter (b) combining the first reaction mixture with a reducing agent under conditions sufficient to produce compound C, or a salt thereof; Compound B is 【Chemistry 5】 The method according to any one of claims 1 to 12, wherein

14. The reducing agent is LiAlH ４ or NaBH ４ The method of claim 13, wherein

15. The reducing agent is NaBH ４ The method according to claim 13 or 14, wherein

16. 16. The method of any one of claims 13 to 15, wherein the conditions sufficient to produce compound C further comprise a second solvent.

17. 17. The method of claim 16, wherein the second solvent is a second polar protic solvent.

18. 18. The method of claim 16 or 17, wherein the second solvent is methanol, ethanol, or isopropanol.

19. The method of any one of claims 16 to 18, wherein the second solvent is methanol.

20. 20. The method of any one of claims 13 to 19, further comprising contacting compound C with a first acid to produce an acid salt of compound C.

21. 21. The method of claim 20, wherein the first acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, or oxalic acid, and the acid salt of compound C is a hydrochloride, hydrobromide, hydroiodide, or oxalate.

22. 22. The method of claim 21, wherein the first acid is oxalic acid and the acid salt of compound C is an oxalate salt.

23. Compound B exists as an acid salt; The method further comprises the step of combining the salt of Compound B with a third organic base, thereby producing the free base of Compound B; The step of mixing the salt of compound B with a third organic base comprises mixing compound B with a third organic base. 【Chemistry 6】 The method according to any one of claims 13 to 22, wherein the method is carried out before mixing.

24. 24. The method of claim 23, wherein the acid salt of compound B is a hydrochloride, a hydrobromide, or a hydroiodide salt.

25. 25. The method of claim 24, wherein the acid salt of compound B is a hydrochloride salt.

26. 26. The method of any one of claims 23 to 25, wherein the third organic base comprises sodium methoxide.

27. (a) combining Compound A with a second acid under conditions sufficient to produce Compound B, or a salt thereof; Compound A is 【Chemistry 7】 The method according to any one of claims 13 to 26, wherein the method is represented by:

28. 28. The method of claim 27, wherein the second acid is hydrochloric acid, hydrobromic acid, or hydroiodic acid.

29. 29. The method of claim 28, wherein the second acid is hydrochloric acid.

30. 30. The method of any one of claims 27-29, wherein the conditions sufficient to produce compound B include a third polar protic solvent.

31. 31. The method of claim 30, wherein the third polar protic solvent is methanol, ethanol, or isopropanol.

32. 32. The method of claim 30 or 31, wherein the third polar protic solvent is isopropanol.

33. The method of any one of claims 27 to 32, wherein compound B is produced as an acid salt.

34. 34. The method of claim 33, wherein the acid salt of compound B is a hydrochloride, a hydrobromide, or a hydroiodide salt.

35. 35. The method of claim 34, wherein the acid salt of compound B is a hydrochloride salt.

36. (c) combining Compound D with a deprotection reagent to form a second reaction mixture, and thereafter (d) exposing said second reaction mixture to conditions sufficient to form Compound I as a free base; Compound I is 【Chemistry 8】 The method according to any one of claims 1 to 35, wherein

37. 37. The method of claim 36, wherein the deprotecting reagent is a third acid.

38. 38. The method of claim 37, wherein the third acid is hydrochloric acid, hydrobromic acid, or hydroiodic acid.

39. 39. The method of claim 38, wherein the third acid is hydrochloric acid.

40. 40. The method of any one of claims 36-39, wherein the conditions sufficient to produce compound I as a free base include a fourth base.

41. 41. The method of claim 40, wherein the fourth base is aqueous ammonia.

42. e) providing a first free base mixture of Compound I as a free base in a fourth organic solvent; f) combining the free base mixture with a first reagent solution comprising a fourth acid and a fifth organic solvent under conditions sufficient to produce a third reaction mixture comprising a salt of Compound I; and g) crystallizing the salt of Compound I from a mixture containing said salt of Compound I. The method of any one of claims 36 to 41, further comprising:

43. 43. The method of claim 42, wherein the crystalline salt is the hydrochloride salt.

44. 43. The method of claim 42, wherein the crystalline salt is a bis(hydrochloride) salt.

45. 45. The method of any one of claims 42 to 44, wherein the fourth organic solvent comprises a fourth polar aprotic solvent.

46. 46. ​​The method of claim 45, wherein the fourth polar aprotic solvent is methyl tert-butyl ether.

47. 47. The method of claim 45 or 46, wherein the fourth organic solvent further comprises a fourth non-polar solvent.

48. 48. The method of claim 47, wherein the fourth non-polar solvent is toluene.

49. The method of any one of claims 42 to 48, wherein the fourth acid is hydrochloric acid.

50. 45. The method of any one of claims 42 to 44, wherein the fifth organic solvent is a fifth polar protic solvent.

51. 51. The method of claim 50, wherein the fifth polar protic solvent is methanol.

52. 52. The method of any one of claims 1 to 51, wherein compound C is used in an amount of at least 5 kg, at least 10 kg, at least 15 kg, at least 20 kg, at least 25 kg, at least 30 kg, at least 35 kg, at least 40 kg, at least 45 kg, at least 50 kg, at least 55 kg, or at least 60 kg.

53. The method according to any one of claims 1 to 52, wherein compound C is used in an amount of at least 50 kg.

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