USOO703416OB2

(12) United States Patent (10) Patent No.: US 7,034,160 B2 Woodward et al. (45) Date of Patent: Apr. 25, 2006

(54) CRYSTALLINE FORMS OF A FACTOR XA (52) U.S. Cl...... 54.6/332: 514/357 INHIBITOR (58) Field of Classification Search ...... 514/351, 514/357: 546/332 (75) Inventors: Rick G. Woodward, Harleysville, PA See application file for complete search history. (US); David S. Teager, Marcus Hook, PA (US) (56) References Cited (73) Assignee: Aventis Pharmaceuticals Inc., U.S. PATENT DOCUMENTS Bridgewater, NJ (US) 6,080,767 A * 6/2000 Klein et al...... 514,357 (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. WO WO 97.241.18 7/1997 OTHER PUBLICATIONS (21) Appl. No.: 10/404,678 Halebian et al. J of Pharm Sci., (1969), 58, pp. 911-929.* (22) Filed: Apr. 1, 2003 Chemical & Engineering news, Feb. 2003, pp. 32-35.* US Pharmacopia, 1995, pp. 1843-1844.* (65) Prior Publication Data Concise Encyclopedia Chemistry, pp. 872-873 (1993).* Brittain et al., Polymorphism in Pharmaceutical Solids, NY: US 2003/0225144 A1 Dec. 4, 2003 Marcel Dekker, Inc., 1999, pp. 183-226, 228-330.* Related U.S. Application Data * cited by examiner (63) Continuation of application No. PCT/US01/31087, Primary Examiner Patricia L. Morris filed on Oct. 4, 2001. (74) Attorney, Agent, or Firm—Joseph D. Rossi (60) Provisional application No. 60/238.316, filed on Oct. 5, 2000. (57) ABSTRACT (30) Foreign Application Priority Data The present invention relates to novel crystalline forms of an inhibitor of Factor Xa, processes for its preparation, com Apr. 10, 2001 (GB) ...... O108903 positions comprising it, and its therapeutic use. (51) Int. Cl. C07D 213/89 (2006.01) 4 Claims, 2 Drawing Sheets U.S. Patent Apr. 25, 2006 Sheet 1 of 2 US 7,034,160 B2 OZZ0||200206||08||

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O C O O C O O r CN Sl US 7,034,160 B2 1. CRYSTALLINE FORMS OF AFACTOR XA INHIBITOR (I) This application is a continuation application of Interna tional Application Number PCT/US01/31087, filed Oct. 4, 2001, which, in turn, is entitled to the benefit of, and claims priority from earlier filed U.S. Application No. 60/238.316, filed Oct. 5, 2000, and Great Britain Application Number 0108903.6, filed Apr. 10, 2001, the contents of all of which are incorporated herein by reference. 10 FIELD OF THE INVENTION The present invention relates to novel crystalline forms of an inhibitor of Factor Xa, and its production and use. 15 BACKGROUND OF THE INVENTION N-(aminomethyl) phenylpropyl amide compounds, includ Factor Xa is the penultimate enzyme in the coagulation ing Compound (I), are disclosed in commonly assigned U.S. cascade. Inhibition of Factor Xa may be achieved, for Pat. No. 6,080,767, which is based on an application that example, by direct complex formation between a suitable claims priority benefit under 35 U.S.C. S. 371 of Interna inhibitor and the enzyme and is therefore independent of the tional application Serial No. PCT/US96/20770 (designating plasma co-factor antithrombin III. Effective factor Xa inhi the United States) filed Dec. 23, 1996, which, in turn, claims bition may be achieved by administering compounds by oral priority benefit of U.S. Provisional application Ser. No. administration, continuous intravenous infusion, bolus intra 60/009,485 filed Jan. 2, 1996. venous administration or any other Suitable route Such that it preferably achieves the desired effect of preventing the 25 Treatment and/or prevention of the foregoing pathological Factor Xa induced formation of thrombin from prothrombin. conditions may be accomplished by administering a thera Anticoagulant therapy is often indicated for the treatment peutically effective amount of Compound (I) to a patient in and prophylaxis of a variety of thrombotic conditions of need of such treatment and/or prevention. Treatment with both the venous and arterial vasculature. In the arterial Such forms of Compound (I) may be accomplished by its use system, abnormal thrombus formation is primarily associ 30 alone, as an ingredient of a pharmaceutical composition, or ated with arteries of the coronary, cerebral and peripheral in combination with one or more other medications. Com vasculature. The diseases associated with thrombotic occlu pound (I) may be administered enterally or parenterally in sion of these vessels include, for example, acute myocardial Solid or liquid dosage forms. infarction (AMI), unstable angina, thromboembolism, acute Crystalline forms of Compound (I) have not been known vessel closure associated with thrombolytic therapy and 35 to exist previously. There exists a need for crystalline forms percutaneous transluminal coronary angioplasty (PTCA), transient ischemic attacks, stroke, intermittent claudication which may exhibit desirable and beneficial chemical and and bypass grafting of the coronary (CABG) or peripheral physical properties. There also exists a need for reliable and arteries. reproducible methods for the manufacture, purification, and Chronic anticoagulant therapy may also be beneficial in formulation of Compound (I) to permit its feasible commer preventing the vessel luminal narrowing (i.e., restenosis) 40 cialization. The present invention is directed to these, as well that often occurs following PTCA and CABG, and in the as other important ends. maintenance of vascular access patency in long-term hemo dialysis patients. With respect to the venous vasculature, SUMMARY OF THE INVENTION pathologic thrombus formation frequently occurs in the veins of the lower extremities following abdominal, knee 45 Accordingly, the present invention is directed, in part, to and hip surgery (deep vein thrombosis, or DVT). DVT novel crystalline forms of Factor Xa inhibitors. Specifically, further predisposes the patient to a higher risk of pulmonary in one embodiment, there are provided novel crystalline thromboembolism. A systemic, disseminated intravascular forms of Compound (I): coagulopathy (DIC) commonly occurs in both vascular systems during septic shock, certain viral infections and 50 cancer. This condition may be characterized by a rapid consumption of coagulation factors and their plasma inhibi (I) tors which may result in the formation of life-threatening clots throughout the microvasculature of several organ sys tems. The indications discussed above include some, but not all, of the possible clinical situations where anticoagulant 55 therapy may be warranted. Those experienced in this field are well aware of the circumstances requiring either acute or chronic prophylactic anticoagulant therapy. Both free Factor Xa and Factor Xa assembled in the prothrombinase complex (Factor Xa, Factor Va, calcium and 60 phospholipid) may be inhibited by N-(aminomethyl) phe nylpropyl amide compounds. A particularly promising N-(aminomethyl) phenylpropyl amide compound is methyl (2R,3R)-2-3-amino(imino)methylbenzyl-3-(4- (1-oxido-4-pyridinyl)benzoylaminobutanoate, i.e., the 65 in particular in pharmaceutically acceptable form. compound of formula (I) (hereinafter referred to as “Com These and other aspects of the invention will become pound (I)): more apparent from the following detailed description. US 7,034,160 B2 3 4 BRIEF DESCRIPTION OF THE DRAWINGS paring the salts. Preferably, the salts of the present invention comprise about one equivalent of acid for about each FIG. 1 is a graphical representation of a differential equivalent of Compound (I). scanning calorimetry thermogram of the 2-butanol hemisol The acid addition salts of Compound (I) may be prepared vate of methyl (2R,3R)-2-3-amino(imino)methylben by dissolving the free base in aqueous or aqueous- Zyl-3-4-(1-oxido-4-pyridinyl)benzoylaminobutanoate Solution or other Suitable solvents containing the appropriate hydrochloride. acid or to which the appropriate acid is added, and isolating FIG. 2 is a graphical representation of an X-ray powder the salt by evaporating the solution, or by reacting the free diffraction pattern of the 2-butanol hemisolvate of methyl base and acid in an organic solvent, in which case the salt (2R,3R)-2-3-amino(imino)methylbenzyl-3-4-(1- 10 may separate directly and/or may be obtained by concen oxido-4-pyridinyl)benzoylaminobutanoate hydrochloride. tration of the solution. In accordance with preferred embodiments of the inven DETAILED DESCRIPTION OF THE tion, Compound (I) may be present in the novel crystals as INVENTION a solvate. A wide variety of solvents may be employed in the 15 preparation of the solvates of Compound (I). Preferred The present invention provides, at least in part, crystals of Solvents include, for example, polar solvents, including Compound (I) (i.e., methyl (2R,3R)-2-3-amino(imino)me polar protic and polar aprotic solvents. In preferred form, the thylbenzyl-3-4-(1-oxido-4-pyridinyl)-benzoyl Solvent employed in the preparation of the Solvates is aminobutanoate) as a novel material, in particular in phar selected from the group consisting of , ethers and maceutically acceptable form. The term “pharmaceutically nitriles. Suitable alcohols for use in the preparation of acceptable', as used herein, refers to those compounds, Solvates of Compound (I) include, for example, methyl materials, compositions, and/or dosage forms which are, alcohol, ethyl alcohol, propyl alcohols, including n-propyl within the scope of Sound medical judgment, Suitable for alcohol and i-propyl alcohol, and butyl alcohols, including contact with the tissues of human beings and animals n-butyl alcohol, t-butyl alcohol, iso-butyl alcohol, and sec without excessive toxicity, irritation, allergic response, or butyl alcohol (i.e., 2-butanol). Preferred among these alco other problem complications commensurate with a reason 25 hols are secondary butyl alcohols, with 2-butanol being able benefit/risk ratio. In certain preferred embodiments, more preferred. The 2-butanol employed in the solvates may Compound (I) is in substantially pure form. The term be (R)-(-)-2-butanol, (S)-(+)-2-butanol or mixtures thereof. “Substantially pure', as used herein, means a compound Suitable ethers for use in the preparation of solvates of having a purity greater than about 90% including, for Compound (I) include, for example, dimethoxymethane, example, about 91%, about 92%, about 93%, about 94%, 30 tetrahydrofuran, dioxanes, including 1,3-dioxane and 1,4- about 95%, about 96%, about 97%, about 98%, about 99%, dioxane, furan, , glycol dimethyl ether, and about 100%. ethylene glycol diethyl ether, diethylene glycol dimethyl Compound (I) may be present in the novel crystals as the ether, diethylene glycol diethyl ether, triethylene glycol free base or as a salt, Solvate and/or hydrate. In accordance diisopropyl ether, anisole, and t-butyl methyl ether. Pre 35 ferred among these ethers are diethyl ether, tetrahydrofuran, with preferred embodiments, Compound (I) is present in the and 1,4-dioxane. novel crystals as a salt, preferably an acid addition salt. A suitable nitrile for use in the preparation of solvates of Acids which may be used to prepare the acid addition salts Compound (I) include, for example, acetonitrile, propioni preferably include those which produce, when combined trile, and butyronitrile. Other solvents suitable for the prepa with the free base, pharmaceutically acceptable salts so that ration of solvates of Compound (I), in addition to those the beneficial properties inherent in the free base may not be 40 exemplified above, would be apparent to one skilled in the vitiated by side effects that may be ascribable to the afforded art, based on the present disclosure. by the acids. Although pharmaceutically acceptable salts of The ratio of Compound (I) to solvent in the solvates may the free base form of Compound (I) are preferred, all acid vary and depends, for example, on the particular solvent addition salts are useful as sources of the free base form even selected and the methods for preparing the solvates. Pref if the particular salt perse is desired only as an intermediate 45 erably, the solvates are monosolvates or hemisolvates, with product as, for example, when the salt is formed only for hemisolvates being preferred. purposes of purification and identification, or when it is used Thus, the present invention is directed, in part, to crys as an intermediate in preparing a pharmaceutically accept talline forms of the potent Factor Xa inhibitor represented as able Salt by ion exchange procedures. Compound (I): Pharmaceutically acceptable salts within the scope of the invention include, for example, those salts derived from the 50 following acids: mineral acids, such as hydrochloric acid, hydrobromic acid, Sulfuric acid, phosphoric acid and Sul (I) famic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic 55 acid, cyclohexylsulfamic acid, quinic acid, and the like. Preferably, the acid addition salt is derived from a mineral acid, with hydrochloric acid and hydrobromic acid being preferred. The corresponding acid addition salts comprise the fol 60 lowing: hydrochloride, hydrobromide, Sulfate, phosphate, Sulfamate, acetate, citrate, lactate, tartarate, malonate, meth anesulfonate, ethanesulfonate, benzenesulfonate, p- Sulfonate, cyclohexylsulfamate and quinate, respectively. The relative amounts of Compound (I) and acid in the acid 65 In preferred form, the novel crystals of the present invention addition salts may vary and depends, for example, on the comprise methyl (2R,3R)-2-3-amino(imino)methylben particular acid selected and the methods employed in pre Zyl-3-4-(1-oxido-4-pyridinyl)benzoylaminobutanoate US 7,034,160 B2 5 6 hydrochloride, 2-butanol hemisolvate, i.e., the compound nances in d6-DMSO at 1730, 165.7, 165. 1, 140.6, 139.0 having Formula (I-i) (hereinafter referred to as “Compound (2C), 138.0, 135.2, 134.4, 134.0, 128.9, 128.4, 128.2 (2C), 127.8, 126.0 (2C), 125.97, 123.9 (2C), 67.1 (*), 51.8, 51.5, 46.6, 33.3, 31.7 (*), 23.0 (*), 17.5, 10.0 (*), wherein * indicates the 2-butanol resonance. Compound (I-i) may also (I-i) be characterized by a differential scanning calorimetry ther mogram having an onset peak at about 175°C. to about 185° C., preferably about 181.0° C. to 184°C., and a peak at about 185°C. to about 192°C. Compound (I-i) may also be 10 characterized by an X-ray powder diffraction pattern which

comprises two or more 20 values selected from 5.0+0.2, 14.8+ 0.2, 15.1+0.2, 15.8+0.2, 16.6+0.2, 17.7+0.2, 17.9+0.2, 19.6+0.2, 24.9+0.2, 25.0+0.2, and 27.0+0.2. Broadly speaking, the novel crystalline forms of Com 15 pound (I) may be prepared by a variety of methods, includ o HCI ing but not limited to, recrystallizing Compound (I), pref 0 1 22-BOH erably in salt form, from a suitable solvent. More preferably, the crystalline form of Compound (I) is obtained directly from a reaction mixture through the addition of a suitable where 2-BuOH is 2-butanol. In certain preferred embodi solvent. In certain preferred embodiments, Compound (I) is ments of the present invention, the 2-BuOH in Compound obtained through either recrystallization or solvent addition (I-i) is (S)-(+)-2-butanol. In certain other preferred embodi using a solvent which is to be incorporated in the Solvate. ments, the 2-BuOH in Compound (I-i) is (R)-(-)-2-butanol. Suitable solvents include those mentioned above in connec In certain other preferred embodiments, the 2-BuOH in tion with the solvates. Compound (I-i) is a combination of (S)-(+)-2-butanol and 25 Compound (I) may be prepared using methods well (R)-(–)-2-butanol. known to the skilled artisan of organic synthesis, as well as The various forms described herein may be distinguish methods taught in commonly assigned U.S. Pat. No. 6,080, able from one another through the use of various analytical 767, which claims priority benefit under 35 U.S.C. S 371 of techniques known to one of ordinary skill in the art. Such International Application Serial No. PCT/US96/20770 (des techniques include, but are not limited to, chiral or achiral 30 ignating the United States) filed Dec. 23, 1996, which, in high pressure liquid chromatography (HPLC), X-ray powder turn, claims priority benefit of U.S. Provisional Patent diffraction (XRD), differential scanning calorimetry (DSC), Application Ser. No. 60/009,485 filed Jan. 2, 1996. The and nuclear magnetic resonance (NMR) spectroscopy. In disclosures of each of these documents are hereby incorpo preferred form, Compound (I-i) may be characterized by 'H rated herein by reference, in their entireties. nuclear magnetic resonance spectrum having resonances in 35 A particularly preferred novel crystalline form of Com DMSO-d6 at 9.6–9.3 (bd, 4H, amidine), 8.55 (d. 1H), 8.38 pound (I) is the 2-butanol hemisolvate hydrochloride salt, (d. 2H), 7.96 (2H), 7.8 7.9 (m, 4H), 7.75 (s, 1H), 7.70 (d. referred to herein as Compound (I-i). Compound (I-i), as 1H), 7.46–7.52 (m, 2H), 4.45 (m, 1H), 4.38 (-OH, 0.5H8), well as other crystalline forms, may be prepared by the 3.50 (s.3H), 3.48 (m, 0.5H), 3.15 (m. 1H), 2.9–3.1 (m, 2H), methods described in Scheme 1. Once the target compound 1.25–1.35 (m, 1 H*) overlapping with 1.29 (d. 3H), 1.0 (d. 40 is made, crystalline forms of Compound (I) may be obtained 1.5H*), 0.8 (t, 1.5H*), wherein * indicates the 2-butanol by recrystallization of the crude, amorphous product or, resonance. Compound (I-i) may be also be characterized by alternatively, by addition of a suitable solvent following the 'C nuclear magnetic resonance spectrum having reso final chemical step of the synthetic process.

o TSOH NH NH O 1. LHMDS, THF, -20° C. He COMe CN 1. 2. Br O (II) 3. CH3CO2H watertoluene

(III)

aq. Na2CO3 (o )-()- CO2H TBTU (IV) NMM DMF US 7,034,160 B2

-continued O O

NH O NH O N --- CHCl2.H2O N o1 se O-N 21 NC Na2 NC

(VI) (V)

1.HC MeOH 2. NH

O O o HCI (from reaction) o 1.22-BOH NH 1.2-BuOH NH O 2. filter NHCI O N 3. seed? crystallize 1. O 1. O O N (isolated) N o1 recrystallize o1 from 2-BuOH

(I) (I-i)

In the above reaction scheme, “TsOH' means p-toluene romethane, bromoform, , bromochloromethane, sulfonic acid, “LHDMS means lithium hexamethyldisi 35 dibromomethane, butyl chloride, , tetra lazide, “THF means tetrahydrofuran, “TBTU” means chloroethylene, , 1,1,1-trichloroethane, O-(1H-benzotriazol-1-yl)-N.N.N',N'-tetramethyluronium 1.1.2-trichloroethane, 1,1-dichloroethane, 2-chloropropane, tetrafluoroborate, “NMM” means N-methyl morpholine, hexafluorobenzene, 1,2,4-trichlorobenzene, o-dichloroben “DMF' means N,N-dimethylformamide, “MMPP” means Zene, chlorobenzene, fluorobenzene, fluorotrichlo magnesium monoperoxyphthalate, and “MeOH' means 40 romethane, chlorotrifluoromethane, bromotrifluoromethane, . carbon tetrafluoride, dichlorofluoromethane, chlorodifluo Compound (II) may be prepared using methods well romethane, trifluoromethane, 1,2-dichlorotetrafluorethane known to the skilled artisan of organic synthesis, as well as and hexafluoroethane. methods taught in commonly assigned U.S. patent applica Suitable hydrocarbon solvents include, for example, ben tion Ser. No. 09/491,548, filed Jan. 26, 2000, which is a 45 Zene, cyclohexane, pentane, hexane, toluene, cycloheptane, continuation of International Patent Application No. PCT/ methylcyclohexane, heptane, ethylbenzene, m-, o-, or p-Xy US99/303.66, filed Dec. 17, 1999, which is, in turn, a lene, octane, indane and nonane. continuation-in-part of U.S. Patent Application Ser. No. Suitable ether solvents include, for example, 60/114,598, filed Dec. 31, 1998, now abandoned. The dis dimethoxymethane, tetrahydrofuran, dioxanes, including closures of each of these documents are hereby incorporated 50 1,3-dioxane and 1,4-dioxane, furan, diethyl ether, ethylene herein by reference, in their entireties. glycol dimethyl ether, ethylene glycol diethyl ether, dieth Procedures for recrystallization of the preferred crystal ylene glycol dimethyl ether, diethylene glycol diethyl ether, line forms of Compound (1) will be readily understood by triethylene glycol diisopropyl ether, anisole, and t-butyl one skilled in the art, once placed in possession of the methyl ether. present disclosure. By way of general guidance, Compound 55 Suitable polar protic solvents include, for example, alco (I) may be suspended and/or stirred in a suitable solvent to hols and glycols, such as methanol, ethanol, 2-nitroethanol, afford a slurry, which may be heated to promote dissolution. 2-fluoroethanol. 2.2.2-trifluoroethanol, ethylene glycol, The term “slurry', as used herein, means a saturated Solution 1-propanol. 2-propanol, 2-methoxyethanol. 1-butanol, 2-bu of Compound (I), which may also contain an additional tanol, i-butyl alcohol, t-butyl alcohol, 2-ethoxyethanol, amount of Compound (I) to afford a heterogeneous mixture 60 diethylene glycol, 1-, 2-, or 3-pentanol, neo-pentyl alcohol, of Compound (I) and a solvent at a given temperature. t-pentyl alcohol, diethylene glycol monomethyl ether, dieth Suitable solvents in this regard include, for example, halo ylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, genated solvents, hydrocarbon solvents, ether solvents, polar , and glycerol. protic solvents, and polar aprotic solvents, and mixtures of Suitable polar aprotic solvents include, for example, dim two or more of these. 65 ethylformamide (DMF), dimethylacetamide (DMAC), 1,3- Suitable halogenated solvents include, for example, car dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), bon tetrachloride, bromodichloromethane, dibromochlo 1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidi US 7,034,160 B2 9 10 none (NMP), formamide, N-methylacetamide, N-methylfor ingly, calculation of the amount of seeds needed depends on mamide, acetonitrile (ACN), dimethylsulfoxide, propioni the size of the seed available and the desired size of an trile, ethyl formate, methyl acetate, hexachloroacetone, average product particle as described, for example, in "Pro , ethyl methyl ketone, ethyl acetate, isopropyl grammed cooling of batch crystallizers. J. W. Mullin and J. acetate, t-butyl acetate, dioxane, sulfolane, N,N-dimethyl Nyvlt, Chemical Engineering Science, 1971, 26, 369-377. propionamide, nitromethane, nitrobenzene and hexameth In general, seeds of small size are needed to effectively ylphosphoramide. Other solvents suitable for the preparation control the growth of crystals in the batch. Seeds of small of slurries of Compound (I), in addition to those exemplified size may be generated by sieving, milling, or micronizing of above, would be apparent to one skilled in the art, based on larger crystals, or by micro-crystallization of solutions. Care the present disclosure. 10 should be taken that milling or micronizing of crystals does If the desired crystalline form is a solvate or hemisolvate, not result in any change in crystallinity from the desired preferred solvents include those which may be complemen crystal form (i.e. change to amorphous or to another poly tary to the desired solvate, i.e., solvents which may be morph). ultimately present in the crystalline lattice itself. Preferred The cooled mixture may be filtered under vacuum, and the among these solvents are the alcohol and ether solvents 15 isolated solids may be washed with a suitable solvent, such discussed above, with alcohols being preferred. These as cold recrystallization solvent, and dried under a include, but are not limited to, alcohol solvents such as purge to afford the desired crystalline form. The isolated methyl alcohol, ethyl alcohol, propyl alcohol, including Solids may be analyzed by a Suitable spectroscopic or n-propyl alcohol and i-propyl alcohol, and butyl alcohol, analytical technique, such as NMR, DSC, XRD, HPLC, or including n-butyl alcohol, t-butyl alcohol, iso-butyl alcohol, the like, to assure formation of the preferred crystalline form and sec-butyl alcohol (i.e., 2-butanol). More preferred of the product. The resulting crystalline form is typically among these solvents are secondary butyl alcohols, with produced in an amount of greater than about 70% isolated 2-butanol, including the R- and S-forms thereof, being even yield, but preferably greater than 90% based on the amount more preferred. In the case of alcholic solvents, a cosolvent, of Compound (I) originally employed in the crystallization Such as water, may be used, if necessary or desired, to aid in 25 procedure. The product may be comilled or passed through dissolution. It will be appreciated that the amount of such a mesh screen to delump the product, if necessary. cosolvent employed may vary and depends on desired yield, Preferred crystalline forms may also be prepared directly purity, and other concerns. By way of general guidance from the reaction medium of the final process step for about 1% of cosolvent may be used based on the amount of preparing Compound (I). This may be achieved, for solvent used in the recrystallization. 30 example, by employing in the final process step a solvent or The amount of solvent employed in the recrystallization mixture of solvents from which Compound (I) may be may vary and depends, for example, on the particular crystallized. Alternatively, preferred crystalline forms may solvent employed, as well as the form of Compound (I), for be obtained by distillation or solvent addition techniques example, free base or acid addition salt. Broadly speaking, which would be apparent to the skilled artisan, once placed the amount of Solvent employed in the recrystallization may 35 in possession of the present disclosure. Preferably, such range from about 1 mL Solvent per gram Compound (I) to techniques may be carried out following the final process about 50 mL solvent per gram Compound (I), and all step for preparing Compound (I) through the addition of a combinations and Subcombinations of ranges and specific solvent suitable for isolating the product in crystalline form. amounts of solvent therein. In preferred form, the amount of Suitable solvents for this purpose include any of those Solvent employed may be from about 3 mL solvent per gram 40 solvents described herein. Compound (I) to about 10 mL solvent per gram Compound By way of general guidance, the reaction mixture may be (1). filtered to remove any undesired impurities, inorganic salts, As noted above, the combination of solvent and Com and the like, followed by washing with reaction or crystal pound (I) may be heated for more effective dissolution of the lization solvent. The resulting solution may be concentrated compound in the solvent, as well as improved conversion to 45 to remove excess solvent or gaseous constituents. If distil the preferred crystalline form. Preferred temperatures in this lation is employed, the ultimate amount of distillate col regard may range from about 30° C. to about the boiling lected may vary, depending on process factors including, for point (i.e., the reflux temperature) of the involved solvent, example, vessel size, stirring capability, and the like, by way and all combinations and Subcombinations of ranges and of general guidance, the reaction solution may be distilled to specific temperatures therein. More preferred temperatures 50 about /10 the original volume before solvent replacement is may range from about 60° C. to about the boiling point of carried out. The reaction may be sampled and assayed to the solvent. By way of example, heating 1 gram of a salt of determine the extent of the reaction and the wt % product in Compound (I), preferably the HCl salt, in about 6 mL accordance with standard process techniques. If desired, 2-butanol at about 85°C. to about 90° C. results in substan additional reaction solvent may be added or removed to tially complete dissolution of Compound (I). Cosolvents, 55 optimize reaction concentration. Preferably, the final con Such as water, may be removed azeotropically, preferably centration is adjusted to about 50 wt % at which point a until crystallization occurs spontaneously. The resulting slurry typically results. mixture of solvent and Compound (I) may be cooled to Solvent exchange may be accomplished through the addi initiate and/or continue crystallization. The mixture may be tion of the desired replacement solvent. If the desired preferably cooled to a temperature which ranges from about 60 crystalline form is a solvate or hemisolvate, preferred sol -20° C. to about 20° C., and all combinations and subcom vents include those which are complementary to the desired binations of ranges and specific temperatures therein. Solvate, i.e., Solvents which may ultimately participate in the Seed crystals may be added to any crystallization mixture crystalline lattice itself. Even more preferred solvents to promote crystallization. As will be clear to the skilled include the polar solvents discussed above, including alco artisan, seeding is used as a means of controlling growth of 65 hols, preferably secondary alcohols, ethers and the like. The a particular polymorph or as a means of controlling the Solvent is typically added to provide a desired concentration particle size distribution of the crystalline product. Accord Such that Compound (I) Substantially completely dissolves. US 7,034,160 B2 11 12 The solution may be filtered and washed to remove addi associated with thrombolytic therapy, percutaneous translu tional impurities or salts which may have precipitated during minal coronary angioplasty (PTCA), transient ischemic solvent replacement, followed by the combination of the attacks, stroke, intermittent claudication, and restenosis. filtrates. The solution may be further distilled to remove as The crystalline forms of Compound (I) described herein much reaction solvent as is practicable. The use of additional thus may be useful for, interalia, inhibiting blood coagula recrystallization solvent may be necessary for continued tion by virtue of their general ability to inhibit the penulti distillation as is well understood by the skilled artisan, once mate enzyme in the coagulation cascade, Factor Xa, rather armed with the present disclosure. than thrombin. Novel crystalline forms of Compound (I) It may be preferable to add solvents directly to the within the scope of the present invention may exhibit reaction vessel without distilling the reaction mixture. Pre 10 marked pharmacological activities according to tests ferred solvents for this purpose are those which may ulti described in the literature, including in vivo tests and in vitro mately participate in the crystalline lattice as discussed tests, the latter of which are believed to correlate to phar above in connection with solvent exchange. Although the macological activity in humans and other mammals. For final concentration may vary depending on desired purity, example, both free Factor Xa and Factor Xa assembled in recovery and the like, the final concentration of (I) in 15 the prothrombinase complex (Factor Xa, Factor Va, calcium solution is preferably about 4% to about 7%. More preferred and phospholipid) may be inhibited. Factor Xa inhibition is about 4% to 6% (I) in 1:6 MeOH:crystallization solvent. may be obtained by direct complex formation between the The reaction mixture may be stirred following solvent inhibitor and the enzyme and is therefore independent of the addition and simultaneously warmed. By way of illustration, plasma co-factor antithrombin III. Effective Factor Xa inhi the reaction mixture may be stirred for about 1 hour while bition may be achieved by administering Compound (I) by warming to about 70° C. The reaction is preferably filtered oral administration, continuous intravenous infusion, bolus hot and washed with either the reaction solvent, the solvent intravenous administration or any other Suitable route Such added or a combination thereof. Seed crystals may be added that it may achieve the desired effect of preventing the to any crystallization Solution to initiate crystallization. Factor Xa induced formation of thrombin from prothrombin. Following crystallization, the mixture may be preferably 25 In addition to their use in anticoagulant therapy, Factor Xa cooled to a temperature ranging from about -20°C. to about inhibitors may be useful in the treatment or prevention of 20°C., and all combinations and Subcombinations of ranges other diseases in which the generation of thrombin may play and specific temperatures therein. The resulting Solid may be a pathologic role. For example, thrombin has been proposed filtered under vacuum, washed with a suitable solvent, such to contribute to the morbidity and mortality of such chronic as cold recrystallization solvent, and dried under a nitrogen 30 and degenerative diseases as arthritis, cancer, atherosclerosis purge to afford the desired crystalline form. The solids may and Alzheimer's disease by virtue of its ability to regulate be analyzed using suitable analytical techniques, including many different cell types through specific cleavage and NMR, DSC, XRD, and HPLC as discussed above. The activation of a cell surface thrombin . Inhibition of resulting crystalline form is typically produced in about 70% Factor Xa may effectively block thrombin generation and isolated yield. The product may be comilled or passed 35 therefore neutralize any pathologic effects of thrombin on through a mesh screen to delump the product. various cell types. The novel crystalline forms of Compound (I) described The methods preferably comprise administering to a herein may be formulated into pharmaceutical compositions patient a pharmaceutically effective amount of the novel and/or employed in therapeutic and/or prophylactic meth crystals of the present invention, preferably in combination ods. These methods include, but are not limited to, the 40 with one or more pharmaceutically acceptable carriers and/ administration of the novel crystalline compound (I), alone or excipients. The relative proportions of active ingredient or in combination with one or more other pharmaceutically and carrier and/or excipient may be determined, for active agents, including agents that may be useful in the example, by the and chemical nature of the treatment of the disorders mentioned herein. If Compound materials, chosen route of administration and standard phar (I) is used in combination with another medication, the 45 maceutical practice. combination of compounds described herein may result in a The novel crystalline forms of Compound (I) may be synergistic combination. Synergy, as described for example administered to a patient, for example, enterally or parenter by Chou and Talalay, Adv. Enzyme Regul. 22:27-55 (1984), ally. The percentage of active ingredient in the pharmaceu occurs when the effect of the compounds when administered tical compositions of the present invention may vary, it in combination is greater than the additive effect of the 50 being only necessary that it should constitute a proportion compounds when administered alone as a single agent. In such that a suitable dosage shall be obtained. Obviously, general, a synergistic effect is most clearly demonstrated at several unit dosage forms may be administered at about the Suboptimal concentrations of the compounds. same time. The dose employed may be determined by the The compounds and pharmaceutical compositions of the physician, and may depend upon the desired therapeutic present invention may be useful in inhibiting Factor Xa. 55 effect, the route of administration and the duration of the Accordingly, the present invention provides methods for the treatment, and the condition of the patient. The dosage of the treatment and/or prevention of a pathological condition that novel form of Compound (I) that will be most suitable for may be capable of being modulated by inhibiting production prophylaxis or treatment may vary with the form of admin of Factor Xa. The term “pathological condition', as used istration, the particular novel form of the compound chosen herein, refers to diseases, disorders and/or conditions in a 60 and the physiological characteristics of the particular patient patient, particularly those in which Factor Xa may play a under treatment. Broadly, Small dosages may be used ini role. The term “patient’, as used herein, refers to animals, tially and, if necessary, increased by Small increments until including mammals, preferably humans. Examples of patho the desired effect under the circumstances is reached. logical conditions that may be capable of being treated with Generally speaking, in the adult, Suitable doses may range the compounds and compositions of the present invention 65 from about 0.01 to about 100 mg/Kg body weight, and all include, for example, acute myocardial infarction (AMI), combinations and Subcombinations of ranges and specific unstable angina, thromboembolism, acute vessel closure doses therein. Preferred doses may be from about 0.01 to US 7,034,160 B2 13 14 about 10 mg/kg body weight per day by inhalation, from 1% to 10% by weight relative to the total weight of the about 0.01 to about 100, preferably 0.1 to 70, more prefer dosage unit. CroScarmelose, crospovidone and Sodium ably 0.5 to 10 mg/Kg body weight per day by oral admin starch glycolate represent examples of a cross-linked cellu istration, and from about 0.01 to about 50, preferably 0.01 to lose, a cross-linked polymer and a cross-linked Starch, 10 mg/Kg body weight per day by intravenous administra respectively. Sodium starch glycolate Swells seven- to tion. In each particular case, the doses may be determined in twelve-fold in less than 30 seconds effectively disintegrating accordance with the factors distinctive to the subject to be the granulations that contain it. treated. Such as age, weight, general state of health and other characteristics which can influence the efficacy of the For oral administration in liquid form, the solvate or medicinal product. 10 crystalline Compound (I) can be combined with any oral, The products according to the invention may be admin non-toxic pharmaceutically acceptable inert carrier Such as istered as frequently as necessary to obtain the desired ethanol, glycerol, water and the like. The liquid composition therapeutic effect. Some patients may respond rapidly to a may contain a Sweetening agent which to make the compo higher or lower dose and may find much weaker mainte sitions more palatable. The Sweetening agent can be selected nance doses adequate. For other patients, it may be neces 15 from a Sugar Such as Sucrose, mannitol, Sorbitol. Xylitol, sary to have long-term treatments at the rate of about 1 to lactose, etc. or a Sugar Substitute such as cyclamate, Sacca about 4 doses per day, in accordance with the physiological harin, aspartame, etc. If Sugar Substitutes are selected as the requirements of each particular patient. Generally, the active Sweetening agent, the amount employed in the compositions product may be administered orally about 1 to about 4 times of the invention may be substantially less than if Sugars are per day. It goes without saying that, for other patients, it may employed. Taking this into account, the amount of Sweet be necessary to prescribe not more than one or two doses per ening agent may range from about 0.1 to about 50% by day. weight, and all combinations and Subcombinations of ranges The crystalline and solvate forms of Compound (I) of this and specific amounts therein. Preferred amounts range from invention may be administered in oral dosage forms such as about 0.5 to about 30% by weight. tablets, capsules (each of which includes Sustained release or 25 The more preferred Sweetening agents are the Sugars and timed release formulations), pills, powders, granules, elixirs, particularly sucrose. The particle size of the powdered tinctures, Suspensions, syrups, and emulsions. Solid dosage Sucrose used has been found to have a significant influence forms (pharmaceutical compositions) Suitable for adminis in the physical appearance of the finished composition and tration may generally contain from about 1 mg to about 1000 its ultimate acceptance for taste. The preferred particle size mg of Compound (I) per dosage unit. 30 For oral administration in solid form such as a tablet or of the Sucrose component when used may range from about capsule, the novel crystals of Compound (I) can be com 200 to less than about 325 mesh US Standard Screen, and all bined with a non-toxic, pharmaceutically acceptable inert combinations and Subcombinations of ranges and specific carrier, such as lactose, starch, Sucrose, glucose, methylcel particle sizes therein. lulose, magnesium Stearate, dicalcium phosphate, calcium 35 Sterile injectable solutions may be prepared by incorpo sulfate, mannitol, sorbitol and the like. rating novel crystalline forms of Compound (I) in the Preferably, in addition to the active ingredient, solid required amounts, in the appropriate solvent, with various of dosage forms may contain a number of additional ingredi the other ingredients enumerated herein, as required, fol ents referred to herein as “excipients.” These excipients lowed by filtered sterilization. Generally, dispersions may be include, among others, diluents, binders, lubricants, glidants 40 prepared by incorporating the sterilized active ingredient and disintegrants. Coloring agents may also be incorporated. into a sterile vehicle which contains the dispersion medium and any other required ingredients. In the case of sterile “Diluents” as used herein, refers to agents which may impart powders for the preparation of sterile injectable solutions, bulk to the formulation to make a tablet a practical size for the preferred methods of preparation may include vacuum compression. Examples of diluents are lactose and cellulose. drying and the freeze drying technique which may yield a “Binders' as used herein, refers to agents that may be used 45 to impart cohesive qualities to the powered material to help powder of the active ingredient, plus any additional desired ensure the tablet remains intact after compression, as well as ingredient from the previously sterile-filtered solution to improve the free-flowing qualities of the powder. thereof. Examples of typical binders include lactose, starch and AS would be apparent to a person of ordinary skill in the various Sugars. “Lubricants', as used herein, have several 50 art, once armed with the teachings of the present disclosure, functions including preventing the adhesion of the tablets to when dissolved, Compound (I) loses its crystalline structure, the compression equipment and improving the flow of the and is therefore considered to be a solution of Compound (I). granulation prior to compression or encapsulation. Lubri All forms of the present invention, however, may be used for cants are in most cases hydrophobic materials. Excessive the preparation of liquid formulations in which Compound use of lubricants is undesired, however, as it may can result 55 (I) may be, for example, dissolved or Suspended. In addition, in a formulation with reduced disintegration and/or delayed the crystalline forms of Compound (I) may be incorporated dissolution of the drug substance. “Glidants, as used herein, into Solid formulations. refers to substances which may improve the flow character Liquid compositions may also contain other components istics of the granulation material. Examples of glidants routinely utilized in formulating pharmaceutical composi include talc and colloidal silicon dioxide. “Disintegrants', as 60 tions. One example of Such components is lecithin. Its use in used herein, refer to Substances or mixtures of Substances compositions of the invention as an emulsifying agent may added to a formulation to facilitate the breakup or disinte range from about 0.05 to about 1% by weight, and all gration of the Solid dosage form after administration. Mate combinations and Subcombinations of ranges and specific rials that may serve as disintegrants include starches, clays, amounts therein. More preferably, emulsifying agents may celluloses, algins, gums and cross-linked polymers. A group 65 be employed in an amount of from about 0.1 to about 0.5% of disintegrants referred to as 'Super-disintegrants' gener by weight. Other examples of components that may be used ally are used at a low level in the Solid dosage form, typically are antimicrobial preservatives, such as benzoic acid or US 7,034,160 B2 15 16 parabens; Suspending agents, such as colloidal silicon diox Tablets ide; antioxidants; topical oral anesthetics; flavoring agents; A large number of tablets may be prepared by conven and colorants. tional procedures so that the dosage unit is 100 mg of active The selection of such optional components and their level ingredient, 0.2 mg of colloidal silicon dioxide, 5 mg of ofuse in the compositions of the invention is within the level 5 magnesium Stearate, 275 mg of microcrystalline cellulose, of skill in the art and will be even better appreciated from the 11 mg of starch and 98.8 mg of lactose. Appropriate coatings working examples provided hereinafter. may be applied to increase palatability or delay absorption. The solvate of crystalline Compound (I) may also be Suspensions coupled with soluble polymers as targetable drug carriers. An aqueous Suspension may be prepared for oral admin Such polymers can include, for example, polyvinylpyrroli 10 dine pyran copolymer, polyhydroxypropylmethacrylamide istration so that each 5 mL contains 25 mg of finely divided phenol, polyhydroxyethyl-aspartamidephenol or polyethyl active ingredient, 200 mg of sodium carboxymethyl cellu ene oxide-polylysine substituted with palmitolyl residues. lose, 5 mg of sodium benzoate, 1.0 g of Sorbitol Solution, Furthermore, crystalline Compound (I) may be coupled to a U.S.P., and 0.025 mg of vanillin. class of biodegradable polymers useful in achieving con 15 Injectable trolled release of a drug, for example, polylactic acid, A parenteral composition Suitable for administration by polyglycolic acid, copolymers of polylactic and polyglycolic injection may be prepared by stirring 1.5% by weight of acid, polyepsilon caprolactone, polyhydroxy butyric acid, active ingredient in 10% by Volume propylene glycol and polyorthoesters, polyacetals, polydihydropyrians, polycy water. The solution may be sterilized by commonly used anoacrylates and crosslinked or amphipathic block copoly techniques. mers of hydrogels. Nasal Spray Gelatin capsules of the solvate or crystalline Compound An aqueous solution may be prepared Such that each mL (I) may contain the Solvate or crystalline Compound (I) and contains 10 mg of active ingredient, 1.8 mg methylparaben, the liquid or solid compositions described herein. Gelatin 0.2 mg propylparaben and 10 mg methylcellulose. The capsules may also contain powdered carriers such as lactose, 25 starch, cellulose derivatives, magnesium Stearate, Stearic Solution may be dispensed into 1 mL vials. acid and the like. Similar diluents can be used to make Lung Inhaler compressed tablets. Both tablets and capsules can be manu A homogeneous mixture of the active ingredient in factured as Sustained release products to provide for con polysorbate 80 may be prepared such that the final concen tinuous release of medication over a period of hours. Tablets 30 tration of the active ingredient will be 10 mg per container can be Sugar coated or film coated to mask any unpleasant and the final concentration of polysorbate 80 in the container taste and to protect the tablet from the atmosphere or enteric will be 1% by weight. The mixture may be dispensed into coated for selective disintegration in the gastrointestinal each can, the valves may be crimped on the can and the track. required amount of propellant, for example, dichlorotet In general, water, a suitable oil, saline, aqueous dextrose 35 rafluoroethane, may be added under pressure. (glucose), and related Sugar Solutions and glycols, such as The preferred crystalline form of Compound (I) may propylene glycol or polyethylene glycols may be suitable serve as component (a) of this invention and can indepen carriers for parenteral solutions. Solutions for parenteral dently be in any dosage form, Such as those described above, Solutions may be prepared by dissolving the Solvate or and can also be administered in various combinations, as crystalline Compound (I) in the carrier and, if necessary, 40 described above. In the following description component (b) adding buffering Substances. Anti-oxidizing agents, such as is to be understood to represent one or more agents as sodium bisulfite, sodium sulfite, or ascorbic acid either alone described herein suitable for combination therapy. or combined, may be Suitable stabilizing agents. Citric acid Thus, Compound (I) may be used alone or in combination and its salts and sodium EDTA may also be employed. with other diagnostic, anticoagulant, antiplatelet, fibrin Parenteral Solutions may also contain preservatives, such as 45 olytic, antithrombotic and/or profibrinolytic agents. For benzalkonium chluoride, methyl- or propyl-paraben and example adjunctive administration of Factor Xa inhibitors chlorobutanol. with standard heparin, low molecular weight heparin, direct Suitable pharmaceutical carriers, excipients and diluents thrombin inhibitors (i.e. hirudin), aspirin, fibrinogen recep are described in Remington's Pharmaceutical Sciences, tor antagonists, streptokinase, urokinase and/or tissue plas Mack Publishing Co., the disclosures of which are hereby 50 minogen activator may result in improved antithrombotic or incorporated herein by reference, in their entireties. Useful thrombolytic efficacy or efficiency. The novel crystals pharmaceutical dosage-forms for administration of the com described herein may be administered to treat thrombotic pounds of this invention can be illustrated as follows: complications in a variety of animals, such as primates, Capsules including humans, sheep, horses, cattle, pigs, dogs, rats and A large number of unit capsules may be prepared by 55 mice. Inhibition of Factor Xa may be useful not only in the filling standard two-piece hard gelatin capsules each with anticoagulant therapy of individuals having thrombotic con 100 mg of powdered active ingredient (i.e., Factor Xa ditions, but also when inhibition of blood coagulation may inhibitor), 150 mg of lactose, 50 mg of cellulose, and 6 mg be required, such as to prevent coagulation of stored whole of magnesium Stearate. blood and to prevent coagulation in other biological samples 60 for testing or storage. Thus, any Factor Xa inhibitor, includ Soft Gelatin Capsules ing the novel crystalline forms of Compound (I) as described A mixture of active ingredient in a digestible oil Such as herein, can be added to or contacted with any medium Soybean oil, cottonseed oil or olive oil may be prepared and containing or Suspected of containing Factor Xa and in injected by means of a positive displacement pump into which it may be desired to inhibit blood coagulation. gelatin to form Soft gelatin capsules containing 100 mg of 65 The novel crystals of the present invention may be used the active ingredient. The capsules may then be washed and in combination with any antihypertensive agent or choles dried. terol or lipid regulating agent, or concurrently in the treat US 7,034,160 B2 17 18 ment of restenosis, atherosclerosis or high blood pressure. Some examples of agents that may be useful in combination -continued with a novel form of Compound (I) according to the present NH2 O invention in the treatment of high blood pressure include, for example, compounds of the following classes: beta-block E o1 ers. ACE inhibitors, calcium channel antagonists and alpha receptor antagonists. Some examples of agents that may be NC C6H5COH useful in combination with a compound according to the invention in the treatment of elevated levels or disregulated lipid levels include compounds known to be 10 HMGCoA reductase inhibitors, or compounds of the fibrate class. (III) Accordingly, components (a) and (b) of the present inven tion may be formulated together, in a single dosage unit (that To a reactor were charged Compound (II) (100.0 g) and is, combined together in one capsule, tablet, powder, or 15 anhydrous THF (320 g). The resulting suspension was liquid, etc.) as a combination product. When component (a) cooled down to -20+3° C. and LiHMDS (475.6 grams, 1.3 and (b) are not formulated together in a single dosage unit, M solution in THF) was added over 55 minutes and stirred the component (a) may be administered at the same time as for 20 minutes at -20+3° C. A solution of C.-bromo-m- component (b) or in any order; for example component (a) tolunitrile in THF (65.1 g in 181 g of THF) was then charged of this invention may be administered first, followed by into the reactor over 40 minutes while maintaining the administration of component (b), or they may be adminis temperature at -20+3° C. and stirred for another 30 minutes. tered in the reverse order. If component (b) contains more Benzoic acid (126.6 grams) was charged as a solid to the that one agent, these agents may be administered together or reactor. Water (1000 g) was then added and mixture distilled in any order. When not administered at the same time, at a 65+3° C. jacket temperature and 200-233 mbar vacuum. preferably the administration of component (a) and (b) 25 After distilling to a constant pot temperature of 57°C. and occurs less than about one hour apart. Preferably, the route constant head temperature of 45° C., the distillation was of administration of component (a) and (b) is oral. Although stopped. Toluene (432 g) was added to the hot solution and it may be preferable that component (a) and component (b) stirred while cooling down to 10+2° C. The resulting sus both be administered by the same route (that is, for example, pension was then filtered and the filter cake washed with both orally) or dosage form, if desired, they may each be water (250 grams) and toluene (432 grams). Compound (III) administered by different routes (that is, for example, one was dried at 45–50° C. at ~350 mbar vacuum under a component of the combination product may be administered nitrogen stream for 24 hours until constant weight. The orally, and another component may be administered intra isolated solid weighed 76.0 grams (62.0% yield). venously) or dosage forms. Pharmaceutical kits which may be useful for the treatment 35 Example 2 of various disorders, and which comprise a therapeutically effective amount of a pharmaceutical composition compris Preparation of Compound (V) ing a novel form of Compound (I) in one or more sterile containers, are also within the ambit of the present inven tion. The kits may further comprise conventional pharma 40 ceutical kit components which will be readily apparent to NH2 O 1. aq. Na2CO3 those skilled in the art, once armed with the present disclo O1 sure. Sterilization of the container may be carried out using s 2. M \ conventional sterilization methodology well known to those NC N COH skilled in the art. 45 The present invention is further described in the following (IV) examples. All of the examples are actual examples. These 3. TBTU examples are not to be construed as limiting the scope of the NMM, DMF appended claims. (III) 50 O EXAMPLES Example 1 NH O Preparation of Compound (III) 55 N uluso1 N N2 NC :

NHTSOH 1. LHMDS, THF, -20° C. 60 (V) lucose 2. Br CN (II) Compound (III) was partitioned between dichlo 3. C6H5CO2H 65 romethane and aqueous sodium carbonate. The organic watertoluene phase (containing the free base of (III)) was washed with additional aqueous sodium carbonate and was distilled under US 7,034,160 B2 19 20 reduced pressure and solvent exchanged with dimethylfor Example 4 mamide (DMF). This solution was assayed for wt/wt content of (III). To a suspension of (IV) (1.0 equivalent vs. (III)) in Preparation of Compound (I) DMF were added 2 equivalents of 4-methylmorpholine and 1.1 equivalents of O-Benztriazol-1-yl-N,N,N',N'-tetram ethyluronium tetrafluoroborate (TBTU). This mixture was stirred at ambient temperature until ester activation was O complete (about 90 minutes). The DMF solution of Com NH O pound (III) (1 equivalent) was added and the resulting 10 solution stirred overnight after which HPLC indicated that the reaction was complete. Water was added at 75° C. and N-N- - - - so the mixture was cooled to crystallize the product. The O 2 NC - mixture was cooled to 5°C., filtered, and the filter cake was washed with water. The product was dried under reduced 15 pressure at 70° C. (VI) Example 3 O

Preparation of Compound (VI)

25

NH O o HCI

N --- 30 (I) MMPP Na2 NC CH2Cl2/H2O -e- To a 200-mL jacketed reaction flask were charged Com pound (VI) (50.0g, 116 mmol) and methanol (50 mL). This mixture was cooled to -5° C. and sealed after establishing 35 a partial vacuum (ca. 100 torr). Anhydrous HCl (52.2g, 1.43 (V) O mol) was added while maintaining the reaction temperature at less than 0° C. The reaction was stirred at 0+1° C. under closed conditions. After 16 hours, the reaction was complete NH O 40 (less than 2 A% (VI) by HPLC). To the intermediate product solution was added anhydrous methanol (100 mL) while N --- maintaining the temperature at less than 5° C. The solution was treated with NH (27.7 g, 1.62 mol) keeping the tem 1N 21 NC perature less than 0°C. Before allowing the mixture to warm 45 to room temperature, a pH check was made of an aliquot dissolved in DI water (a pH of 8–10 indicates a sufficient charge of ammonia). The reaction was stirred at 20° C. overnight at which point the reaction was complete. (VI) 50 Example 5 In a well-stirred reactor, 45g of Compound (V) in 450 mL Preparation of Compound (I-i) by Solvent Addition dichloromethane was reacted for at least 5 hours with 61 g of magnesium monoperoxyphthalate (66.4% based on avail able oxygen, 1.5 eq.) in 450 g of water until the reaction was 55 complete. The phases were separated and the organic phase O was washed successively with equal volumes of water, a 5% aqueous Sodium bicarbonate Solution, and water. The result NH O ing solution was concentrated to an approximately 40 wt % 60 N -sul 1.2-BuOH solution and diluted with 180 g of methyl isobutyl ketone N H s o 2. filter NH4 (MIBK). Further distillation to remove residual dichlo o1 N 2 HN 3. seed crystallizery romethane, seeding with appropriate crystals, and cooling gave the product as a crystalline Solid. The crystals were o HCI filtered, rinsed with 30 g of MIBK, and dried at 50° C. under 65 reduced pressure to give 41.8g of Compound (VI) (89.3% (I) yield). US 7,034,160 B2 21 22 the water and crystallization began to occur spontaneously. -continued The resulting mixture was cooled to continute crystalliza O o HCI tion. The crystals were filtered, washed with 2-butanol, and 0 1 22-BOH dried overnight at 20 p.s. i. Vacuum with a nitrogen purge at 60° C. The dried crystals of Compound (I-i) weighed 48.3 NH O grams (91% yield). N --- Physical Data of Compound (I-i) NH s H NMR (DMSO-d6) 9.6-9.3 (bd, 4H, amidine), 8.55 (d. N 1H), 8.38 (d. 2H), 7.96 (2H), 7.8-7.9 (m, 4H), 7.75 (s, 1H), - N4 HN 10 7.70 (d. 1H), 7.46–7.52 (m, 2H), 4.45 (m. 1H), 4.38 (-OH, 0.5H), 3.50 (s.3H), 3.48 (m, 0.5H), 3.15 (m. 1H), 2.9–3.1 (m. 2H), 1.25–1.35 (m, 1 H*) overlapping with 1.29 (d. 3H), 1.0 (d. 1.5H*), 0.8 (t, 1.5H*), wherein * indicates the (I-i) 2-butanol resonance. 15 'C NMR (d6-DMSO) at: 173.0, 165.7, 165. 1, 140.6, To the ammonium chloride slurry from Example 4 was 139.0 (2C), 138.0, 135.2, 1344, 134.0, 128.9, 128.4, 128.2 added 2-butanol (840 mL), and the resulting mixture was (2C), 127.8, 126.0 (2C), 125.97, 123.9 (2C), 67.1 (*), 51.8, stirred for 1 hour while warming to 70° C. The ammonium 51.5, 46.6, 33.3, 31.7 (*), 23.0 (*), 17.5, 10.0 (*), wherein chloride was removed by hot filtration and the cake was * indicates the 2-butanol resonance. washed with a solution of 20 mL methanol in 160 mL Differential Scanning Calorimetry (DSC) (heating from 2-butanol. The filtrates were combined and 0.5 g of seed 25.0° C. to 220.0° C. at 10.0°C/min.): Batch 1: onset peak crystals were added. The mixture was allowed to stir over at 176.4° C., peak at 185.3°C., and an endotherm of about night at ambient temperature. The slurry was cooled to -15° 76 J/g: Batch 2: onset peak at 183.2°C., peak at 191.3°C., C. and held for 2 hours to ensure complete crystallization. and an endotherm of 101.9 J/g. The solid was filtered and the reactor and cake were washed 25 X-ray powder diffraction (XRD): 20 values at 5.0, 14.8, with 165 mL of 2-butanol. The solid was dried under 15.1, 15.8, 16.6, 17.7, 17.9, 19.6, 24.9, 25.0, and 27.0. vacuum at 45–50 C with a nitrogen bleed giving 44.3 g Elemental analysis: Calculated: C 62.36%, H 6.20%, N (73.2%) of Compound (I-i) as an off-white crystalline solid. 10.77%, C16.82%. Found: C 62.33%, H 6.19%, N 10.64%, Cl 6.70% Mass spec: ion spray found M+ at 477 Example 6 30 The disclosures of each patent, patent application and publication cited or described in this document are hereby Preparation of Compound (I-i) by Solvent incorporated herein by reference, in their entirety. As will be Replacement apparent to those skilled in the art from the foregoing description, various modification of the invention, in addi After carrying out the synthesis of Compound (I), as set 35 tion to those described herein, are also intended to fall within forth above in Example 4, ammonium chloride was removed the scope of the appended claims. from the reaction mixture by filtration and the resulting filter What is claimed is: cake was washed with anhyrous methanol. The resulting 1. A crystalline form of (2R,3R)-2-3-amino(imino)me Solution was concentrated to remove methanol and residual thylbenzyl-3-4-(1 -oxido-4-pyridinyl)benzoylamino ammonia to approximately /10 the original reaction Volume. 40 butanoate hydrochloride, 2-butanol hemisolvate according The reaction mixture was sampled and assayed for wt % of to the formula Compound (I). Methanol was added to adjust the final concentration to 50 wt % of Compound (I). To the resulting slurry was added 2-butanol and the mixture was stirred for one hour. The precipitated ammonium chloride was washed with a solution of methanol and 2-butanol. The filtrates were 45 combined and further concentrated, and additional 2-butanol

was added until the methanol was reduced to a low level. The overhead temperature during the distillation was about 98° C. Seed crystals were added to initiate crystallization. The mixture was allowed to cool to 20° C. over 2 hours and 50 was maintained at that temperature for 2 hours to ensure complete crystallization. The solids were isolated by filtra tion, and the reactor and filter cake were washed two times with 2-butanol. The solids were dried in a vacuum oven at oHC o1,22-BOH. 50 to 60° C. to give Compound (I-i) as an off-white solid in 55 70% yield. 2. The crystalline form of claim 1 having an X-ray powder Example 7 diffraction pattern which comprises two or more 20 values selected from 5.0+0.2, 14.8+0.2, 15.1+0.2, 15.8+0.2, Preparation of Compound (I-i) through 60 16.6+0.2, 17.7+0.2, 17.9+0.2, 19.6+0.2, 24.9+0.2, 25.0+0.2, Recrystallization and 27.0.0.2. 3. The crystalline form according to claim 1 wherein Compound (I) (50 g) as an amorphous hydrochloride salt 2-BuOH is (S)-(+)-2-butanol. (water content 1.2 wt %) was suspended in 2-butanol (300 4. The crystalline form according to claim 1, wherein g) containing water (3 g). The mixture was heated to boiling 65 2-BuOH is (R)-(-)-2-butanol. and dissolution occurred at about 85° C. to 90° C. The sample was concentrated slightly to azeotropically remove k k k k k