NOVEL CRYSTAL FORMS OF 9-SUBSTITUTED HYPOXANTHINE DERIVATIVES

Abstract: The bifunctional compound N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt occurs in a number of crystal forms which differ in their stability. Of these forms, the most stable is designated Type I, which is a monopotassium salt monophydrate in which the water is held as water of crystallization. Other crystalline forms, designated Type II and Type III, convert to Type I. Methods are disclosed for the synthesis of Type I and for the conversion of Type II or Type III to Type I.

Description: NOVEL CRYSTAL FORMS OF 9-SUBSTITUTED HYPOXANTHINE DERIVATIVES FIELD OF THE INVENTION This invention is directed to novel crystal forms and improved synthetic methods for 9- substituted hypoxanthine compounds, particularly the 9-substituted hypoxanthine derivative 4- [[3-(l,6-dihydro-6-oxo-9H-purin-9-yl)-l-oxopropyl]amino]benzoic acid monopotassium salt (AIT-082), also designated N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, monopotassium salt. BACKGROUND OF THE INVENTION There is a need for compounds that are bifunctional and that can interact with multiple receptors on the surface of different cell types. There is also a particular need for compounds that bypass the blood-brain barrier so that the activities of such compounds can be exerted in the central nervous system, such as for the treatment of diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), and other neurodegenerative diseases. A number of such compounds and methods for synthesizing them are disclosed in U.S. Patent No. 5,091,432 to Glasky, incorporated herein by this reference. This includes a number of bifunctional compounds that cross the blood-brain barrier. Exemplary multifunctional compounds are formed from a first biologically active chemical moiety having immunological activity and a second biologically active chemical moiety having neurological activity. An exemplary immunologically active chemical moiety is hypoxanthine or purin-6(lH)-one. An additional benefit to the utilization of hypoxanthine is its structural relationship to inosine, the only purine known to cross the blood brain barrier. Hypoxanthine can be linked by a chemical bridging group, such as propionic acid or butyric acid, to a wide variety of neurologically active chemical moieties to produce a variety of compounds including 4-[[3-(l,6-dihydro-6- oxo-9H-purin-9-yl)-l-oxopropyl]amino]benzoic acid, also referred to as N-4-carboxyphenyl-3- (6-oxohydropurin-9-yl) propanamide. Although synthetic methods for these bifunctional compounds, including 4-[[3-(l,6- dihydro-6-oxo-9H-purin-9-yl)-l-oxopropyl]amino]benzoic acid or N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide hydrochloride were described in U.S. Patent No. 5,091,432 to Glasky, there is a need for an improved synthetic method for these compounds. There is a particular need for a more efficient synthesis that provides higher yields and a more consistent synthesis that provides fewer side reactions as well as providing a pure and stable product. During compound synthesis a variety of different crystalline forms of the same drug substance (polymorphism) may result. The existence of such polymorphisms is significant because different crystalline forms may have different solubility in water and thus different degrees of bioavailabihty in terms of pharmacokinetics (solubility) and pharmacodynamics of the compound. Therefore, compound polymorphism must be carefully checked as individual crystal structures could lead to differences in bioavailabihty. Therefore, there is a particular need for an analysis of the polymoφhic forms of compound products and their interconversion processes. There is also a particular need for methods for producing the most stable form of compounds such as 4-[[3-(l,6-dihydro-6-oxo-9H-purin-9-yl)-l-oxopropyl]amino]benzoic acid monopotassium salt or N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt and for converting other crystalline and stable forms of the compound into the most stable form. All of the stable forms of this compound are pharmaceutically acceptable. SUMMARY One aspect of the present invention is a crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate that has a solubility of about 5% (w/v) in water at 25°C, has an infrared spectrum peak from 1674.1 cm "1 to about 1675.7 cm "1 as measured by Fourier-transform infrared spectroscopy, and that has an X-ray powder pattern that is distinct from the X-ray powder pattern of any other crystalline form of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt, the crystalline form being designated Type I. Another aspect of the present invention is a crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate that has a solubility of about 10% (w/v) in water at 25°C, has an infrared spectrum peak of about 1693.4 cm "1 as measured by Fourier-transform infrared spectroscopy, and that has an X-ray powder pattern that is distinct from the X-ray powder pattern of any other crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, the crystalline form being designated Type II. Yet another aspect of the present invention is a substantially anhydrous crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate that has less than about 1.0% water as measured by the Karl Fischer titration process, that has an infrared spectrum peak of about 1687.8 cm "1 and that has an X-ray powder pattern that is distinct from the X-ray powder pattern of any other crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, the crystalline form being designated Type III. Another aspect of the present invention is a method for synthesizing the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate comprising the steps of: (1) reacting the free acid of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide with potassium hydroxide in water; and (2) precipitating the product of step (1) with ethanol to yield the Type I crystalline form. Yet another aspect of the present invention is another method for synthesizing the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate comprising the steps of: (1) reacting N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide ethyl ester monoacetate with potassium hydroxide to produce N-4-carboxyphenyl-3-(6-oxohydropurin-9- yl) propanamide in a crystalline form distinct from Type I; and (2) reacting the product of step (1 ) with a mixture of ethanol and water containing a low concentration of potassium hydroxide to yield the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate. Still another aspect of the present invention is a method for converting a distinct crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, designated Type II, to the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate comprising the step of equilibrating the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide designated Type II with water vapor at a temperature in a range from about 60°C to about 80°C for a time period of from about 36 hours to about 60 hours to yield the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3- (6-oxohydropurin-9-yl) propanamide monohydrate. Preferably, in this method, the temperature is about 70°C and the time period is about 48 hours. Yet another aspect of the present invention is a method for converting a distinct crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, designated Type III, to the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate comprising the step of equilibrating the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide designated Type III with water vapor at about 25°C to yield the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide monohydrate. BRIEF DESCRIPTION OF THE DRAWINGS The following invention will become better understood with reference to the specification, appended claims, and accompanying drawings, where: Figure 1 is a diagram showing the interconversions between the Type I, Type II, and Type III crystal structures for N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt; Figure 2 is a graph showing the solubility of the Type II form of N-4-carboxyphenyl-3- (6-oxohydropurin-9-yl) propanamide monopotassium salt as a function of increasing temperature; Figure 3 shows X-ray diffraction powder patterns of various crystal forms of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt: (a) Type II (designated Am 927/a) and Type I (designated DD-477-21-5); (b) Type I (designated DD-477- 21-5); (c) Type II (designated Am 927/a RT) after incubation of Type II of (a) at 25°C; Type I (designated DD-477-21-5) after incubation of Type I of (a) at 25 °C; (d) Type I (designated Am 927/a 70°C) after incubation of Type II of (a) at 70°C; Type I (designated DD-477-21-5 70°C) after incubation of Type I at 70°C; (e) Type I containing admixture of dipotassium salt (designated Am 964/b); (f) Type I (designated NTA-107/B); (g) Type II (designated NTA- 107/A); (h) Type I as in (f) (designated NTA 107-B); Type III (designated NTA-107-B/2) produced by heating of Type I under vacuum at 140°C; Figure 4 shows Fourier-transform infrared spectra of various crystal forms of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide: (a) Type I (designated DD-477-21-5); (b) Type I, second preparation (designated NTA- 107-B); (c) Type I with admixture of dipotassium salt (designated Am 964/b); (d) Type II (designated Am 972/a); (e) Type II, second preparation (designated NTA- 107- A); Figure 5 shows differential scanning calorimetric (DSC) measurements on various crystal forms of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide: (a) Type I (designated NTA- 107-B); (b) Type I, second preparation (designated DD-477-21-5); (c) Type II (designated NTA- 107- A); Figure 6 shows thermogravimetric (TG) plots of mass against temperature for various crystal forms of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide: (a) Type I (designated NTA- 107-B); (b) Type I, second preparation (designated DD-477-21-5); (c) Type II (designated NTA- 107- A); (d) Type I with admixture of dipotassium salt (designated AM 964/b); Figure 7 shows X-ray diffraction powder patterns of various crystal forms of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt: (a) Type III (designated NTA- 107-B/a); (b) Type I (designated NTA- 107-B); Figure 8 shows the Fourier-transform infrared spectrum of the Type III crystal form of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide (sample designated NTA- 107-B/a); Figure 9 shows the DSC measurement on the Type III crystal form of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide (sample designated NTA- 107-B/a); and Figure 10 shows the TG measurement on the Type III crystal form of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide (sample designated NTA- 107-B/a). DESCRIPTION The compound N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt occurs in a number of different crystal forms. These forms reflect different degrees of hydration. As detailed below, there are three crystalline forms of these compound, designated as Type I, Type II, and Type III. These forms can be interconverted, also as detailed below. Type I is a monohydrate in which the carboxyl group is in the form of a potassium salt. From the standpoint of stability, Type I is the most desirable crystal form. Therefore, it is important that manufacturing processes be developed that either result in Type I or convert other forms to Type I. Preferably, Type I is prepared under conditions that suppress deprotonation of the hydroxyl group (as a tautomer of the oxo group) of the pyrimidine ring of the molecule. If Type I is prepared at a pH value greater than about 8.5, the hydroxyl group of the pyrimidine begins to deprotonate and the crystal contains a slight excess of potassium as a counterion. This leads to a variant to Type I which has a distinguishable X-ray powder pattern with an additional line. Type II is also in the form of a monopotassium salt, but the water of hydration is not so strongly bound in the crystal lattice as that of Type I and may be considered as "adherent" water. Type III is also in the form of a monopotassium salt, but lacks both the "adherent" water and the water of crystallization. Type III is virtually anhydrous. The forms can be interconverted. Type II can be converted into Type I by equilibration of Type II in a humid chamber at 70°C or by heating in a solution of water and ethanol at 70°C. Type I can be converted to Type III by strong drying conditions, such as heating under vacuum at 140°C. Type III can be converted back to Type I by humidification at 25°C. Type I is stable under treatment at either 25°C or 70°C in a humid chamber. Therefore, one aspect of the present invention is a distinct crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate. This crystalline form is designated as Type I. This crystalline form has a solubility of about 5% (w/v) in water at 25°C. This crystalline form also has an infrared spectrum peak from about 1674.1 cm "1 to about 1675.7 cm '1 as measured by Fourier-transform infrared spectroscopy. This crystalline form also has an X-ray diffraction powder pattern that is distinct from the X-ray diffraction powder pattern of any other form of N-4-carboxyphenyl- 3-(6-oxohydropurin-9-yl) propanamide monopotassium salt. This X-ray diffraction powder pattern is shown in Figure 3(b), Figure 3(c), Figure 3(d), Figure 3(f), and Figure 3(h). Another aspect of the present invention is a method for synthesizing the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate. In general, this method comprises: (1) reacting the free acid of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide with potassium hydroxide in water; and (2) precipitating the product of step (1) with ethanol to yield the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate. Preferably, this reaction takes place at about 0°C to about 80°C. Still another aspect of the present invention is another method for synthesizing the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate. In general, this method comprises: (1) reacting N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide ethyl ester monoacetate with potassium hydroxide in water and then with hydrogen chloride to produce the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide in a crystalline form that is distinct from Type I; and (2) reacting the product of step (1) with an ethanol- water mixture containing a small proportion of potassium hydroxide to produce the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate. Preferably, the concentration of potassium hydroxide in the ethanol-water mixture is about 0.1%. In this procedure, the crystalline form of the monopotassium salt of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide is initially in a form that is distinct from Type I. This form is designated Type II. In Type II, the molecule is not in the form of a monohydrate and does not contain water of crystallization, as detailed above. It is possible to interconvert Type I and Type II, as described above and in the Example. Still another aspect of the present invention is a method of converting the Type II crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide into the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl- 3-(6-oxohydropurin-9-yl) propanamide monohydrate. This method comprises heating the Type II crystalline form in the presence of water vapor at a temperature of from about 60°C to about 80°C for a period of from about 36 hours to about 60 hours. Preferably, the temperature is about 70°C and the period of the interconversion is about 48 hours. This conversion results in the insertion of water of hydration into the crystal lattice. Another aspect of the present invention is the Type II crystalline form. The Type II crystalline form has a solubility of about 10% (w/v) in water at 25 °C, has an infrared spectrum peak of about 1693.4 cm "1 as measured by Fourier-transform infrared spectroscopy, and that has an X-ray powder pattern that is distinct from the X-ray powder pattern of any other crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide. The X-ray diffraction powder pattern for Type II is shown in Figure 3(a), 3(c), and 3(g). There is yet another crystal form of the monopotassium salt of N-4-carboxyphenyl-3- (6-oxohydropurin-9-yl) propanamide, designated Type III. Type III is substantially anhydrous, having a water content of less than about 1.0% as measured by Karl Fischer titration or by thermogravimetric analysis. Type III has an X-ray diffraction powder pattern distinct from that of Type I or Type II. Type III has an infrared spectrum peak of about 1687.8 cm "1 as measured by Fourier-transform infrared spectroscopy. Type III can be converted into Type I by incubation in the presence of water vapor at a temperature of about 25°C, while Type I can be converted into Type III only by drying in a vacuum oven at a temperature of about 140°C. Type III can be readily converted back to Type I by incubation in a humid chamber at 25°C. The X-ray diffraction powder pattern of Type III is shown in Figure 3(h) and in Figure 7(a). Therefore still another aspect of the present invention is a method of converting the Type III crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide into the Type I crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate by incubating the Type I crystals at a temperature of about 25°C in the presence of water vapor. The pattern of interconversions of Type I, Type II, and Type III crystal structures is shown in Figure 1. The invention is illustrated by the following Example. This Example is presented for exemplification only and are not intended to limit the invention. EXAMPLE Polymorphic Forms of Crystals of N-4-Carboxyphenyl-3-(6-Oxohydropurin-9-yl) Propanamide Monopotassium Salt Materials and Methods The occurrence of polymoφhic forms can be examined by various physico-chemical investigation techniques. The solubility measured under defined conditions can provide information regarding the occurrence of different crystal structures. However, the preferred technique for the investigation of the polymoφhism of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide monopotassium salt is the analysis of the X-ray diffraction powder diagrams, which allows the distinct crystal structures to be clearly distinguished by their characteristic powder patterns. The powder pattern, position, intensity and shaφness of the bands are dependent upon the crystal structure and the fine arrangement of the atoms in the crystal lattice, which in turn are both influenced by the content of crystal water. For this study, material from different batches of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt was used, either in the original or in a dry or wet state (Table 1). The water content was determined by Karl Fischer (KF) titration (Titroprocessor Metrohn 682 and Karl-Fischer Titrator Metrohm 701 Titrino) or thermogravimetric (TG) analysis (Mettler TG 50 Thermobalance). X-ray powder patterns were measured with a γ-Generator Philips PW 1120/90 equipped with a CuK α Guinier-de Wolff Camera No. 11 (Enraf-Nonius). Characterization of the thermal stability (loss of water, melting point) of different polymoφhic forms was performed by differential scanning calorimetric (DSC) measurements (Mettler DSC 821) and infrared spectra (IR) were recorded on a Fourier-transform (FT) infrared spectrophotometer (Perkin Elmer FT-IR Spectrometer Paragon 1000). Synthesis A. Via Free Acid C,5H l3 N5θ4 C15H12KN5O4XH2O Mol Wt- 32730 Mol Wt 38341 [138117-50-7] [192564-13-9] ATT-082 free acid A1T-082 mono-hydrate (type I) Studies with the N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt revealed that the Type I crystal structure is obtained by the synthetic route depicted above. Type I form is identical to the crystal structure of the reference compound lot DD-477-21-5 B. Via Ethyl Ester Monoacetate CJSHUKN . [no CAS-No., without AcOH: 138117-79-0] Mol. Wt: 365.39 AIT-082 ethyl ester mono-acetate [192564-13-9] AIT-082 (type II) C 15 H 12 KΪ 04 Cj 5 H l2 KN 5 θ 4 xH 2 0 Mol Wt 365 39 Mol Wt 383 41 [192564-13-9] [192564-13-9] AIT-082 (type II) AIT-082 mono-hydrate (type I) The hydrolysis of the N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide ethyl ester and the subsequent isolation of the N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt affords the Type II crystal structure which must be converted to the Type I modification at elevated temperature (70°C) in a solvent mixture of ethanol/water in order to get the N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt monohydrate (Type I). Without addition of water no interconversion of the crystal structure of Type II to Type I was observed. The synthetic route depicted above is preferably used in the manufacturing process and preferably includes a small amount of KOH in the final interconversion step in order to meet the solubility requirements of the active pharmaceutical ingredient. Results Polymorphism Starting from different crystal structures, hydration and dehydration studies were performed (Figure 1, Table 2). The crystal structure designated Type I (lot NTA-107-B) is obtained by equilibration of Type II (lot NTA- 107- A) with water vapor in a humid chamber, whereas Type III was generated by dehydration of Type I in a vacuum drying oven at elevated temperature (Tables 1 and 2). The X-ray powder patterns of the Type I and II crystal structures are not sensitive to small changes in the water content and appear uniform from batch to batch. Starting from Type I, humidification leads to unchanged Type I, whereas under strong drying conditions (temperature and vacuum) Type III was obtained. The latter process is fully reversible and Type III turns completely into Type I upon humidification at 25°C. Incubation of the Type I and II crystal structures in a humid chamber (H 2 O vapor) at 25°C and 70°C for 48 h resulted in the interconversion of Type II into Type I crystal structure only at elevated temperature, whereas Type I remains unchanged. Heating of AIT-082 Type II in a solution containing water and ethanol to 70°C leads to the interconversion of Type II into Type I crystal structure. The well-resolved powder diagrams of these two forms indicate well ordered crystal lattice structures (Figure 3). The Type I crystal structure contains about 5.8% (w/w) water and is only slightly hygroscopic. The manufacturing process leads to a water- soluble (5% w/v) crystalline product of the monopotassium salt of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide monohydrate Type I (active pharmaceutical ingredient). N- 4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt exists, as known so far, in three different crystal structures (Types I-III) depending upon the water content. The corresponding powder diagrams exhibit well-resolved patterns with a strong peak intensity against a relatively low background. These powder patterns are shown in Figure 3. If the K-salt formation is performed at a pH value higher than 8.5, the oxo-group (hydroxyl-group) of the pyrimidine ring is also prone to salting. Samples which have been prepared under these reaction conditions contain a slight excess of K (di-K-salt of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide free acid) and show an extra band in the powder diagrams (Table 1 and Figure 3(e)). In addition to X-ray powder diffraction, IR-spectroscopy can also be used to distinguish the Type I and II crystal structure polymoφhisms (Figure 4). Type I (same as the reference compounds lot DD-477-21-5 and lot 1024.D.96.2) exhibits a characteristic IR-band centered at from about 1674.1 cm "1 to about 1675.7 cm "1 , whereas the corresponding band of the Type II crystal structure is shifted to 1692 cm "1 . Incubation of the Type II crystal structure in a humid chamber (H 2 O) at 25°C and 70°C for 48 h resulted in an interconversion of Type II into Type I only at elevated temperatures. These findings might be explained by the incoφoration of water into the crystal lattice, a process which requires a relatively high energy input. It is interesting to note, that the determination of the water content of Type I and II by two different analytical methods (KF and TG) reveals a different water content only for Type I (Table 1). Tightly bound (structural) crystal water in the Type I crystal lattice might be a possible explanation for this phenomenon. DSC spectra of Type I and II are different showing a slow evaporation of the tightly-bound crystal water in Type I (Figure 5). In contrast, the Type II structure probably contains only "adherent" water which can be evaporated under normal TG conditions (Figure 6). The residual organic solvent (ethanol) content of both samples is rather low in both structures. The water content of crystalline N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt lies in a relatively broad range between 5-12% (w/w) depending on the type of crystal structure. A difference corresponding to one equivalent of crystal water was found in the Type I form of the monopotassium salt of N-4-carboxyphenyl-3- (6-oxohydropurin-9-yl) propanamide monohydrate upon determination of the water content by KF and TG. This result shows that a 1 : 1 stoichiometric mixture of structurally bound water and N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt requires relatively strong drying conditions to remove the water (temperature and vacuum) because the water molecules are in a structurally tightly-bound form. It is not known whether the two polymoφhic forms Type I and II of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt show a varying solubility behavior in aqueous solutions. Under controlled conditions of the manufacturing (e.g. hydrolysis of the N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide ethyl ester, drying process) only one single consistent Type I crystal structure with a characteristic powder pattern is obtained. Incubation of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt Type I and II at 70°C with water vapor does not significantly alter the water content of either sample. Results from the interconversion studies demonstrated that the manufacturing process for N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt monohydrate results in a product with a uniformly stable crystal structure. Solubility N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt monohydrate Type I exhibits a good solubility in water (5% (w/v) at 25°C). In aqueous solutions prepared with material which was synthesized by the route designated "direct crystallization" a fine microcrystalline precipitate of the N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide free acid was formed after 2-3 min. Although the assay of K is 1.0 equivalent and although direct crystallized K-salt is isolated at equal or slightly higher pH (pH 8.7) than K-salt synthesized from N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide free acid (pH 8.5), only the latter is completely soluble in water. The precipitation of the free acid form can be explained by the occurrence of an equilibrium between the mono-K-salt and the di-K-salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide free acid (2 mono-K-salt<-»di-K-salt + free acid) and the extreme low solubility of the free acid in water. The free acid precipitates and generates the di-K salt until its concentration is high enough to keep the precipitation and dissolution of the free acid at equilibrium. The solubility of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide Type II (lot Am 960/a) in aqueous solution was measured as a function of temperature (Figure 2). In the solubility curve a plateau is reached at 70°C (17% w/w). Further characterization of the Type III crystalline form was done as described above for Types I and II. The X-ray diffraction powder pattern of Type III is shown in Figure 7(a); Figure 7(b) shows the X-ray diffraction powder pattern of Type I for comparison. The Fourier-transform infrared spectrum of the Type III crystal structure is shown in Figure 8. The peak of the infrared spectrum is at 1687.8 cm "1 . The DSC plot of the Type III crystal form is shown in Figure 9. The TG plot of the Type III crystal form is shown in Figure 10. Analysis of the data of Figure 10 yields a figure of 0.91% for the amount of water present, in close agreement with the value of 0.87% obtained for the Type III crystal structure by Karl Fischer titration. Conclusion The occurrence of different crystalline forms of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide monopotassium salt (polymoφhism) can be attributed to the different crystal water content of the substance (Table 1). The different crystal structures, designated Type I, II and III, can be easily identified by their characteristic X-ray powder patterns which are of good quality. Powder diagrams of different batches of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt monohydrate of Type I show uniform patterns in terms of peak position and intensity. Interconversions of the crystal structures can be achieved by drying or humidification. The fully hydrated crystal structure of Type I containing one crystal water is not susceptible to small changes in total water content (bound crystal water and "adherent" water) with regard to its powder pattern. TABLE 1 TABLE 2 n.a. = not available ADVANTAGES OF THE INVENTION The synthetic methods of the present invention provide an efficient and economical method of synthesizing 9-substituted hypoxanthine derivatives, particularly N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monopotassium salt, in a crystalline form that provides high stability and purity along with pharmaceutical acceptability. These methods use available starting materials, require a minimum number of steps, and provide a product of high purity and bioavailabihty and consistent crystal structure with a minimum of purification. Methods were developed to synthesize different crystal types of the final product and to transform them into the most stable type. The procedure avoids side reactions that complicate isolation and purification of the final product. Although the present invention has been described in considerable detail, with reference to certain preferred versions thereof, other versions and embodiments are possible. Therefore, the scope of the invention is determined by the following claims.

Claims: We claim: 1. A crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide monohydrate that has a solubility of about 5% (w/v) in water at 25°C, has an infrared spectrum peak from about 1674.1 cm "1 to about 1675.7 cm "1 as measured by Fourier-transform infrared spectroscopy, and that has an X-ray powder pattern that is distinct from the X-ray powder pattern of any other crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, the crystalline form being designated Type I. 2. A method for synthesizing the crystalline form of the monopotassium salt of N- 4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate of claim 1 comprising the steps of: (a) reacting the free acid of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide with potassium hydroxide in water; and (b) precipitating the product of step (a) with ethanol to yield the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide of claim 1. 3. A method for synthesizing the crystalline form of the monopotassium salt of N- 4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate of claim 1 comprising the steps of: (a) reacting N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide ethyl ester monoacetate with potassium hydroxide to produce N-4-carboxyphenyl-3-(6-oxohydropurin-9- yl) propanamide in a crystalline form distinct from the crystalline form of claim 1; and (b) treating the product of step (a) with a mixture of ethanol and water containing a low concentration of potassium hydroxide to yield the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate of claim 1. 4. A method for converting a distinct crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, designated Type II, to the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate of claim 1 comprising the step of equilibrating the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide designated Type II with water vapor at a temperature in a range from about 60°C to about 80°C for a time period of from about 36 hours to about 60 hours in the presence of water vapor to yield the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide monohydrate of claim 1. 5. The method of claim 4 wherein the temperature is about 70°C. 6. The method of claim 4 wherein the time period is about 48 hours. 7. The method of claim 5 wherein the time period is about 48 hours. 8. A method for converting a distinct crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, designated Type III, to the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate of claim 1 comprising the step of equilibrating the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide designated Type III with water vapor at about 25°C to yield the crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate of claim 1. 9. A crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6- oxohydropurin-9-yl) propanamide monohydrate that has a solubility of about 10% (w/v) in water at 25°C, has an infrared spectrum peak of about 1693.4 cm "1 as measured by Fourier- transform infrared spectroscopy, and that has an X-ray powder pattern that is distinct from the X-ray powder pattern of any other crystalline form of the monopotassium salt of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, the crystalline form being designated Type II. 10. A substantially anhydrous crystalline form of the monopotassium salt of N-4- carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide monohydrate that has less than about 1.0% water as measured by the Karl Fischer titration process, has an infrared spectrum peak of about 1687.8 cm-1 as measured by Fourier-transform infrared spectroscopy, and that has an X- ray powder pattern that is distinct from the X-ray powder pattern of any other crystalline form of the monopotassium salt of N-4-carboxyphenyl-3-(6-oxohydropurin-9-yl) propanamide, the crystalline form being designated Type III.

Inventors: BOLLINGER HEINRICH, GLASKY ALVIN, MUELLER HANS RUDOLF, BOLLINGER, HEINRICH, GLASKY, ALVIN, MUELLER, HANS, RUDOLF
Applicants: BOLLINGER HEINRICH, GLASKY ALVIN, MUELLER HANS RUDOLF, NEOTHERAPEUTICS INC, BOLLINGER, HEINRICH, GLASKY, ALVIN, MUELLER, HANS, RUDOLF, NEOTHERAPEUTICS, INC.
Application year: 2001
Main classification: C07D473
IPC codes: A61K31, A61P25, C07D473