A NOVEL CRYSTALLINE FORM OF DISODIUM N-[4-[2-(2-AMINO-4,7-DIHYDRO-4-OXO-3H-PYRROLO[2,3-D]-PYRIMIDIN-5-YL)ETHYL]BENZOYL]-L-GLUTAMIC ACID SALT AND PROCESSES THEREFOR

Abstract: The invention relates to the field of organic and pharmaceutical chemistry and provides an improved process for preparing the multi-targeted antifolate N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid and crystals thereof.

Description: A NOVEL CRYSTALLINE FORM OF DISODIUM N-[4-[2-(2-AMTNO-4,7- D YDRO-4-OXO-3H-PYRROLO[2,3-D]-PYRIMIDrN-5-YL)ETHYL]BENZOYL]-L- GLUTAMIC ACID SALT AND PROCESSES THEREFOR Field of the Invention The present invention relates to the field of pharmaceutical and organic chemistry and provides for a novel crystal form of the multi-targeted antifolate disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L- glutamic acid salt (hereinafter MTA) and processes therefor. State of the Art Pyrrolo[2,3-d]pyrimidine based antifolates have been used for a number of years as chemotherapeutic agents in the treatment of cancer. A number of such pyrrolo[2,3-d]pyrimidine based antifolates are known (see: for example, U.S. Patents 4,997,838; 5,106,974; 5,939,420; and 5,877,178, incorporated by reference herein), as are processes for preparing the same (see for example, U.S. Patents 5,416,211, 5,344,932 and 5,539,113, incorporated by reference herein and hereinafter referred to as '211 Patent, '932 Patent, and '113 Patent). The pyrrolo[2,3-d]pyrimidine disodium salt, as represented by formula I: is referred to herein as MTA. Extensive research and evaluation has revealed that MTA is a potent inhibitor of several folate -requiring enzymes, including thymidine synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase. MTA is currently in clinical trials for use as an anticancer treatment in patients exhibiting a wide variety of solid tumors. The process by which MTA is produced needs to be one that is amenable to large scale production. Additionally, it is desirable that the product should be in a form that is readily filterable and easily dried. Finally, it is economically desirable that the product be stable for extended periods of time without the need for specialized storage conditions. We have now surprisingly and unexpectedly found that MTA can be prepared in crystalline form. Thus, the present invention provides MTA in the new crystalline form designated disodium MTA Hydrate Form I. Furthermore, the present invention provides a process for preparing disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid salt . The process described in the '211 Patent teaches preparing the disodium salt of formula I by treating the acid of formula II with a base (Scheme I). There are several disadvantages with this process. For example, the acid of formula π is highly toxic requiring special handling measures and equipment. Also, isolation of the acid of formula U requires a difficult pH operation and a filtration, which is time-consuming and costly. Scheme I We have surprisingly and unexpectedly found that the disodium salt form of MTA may be prepared by a process which avoids the toxicity problem, a difficult pH operation, and a costly, time-consuming filtration process. Unexpectedly, the present improved process for making MTA provides a number of advantages, e.g. it avoids the isolation and subsequent dispensing of the acid of formula π, thus avoiding the problems previously discussed. Also, the amount of solvent used in the present process is reduced by about 30 percent over the process described in Patent '211. The improved process of the present invention for preparing MTA comprises reacting the 5-substituted pyrrolo[2,3-d]pyrimidine intermediate of the formula in with sodium hydroxide and an appropriate solvent according to Scheme π, where R is a carboxy protecting group. Scheme π Summary of the Invention The present invention provides a novel hydrate crystal form of disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L- glutamic acid salt ("disodium MTA Hydrate Form I"), having a characteristic X-ray diffraction pattern, which comprises the following intensities corresponding to d spacings: 18.66 ±0.04 and/or 9.33 ±0.04 when obtained at 22 ±2°C at ambient % relative humidity from a copper radiation source. The present invention further contemplates a process for preparing a compound of formula I: comprising reacting a 5-substituted pyrrolo[2,3-d]pyrimidine of the formula m, wherein R is carboxy protecting groups or a salt thereof with sodium hydroxide. The invention further provides a method of use of the compound of disodium MTA Hydrate Form I for the manufacture of a medicament for the treatment of cancer. The invention further provides for a process for preparing a medicament comprising combining crystalline disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H- pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt in an aqueous solution. The invention further provides for a formulation comprising crystalline disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L- glutamic acid salt in association with a pharmaceutically acceptable carrier. The invention further provides for a process for the preparation of crystalline disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid salt comprising crystallizing disodium N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt from an appropriate solvent. The invention further provides for the preparation of disodium MTA Hydrate Form I, which comprises adjusting the pH of an aqueous solution of disodium N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt from about 6.5 to about 9.5 and precipitating disodium MTA Hydrate Form I from the pH adjusted aqueous solution. The invention further provides an article of manufacture comprising packaging material and a composition comprising crystalline disodium N-[4-[2-(2-amino-4,7- dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt contained within said packaging material, wherein said crystalline salt is effective in the treatment of cancer and a label which indicates that said crystalline salt can be used in the treatment of cancer. BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts a representative XRD pattern of disodium MTA Hydrate Form I. Figure 2 is a representative solid state NMR spectrum of disodium MTA Hydrate Form I. Figures 3 depicts a representative FT-IR spectra for disodium MTA Hydrate Form I. Detailed Description of the Invention Throughout this document, all temperatures are in degrees Celsius and all expressions of proportion, percentage, and the like, are in weight units, except for solvents or mixtures thereof which are in volume units. The term "hydrate" as used herein describes the crystalline lattice of disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]-pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid salt, which can contain variable amounts of water, from about 0.01 to about 3 equivalents of water, depending upon the relative humidity in the storage conditions. Preferably, disodium MTA Hydrate Form I contains from about 2 to about 3 equivalents of water, most preferred is 2.4-2.6 equivalents of water. The term "cancer" as used herein describes a disease state well known in the art wherein the tumor responds to treatment with an antifolate drug. Such disease states include, but are not limited to, colorectal, breast, cervical, acute myeloid leukemia (ALM), acute lymphoblastic leukemia (ALL), nonsmall-cell lung cancer, bladder, head and neck, non-Hodgkin's lymphoma, sarcoma, prostate, melanoma, mesothelioma, gastrointestinal tract, stomach, rectal, colorectal, ovarian, pancreatic, lung, hepatoma, malignant fibrous histiocytoma, and oropharyngeal. See Ann L. Jackman, Antifolate Drugs in Cancer Therapy. Humana Press, Totowa, N.J., 1999 As used herein, the term "effective amount" refers to an amount of a compound or drug, which is capable of performing the intended result. For example, an effective amount of disodium MTA Hydrate Form I that is administered in an effort to reduce tumor growth is that amount which is required to reduce tumor growth. The term "ambient % relative humidity" as used herein describes a range of humidity of about 20% to about 50% relative humidity. The term "carboxy protecting group" as used herein refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound. The species of carboxy-protecting group employed is not critical so long as the derivatized carboxylic acid is labile accessible and is stable to conditions up to its removal and can be removed by the action on sodium hydroxide. See E. Haslam, Protective Groups in Organic Chemistry, J.G.W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T.W. Greene, Protective Groups in Organic Synthesis. John Wiley and Sons, New York, N.Y., 1981, Chapter 5. A related term is "protected carboxy," which refers to a carboxy-protecting groups. Preferred esters include, straight or branched Ci-Cό alkyl esters, preferably methyl ester or ethyl ester. The compounds of formula HI, exist as acid addition salts formed with a wide variety of inorganic and organic acids. The acid salts of the formula HJ can be prepared by methods known in the art, for example, according to the '211 Patent, incorporated by reference herein. Typical acids which can be used include sulfuric, hydrochloric, hydrobromic, phosphoric, hypophosphoric, hydroiodic, sulfamic, citric, acetic, maleic, malic, succinic, tartaric, cinnamic, benzoic, ascorbic, mandelic, p-toluenesulfonic, benzenesulfonic, methanesulfonic, trifluoroacetic, hippuric and the like. The preferred salts are those formed with p-toluenesulfonic acid, hydrochloric acid or acetic acid. The process of Scheme II may be performed by reacting a 5-substituted pyrrolo[2,3-d]pyrimidine acid and formula HI, wherein R is carboxy protecting groups or a salt thereof with sodium hydroxide. At least 2 w/v (g mL) equivalents of sodium hydroxide are needed when using Form II and the salt of Form LLT. When the salt of Form HI is used an additional equivalence of sodium hydroxide is required. Preferably from about 3 to 5 equivalents of the sodium hydroxide is required. For the ease of process about 3 to 10 equivalents are employed, preferably from about 4 to about 6 equivalents. More preferred is from about 3 to about 10 equivalents, most preferably from about 4 to about 7 equivalents are employed. A typical reaction is carried out in the presence of an appropriate solvent. Generally the use of an aqueous solvent is suitable. The process of Scheme II could be carried out under anhydrous conditions, however, water or an aqueous solution is preferable. Such aqueous solutions contain water and a water miscible solvent such as acetone, acetonitrile, dimethyl formamide, ethanol, methanol, isopropanol, and tetrahydrofuran. The process can be can be carried out at ambient or elevated temperatures, preferably maintaining the solution from about 40 to 70°C. In a preferred embodiment in Scheme II a crystalline form can be collected by lypho zation, evaporation and recrystallization Additionally, the present invention provides a process for the preparation of crystalline disodium N-[4-[2-(2-amιno-4,7-dιhydro-4-oxo-3H-pyrrolo[2,3-d]-pynmιdm-5- yl)ethyl]benzoyl]-L-glutamιc acid salt which compπses crystallizing MTA from a solution under conditions which yield crystalline disodium N-[4-[2-(2-amιno-4,7-dιhydro- 4-oxo-3H-pyrrolo[2,3-d]-pyπmιdιn-5-yl)ethyl]benzoyl]-L-glutamιc acid salt Crystalline disodium N-[4-[2-(2-amιno-4,7-dιhydro-4-oxo-3H-pyrrolo[2,3-d]- pyπmιdm-5-yl)ethyl]benzoyl]-L-glutamιc acid salt may be prepared by adjusting the pH of an aqueous solution of disodium N-[4-[2-(2-amιno-4,7-dιhydro-4-oxo-3H-pyrrolo[2,3- d]-pyπmιdιn-5-yl)ethyl]benzoyl]-L-glutamιc acid salt from about 5 to about 12 and precipitating crystalline disodium N-[4-[2-(2-amιno-4,7-dιhydro-4-oxo-3H-pyrrolo[2,3- d]-pyπmιdιn-5-yl)ethyl]benzoyl]-L-glutamιc acid salt from the pH adjusted aqueous solution. In the process according to the invention, an aqueous solution is defined as about 1 to about 20, preferably, 3 to about 10 volumes, more preferably from about 4 to about 7 volumes, of water and an appropπate solvent The skilled artisan will appreciate that numerous solvents may be employed so long as the solvent in conjunction with the water dissolves the disodium MTA salt, such solvents include, but are not limited to alcohols, dimethylsulfoxide, acetonitrile, dimethyl formamide, tetrahydrofuran and acetone The temperature of the solution should be maintained at about room temperature to about the boiling point of the solution, preferably from about 60°C to about 70°C. The pH of the solution may be adjusted to the desired pH through the use of acid and base buffers Acid and base buffers are well known to the skilled artisan and are commercially available The methods in which crystals are precipitated from the solution are well known to the skilled artisan and are not cπtical to the process of the present invention For example, an anti-solvent may be added, the solution may be cooled, or the solution may be seeded. For additional methods of precipitating crystals see, for example, A.E Nielsen, Treatise on Analytical Chemistry, 2 nd ed., Part I, vol. 3, Chapter 27, 1983 The precise conditions under which crystalline disodium MTA Hydrate Form I is formed may be empirically determined and it is only possible to give a number of methods that have been found to be suitable in practice. In a preferred embodiment in Scheme II the crystalline disodium MTA Hydrate Form I can be prepared by adjusting the pH of an aqueous solution of the disodium MTA salt from about 6.5 to about 9.5, preferably about 7.5 to about 8.5, most preferably about 8.0 and precipitating crystalline disodium MTA Hydrate Form I from the pH adjusted aqueous solution. In the process according to the invention, an aqueous solution is defined as about 1 to about 20, preferably, 3 to about 10 volumes, more preferably from about 4 to about 7 volumes, of water and an appropπate solvent. The skilled artisan will appreciate that numerous solvents may be employed so long as the solvent in conjunction with the water dissolves the disodium MTA salt. The temperature of the solution should be maintained at about room temperature to about the boiling point of the solution, preferably from about 60°C to about 70°C . The methods in which crystals are precipitated from the solution are well known to the skilled artisan and are not cπtical to the process of the present invention. For example, an anti-solvent may be added, the solution may be cooled, or the solution may be seeded. For additional methods of precipitating crystals see, for example, A.E. Nielsen, Treatise on Analytical Chemistry, 2 nd ed., Part I, vol. 3, Chapter 27, 1983. Preferably an anti-solvent, such as ethanol, isopropanol, acetonitrile, or acetone, is added to the pH adjusted aqueous solution. The following Preparations and Examples further illustrate a process for the preparation of the compounds of formula HI and disodium MTA Hydrate Form I. These Preparations and Examples are not intended to be limiting to the scope of the invention in any respect and should not be so construed. Preparation 1 Methyl 4-(4-tπmethylsιlyloxy-3-butenyl)benzoate To 3.65 g (17.7 mmol) of 4-(4-carbomethoxyphenyl)butanal and 3.43 g (21.2 mmol) of 1,1,1,3,3,3-hexamethyldιsιlazane m 177 ml of methylene chloπde in a nitrogen atmosphere was added 3.89 g (19.5 mmol) tπmethylsilyl iodide at -15°C over 2 minutes The mixture was stirred for 10 minutes then allowed to come to room temperature After 2 hours, the excess reagent was quenched by addition of 100 ml water. The layers were separated and the organic phase dπed over sodium sulfate. The solvent was removed by vacuum concentration to give 5.0 g of methyl 4-(4-tπmethylsιlyloxy-3-butenyl)benzoate m a yield of 100 percent. Thin Layer Chromatography (TLC) analysis (silica; hexane - ethyl acetate 3:2) indicated that the above product was substantially pure, b.p. 170°C @ 0.12 torr. *H NMR (CDC1 3 ) δ 0.15 (s, 9H), 2.41 (q, J = 7.2 Hz, 2H), 2.69 (m, 2H), 3.89 (s, 3H), 4.47 (q, J = 7.2 Hz, IH), 6.15 (m, IH), 7.25 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H); 13 C NMR (CDCI3) δ -0.60, 25.0, 35.9, 51.8, 109.8, 128.4, 128.5, 129.6, 138.6, 148.0, 167.1; FDMS 279 (90), 278 (M+ 100%) 280, 251, 226; An analytical sample was obtained by flash chromatography (silica; hexane - ethyl acetate 3:2). Anal, for C15H22O3S1, Calcd: C, 64.71; H, 7.96; Found: C, 64.90; H, 8.05. Preparation 2 2-bromo-4-(4-carbomethoxyphenyl)butanal To 4.46 g (16 mmol) of methyl 4-(4-tπmethylsιlyloxy-3-butenyl)benzoate, prepared in Preparation 1, in 16 ml carbon tetrachloπde at -20°C was slowly added 2.56 g (16 mmol) of bromine in 16 ml carbon tetrachloπde over 4 hours. The mixture was allowed to come to room temperature then decanted from a small amount of insoluble mateπal. The solvent was removed by vacuum rotary evaporation to give 4.60 g of 2- bromo-4-(4-carbomethoxyphenyl)butanal Chromatography puπfication (silica; hexane - ethyl acetate 7:3) of the above product gave 4.0 g in a yield of 87.8 percent *H NMR (CDCI3) δ 2.24 (m, IH), 2,36 (m, IH), 2.81 (m, IH), 2.90 (m, IH), 3.90 (s, 3H), 4.16 (m, 1H), 7.27 (d, / = 8.2 Hz, 2H), 7.97 (d, J = 8.2 Hz, 2H), 9.46 (d, J = 2.1 Hz, IH); 13 C NMR (CDC1 3 ) δ 32.5, 32.7, 51.7, 54.3, 128.3, 128.4, 129.8, 145.0, 166.6, 192.0. Preparation 3 4-(2-[2-Amιno-4-Oxo-3,7-Dιhydropyrrolo[2, 3 d] Pyπmιdιn-5-yl]ethyl)Benzoιc Acid Methyl Ester To 1.69 g (13.4 mmol) of 2,4-dιamιno-6-hydroxypyπmιdιne, 2.20 g (26.8 mmol) of sodium acetate and 20 ml of water at 80°C were added 3.82 g (13.4 mmol) of 2-bromo- 4-(4-carbomethoxyphenyl)butanal, prepared m Preparation 2, in 7 ml of methanol over 5 minutes Most of the suspended mateπal dissolved and formed a precipitate. The mixture was maintained at 80°C for 5 minutes, cooled to room temperature and stirred for 30 minutes. The mixture was filtered, washed with water, and dπed for 18 hours at 50°C @ 10 ton to provide 3.32 g of 4-(2-[2-amιno-4-oxo-3,7-dιhydropyrrolo[2, 3 d] pynmidin- 5-yl]ethyl)benzoιc acid methyl ester (m.p. >220°C) in a yield of 79.4 percent. The remaining filtrate was cooled to 5°C and filtered to provide an additional 0.069 g of the above product, for a combined yield of 81 percent. *H NMR (DMSO-d6) δ 2.80 (m, 2H), 2.93 (m, 2H), 3.78 (s, 3H), 5.97 (s, 2H), 6.26 (d, J = 2.0 Hz, IH), 7.28 (d, J = 8.2 Hz, 2H), 7.80 (d, 7 = 8.2 Hz, 2H), 10.12 (s, IH), 10.58 (s, IH); FDMS 312 (M+), Anal, for C16H16N4O3, Calcd: C, 61.53; H, 5.16; N, 17.94; Found: C, 61.79; H, 5.33; N, 17.66. Preparation 4 4-[2-(2-Amιno-4,7-dιhydro-4-oxo-3H-pyrrolo[2,3-d] pyπmιdιn-5-yl)ethyl]benzoιc acid A mixture of 3.17 g (10.15 mmol) of 4-[2-(2-ammo-4,7-dιhydro-4-oxo-3H- pyrrolo[2,3-d]pyπmιdιn-5-yl)ethyl]benzoιc acid methyl ester, prepared in Example 3, in 30 ml (30 meq) of IN aqueous sodium hydroxide and 5 ml methanol was stirred for 20 hours at room temperature Tetrahydrofuran (5 ml) was added and the mixture was stiπed for 4 hours then neutralized with 30 ml of IN aqueous hydrochloπc acid. The resulting precipitate was separated by filtration, washed with water (20 ml) and dπed in a vacuum oven at 50°C to obtain 2.65 g of the above product in a yield of 87 percent. J H NMR (DMSO-d 6 ) δ 2.45 (m, 2H), 2.91 (m, 2H), 5.99 (s, 2H), 6.27 (d, J = 2.0 Hz, IH), 7.27 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.2 Hz, 2H), 10.14 (s, IH), 10.59 (d, J = 2.0 Hz, IH), 13.2 (bs, IH). Preparation 5 N-[4-[2-[2-Amino-4,7-dihydro-4-oxo-3H-pyπolo[2,3-d]pyrimidin-5- yl]ethyl]benzoyl]glutamic acid dimethyl ester To 2.00 g (6.74 mmol) of 4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoic acid, prepared in Preparation 4, in 23 ml dimethylformamide under nitrogen was added 1.40 g (13.8 mmol) of N- methylmorpholine and 1.17 g (6.70 mmol) of 4-chloro-2,6-dimethoxytriazine. The formation of the active ester was monitored by the HPLC analysis of aliquots. After 40 minutes at room temperature 0.70 g (6.9 mmol) of N-methylmorpholine was added followed by 1.56 g (7.37 mmol) L- glutamic acid dimethyl ester hydrochloride. After 30 minutes HPLC analysis indicated substantially complete consumption of the active ester and formation of N-[4-[2-[2-Amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5- yljethyl] benzoyl]glutamic acid dimethyl ester. The reaction mixture was filtered and the above product concentrated and purified by silica chromatography (elution 20/80 percent methanol : methylene chloride). The pure fractions were pooled and provided 1.30 g of the above product in a yield of 43 percent. *H NMR (DMSO-d 6 ) δ 2.01 (m, 2H), 2.40 (t, J = 7.4 Hz, 2H), 2.80 (m, 2H), 2.92 (m, 2H), 3.53 (s, 3H), 3.59 (s, 3H), 4.40 (m, IH), 5.99 (s, 2H),6.26 (d, J = 1.9 Hz, IH), 7.23 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 8.62 (d, J = 7.5 Hz, IH), 10.15 (s, IH), 10.57 (d, J = 1.9 Hz, IH). Preparation 6 N-4-(3-oxoprop-l-yl)-benzoyl-L-glutamιc acid diethyl ester A mixture of 310 mg (1.38 mmol) of palladium acetate, 14.88 g (46.2 mmol) of tetrabutylammonium bromide, and 9.70 g (115 mmol) of sodium bicarbonate in 160 ml of dimethylformamide was degassed under vacuum and restored in a nitrogen atmosphere. Then 20.0 g (46.2 mmol) of 4-ιodobenzoyl-L-glutamιc acid and 4.01 g (69.2 mmol) of allyl alcohol were added and the resulting mixture heated to 52°C. After 20 hours TLC (silica; hexane - ethyl acetate 6:4) indicated that the reaction was substantially complete The reaction mixture was added to 100 ml of water, 100 ml of ethyl acetate, and 2 0 g of activated carbon. This mixture was filtered and mixed with 100 ml of water and 100 ml of methyl t-butyl ether. The aqueous phase was extracted with 100 ml of methyl t-butyl ether. The organic phase was washed with 2 X 100 ml of water, 50 ml of saturated sodium chloπde and dπed over sodium sulfate and activated carbon. Vacuum removal of the solvents under vacuum gave 11.5 g of N-4-(3-oxoprop-l-yl)-benzoyl-L-glutamιc acid diethyl ester in a yield of 69 percent. Chromatography of a 2.15 g portion of the above the crude product gave 1.79 g of puπfied mateπal, m.p. 82°C-83°C. [α]589 -15 7°, [αJ365 - 47.6° (c 1, EtOH); J H NMR (CDCI3) δ 1.22 (t, 7 = 7.1 Hz, 3H), 1.31 (t, 7 = 7.1 Hz, 3H), 2.15 (m, IH), 2.32 (m, IH), 2.46(m, 2H), 2.81 (t, 7 = 7 4 Hz, 2H), 3.00 (t, 7 = 7.4 Hz, 2H), 4.11 (q, 7 = 7.1 Hz, 2H), 4.23 (q, 7 = 7.1 Hz, 2H), 4.78 (m, IH), 7.00 (d, 7 = 7.4 Hz, IH), 7.27 (d, 7 = 8.1 Hz, 2H), 7.75 (d, 7 = 8.1 Hz, 2H) 9.82 (d, 7 = 1.1 Hz, IH). Preparation 7 N-4-(l-methoxy-l-buten-4-yl)benzoyl-L-glutamιc acid diethyl ester To 1.18 g (3.45 mmol) of methoxymethyltπphenyl phosphonium chloπde in 10 ml of dry toluene at 0°C under nitrogen was added 3.1 ml (3.1 mmol) of 1M potassium t- butoxide in tetrahydrofuran over 20 minutes. After stirπng for 10 minutes 1.14 g (3.13 mmol) of the N-4-(3-oxoprop-l-yl)-benzoyl-L-glutamιc acid diethyl ester, prepared in Preparation 9, in 10 ml of toluene were added over 10 minutes. After 1 hour the reaction mixture was poured into 20 ml of ethyl acetate, washed with 2 X 10 ml of water and dπed over magnesium sulfate. Vacuum removal of the solvent gave 2.1 g of the above product. Chromatography (silica gel; ethyl acetate - hexane 6:4) gave 0.31 g of a mixture of geometπc isomer products in a combined yield of 25 percent. J H NMR (CDCI3) δ 1.22 (t, 7 = 7.1 Hz, 3H) 1.31 (t, 7 = 7.1 Hz, 3H), 2.1-2.5 (m, 6H), 2.70 (m, 2H), 3 48 (s), 3.56 (s), total 3H, 4.12 (m, 2H), 4.24 (q, 7 = 7.1 Hz, 2H), 4.32 (m), 4.73(m) total IH, 4 81 (m, IH), 5 87 (d, 7 = 6.0 Hz), 6.28 (d, 7 = 12.7 Hz), total IH, 7.26 (m, 2H), 7.74 (m, 2H) Preparation 8 4-(2-[2-amιno-4(lH)-oxo-4,7-dιhydropyπolo[2,3-d]pyπmιdιn-5-yl]ethyl)benzoyl-L- glutamic acid diethyl ester To 300 mg (0.766 mmol) of the N-4-(l-methoxy-l-buten-4-yl)benzoyl-L-glutamιc acid diethyl ester, prepared in Preparation 10, 3.0 ml of acetonitπle and 3.0 ml of water, stirred at room temperature, was added 122 mg (0.766 mmol) of bromine in 1 ml of acetonitπle. To this solution was added 188 mg (2.3 mmol) of sodium acetate and 0.97 mg (0.77 mmol) of 2,4-dιamιno-6-hydroxypyπmιdιne and the resulting mixture was heated to 60°C for 18 hours, cooled, then concentrated under vacuum. The resulting residue was tπturated 2 x 5 ml of water and decanted. Ethanol (5 ml) and 440 mg (2.3 mmol) of p-toluenesulfomc acid monohydrate were added. After heating under reflux for 20 minutes the mixture was cooled to room temperature, filtered. The precipitate was washed with ethanol (2 x 5ml) and dπed to obtain 210 mg a p-toluenesulfonate salt of the above product in a yield of 42 percent. Chromatography gave a 95 percent pure product. Preparation 9 N-4-(l-methoxy-l-buten-4-yl)benzoyl-L-glutamιc acid diethyl ester To 1.18 g (3.45 mmol) of methoxymethyltπphenyl phosphonium chloπde m 10 ml of dry toluene at 0°C under nitrogen was added 3.1 ml (3.1 mmol) of 1M potassium t- butoxide m tetrahydrofuran over 20 minutes After stirring for 10 minutes 1 14 g (3.13 mmol) of the N-4-(3-oxoprop-l-yl)-benzoyl-L-glutamιc acid diethyl ester, prepared in Preparation 9, in 10 ml of toluene were added over 10 minutes. After 1 hour the reaction mixture was poured into 20 ml of ethyl acetate, washed with 2 X 10 ml of water and dπed over magnesium sulfate Vacuum removal of the solvent gave 2.1 g of the above product. Chromatography (silica gel, ethyl acetate - hexane 6:4) gave 0.31 g of a mixture of geometπc isomer products m a combined yield of 25 percent ! H NMR (CDCI3) δ 1.22 (t, 7 = 7.1 Hz, 3H) 1 31 (t, 7 = 7 1 Hz, 3H), 2.1-2.5 (m, 6H), 2.70 (m, 2H), 3 48 (s), 3.56 (s), total 3H, 4.12 (m, 2H), 4.24 (q, 7 = 7.1 Hz, 2H), 4.32 (m), 4.73(m) total IH, 4 81 (m, IH), 5.87 (d, 7 = 6.0 Hz), 6.28 (d, 7 = 12 7 Hz), total IH, 7.26 (m, 2H), 7 74 (m, 2H) The solvents employed in the process of converting the 5-substituted pyrrolo[2,3- d]pyπmιdιne intermediate of the formula HI to MTA include water, which may be combined with a miscible organic solvent such as ethanol, acetonitπle, acetone, isopropyl alcohol or the like Suitable bases for this same conversion include any inorganic base, such as potassium hydroxide and preferably sodium hydroxide. As the skilled artisan would appreciate, the salt form of the 5-substituted pyrrolo[2,3-d]pyπmιdιne depends on the base used For example sodium hydroxide with convert the compound of formula HI to the disodium salt, whereas potassium hydroxide will form the dipotassium salt form of the compound of formula EQ. The following Examples further illustrate the present invention The Examples are not intended to be limiting to the scope of the invention m any respect and should not be so construed. Example 1 — Compound of Formula HI 4-(2-[2-amιno-4(lH)-oxo-4,7-dιhydropyrrolo[2,3-d]pyπmidιn-5-yl]ethyl)benzoyl-L- glutamic acid diethyl ester To 300 mg (0.766 mmol) of the N-4-(l-methoxy-l-buten-4-yl)benzoyl-L-glutamιc acid diethyl ester, prepared in Preparation 1, 3.0 ml of acetomtπle and 3.0 ml of water, stiπed at room temperature, was added 122 mg (0 766 mmol) of bromine in 1 ml of acetonitπle. To this solution was added 188 mg (2.3 mmol) of sodium acetate and 0 97 mg (0 77 mmol) of 2,4-dιamιno-6-hydroxypyπmιdιne and the resulting mixture was heated to 60°C for 18 hours, cooled, then concentrated under vacuum. The resulting residue was tπturated 2 x 5 ml of water and decanted. Ethanol (5 ml) and 440 mg (2.3 mmol) of p-toluenesulfonic acid monohydrate were added. After heating under reflux for 20 minutes the mixture was cooled to room temperature, filtered. The precipitate was washed with ethanol (2 x 5ml) and dπed to obtain 210 mg a p-toluenesulfonate salt of the above product in a yield of 42 percent. Chromatography gave a 95 percent pure product Example 2 Disodium salt of N-[4-[2-(2-amιno-4,7-dιhydro-4-oxo-3H-pyrrolo[2,3-d]-pyπmιdιn-5- yl)ethyl]benzoyl]-L-glutamιc acid— disodium MTA Hydrate Form I Into a 500 ml flask was placed 5.00 g (7.6 mmol) of a compound of formula HI, prepared as in Example 1, and 33.5 ml of IN NaOH. The reaction mixture was stiπed for 30 minutes and then 16.5 ml of de-ionized water was added. The pH of the mixture was adjusted to 7.7. The temperature of the solution was held at 60-70° as 340 ml of 3A ethanol was added. The resulting light blue solution was allowed to cool to room temperature and the resulting slurry was stiπed for 1 hour. The slurry was filtered, washed with 3A ethanol, and the solids were dπed at 40°C in a vacuum oven. Yield = 3.89 g (100%), water = 9.1%. Example 3 Disodium salt of N-[4-[2-(2-amιno-4,7-dιhydro-4-oxo-3H-pyπolo[2,3-d]-pynmιdιn-5- yl)ethyl]benzoyl]-L-glutamιc acid— disodium MTA Hydrate Form I Into a 500 ml Erlenmeyer flask was placed 5.00 g (7.6 mmol) of the diethyl ester pTsOH salt and 33.5 ml of IN NaOH. The reaction mixture was stiπed until all the solids had dissolved (about 30 minutes). Additional water (16.5 ml) was added and the pH of the mixture was adjusted to 8.0 using dilute HC1 and dilute NaOH. The resulting solution was heated to 45-50°C and then 225 ml of acetone was added. The resulting slurry was cooled to room temperature and filtered. The wetcake was washed with acetone and the solids were dried in a vacuum oven at 40°C. Yield = 4.22 g, water = 17.7 %. Example 4 Disodium salt of N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyπolo[2,3-d]-pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid— disodium MTA Hydrate Form I Into a 500 ml Erlenmeyer flask was placed 5.00 g (7.6 mmol) of the diethyl ester pTsOH salt and 33.5 ml of IN NaOH. The reaction mixture was stiπed until all the solids had dissolved (about 30 minutes). Additional water (16.5 ml) was added and the pH of the mixture was adjusted to 8.5 using dilute HC1 and dilute NaOH. The resulting solution was heated to 60-65°C and then 225 ml of isopropanol was added. The resulting slurry was cooled to room temperature and filtered. The wetcake was washed with acetone and the solids were dried in a vacuum oven at 40°C. Yield = 3.85 g, water = 11.7 %. A number of methods are available to characterize crystalline forms of organic compounds. Among these methods are differential scanning calorimetry, solid state NMR spectrometry, infra-red spectroscopy, and x-ray powder diffraction. The x-ray powder diffraction pattern is very useful for distinguishing between different crystalline forms of a compound. It is well known in the crystallography art that, for any given crystal form, the relative intensities of the diffraction peaks may vary due to a number of factors, including the effects of preferred orientation which result from a particular crystal morphology, and particle size. Where the effects of prefeπed orientation and/or particle size are present, peak intensities (that is, the I/Io value) are altered, but the characteristic peak positions of the polymorph are unchanged. See, e.g., The United States Pharmacopoeia #23, National Formulary #18, pages 1843-1844, 1995. X-ray powder diffraction analysis can be readily performed as follows. After lightly gπndmg the sample with an agate mortar and pestle, the sample is loaded into a sample holder for the x-ray powder diffraction measurement. The x-ray powder diffraction patterns are measured using a Siemens D5000 x-ray powder diffractometer equipped with a CuKα source (α=1.54056A) operated at 50 kV and 40 mA using a Kevex solid-state silicon lithium detector. Interplanar spacings and peak intensities for the most prominent features were measured using a double-den vative peak picking method. Disodium MTA Hydrate Form I has a typical XRD pattern with interplanar spacings (d) in Angstroms and typical relative intensities (I/I 0 ) as shown in Table I. The error of measurement is +/- 0.04 A. X-ray peaks with I/Ij of 10% or greater were reported m Table 1, below. The cutoff was chosen arbitraπly. The intensities are reported normalized to the most intense line. The effects of preferred oπentation can be greatly reduced using a sample that is prepared in a manner that minimizes this effects, such as the use of a well ground sample. Disodium MTA Hydrate Form I is characteπzed by X-ray diffraction pattern which compπses intensities corresponding to the following d spacings: 18.66 and/or 9.33 +/-0.04 A when obtained at 22 ±2°C and at ambient % relative humidity using a copper radiation source. Preferably, a properly prepared sample of disodium MTA Hydrate Form I may be characteπzed as having an X-ray diffraction pattern which compπses peaks coπesponding to the following d spacings: 18.66, 9.33 and/or 4.92 +/- 0.04 A when obtained at 22 ±2°C and 31 ±10% relative humidity from a copper radiation source. Table 1: d-spacing I/Io 18.66 100 11.38 18 9.33 69 8.71 11 8.44 24 6.22 28 5.92 17 5.69 55 5.59 10 5.14 11 4.92 49 4.75 24 4.66 22 4.59 16 4.26 12 3.87 52 3.80 12 3.72 38 3.43 19 3.29 25 3.13 10 3.11 16 3.08 18 2.95 11 Additionally, as the skilled artisan would appreciate, disodium MTA Hydrate Form I may also be characterized by solid state NMR spectroscopy. Solid state NMR (l^C) analysis can be carried out using a Varian Unity 400 MHz spectrometer operating at a carbon frequency of 100.580 MHz, equipped with a complete solids accessory and Varian 7 mm VT CP/MAS probe. Acquisition parameters were as follows: 90° proton r.f. pulse width 4.0 μs, contact time 1.0 ms, pulse repetition time 5 s, MAS frequency 7.0 kHz, spectral width 50 kHz, and acquisition time 50 ms. Solid state ^C chemical shifts reflect not only the molecular structure of disodium MTA Hydrate Form I, but also the electronic environment of the molecule in the crystal. The diagnostic ^C resonances for disodium MTA Hydrate Form I were processed in D2O and are reported in Table 2. Table 2: Solid State 13 C NMR Data for disodium MTA Hydrate Form I The assignments for the infrared (IR) and Raman spectra of disodium MTA Hydrate Form I are presented below. In the IR analysis of this compound the KBr pellet spectrum was compared to the mull spectrum. No significant differences were observed between the two spectra showing that no ion exchange appears to be taking place during the KBr pellet formation. Assignments were based on review of model compounds. The following HI assignments were made on a KBr pellet of this compound. The IR bands are found at 3496 cm "1 , 3434 cm "1 , 3391 cm "1 , 3229 cm "1 , 3071 cm "1 , 3009 cm "1 1623 cm "1 , 1591 cm "1 and 1569 cm "1 and the Raman bands at 3108 cm '1 , 3070 cm "1 , 1612 cm "1 , 675 cm "1 , 677 cm "1 and 1569 cm "1 . The present invention also includes methods employing pharmaceutical formulations which contain, as the active ingredient, disodium MTA Hydrate Form I or a crystalline form of disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]- pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt, in association with pharmaceutical carriers. A skilled artisan would know of such formulations and their manufacture, see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, (16th ed. 1980). The formulations are preferably formulated in a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. The crystals are effective over a wide dosage range. For example, dosages per day normally fall within the range of about 0.5 to about 30 mg/kg of body weight. However, it will be understood that the amount of the crystal actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual crystal administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several smaller doses for administration throughout the day. The crystals of the present invention can be administered alone or in the form of a pharmaceutical composition in combination with pharmaceutically acceptable carriers or excipients, the proportion and nature of which are determined by the solubility and chemical properties of the compound selected, the chosen route of administration, and standard pharmaceutical practice. In another embodiment, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of the crystal in admixture or otherwise in association with one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art. The carrier or excipient may be a solid, semi-solid, or liquid material, which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art. The pharmaceutical composition may be adapted for oral, inhalation, parenteral, or topical use and may be administered to the patient in the form of tablets, capsules, aerosols, inhalants, suppositories, solution, suspensions, or the like. The crystals of the present invention may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the crystals may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 4% of the compound of the present invention, the active ingredient, but may be varied depending upon the particular form and may conveniently be between 4% to about 70% of the weight of the unit. The amount of the crystal present in compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention may be determined by someone skilled in the art. The tablets, pills, capsules, troches and the like may also contain one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; and sweetening agents such as sucrose or saccharin may be added or a flavoring agent such as peppermint, methyl salicylate or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil. Other dosage unit forms may contain other various materials, which modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the present compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used. For the puφose of parenteral therapeutic administration, the crystals of the present invention may be incoφorated into a solution or suspension. These preparations should contain at least 0.1% of a crystal of the invention, but may be varied to be between 0.1 and about 50% of the weight thereof. The amount of the crystalline disodium N-[4-[2-(2- amino-4,7-dihydro-4-oxo-3H-pyπolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt present in such compositions is such that a suitable dosage will be obtained. Prefeπed compositions and preparations are able to be determined by one skilled in the art. The crystals of the present invention may also be administered by inhalation, such as by aerosol or dry powder. Delivery may be by a liquefied or compressed gas or by a suitable pump system, which dispenses the compounds of the present invention or a formulation thereof. Formulations for administration by inhalation of compounds of formula (1) may be delivered in single phase, bi-phasic, or tri-phasic systems. A variety of systems are available for the administration by aerosol of the compounds of formula (1). Dry powder formulations are prepared by either pelletizing or milling the compound of formula (1) to a suitable particle size or by admixing the pelletized or milled compound of formula (1) with a suitable carrier material, such as lactose and the like. Delivery by inhalation includes the necessary container, activators, valves, subcontainers, and the like. Preferred aerosol and dry powder formulations for administration by inhalation can be determined by one skilled in the art. The crystals of the present invention may also be administered topically, and when done so the carrier may suitably comprise a solution, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Topical formulations may contain a concentration of the formula (1) or its pharmaceutical salt from about 0.1 to about 10% w/v (weight per unit volume). The solutions or suspensions may also include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, such as sodium chloride and mannitol, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials mane of glass or plastic. The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. "Active ingredient" means either disodium MTA Hydrate Form I or a crystalline form of disodium N-[4-[2-(2-amino-4,7-dihydro-4- oxo-3H-pyπolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt. Formulation Example 1 Hard gelatin capsules containing the following ingredients are prepared: Quantity Ingredient (mg/capsule) Active ingredient 30.0 Starch 305.0 Magnesium stearate 5.0 The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities. Formulation Example 2 A tablet formula is prepared using the ingredients below: Quantity Ingredient (mg/tablet) Active ingredient 25.0 Cellulose, microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0 The components are blended and compressed to form tablets, each weighing 240 mg. Formulation Example 3 A dry powder inhaler formulation is prepared containing the following components: Ingredient Weight % Active ingredient 5 Lactose 95 The active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance. Formulation Example 4 Tablets, each containing 30 mg of active ingredient, are prepared as follows: Quantity Ingredient (mg/tablet) Active ingredient 30.0 mg Starch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyπolidone (as 10% solution in water) 4.0 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc l.Q mg Total 120 mg The active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyπolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve. The granules so produced are dried at 50-60°C and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg. Formulation Example 5 Capsules, each containing 40 mg of medicament are made as follows: Quantity Ingredient (mg/capsule) Active ingredient 40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mg Total 150.0 mg The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities. Formulation Example 6 Suppositories, each containing 25 mg of active ingredient are made as follows: Ingredient Amount Active ingredient 25 mg Saturated fatty acid glycerides to 2,000 mg The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool. Foπriulation Example 7 Suspensions, each containing 50 mg of medicament per 5.0 ml dose are made as follows: Ingredient Amount Active ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purified water to 5.0 ml The active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume. Formulation Example 8 Capsules, each containing 15 mg of medicament, are made as follows: Quantity Ingredient (mg/capsule) Active ingredient 15.0 mg Starch 407.0 mg Magnesium stearate 3.0 mg Total 425.0 mg The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425 mg quantities. Formulation Example 9 An intravenous formulation may be prepared as follows: Ingredient Ouantity Active ingredient 250.0 mg Isotonic saline 1000 ml Formulation Example 10 A topical formulation may be prepared as follows: Ingredient Quantity Active ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g White Soft Paraffin to 100 g The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incoφorated and stiπed until dissolved. The active ingredient is added and stirring is continued until dispersed. The mixture is then cooled until solid. Formulation Example 11 Subl gual or buccal tablets, each containing 10 mg of active ingredient, may be prepared as follows: Quantity Ingredient Per Tablet Active ingredient 10.0 mg Glycerol 210.5 mg Water 143.0 mg Sodium Citrate 4.5 mg Polyvmyl Alcohol 26.5 mg Pol yvinylpyπo done 15.5 mg Total 410.0 mg The glycerol, water, sodium citrate, polyvmyl alcohol, and polyvinylpyrro done are admixed together by continuous stirπng and maintaining the temperature at about 90°C. When the polymers have gone into solution, the solution is cooled to about 50-55°C and the active ingredient is slowly admixed. The homogenous mixture is poured into forms made of an inert mateπal to produce a drug-containing diffusion matπx having a thickness of about 2-4 mm This diffusion matπx is then cut to form individual tablets having the appropπate size. Another preferred formulation employed m the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent 5,023,252, issued June 11, 1991, herein incoφorated by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents

Claims: We claim: 1. A process for preparing a compound of formula I: comprising reacting a 5-substituted pyπolo[2,3-d]pyrimidine of the formula HI, wherein R is carboxy protecting groups: with sodium hydroxide. 2. The process of Claim 1 wherein the 5-substituted pyπolo[2,3-d]pyrimidine is the acid salt of formula V: and X is an anion. 3. The process of Claim 1 or Claim 2, wherein the carboxy protecting groups are selected from the Ci-Cό alkyl esters. 4. The process of claim 3 wherein the Ci-Cό alkyl ester is selected from methyl ester or ethyl ester. 5. The process of Claim 1, Claim 2, Claim 3, or Claim 4 wherein the 5-substituted pyπolo[2,3-d]pyrimidine acid salt is 6. A novel hydrate crystal form of disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo- 3H-pyπolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt ("disodium MTA Hydrate Form F), having an X-ray diffraction pattern, which comprises the following peaks coπesponding to d spacing: 18.66 ±0.04 when obtained at 22 ±2°C and ambient % relative humidity from a copper radiation source. 7. A novel hydrate crystal form of disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo- 3H-pyπolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt ("disodium MTA Hydrate Form I"), having an X-ray diffraction pattern, which comprises the following peaks coπesponding to d spacing: 9.33 ±0.04 when obtained at 22 ±2°C and ambient % relative humidity from a copper radiation source. 8. A novel hydrate crystal form of disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo- 3H-pyrrolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt ("disodium MTA Hydrate Form I"), having an X-ray diffraction pattern, which comprises the following peaks coπesponding to d spacings: 18.66 ±0.04 and 9.33 ±0.04 when obtained at 22 ±2°C and ambient % relative humidity from a copper radiation source. 9. The novel hydrate crystal form of any one of claims 6 - 8 further comprising the d spacing 4.92 ±0.04. 10. A method of use of the compound of Claim 6, Claim 7, Claim 8, or Claim 9 for the manufacture of a medicament for the treatment of cancer. 11. The method of use of Claim 10 wherein the cancer is mesothelioma. 12. A process for preparing a medicament comprising combining crystalline disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyπolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L- glutamic acid salt with a buffer in an aqueous solution. 13. The process of Claim 12 wherein the crystal form is the compound of Claim 6, Claim 7, Claim 8, or Claim 9. 14. A formulation comprising crystalline disodium N-[4-[2-(2-amino-4,7-dihydro-4- oxo-3H-pyπolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt in association with a pharmaceutically acceptable carrier. 15. The formulation of Claim 14 wherein the crystal form is the compound of Claim 6, Claim 7, Claim 8, or Claim 9. 16. A process for the preparation of the compound of Claim 6, Claim 7, Claim 8, or Claim 9 comprising crystallizing disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H- pyπolo[2,3-d]-pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt from an appropriate solvent. 17. The process of Claim 16 which comprises adjusting the pH of an aqueous solution of disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyπolo[2,3-d]-pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid salt from about 6.5 to about 9.5 and precipitating disodium MTA Hydrate Form I from the pH adjusted aqueous solution. 18. The process of Claim 17, which further comprises formulating the disodium MTA Hydrate Form I with a diluent to form a medicament. 19. The process of any one of Claims 17 through 18 wherein the pH is adjusted to from about 7.5 to about 8.5. 20. The process of any one of Claims 17 through 19 wherein the pH is adjusted to about 8.0. 21. An article of manufacture comprising packaging material and a composition comprising crystalline disodium N-[4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]- pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid salt contained within said packaging material, wherein said crystalline salt is effective in the treatment of cancer and wherein said packaging material comprises a label which indicates that said crystalline salt can be used to treat cancer. 22. The article of manufacture of Claim 21 wherein the crystalline form is the compound of Claim 6, Claim 7, Claim 8, or Claim 9. 23. The article of manufacture of Claim 21 or Claim 22 wherein the cancer is mesothelioma.

Inventors: CHELIUS ERIK CHRISTOPHER, OSBORNE LINDA MARIE, SNOREK SHARON VAN DEN BERGHE, CHELIUS, ERIK, CHRISTOPHER, OSBORNE, LINDA, MARIE, SNOREK, SHARON, VAN DEN BERGHE
Applicants: CHELIUS ERIK CHRISTOPHER, LILLY CO ELI, OSBORNE LINDA MARIE, SNOREK SHARON VAN DEN BERGHE, STEPHENSON GREGORY ALAN, WARNER ANNE MARIE, CHELIUS, ERIK, CHRISTOPHER, ELI LILLY AND COMPANY, OSBORNE, LINDA, MARIE, SNOREK, SHARON, VAN DEN BERGHE, STEPHENSON, GREGORY, ALAN, WARNER, ANNE, MARIE
Application year: 2000
Main classification: C07D487
IPC codes: A61P35, C07D487