CRYSTALLINE ANTIFUNGAL GLYCINE ESTER POLYMORPH Background of the Invention This invention relates to a crystalline polymorph of (-)-2S- [4- [4- [4- [4- [ [ (R-CIS)-5- (2, 4-difluorophenyl) tetrahydro-5- (lH-1, 2, 4-triazol-1- ylmethyl)-3-furanyl] methoxy] phenyl]-1-piperazinyl] phenyl-4, 5-dihydro-5- oxo-lH-1, 2, 4-triazol-1-yl]-1 (S)-methylbutyl aminoacetate represented by the formula I (hereinafter"the compound of formula I") pharmaceutical compositions containing such a polymorph and methods of using such a polymorph to treat fungal infections in mammals.

International Publication Number WO 96/38443 (published 5December 1996) disclose methods of making the compound of formula I and using it to treat antifungal infections in mammals. The synthetic procedure disclosed in WO 96/38443 produces the compound of formula I as an amorphous solid. WO 96/38443 does not disclose, or even suggest the possible existence of a crystalline polymorph of the compound of formula I.

To prepare pharmaceutical compositions containing the compound of formula I for administration to mammals in accordance with exacting health registration requirements of the U. S. and international health registration authorities, e. g. the FDA's Good Manufacturing Practices ("GMP") requirements, there is a need to produce the compound of formula I in as pure a form as possible, especially a form having constant physical properties.

Summary of the Invention We have discovered that the compound of formula I can exist in the form of a crystalline polymorph, having distinctly different physical properties compared to the amorphous form.

Accordingly, this invention provides a crystalline polymorph of (-)-2S- [4- [4- [4- [4- [ [ (R-CIS)-5- (2, 4-difluorophenyl) tetrahydro-5- (1H-1, 2, 4- triazol-1-ylmethyl)-3-furanyl] methoxy] phenyl]-1-piperazinyl] phenyl-4, 5- dihydro-5-oxo-lH-1, 2, 4-triazol-1-yl]-1 (S)-methylbutyl aminoacetate represented by the formula I and characterized by the following x-ray powder diffraction pattern expressed in terms of"d"spacing and relative intensities ("RI") : d spacing ( 0. 04) RI 5. 08 Strong 4. 09 Strong 3. 44 Very Strong This invention also provides a crystalline polymorph of (-)-2S- [4- [4- [4- [4- [ [ (R-CIS)-5- (2, 4-difluorophenyl) tetrahydro-5- (lH-1, 2, 4-triazol-1- ylmethyl)-3-furanyl]methoxy]phenyl]-1-piperazinyl]phenyl-4,5 -dihydro-5- oxo-1H-1, 2, 4-triazol-1-yl]-1 (S)-methylbutyl aminoacetate characterized by the following x-ray powder diffraction pattern expressed in terms of"d" spacings and relative intensities ("RI") (S=strong, M=medium, W=weak, V=very, D=diffuse and VWD, for example = very weak diffuse) : relative d spacing intensi 14.50 W 12.10 V W D 10.24 V W 9.68 W 9.29 V W D 8.77 W 8. 38 VWD 7.08 W 6.67 V W 6.21 M 6. 00 V W 5.76 V W 5.62 V W 5.45 V W 5.19 W D 5.08 S 4.96 V W D 4. 87 VW D 4. 74 V W 4.58 W 4.53 V W 4.46 V W 4.40 V W D 4.28 V W 4.21 W 4.09 S 3.97 V W 3.87 M 3.80 M 3.66 V W relative d spacing intensity 3.59 M 3. 54 V W 3.44 V W 3. 41 MD 3. 35 V W 3.24 W 3. 15 V W 3.10 V W 3.00 W D 2. 94 V W .290 V W 2. 87 VWD 2.82 W 2. 78 VWD 2. 74 VWD 2.67 V W D 2.66 V W D 2. 57 VWD 2.54 W D 2.52 V W D 2.50 W 2. 43 VWD 2.42 V W D 2. 37 VWD 2. 34 VWD 2.31 V W D 2.30 V W D 2.29 V W D 2.27 V W D This invention further provides pharmaceutical composition containing the crystalline polymorph of the compound of formula I and methods of treating and/or preventing fungal infections using such crystalline polymorph BRIEF DESCRIPTION OF THE FIGURES Figure 1 presents a characteristic x-ray powder diffraction pattern of the cyrstalline polymorph of the compound of formula I [Vertical Axis : Intensity (CPS, counts (square root)) L Horizontal Axis : Two Theta (degrees)].

Figure 2 presents a characteristic infrared spectrum of the crystalline polymorph of the compound of formula I in a potassium bromide pellet [Vertical Axis ; Transmittance (Percent) ; Horizontal Axis : wavenumber (cm~1)] Figure 3 presents a characteristic differential scanning calorimetry therogram of the crystalline polymorph of the compound of formula I [on a DuPont 2100 : Thermal Analysis under a nitrogen atmosphere ; 10° C/min scan rate ; single endotherm, onset temperature : 186. 33° C ; Vertical Axis ; Heat Flow in cal/sec/g ; Horizontal Axis : Temperature in degrees centigrade].

Figure 4 presents a characteristic 1H nmr spectrum of the crystalline polymorph of the compound of formula I (Varian XL400) in CDC13 at 400 MHz with TMS as an internal standard.

Detailed Description of the Invention The crystalline polymorph of (-)-2S- [4- [4- [4- [4- [ [ (R-CIS)-5- (2, 4- difluorophenyl) tetrahydro-5- (lH-1, 2, 4-triazol-1-ylmethyl)-3-furanyl]- methoxy] phenyl]-1-piperazinyl] phenyl-4, 5-dihydro-5-oxo-lH-1, 2, 4-triazol-l- yl]-1 (S)-methylbutyl aminoacetate represented by the formula I (hereinafter "the compound of formula I") provides an antifungal drug having the following advantages compared to its amorphous form in the prior art : lower impurity content and more consistent product quality i. e., more consistent physical characteristics including more consistent color, rate of dissolution and ease of handling ; as well as a longer term stability.

All of these advantages inherent in the crystalline polymorphic form of the compound of formula I would be useful in the development and approval of a pharmaceutical product containing the antifungal drug of formula I.

The crystalline polymorphic form of the compound of formula I may be formed by crystallizing the compound of formula I (as the free base) using a solvent system such as acetonitrile, tetrahydrofuran ("THF"), THF in combination with heptane or isopropyl acetate in a v/v ratio of 1. 5 : 1 to : 1. 5, preferably about 1 : 1 ; and methylene chloride in combination with hexane in a volume/volume (v/v) ratio of 0. 2 : 1 to 0. 75 : 1, preferably about 0. 25 : 1. The solvent or solvent system were typically healed to reflux and slowly cooled to room temperature (20°-25°C) or even 0° (with THF or THF/heptane for 1-3 days.

The complete infrared spectrum of the crystalline polymorph of the compound of formula I taken in the form of a potassium bromide pellet is characterized by the following peaks : frequency (cm''h 3501 1351 3435 1327 3391 1301 3144 1271 3131 1248 3119 1230 3073 1185 3976 1157 2967 1137 2947 1119 2936 1105 2920 1075 2867 1038 2845 1028 2814 974 2767 946 1734 887 frequency (cm~1 ! 1686 858 1615 825 1601 818 1558 789 1522 738 1512 680 1497 659 1450 585 1423 548 1390 530 The infrared spectra were obtained on a Mattson Galaxy 6021 FTIR spectrometer. The potassium bromide pellets were prepared in accordance with the USP procedure <197K>, U. S. Pharmacopeia, National Formulary, USP XXIII, NF XVIII.

The x-ray powder diffraction patterns were measured on a Philips APD3720 automated diffractometer system (model PW 1800). The radiation source was copper (K-alpha) and the long fine focus tube connected to a Philips XRG 3100 x-ray generator operated at 45 KV and 40 mA. The take-off angle was 6 degrees and a graphite monochromator as used. A scintillation detector was employed and data was acquired with a scan rate of 0. 025 degrees per second, a step size of 0. 010 and a step time of 40 seconds per degree.

The x-ray powder diffraction pattern distinctive for the crystalline polymorph of the compound of formula I expressed in terms of the following distinctive"d"spacing and relative intensties ("RI") is provided hereinbelow : "d"spacings ( 0. 04) RI 5. 08 Strong 4. 09 Strong 3. 44 Very Strong A more complete x-ray powder diffraction pattern for the crystalline polymorph of the compound of formula I expressed in"d" spacings and relative intensities ("RI") with V, M, W, V, and D defined as herein above is provided hereinbelow : relative d spacing intensity 14.50 W 12.10 V W D 10. 24 VW 9.68 W 9.29 V W D 8.77 W 8. 38 VWD 7.08 W 6.67 V W 6.21 M 6. 00 V W 5.76 V W 5.62 V W 5.45 V W 5.19 W D 5.08 S 4.96 V W D 4. 87 VW D 4. 74 VW 4.58 W 4.53 V W 4.46 V W 4.40 V W D 4.28 V W 4.21 W 4.09 S 3.97 V W 3.87 M 3.80 M 3. 66 V W relative d spacing intensity 3.59 M 3. 54 V W 3.44 V S 3. 41 MD 3.35 V W 3.24 W 3. 15 V W 3.10 V W 3.00 W D 2. 94 V W 2.90 V W 2. 87 VWD 2.82 W 2.78 V W D 2. 74 VWD 2.67 V W D 2.66 V W D 2. 57 VWD 2. 54 WD 2.52 V W D 2.50 W 2.43 V W D 2.42 V W D 2.37 V W D 2.34 V W D 2.31 V W D 2.30 V W D 2.29 V W D 2.27 V W D Pharmaceutical Compositions Pharmaceutical compositions of this invention may contain in addition to an anti-fungally effective amount of crystalline polymorph of the compound of formula I as the active ingredient, inert pharma-ceutically acceptable carriers that may be solids or liquids. Solid form compositions include powders, tablets, dispersible granules, capsules, cachets, and suppositories. A solid carrier can be one or more substances which may also act as diluants, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegration agents ; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active compound. In the tablet the active compound is mixed with carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from about 5 to about 20 percent of the active ingredient. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methycelulose, sodium carboxymethyl-cellulose, a low melting wax. cocoa butter and the like. The term"compositions"is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component (with or without other carriers) is surrounded by carrier, which is thus in association with it. Similarly, caches are included. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.

Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for topical administration. Liquid preparations can also be formulated in solution in aqueous polyethylene glycol solution. Aqueous solutions suitable for oral use can be prepared by adding the active component in water and adding suitable colorants, flavors, stabilizing, sweetening, solubilizing and thickening agents as desired. Aqueous suspensions suitable for oral use can e made by dispersing the finely divided active component in water with viscous material, i. e., natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other well-known suspending agents.

Topical formulation useful for nasal or ophthalmic administration are also contemplated. Topical formulation suitable for nasal administration may be solutions or suspensions. Ophthalmic formulations may be solutions, suspension or ointments. Ointments usually contain lipophilic carriers such as mineral oil and/or petrolatum.

Solution for ophthalmic administration may contain sodium chloride, acid and/or base to adjust the pH as well as purified water and preservatives, which normally contains a non-toxic, pharmaceutically acceptable topical carrier may be applied daily to the affected skin until the condition has improved.

The anti-fungally effective amount of the crystalline polymorph of the compound of formula I for topical administration varies from 0. 1 to 20% by weight of the total pharmaceutical composition, which normally contains one or more non-toxic, pharmaceutically acceptable topical carriers ; the pharmaceutical composition may be applied daily to the affected area of the skin until the fungal infection has been irradiated The preferred amount varies from 0. 5 to 10% by weight of the total pharmaceutical composition.

The anti-fungally effective amount of the crystalline polymorph of the compound of formula I for oral administration varies from about 1 to 30 mg/day, more preferably about 1 to 20 mg/day and most preferably about 1 to 10 mg/day in single or divided doses.

Parenteral forms to be injected intravenously, intramuscularly, or subcutaneously are usually in the form of a sterile soluon, and may contain salts or glucose to make the solution isotonic.

In general, the parenteral dosage for humans for antifungal use ranges from about 0. 25 mg per kilogram of body weight per day to about 20 mg kilogram of body weight per day being preferred.

The exact amount, frequency and period of administration of the compound of the present invention for antifungal use will vary, of course, depending upon the sex, age and medical condition of the patent as well as the severity of the fungal infection as determined by the attending clinician.

GENERAL EXPERIMENTAL The compounds of this invention are prepared in accordance with the following Examples using commercially available starting materials.

EXAMPLE 1 PREPARATION OF (-)-2 (S)-4 [-4 [-4 [-4 [ [ (R-CIS)-5-2, 4-DIFLUOROPHENYL)- TETRAHYDRO-5- (1H-1, 2, 4-TRIAZOL-1-YLMETHYL)-3-FURANYL] METHOXY] PHENYL]-1-PIPERAZINYL] PHENYL-4, 5-D1HYDRO-5-OXO-1H- 1, 2, 4-TRIAZOLolYL]-l (S)-METHYL BUTYL [[(PHENYLMETHOXY)- CARBONYL] AMINO] ACETATE To a cold solution (0-5°C) containing 2 kg of the product produced in accordance with Example 32 of International Patent Publication No. WO 95/17407 published 29 June 1995. 1. 02 kg of CBZ-glycine, and 1. 13 kg of dimethylaminopyridine in 18. 8 L of methylene chloride was dropwise added a solution of 0. 92 kg of p-toluene sulfonyl chloride in 3. 7 L of methylene chloride. The mixture was allowed to warm to room temperature and agitated for 12 to 16 hours. When the reaction was complete the mixture was quenched with approximately 1. 3 L of 1 N sulfuric acid to a final pH of about 1. 5. The layers were separated and the organic layer was washed sequentially with 0. 65 L of IN sulfuric acid, 4 L of 9% aqueous sodium bicarbonate solution and 3. 75 L of an 18% aqueous sodium chloride solution. The organic layer was treated with 0. 1 kg of darco and 0. 1 kg of supercel, filtered, and then diluted into 70 L of warm isopropyl alcohol. The volume of the solution was reduced to 40 L by distillation, diluted with 20 L of isopropyl alcohol, then reduced to 40 L of volume by distillation. While maintaining the temperature at about 60°C, about 11. 5 L of acetone was added to the solution and the mixture was cooled slowly to about 0 to 5°C for a period of 6 hours. The crystals were collected by filtration, then slurried into 40 L of isopropyl alcohol, heated to about 80°C, and cooled to about 60°C. About 4 L of acetone was added to the solution and the solution was cooled slowly to about 0 to 5°C for a period of 6 hours. The white crystals were collected by filtration and dried in an oven at 50-55°C for 12 hours to yield 2. 1 kg of product. mp 74- 76°C.

EXAMPLE 2 PREPARATION OF (-)-2 (S)- [-4 [-4 [-4 [4-[[(R-CIS)-5-(2, 4-DIFLUORO-PHENYL)- TETRAHYDRO-5- (IH1, 2, 4-TRIAZOL-I-YLMETHYL) 3-FURANYL] METHOXY] PHENYL]-1-PI PERAZI NYL] PH ENYL-4, 5-DI HYD RO-5-OXO-IH-1, 2, 4-TRIAZOL- 1YL]-1 (S)-METHYLBUTYL AMINOACETATE AND CRYSTALLIZATION THEREOF from THF/ISOPROPYL ACETATE To 21 g of 5% palladium on carbon (50% water wet) in an autoclave was charged a solution of 300 g of Example 1 in 6. 6 L of tetrahydrofuran. The autoclave was inerted with vacuum and filled with 100 to 115 psig of hydrogen gas. The mixture was warmed to 60 to 65°C and agitated for 12 to 16 hours. When the reaction was complete, the catalyst was removed by filtration, and the distillate was concentrated to about 3 L by distillation.

About 1. 2 L of isopropyl acetate was added to the solution while maintaining a temperature of 65 to-70°. The mixture was slowly cooled to 0 to sod and agitated for about 6 hours. The crystals were filtered, washed with 1. 5 L of isopropyl acetate, and dried in an oven at 70°C for 24 hours to yield about 220 g of product. mp 186-188°C.

EXAMPLE 3 CRYSTALLIZATION of (-)-2 (S)- [-4 [-4 [-4 [4- [ [ (R-CIS)-5- (2, 4-DIFLUORO- PHENYL)-TETRAHYDRO-5- (IH1, 2, 4-TRIAZOL-I-YLMETHYL) 3- FURANYL] METHOXY] PHENYL]-1-PIPERAZINYL] PHENYL-4, 5-DIHYDRO-5- OXO-IH-1, 2, 4-TRIAZOL-1YL]-1 (S)-METHYLBUTYL AMINOACETATE from ACETONITRILE To 0. 8 kg of the product of Example 2 was added 32 L of acetonitrile at room temperature. The agitated mixture was refluxed at 80-82°C until a clear solution was obtained. The solution was cooled to 65-70°C, held at that temperature, filtered, and then concentrated to about 8 L of volume. the solution was cooled to 20 to 25°C over a two hour period, held at 20-25°C for about 16 hours, and then filtered to collect the crystalline product. The filtrate was washed with 2. 4 L of acetonitrile and then dried in vacuum oven at 55-60°C for 24 hours to yield 0. 7 kg of crystalline product. mp 187- 189°C.

EXAMPLE 4 PREPARATION OF (-)-2 (S)- [-4 [-4 [-4 [4- [ [ (R-CIS)-5- (2, 4-DIFLUORO-PHENYL)- TETRAHYDRO-5- (1H1, 2, 4-TRIAZOL-1-YLMETHYL) 3-FURANYL]- METHOXY] PHENYL]-1-PIPERAZINYL] PHENYL-4, 5-DIHYDRO-5-OXO- 1H-1, 2, 4-TRIAZOL-1YL]-1 (S)-METHYLBUTYL AMINOACETATE AND CRYSTALLIZATION THEREOF from THF solvent To 100 mL of THF was charged 5. 0 g of the product of Example 1 and 2. 0 g of charcoal. After agitation for 15 mins, the mixture was filtered through celite and then charged into 100 mL of THF containing 2. 0 g of 20% palladium hydroxide on charcoal (50% wet). The mixture was subjected to 20 psi hydrogen pressure in a Parr apparatus for 36 hours. The catalyst was removed by filtration through celite and then slurried with 5 g of silica gel.

The silica gel was filtered to yield a pale green solution. The solution was cooled to 0°C for about 3 days to yield granular white crystals. Filtration and drying under vacuum gave 3. 6 g of product. mp 185-188°C.

EXAMPLE 5 PREPARATION OF (-)-2 (S)- [-4 [-4 [-4 [4- [ [ (R-CIS)-5- (2, 4-DIFLUORO-PHENYL)- TETRAHYDRO-5- (1H1, 2, 4-TRIAZOL-1-YLMETHYL) 3-FURANYL]- METHOXY] PHENYL]-1-PIPERAZINYL] PHENYL-4, 5-DIHYDRO-5-OXO-1H- 1, 2, 4-TRIAZOL-1YL]-1 (S)-METHYLBUTYL AMINOACETATE AND CRYSTALLIZATION from THF-HEPTANE solvent Into a Parr vessel was charged 0. 1 g of 20% palladium hydroxide on charcoal (50% wet), 10 mL of THF, 1. 15 g of the product of Example 1 and 0. 1 ml of acetic acid. The mixture was subjected to 30 psi hydrogen pressure in a Parr apparatus for 24 hours. The catalyst was removed by filtration through celite and then concentrated to about 4 mL of solution. To this solution at room temperature was added about 5 mL of heptane. The solution was cooled to 0°C for about 3 days to yield granular white crystals. Filtration and drying under vacuum gave 0. 6 g of product. mp 184-187°C.

EXAMPLE 6 CRYSTALLIZATION of (-)-2 (S)- [-4 [-4 [-4 [4- [ [ (R-CIS)-5- (2, 4-DIFLUORO- PHENYL)-TETRAHYDRO-5- (IH1, 2, 4-TRIAZOL-1-YLMETHYL) 3- FURANYL] METHOXY] PHENYL]-1-PIPERAZINYL] PHENYL-4, 5- DIHYDRO-5-OXO-1H-1, 2, 4-TRIAZOL-1YL]-1 (S)-METHYLBUTYL AMINOACETATE (SCH 60175-C) from METHYLENE CHLORIDE/HEXANE solvent To 12. 5 mL of methylene chloride was added 2. 5 g of the product of Example 2 at room temperature. The agitated mixture was heated to reflux until a clear solution was obtained. The solution was added to 50 mL of refluxing hexane. The solution was distilled to remove methylene chloride, cooled to room temperature, and then filtered to collect the crystalline product. The filtrate was dried in vacuum oven at 85°C for 24 hours to yield 2. 0 g of crystalline product.