WO2001007437A1

WO2001007437A1 - Crystal forms of 1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone

Info

Publication number: WO2001007437A1
Application number: PCT/US2000/019423
Authority: WO
Inventors: Richard J. Varsolona; James A. Mccauley
Original assignee: Merck & Co., Inc.
Priority date: 1999-07-21
Filing date: 2000-07-17
Publication date: 2001-02-01
Also published as: AU6105000A

Abstract

The present invention is directed to the crystal forms of 1-(3-Chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone, which may inhibit farnesyl-protein transferase, and the process for the preparation of these crystal forms.

Description

TITLE OF THE INVENTION

CRYSTAL FORMS OF l-(3-CHLOROPHENYL)-4-[l-(4-CYANOBENZYL)-

5-IMIDAZOLYLMETHYL1-2-PIPERAZINONE

BACKGROUND OF THE INVENTION

The Ras proteins are a family of guanine nucleotide binding GTPases that play a pivotal role in mediating cell growth, differentiation and development. (Barbacid, Annual Review of Biochemistry , Vol. 56, p. 779 (1987)). In mammalian cells, there are three ras genes that encode four Ras proteins, H, N, KA and KB-Ras. (E.C. Lerner et al., Anti-Cancer Drug Design, Vol. 12, pp. 229-238 (1997)). Mutations in Ha-rαs, Ki-ras and N-ras, and the overexpression of Ras has been observed in approximately 30% of all human cancer tissues. (Lerner et al., S.L. Graham, Exp. Opin. Ther. Patents, Vol. 5, no. 12, pp. 1269-1285 (1995); T. Hiwasa, Oncology Reports, Vol. 3, pp. 7-14 (1996); S.L. Graham and T.M. Williams, Exp. Opin. Ther. Patents, Vol. 6, no. 12, pp. 1295-1304 (1996)). Although several steps are involved in modifying Ras proteins, farnesylation is the only step which is required and sufficient for Ras transforming activity. (E.C. Lerner et al.) Therefore, farnesyltransferase (FTase) serves as an attractive target for the development of a potential new class of anti-cancer agents. (E.C. Lerner et al.) It has been noted that routes to inhibitors of Ras farnesylation are apparent from an examination of the substrate specificities of the enzyme. One can design analogs either of the lipid, or of the peptide sequence to which the lipid is transferred. Such compound must be stable, and readily cross the cell membrane to gain access to the cytosolic transferase. (J. E. Buss and J.C. Marsters, Jr., Chemistry and Biology, Vol. 2, pp. 787-791 (1995)).

Compounds that incorporate substituted 5-imidazolyl- methyl-2-piperazinone moieties have been observed to be farnesyl- transferase inhibitors. (WO 96/30343 published on October 30, 1996). This invention relates to the crystal forms of the free base of l-(3- chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone which inhibits farnesyl-protein transferase. In particular, this invention is directed to the anhydrous and isopropanol (IPA) solvate crystal forms

1 - of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2- piperazinone.

SUMMARY OF THE INVENTION This invention relates to the crystal forms of l-(3- chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone which inhibits farnesyl-protein transferase. In particular, this invention is directed to Form III, which is the anhydrous crystal form of l-(3- chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone, and Form IV, which is the IPA solvate of l-(3-chlorophenyl)-4-[l-(4- cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts the DSC Curve (open cup) for Form III, the anhydrous crystal form of l-(3-Chlorophenyl)-4-[l-(4-cyanobenzyl)-5- imidazolylmethyl]-2-piperazinone.

FIG. 2 depicts the X-ray powder diffraction pattern for Form

III, the anhydrous crystal form of l-(3-Chlorophenyl)-4-[l-(4-cyanobenzyl)-

5-imidazolylmethyl]-2-piperazinone. FIG. 3 depicts the DSC Curve (open cup) for Form IV, the

IPA solvate of l-(3-Chlorophenyl)-4-[l-(4-cyanobenzyl)-5- imidazolylmethyl] -2-piperazinone .

FIG. 4 depicts the X-ray powder diffraction pattern for Form rV, the IPA solvate of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5- imidazolylmethyl]-2-piperazinone.

FIG. 5 depicts the solid state 13 C nuclear magnetic resonance spectrum [¹³C CP/MAS NMR] for Form III, the anhydrous crystal form of l-(3-Chlorophenyl)-4-[l-(4-cyanobenzyl)-5- imidazolylmethyl] -2-piperazinone . FIG. 6 depicts the solid state 13 nuclear magnetic resonance spectrum [¹³C CP/MAS NMR] for Form IV, the IPA solvate of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2- piperazinone.

2 - DETAILED DESCRIPTION OF THE INVENTION

This invention relates to two crystal forms, Crystal Form III and Crystal Form IV, of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5- imidazolylmethyl] -2-piperazinone, which may inhibit farnesyl-protein transferase, and the process for the preparation of these crystal forms. As used herein, the terms "Crystal Form III" or "Form III" represent an anhydrous crystal form of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5- imidazolylmethyl] -2-piperazinone. As used herein, the terms "Crystal Form IV" or "Form IV" represents an isopropanol (IPA) solvate crystal form of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2- piperazinone.

Another embodiment of this invention is related to the preparation of Crystal Forms III and IV. Crystal Form III may be prepared by mixing the monohydrate form of l-(3-chlorophenyl)-4-[l- (4-cyanobenzyl)-5-imidazolylmethyl] -2-piperazinone with a solvent for about 20 to about 36 hours at ambient temperature. Any solvent may be used so long as it does not form a solvate with l-(3-chlorophenyl)-4-[l- (4-cyanobenzyl)-5-imidazolylmethyl] -2-piperazinone and the water content is below the thermodynamically stable region for the monohydrate at a given temperature. Types of solvents that may be used include, but are not limited to, ethanol, n-propanol, butanol, acetonitrile, acetone, ethyl acetate and tetrahydrofuran. Preferably, acetonitrile is used. Crystal Form III may then be isolated. Preferably, isolation of the crystalline form is accomplished via filtration techniques. Crystal Form IV may be prepared by mixing the monohydrate form of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5- imidazolylmethyl] -2-piperazinone with isopropanol for about 20 to about 36 hours at ambient temperature, and then isolating the crystalline form. Preferably, isolation of the crystalline form is accomplished via filtration techniques.

Another embodiment of this invention is related to a pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of Crystal Form III or Crystal Form IV. Additionally, the instant invention is related to a pharmaceutical composition made by combining Crystal Form III or Crystal Form IV and a pharmaceutically acceptable carrier.

Abbreviations used throughout the specification include:

ACN acetonitrile

Ac2θ acetic anhydride

Boc t-Butoxycarbonyl

CBz carbobenzyloxy

DBU l,8-diazabicyclo[5.4.0]undec-7-ene

DEAD diethylazodicarboxylate

DEM diethoxymethane

DLAD diisopropylazodicarboxylate

DIEA diisopropyleth lamine

DPAD dipiperidineazodicarbonyl

DMA dimethyl acetamide

DMAP 4-Dimethylaminopyridine

DME 1 ,2-Dimethoxyethane

DMF Dimethylformamide

DMPU l,3-Dimethyl-3,4,5,6-tetrahydro-2(lH)-pyrimidinone

DMSO dimethyl sulfoxide

EDC l-(3-dimethylaminopropyl)-3-ethyl-carbodiimide- hydrochloride

Et3N triethylamine

EtOAc ethyl acetate

FAB Fast atom bombardment

HMTA hexamethylenetetramine

HOBT 1-Hydroxybenzotriazole hydrate

HOOBT 3-Hydroxy-l,2,2-benzotriazin-4(3H)-one

HPLC High-performance liquid chromatography

IPA isopropanol or 2-propanol

MCPBA m-Chloroperoxybenzoic acid

MeCN acetonitrile

MEK methyl ethyl ketone

MIBK methyl isobutyl ketone

4 - MsCl methanesulfonyl chloride

MsOH methanesulfonic acid

MTBE methyl-t-butyl-ether

NaHMDS sodium bis(trimethylsilyl)amide

NMP l-Methyl-2-pyrrolidinone

ODCB ortho-dichlorobenzene

Py pyridine

TFA trifluoroacetic acid

THF tetrahydrofuran

TsOH toluene sulfonic acid

SYNTHESIS

l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]- 2-piperazinone ^#H2θ may be prepared using the techniques described in

U.S. Patent Number 5,856,326, which issued on January 5, 1999, or U.S.

Serial Nos. 09/338,643, 09/338,064 or 09/338,065, which were co-filed on

June 23, 1999. These patents and pending patent applications are herein incorporated by reference. The following examples further illustrate the preparation of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2- piperazinone *H2θ, the identification of the crystal forms referred to as Form III and Form IV and the process for preparing these crystal forms, and, as such, are not to be considered or construed as limiting the invention recited in the appended claims.

EXAMPLE 1

Preparation of p-Cyanobenzylamine • H. PO salt A slurry of HMTA in 2.5 L EtOH was added gradually over about 30 min to about 60 min to a stirred slurry of cyanobenzyl -bromide in 3.5 L EtOH and maintained at about 48-53°C with heating & cooling in a 22L neck flask (small exotherm). Then the transfer of HMTA to the reaction mixture was completed with the use of 1.0 L EtOH. The reaction mixture was heated to about 68-73°C and aged at about 68-73°C for about 90 min. The reaction mixture was a slurry containing a granular precipitate which quickly settled when stirring stopped.

The mixture was cooled to a temperature of about 50°C to about 55 °C. Propionic acid was added to the mixture and the mixture was heated and maintained at a temperature of about 50°C to about 55°C. Phosphoric acid was gradually added over about 5 min to about 10 min, maintaining the reaction mixture below about 65 °C to form a precipitate- containing mixture. Then the mixture was gradually warmed to about 65°C to about 70°C over about 30 min and aged at about 65°C to about 70°C for about 30 min. The mixture was then gradually cooled to about 20-25 °C over about 1 hour and aged at about 20-25°C for about 1 hour. The reaction slurry was then filtered. The filter cake was washed four times with EtOH, using the following sequence, 2.5 L each time. The filter cake was then washed with water five times, using 300 mL each time. Finally, the filter cake was washed twice with MeCN (1.0 L each time) and the above identified compound was obtained.

EXAMPLE 2

Preparation of 4-Cyanobenzylamine Hydrochloride via Hexamethylene-tetrammonium salt

A 72 liter vessel was charged with 190 proof ethanol (14.4 L) followed by the addition of 4-cyanobenzylbromide (2.98 kg) and HMTA (2.18 kg) at ambient temperature. The mixture was heated to about 72-75°C over about 60 min. On warming, the solution thickens and additional ethanol (1.0 liter) was added to facilitate stirring. The batch was aged at about 72-75°C for about 30 min.

The mixture was allowed to cool to about 20°C over about 60 min, and HCl gas (2.20 kg) was sparged into the slurry over about 4 hours during which time the temperature rose to about 65°C. The mixture was heated to about 70-72°C and aged for about 1 hour. The slurry was cooled to about 30°C and ethyl acetate (22.3 L) added over about 30 min. The slurry was cooled to about -5°C over about 40 min and aged at about -3 to about -5°C for about 30 min. The mixture was filtered and the crystalline

- 6 - solid was washed with chilled ethyl acetate (3 x 3 L). The solid was dried under an N2 stream for about 1 hour before charging to a 50 liter vessel containing water (5.5 L). The pH was adjusted to about 10-10.5 with 50% NaOH (4.0 kg) maintaining the internal temperature below about 30°C. At about 25°C, methylene chloride (2.8 L) was added and stirring continued for about 15 min. The layers were allowed to settle and the lower organic layer was removed. The aqueous layer was extracted with methylene chloride (2 x 2.2 L). The combined organic layers were dried over potassium carbonate (650 g). The carbonate was removed via filtration and the filtrate concentrated in vacuo at about 25 °C to give a free base as a yellow oil.

The oil was transferred to a 50 liter vessel with the aid of ethanol (1.8 L). Ethyl acetate (4.1 L) was added at about 25°C. The solution was cooled to about 15°C and HCl gas (600 g) was sparged in over about 3 hours, while keeping batch temperature below about 40°C. At about 20-25°C, ethyl acetate (5.8 L) was added to the slurry, followed by cooling to about -5°C over about 1 hour. The slurry was aged at about -5°C for about 1 hour and the solids isolated via filtration. The cake was washed with a chilled mixture of EtOAc/EtOH (9:1 v/v) (1 x 3.8 L), then the cake was washed with chilled EtOAc (2 x 3.8 L). The solids were dried in vacuo at about 25°C to provide the above-titled compound.

¹H NMR (250 MHz, CDCI3) δ 7.83-7.79 (d, 2H), 7.60-7.57 (d, 2H), 4.79 (s, 2H), 4.25 (s, 2H); 13C NMR (62.9 MHz, CDCI3) δ 149.9, 139.8, 134.2, 131.2, 119.7, 113.4, 49.9, 49.5, 49.2, 48.8, 48.5, 48.2, 43.8.

EXAMPLE 3

Preparation of l-(4-Cvanobenzyl)-2-Mercapto-5-Hvdroxymethylimidazole 7% water in acetonitrile (50 mL) was added to a 250 mL roundbottom flask. Next, an amine phosphate salt (12.49 g), as described in Example 1, was added to the flask. Next potassium thiocyanate (6.04 g) and dihydroxyacetone (5.61 g) was added. Lastly, propionic acid (10.0 mL) was added. Acetonitrile/water 93:7 (25 mL) was used to rinse down the sides of the flask. This mixture was then heated to 60°C, aged for about 30 minutes and seeded with 1% thioimidazole. The mixture was then aged for about 1.5 to about 2 hours at 60°C. Next, the mixture was heated to 70°C, and aged for 2 hours. The temperature of the mixture was then cooled to room temperature and was aged overnight. The thioimidazole product was obtained by vacuum filtration. The filter cake was washed four times acetonitrile (25 mL each time) until the filtrates became nearly colorless. Then the filter cake was washed three times with water (approximately 25-50 mL each time) and dried in vacuo to obtain the above-identified compound.

EXAMPLE 4

Preparation of l-(4-Cyanobenzyl)-5-Hydroxymethylimidazole

A 1L flask with cooling/heating jacket and glass stirrer (Lab- Max) was charged with water (200 mL) at 25°C. The thioimidazole (90.27 g), as described in Example 3, was added, followed by acetic acid (120 mL) and water (50 mL) to form a pale pink slurry. The reaction was warmed to 40°C over 10 minutes. Hydrogen peroxide (90.0 g) was added slowly over 2 hours by automatic pump maintaining a temperature of 35-45°C. The temperature was lowered to 25°C and the solution aged for 1 hour. The solution was cooled to 20°C and quenched by slowly adding 20% aqueous Na2SO3 (25 mL) maintaining the temperature at less than 25°C. The solution was filtered through a bed of DARCO G-60 (9.0 g) over a bed of SolkaFlok (1.9 g) in a sintered glass funnel. The bed was washed with 25 mL of 10% acetic acid in water.

The combined filtrates were cooled to 15°C and a 25% aqueous ammonia was added over a 30 minute period, maintaining the temperature below 25°C, to a pH of 9.3. The yellowish slurry was aged overnight at 23°C (room temperature). The solids were isolated via vacuum filtration. The cake (100 mL wet volume) was washed with 2 x 250 mL 5% ammonia (25%) in water, followed by 100 mL of ethyl acetate. The wet cake was dried with vacuum/N2 flow and the above- titled compound was obtained.

- 8 ¹H NMR (250 MHz, CDCI3): δ 7.84-7.72 (d, 2H), 7.31-7.28 (d, 2H), 6.85 (s, 1H), 5.34 (s, 2H), 5.14-5.11 (t, 1H), 4.30-4.28 (d, 2H), 3.35 (s, 1H).

EXAMPLE 5

Preparation of l-(4-cvanobenzyl)-5-chloromethyl imidazole HCl salt l-(4-Cyanobenzyl)-5-hydroxvmethylimidazole (1.0 kg), as described above in Example 4, was slurried with DMF (4.8 L) at 22°C and then cooled to -5°C. Thionyl chloride (390 mL) was added dropwise over 60 min during which time the reaction temperature rose to a maximum of 9°C. The solution became nearly homogeneous before the product began to precipitate from solution. The slurry was warmed to 26°C and aged for l h.

The slurry was then cooled to 5°C and 2-propanol (120 mL) was added dropwise, followed by the addition of ethyl acetate (4.8 L). The slurry was aged at 5°C for 1 h before the solids were isolated and washed with chilled ethyl acetate (3 x 1 L). The product was dried in vacuo at 40°C overnight to provide the above-titled compound.

^XH NMR (250 MHz DMSO-d6): δ 9.44 (s, 1H), 7.89 (d, 2H, 8.3 Hz), 7.89 (s,

1H), 7.55 (d, 2H, 8.3 Hz), 5.70 (s, 2H), 4.93 (s, 2H). ¹³C NMR (75.5 MHz DMSO-dβ): δ_c 139.7, 137.7, 132.7, 130.1, 128.8, 120.7, 118.4, 111.2, 48.9,

33.1.

EXAMPLE 6

Preparation of l-(4-Cyanobenzyl)-5-Chloromethyl Imidazole HCl salt via addition of Hydroxymethylimidazole to Vilsmeier Reagent To an ice cold solution of dry acetonitrile (3.2 L, 15 L/Kg hydroxymethylimidazole) was added 99% oxalyl chloride (101 mL, 1.15 mol, 1.15 equiv.), followed by dry DMF (178 mL, 2.30 mol, 2.30 equiv.), at which time vigorous evolution of gas was observed. After stirring for about 5 to 10 min following the addition of DMF, solid hydroxymethylimidazole (213 g, 1.00 mol), as described above in Example 4, was added

- 9 gradually. After the addition, the internal temperature was allowed to warm to a temperature of about 23°C to about 25°C and stirred for about 1 to 3 hours. The mixture was filtered, then washed with dry acetonitrile (400 mL displacement wash, 550 mL slurry wash, and a 400 mL displace- ment wash). The solid was maintained under an N2 atmosphere during the filtration and washing to prevent hydrolysis of the chloride by adventitious H2O. This yielded the crystalline form of the chloromethylimidazole h drochloride .

^XH NMR (250 MHz DMSO-dβ): δ 9.44 (s, 1H), 7.89 (d, 2H, 8.3 Hz), 7.89 (s,

1H), 7.55 (d, 2H, 8.3 Hz), 5.70 (s, 2H), 4.93 (s, 2H). ¹³C NMR (75.5 MHz DMSO-d6): δ_c 139.7, 137.7, 132.7, 130.1, 128.8, 120.7, 118.4, 111.2, 48.9,

33.1.

EXAMPLE 7

Preparation of l-(4-Cyanobenzyl)-5-Chloromethyl Imidazole HCl salt via addition of Vilsmeier Reagent to Hydroxymethylimidazole To an ice cold solution of dry DMF (178 mL, 2.30 mol, 2.30 equiv.) in dry acetonitrile (2.56 L, 12 L/Kg Hydroxymethylimidazole) was added oxalyl chloride (101 mL, 1.15 mol, 1.15 equiv). The heterogeneous mixture in the reagent vessel was then transferred to a mixture of hydroxymethylimidazole (213 g, 1.00 mol), as described above in Example 4, in dry acetonitrile (1.7 L, 8 L/Kg hydroxymethylimidazole). Additional dry acetonitrile (1.1 - 2.3 L, 5-11 L/Kg hydroxymethylimidazole) was added to the remaining solid Vilsmeier reagent in the reagent vessel. This, now nearly homogenous, solution was transferred to the reaction vessel at T| 2 +6°C. The reaction vessel temperature was warmed to a temperature of about 23°C to about 25°C and stirred for about 1 to 3 hours. The mixture was then cooled to 0°C and aged 1 h. The solid was filtered and washed with dry, ice cold acetonitrile (400 mL displacement wash, 550 mL slurry wash, and a 400 mL displacement wash). The solid was maintained under a N2 atmosphere during the filtration and washing

- 10 - to prevent hydrolysis of the chloride by adventitious H2O. This yielded the crystalline form of the chloromethylimidazole hydrochloride.

EXAMPLE 8

Preparation of the amide alcohol

At 22°C, 3-chloroaniline (50.0 g) was combined with 460 ml isopropyl acetate and 20% aqueous potassium bicarbonate (72.5 g dissolved in 290 ml water). The biphasic mixture was cooled to 5°C and chloroacetyl chloride (42 ml) was added dropwise over 30 minutes, keeping the internal temperature below 10°C. The reaction mixture was warmed to 22°C over 30 min. The aqueous layer was removed at 22°C and ethanolamine (92 ml) was added rapidly. The reaction mixture was warmed to 55°C over 30 minutes and aged for 1 hour. At 55°C, 140 ml water was added with 30 ml isopropyl acetate to the reaction mixture. The biphasic reaction mixture was agitated for 15 minutes at 55°C. The layers were allowed to settle and the aqueous layer was removed. The organic layer was cooled to 45°C and seed was added. The mixture was cooled to 0°C C over 1 hour and aged for 1 hour. The solids were filtered and washed with chilled isopropyl acetate (2 x 75 ml). The solids were dried in vacuo at 40°C for 18 hours to provide the above-identified amide alcohol.

NMR (300 MHz; DMSO-d6) δ 7.85 (t, 1H 2.0 Hz), 7.52 (m, 1H), 7.32 (t, 1H, 8.0 Hz), 4.5-4.8 (br s, 1H), 3.47 (t, 1H, 5.5 Hz), 3.30 (s, 1H), 2.60 (t, 1H 5.0 Hz).

¹³C NMR (75.4 MHz; DMSO-d6) δ_c 170.9, 140.1, 133.0, 130.3, 122.8 118.5, 117.5, 60.3, 52.7, 51.5.

11 EXAMPLE 9

Synthesis of l-(3-Chlorophenyl)-2-Piperazinone Hydrochloride with DPAD An amide alcohol, as described above in Example 8, was slurried with THF (37 ml) at 22°C, followed by the addition of tributyl phosphine (8.7 ml). The mixture was cooled to 0°C and the DPAD was added in portions over 15 min. The slurry was aged at 0-5 °C for 30 minutes, warmed to 25°C and aged for 18 hours. The reaction mixture was filtered and the cake was washed with THF (2 x 25 ml). The filtrate was concentrated in vacuo at < 35°C and combined with 50 ml of 2- propanol. The solution was cooled to 5°C, seeded with authentic material and treated with ethanol HCl (2.6 ml; 8.4M solution) dropwise over 20 min. The resulting slurry was recooled to 10°C and aged for 1 hour. The solids were isolated and the cake and flask rinsed with chilled 2-propanol (2 x 10 ml). The product was dried in vacuo at 40°C for 18 hours to provide the above-titled compound.

1H NMR (300 MHz; DMSO-d₆) δ 10.24 (br s, 2H), 7.50 - 7.30 (m, 4H), 3.92 (t, 2H, 5.5 Hz), 3.84 (s, 2H), 3.51 (t, 5.5 Hz); ¹³C NMR (75.4 MHz; DMSO- d₆) δ_c 162.1, 142.6, 132.9, 130.7, 127.0, 126.1, 124.54, 46.1, 44.9, 39.8.

EXAMPLE 10

Synthesis of l-(3-Chlorophenyl)-2-Piperazinone Hydrochloride with DIAD 58 mL of EtOAc was charged to an N₂-purged flask. Tributylphosphine (28.3 mL, 113.8 mmol) was added, via syringe, and the solution was cooled to about -10°C. DIAD (22.4 mL, 113.8 mmol) was added dropwise over 30 minutes, maintaining the temperature at < 0°C. The above mixture was cannulated into a slurry of an amide alcohol (20.0 g, 87.5 mmol), as described above in Example 8, in 117 mL EtOAc over 20 minutes, maintaining the temperature at < 0°C. The reaction was warmed to room temperature over 25 minutes. 99% conversion was observed by LC assay. Water (0.55 mL) was then added, and the reaction

12 was warmed to 40°C. The solution was seeded with 200 mg of authentic material, and 1.0 eq. HCl (4.0 N in abs. EtOH) was added dropwise over 2 hours. The slurry was cooled to 0°C over 2 hours and aged at 0°C for 1 hour. The mixture was filtered, and the cake was washed with chilled EtOAc (3x16 mL). The cake was dried in vacuo overnight at 40°C to afford the above-titled compound.

EXAMPLE 11

Preparation of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5- imidazolylmethyll -2-piperazinone • HgO

A 50 L four-neck flask, equipped with a mechanical stirrer, cooling bath, teflon-coated thermocouple, and nitrogen inlet was charged with 4.0 L of acetonitrile. Then 4-cyanobenzyl-chloromethylimidazole hydrochloride, as described in Example 6 or 7 (958 g, 3.36 mol), piperazinone hydrochloride, as described in Example 9 or 10, (883 g, 3.54 mol), and the remaining 1.25 L of acetonitrile were added to the flask at room temperature. Diisopropylethylamine (1.99 L, 11.4 mol) was added to the mixture. The bulk of the solid dissolved immediately upon addition of diisopropylethylamine, leaving a slightly turbid solution.

After stirring 30 min, the solution was cooled to 0°C over 60 min. The solution was stirred 26 h at 0°C, then warmed to 20°C over 20 min. Water (2 L) was added to the slightly turbid solution over 20 min. Authentic seed was added to 8 L of water, which was subsequently added to the solution over 70 min. Additional water (17 L) was added over 90 min, and the mixture was aged 60 min thereafter. The temperature throughout the addition and aging was from about 20°C to about 22°C. The mixture was filtered through a polypropylene filter pot. The crystals were washed with 1:5 acetonitrile/water. The crystalline solid was dried by passage of nitrogen through the filter cake (36 h) to provide the above- titled compound.

13C NMR (62.9 MHz, CDC1₃): δ 165.2, 142.7, 142.1, 139.4, 134.8, 133.0, 131.0, 130.2, 127.3, 127.1, 126.3, 126.0, 123.9, 118.1, 112.0, 57.7, 50.6, 49.9, 148.8, 148.3.

13 - EXAMPLE 12

Preparation of Crystal Form III of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)- 5-imidazolylmethvn -2-piperazinone l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]- 2-piperazinone *H2θ, prepared as described in Example 11, (2.5598 g) was added to a 25 mL Erlenmeyer flask. Next, 12.0 mL of acetone was added and the solution was stirred for about 2 min. at ambient tempera- ture. Once a precipitate was observed, the solution was stirred in an ice bath for about 1 hour. The mother liquor was then filtered off and the solids were washed with about 5 mL of pre-chilled acetone. The solids were then dried and collected to obtain the above titled compound.

Solid state x-ray powder diffraction (XRPD) pattern obtained with a CuKα x-ray radiation using a Phillips Diffractometer APD3720 gave the following d-spacings: 7.47, 6.59, 6.21, 5.39, 5.05, 4.84, 4.13, 4.09, 3.85, 3.76, 3.72, 3.68, 3.28, 3.20, 3.10, 3.07 and 3.04 angstroms.

EXAMPLE 13

Preparation of Crystal Form IV of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-

5-imidazolylmethyll-2-piperazinone l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]- 2-piperazinone *H2θ, prepared as described in Example 11, (10.341 g) was mixed with 150 mL of IPA in a 250 mL Erlenmeyer flask and the solution was stirred at ambient temperature for about 5 minutes. Optionally, authentic seed (50 mg) can be added and the solution stirred again. The mother liquor was filtered off and the cake was washed with about 20 mL of IPA at ambient temperature. The solids were then dried and the above-titled compound was collected.

14 Solid state x-ray powder diffraction (XRPD) pattern obtained with a CuKα x-ray radiation using a Phillips Diffractometer APD3720 gave the following d-spacings: 12.51, 6.02, 5.62, 5.15, 4.94, 4.72, 4.34, 4.16, 3.99, 3.90, 3.75, 3.58, 3.50, 3.37, 3.22, 3.17 and 3.13 angstroms.

DIFFERENTIAL SCANNING CALORIMETERIC (DSC)

The thermal properties of the crystal forms were characterized on a DSC Model 910 (DuPont Instruments) with data analysis on a thermal analyzer Model 1090 (DuPont Instruments). The DSC Curve for Form III (FIG. 1), when heated at a rate of 10°C/min in an open cup under a nitrogen atmosphere, shows an endotherm due to melting, with an extrapolated onset temperature of about 150°C, a peak temperature of about 151°C, and an associated heat of about 95 Joules/gram. The DSC Curve for Form IV (FIG. 3), when heated at a rate of 10°C/min in an open cup under a nitrogen atmosphere, shows a first endotherm, due to the loss of the isopropanol of the solvation, with an extrapolated onset temperature of about 59°C, a peak temperature at about 75°C, and an associated heat of about 96 Joules/gram. Form IV also exhibited an exotherm, due to the recrystallization of Form III, with a peak temperature of about 83°C and an associated heat of about 9 Joules/gram. Then Form IV exhibited a second endotherm, due to the melting of Form III, with an extrapolated onset temperature of about 149°C, a peak temperature of about 150°C, and an associated heat of about 86 Joules/gram.

The temperatures given above may vary approximately ± 1°C, while the associated heat may vary approximately ±5%.

X-RAY POWDER DIFFRACTION (XRPD) X-ray powder diffraction (XRPD) patterns were recorded using a Phillips Diffractometer APD3720 with copper tube K alpha radiation.

- 15 - For Crystal Form III, the solid state XRPD pattern (FIG. 2) had the following d-spacings: 7.47, 6.59, 6.21, 5.39, 5.05, 4.84, 4.13, 4.09, 3.85, 3.76, 3.72, 3.68, 3.28, 3.20, 3.10, 3.07 and 3.04 angstroms.

For Crystal Form IV, the solid state XRPD pattern (FIG. 4) had the following d-spacings: 12.51, 6.02, 5.62, 5.15, 4.94, 4.72, 4.34, 4.16, 3.99, 3.90, 3.75, 3.58, 3.50, 3.37, 3.22, 3.17 and 3.13 angstroms.

SOLID STATE ¹³C NUCLEAR MAGNETIC RESONANCE SPECTRUM. C CP/MAS NMR1 Solid-state ¹³C nuclear magnetic resonance (NMR) spectra were acquired on a Bruker DSX 400WB NMR system operating at 100.6 MHz for ¹³C and 400.1 MHz for 1H using the CP/MAS technique with variable-amplitude cross-polarization and total sideband suppression. A total of 462 scans were collected for each spectra with a contact time of 2.0 msec and recycle delay of 120 seconds. A line broadening of 20 Hz was applied to both spectra before FT was performed. Chemical shifts are reported on the TMS scale using the carbonyl carbon of glycine (176.03 ppm) as a secondary reference. Assignment of the solid-state ¹³C resonances were accomplished using solution-state ¹³C data received from a Bruker AMX-400 high-resolution NMR spectrometer. To assist in the assignment of ¹³C solid-state resonances, interrupted decoupling experiments were performed in which the 1H decoupling was turned off for 70 μsec (Interrupted Decoupling CP/MAS).

FIG. 5 and FIG. 6 depict the solid-state ¹³C CP/MAS NMR spectra for Crystal Forms III and IV, respectively. Table 1 lists the chemical shifts for the two crystal forms and compares them to the corresponding ¹³C solution-state values. Exact assignment of solid-state resonances between 120-140 ppm as well as 40-50 ppm becomes difficult due to the peak overlap in this region as well as broadening due to residual dipolar coupling between ¹³C and either directly bonded quad- rupolar 14N nuclei (Harris, R. K.; Olivieri, A. C, Progr. NMR Spectrosc. 1992, 24, 435) or 35/37C1 nuclei (Olivieri, A. C; Elguero, J.; Sobrados, I.; Cabildo, P.; Calramunt, M., J. Phys. Chem. 1994, 98, 5207-5211; and Cravero, R. M.; Fernandez, C; Gonzalez-Sierra, M.; Olivieri, A. C,

- 16 J. Chem. Soc. Chem. Commun. 1993, 1253).

By comparing the ¹³C CP/MAS spectra for Form IV to Form III, it becomes apparent that Form TV displays more ¹³C peaks than does Form III. Three additional peaks in the aliphatic ¹³C region of the Form TV CP/MAS spectra can be attributed to isopropanol (25.3, 26.5, and 63.2 ppm). The presence of solvent peaks in the CP/MAS spectra indicates the incorporation of isopropanol into the solid structure of Form IV.

17

¹³C Chemical Shifts fppml

Carbon Solution Solid-State Solid-State No. (CD CN) Form III Form IV

' doublet due to residual dipolar coupling to ¹⁴N,

^" doublet due to residual dipolar coupling to ^{35 37}Q

'^* confirmed through Interrupted Decoupling CP/MAS experiments

18 As used herein, the term "composition" is intended to encompass a product that contains a crystal form of the instant invention. It is also intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.

The compositions of the instant invention are useful as pharmaceutical agents for mammals, especially for humans. These compositions may be administered to patients for use in the treatment of cancer. Examples of the type of cancer which may be treated with the compositions of this invention include, but are not limited to, colorectal carcinoma, exocrine pancreatic carcinoma, myeloid leukemias and neurological tumors. Such tumors may arise by mutations in the ras genes themselves, mutations in the proteins that can regulate Ras activity (i.e., neurofibromin (NF-1), neu, src, abl, lck, fyn) or by other mechanisms. The compositions of the instant invention inhibit farnesyl- protein transferase and the farnesylation of the oncogene protein Ras. The compositions of the instant invention may also inhibit tumor angiogenesis, thereby affecting the growth of tumors (J. Rak et al. Cancer Research, 55: 4575-4580 (1995)). Such anti-angiogenesis properties of the instant compositions may also be useful in the treatment of certain forms of vision deficit related to retinal vascularization.

The compositions of the instant invention are also useful for inhibiting other proliferative diseases, both benign and malignant, wherein Ras proteins are aberrantly activated as a result of oncogenic mutation in other genes (i.e., the Ras gene itself is not activated by mutation to an oncogenic form) with said inhibition being accomplished by the administration of an effective amount of the compositions of the invention to a mammal in need of such treatment. For example, a component of NF-1 is a benign proliferative disorder.

The compositions of the instant invention may also be useful in the treatment of certain viral infections, in particular in the treatment of hepatitis delta and related viruses (J.S. Glenn et al. Science, 256:1331- 1333 (1992).

19 The compositions of the instant invention are also useful in the prevention of restenosis after percutaneous transluminal coronary angioplasty by inhibiting neointimal formation (C. Indolfi et al. Nature medicine, 1:541-545(1995). The compositions of the instant invention may also be useful in the treatment and prevention of polycystic kidney disease (D.L. Schaffner et al. American Journal of Pathology, 142:1051-1060 (1993) and B. Cowley, Jr. et a FASEB Journal, 2:A3160 (1988)).

The compositions of the instant invention may also be useful for the treatment of fungal infections.

The compositions of the instant invention may also be useful as inhibitors of proliferation of vascular smooth muscle cells and therefore useful in the prevention and therapy of arteriosclerosis and diabetic vascular pathologies. The compositions of the instant invention may also be useful in the prevention and treatment of endometriosis, uterine fibroids, dysfunctional uterine bleeding and endometrial hyperplasia.

In such methods of prevention and treatment, the compositions of the instant invention may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated. For example, the compositions may be useful in further combination with drugs known to supress the activity of the ovaries and slow the growth of the endometrial tissue. Such drugs include but are not limited to oral contraceptives, progestins, danazol and GnRH (gonadotropin-releasing hormone) agonists. Administration of the compositions of the instant invention may also be combined with surgical treatment of endometriosis (such as surgical removal of misplaced endometrial tissue) where appropriate.

The compositions of the instant invention may also be useful as inhibitors of corneal inflammation. These compositions may improve the treatment of corneal opacity which results from cauterization-induced corneal inflammation. The compositions of the instant invention may also be useful in reducing corneal edema and neovascularization. (K. Sonoda et al., Invest. Ophthalmol. Vis. ScL, 1998, vol. 39, p 2245-2251).

- 20 The compositions of the instant invention may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated. For example, the compositions of the instant invention may also be co-administered with other well known cancer therapeutic agents that are selected for their particular usefulness against the condition that is being treated. Included in such combinations of therapeutic agents are combinations of the compositions of the instant invention and an antineoplastic agent. It is also understood that such a combination of antineoplastic agent and compositions of the instant invention may be used in conjunction with other methods of treating cancer and/or tumors, including radiation therapy and surgery.

Examples of an antineoplastic agent include, in general, microtubule-stabilizing agents ( such as paclitaxel (also known as Taxol®), docetaxel (also known as Taxotere®), epothilone A, epothilone B, desoxyepothilone A, desoxyepothilone B or their derivatives); microtubule- disruptor agents; alkylating agents, anti-metabolites; epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes; biological response modifiers and growth inhibitors; hormonal/anti-hormonal therapeutic agents and haematopoietic growth factors.

Example classes of antineoplastic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the taxanes, the epothilones, discodermolide, the pteridine family of drugs, diynenes and the podophyllotoxins. Particularly useful members of those classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro-methotrexate, mitomycin C, porfiromycin, trastuzumab (Herceptin™), 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin or podo-phyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine, paclitaxel and the like. Other useful antineoplastic agents include estramustine, cisplatin, carboplatin, cyclophosphamide,

- 21 bleomycin, tamoxifen, ifosamide, melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives, interferons and interleukins.

The preferred class of antineoplastic agents is the taxanes and the preferred antineoplastic agent is paclitaxel.

Radiation therapy, including x-rays or gamma rays which are delivered from either an externally applied beam or by implantation of tiny radioactive sources, may also be used in combination with the instant compositions alone to treat cancer.

Additionally, compositions of the instant invention may also be useful as radiation sensitizers, as described in WO 97/38697, published on October 23, 1997, and herein incorporated by reference. The compositions of the instant invention may also be useful in combination with other inhibitors of parts of the signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation. Thus, the compositions of the instant invention may be utilized in combination with farnesyl pyrophosphate competitive inhibitors of the activity of farnesyl-protein transferase or in combination with a compound which has Raf antagonist activity. The compositions of the instant invention may also be co-administered with compounds that are selective inhibitors of geranylgeranyl protein transferase.

In particular, the compounds disclosed in the following patents and publications may be useful as farnesyl pyrophosphate- competitive inhibitor component of the instant composition: U.S. Serial Nos. 08/254,228 and 08/435,047. Those patents and publications are incorporated herein by reference.

The compositions of the instant invention may also be useful in combination with an integrin antagonist for the treatment of cancer, as described in U.S. Serial No. 09/055,487, filed April 6, 1998, which is incorporated herein by reference.

As used herein the term an integrin antagonist refers to compounds which selectively antagonize, inhibit or counteract binding of

22 - a physiological ligand to an integrin(s) that is involved in the regulation of angiogenisis, or in the growth and invasiveness of tumor cells. In particular, the term refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβ3 integrin, which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβ5 integrin, which antagonize, inhibit or counteract binding of a physiological ligand to both the αvβ3 integrin and the αvβδ integrin, or which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the αlβl, α2βl, αδβl, α6βl and α6β4 integrins. The term also refers to antagonists of any combination of αvβ3 integrin, αvβδ integrin, αlβl, α2βl, αδβl, α6βl and α6β4 integrins. The instant compounds may also be useful with other agents that inhibit angiogenisis and thereby inhibit the growth and invasiveness of tumor cells, including, but not limited to angiostatin and endostatin.

The compositions of the instant invention may also be useful in combination with an inhibitor of 3-hydroxy-3-methylglutaryl- CoA reductase (HMG-CoA reductase) for the treatment of cancer. Compounds which have inhibitory activity for HMG-CoA reductase can be readily identified by using assays well-known in the art. For example, see the assays described or cited in U.S. Patent 4,231,938 at col. 6, and WO 84/02131 at pp. 30-33. The terms "HMG-CoA reductase inhibitor" and "inhibitor of HMG-CoA reductase" have the same meaning when used herein. Examples of HMG-CoA reductase inhibitors that may be used include but are not limited to lovastatin (MEVACOR®; see US Patent No. 4,231,938; 4,294,926; 4,319,039), simvastatin (ZOCOR®; see US Patent No. 4,444,784; 4,820,850; 4,916,239), pravastatin (PRAVACHOL®; see US Patent Nos. 4,346,227; 4,537,859; 4,410,629; 5,030,447 and 5,180,589), fluvastatin (LESCOL®; see US Patent Nos. 5,354,772; 4,911,165; 4,929,437; 5,189,164; 5,118,853; 5,290,946; 5,356,896), atorvastatin (LIPITOR®; see US Patent Nos. δ,273,995; 4,681,893; 5,489,691; 5,342,9δ2) and cerivastatin (also known as rivastatin and BAYCHOL®; see US Patent No. 5,177,080). The

- 23 structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (5 February 1996) and US Patent Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention. An illustration of the lactone portion and its corresponding open-acid form is shown below as structures I and II.

actone Open-Acid I II

In HMG-CoA reductase inhibitors where an open-acid form can exist, salt and ester forms may preferably be formed from the open- acid, and all such forms are included within the meaning of the term "HMG-CoA reductase inhibitor" as used herein. Preferably, the HMG-CoA reductase inhibitor is selected from lovastatin and simvastatin, and most preferably simvastatin. Herein, the term "pharmaceutically acceptable salts" with respect to the HMG-CoA reductase inhibitor shall mean non- toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base, particularly those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and tetra- methylammonium, as well as those salts formed from amines such

24 as ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine. N-benzylphenethylamine, 1-p-chlorobenzyl- 2-pyrrolidine-l'-yl-methylbenzimidazole, diethylamine, piperazine, and tris(hydroxvmethyl)aminomethane. Further examples of salt forms of HMG-CoA reductase inhibitors may include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxvnapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate.

Ester derivatives of the described HMG-CoA reductase inhibitor compounds may act as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, may cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy.

Similarly, the compositions of the instant invention may be useful in combination with agents that are effective in the treatment and prevention of NF-1, restenosis, polycystic kidney disease, infections of hepatitis delta and related viruses and fungal infections.

If formulated as a fixed dose, such combination products employ the combinations of this invention within the dosage range described above and the other pharmaceutically active agent(s) within its approved dosage range. Combinations of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a multiple combination formulation is inappropriate.

25 - DOSAGE FORMS

The compositions of the instant invention can be administered for the treatment of cancer or the inhibition of farnesyl- δ protein transferase, according to the invention, by any means that effects contact of the active ingredient compound with the site of action in the body of a warm-blooded animal. For example, administration, can be parenteral, i.e., subcutaneous, intravenous, intramuscular or intra peritoneal. Alternatively, or concurrently in some cases administration

10 can be by the oral route.

The compositions of the instant invention can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but lδ are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

For the purpose of this disclosure, a warm-blooded animal is a member of the animal kingdom possessed of a homeostatic mechanism 0 and includes mammals and birds.

The dosage administered will be dependent on the age, health and weight of the recipient, the extent of disease, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. Usually, a daily dosage of active ingredient compound δ will be from about 1 to about 2000 milligrams per day. Ordinarily, from about 10 to about lδOO milligrams per day in one or more applications is effective to obtain desired results. These dosages are the effective amounts for the treatment of cancer and for the inhibition of farnesyl- protein transferase. 0 The crystal forms of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The crystal forms can be administered orally or

- 26 parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.

When a composition containing a crystal form of the instant invention is administered into a human subject, the daily dosage will δ normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms.

In one exemplary application, a suitable amount of composition is administered to a mammal undergoing treatment for 10 cancer. Administration occurs in an amount between about 0.1 mg/kg of body weight to about 60 mg/kg of body weight per day, preferably of between O.δ mg/kg of body weight to about 40 mg/kg of body weight per day.

The active ingredient can be administered orally in solid lδ dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs syrups, and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.

Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium 0 stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the δ atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose 0 (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as butylated hydroxyanisole,

- 27 sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propylparaben, δ and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

Useful pharmaceutical dosage-forms for administration of the 10 compounds of this invention can be illustrated as follows:

CAPSULES

A large number of unit capsules are prepared by filling lδ standard two-piece hard gelatin capsules each with about 100 milligrams of powdered active ingredient, about lδO milligrams of lactose, about δO milligrams of cellulose, and about 6 milligrams magnesium stearate.

SOFT GELATIN CAPSULES 0

A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing about 100 milligrams of the active ingredient. The capsules δ are washed and dried.

TABLETS

A large number of tablets are prepared by conventional 0 procedures so that the dosage unit is about 100 to about δOO milligrams of active ingredient, about 4 milligrams of magnesium stearate, about 27 milligrams of Carbopol^® 974P, and about 13δ milligrams of sodium phosphate dibasic. Appropriate coatings may be applied to increase palatability or delay absorption. δ

28

Claims

INJECTABLEA parenteral composition suitable for administration by injection is prepared from about δ.O grams of the active ingredient, δ about 174 milligrams of citric acid monohydrate, about δO milligrams of trisodium citrate dihydrate, about 400 milligrams of sodium chloride, and sufficient water for injection to make a total volume of about 100 milliliters.10 SUSPENSIONAn aqueous suspension is prepared for oral administration so that each about δ milliliters contain 100 milligrams of finely divided active ingredient, about 100 milligrams of sodium carboxymethyl lδ cellulose, about 5 milligrams of sodium benzoate, about 1 gram of sorbitol solution, U.S. P., and about .025 milliliters of vanillin.29 WHAT IS CLAIMED IS:

1. Crystal Form III, an anhydrous crystal form of l-(3- chlorophenyl)-4-[l-(4-cyanobenzyl)-δ-imidazolylmethyl]-2-piperazinone, which is characterized by a solid state x-ray powder diffraction (XRPD)

5 pattern having the following d-spacings: 7.47, 6.δ9, 6.21, δ.39, δ.Oδ, 4.84, 4.13, 4.09, 3.8δ, 3.76, 3.72, 3.68, 3.28, 3.20, 3.10, 3.07 and 3.04 angstroms.

2. Crystal Form III, the anhydrous crystal form of l-(3- chlorophenyl)-4-[l-(4-cyanobenzyl)-δ-imidazolylmethyl]-2-piperazinone 0 according to Claim 1, which is further characterized by an endotherm with an extrapolated onset temperature of about lδ0°C and a peak temperature of about lδl°C with an associated heat of about 9δ Joules/gram.

5 3. Crystal Form IV, an IPA solvate of l-(3-Chlorophenyl)-

4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone, which is characterized by a solid state x-ray powder diffraction (XRPD) pattern having the following d-spacings: 12.51, 6.02, 5.62, 5.15, 4.94, 4.72, 4.34, 4.16, 3.99, 3.90, 3.75, 3.58, 3.δ0, 3.37, 3.22, 3.17 and 3.13 angstroms. 0

4. Crystal Form IV, the IPA solvate of l-(3- Chlorophenyl)-4- [ l-(4-cyanobenzyl)-δ-imidazolylmethyl] -2-piperazinone according to Claim 3, which is further characterized by a first endotherm with an extrapolated onset temperature of about δ9°C and a peak δ temperature at about 7δ°C, with an associated heat of about 96

Joules/gram, an exotherm with a peak temperature of about 83°C and an associated heat of about 9 Joules/gram, and a second endotherm with an extrapolated onset temperature of about 149°C and a peak temperature of about lδ0°C with an associated heat of about 86 0 Joules/gram.

5. Crystal Form III, the anhydrous crystal form of l-(3- chlorophenyl)-4-[l-(4-cyanobenzyl)-δ-imidazolylmethyl]-2-piperazinone according to Claim 1, which is further characterized by solid-state 13Q

- 30 - CP/MAS NMR spectra of the upfield and downfield regions having 1 C chemical shifts in parts per million as shown in the table below:

¹ C Chemical Shifts Tppml

Carbon Solution Solid-State

- 31

6. Crystal Form IV, the IPA solvate of l-(3- Chlorophenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone according to Claim 3, which is further characterized by solid-state CP/MAS NMR spectra of the upfield and downfield regions having

chemical shifts in parts per million as shown in the table below:

- 32

13 C Chemical Shifts Tppml Carbon Solution Solid-State

No. CDjCN) Form IV

^* doublet due to residual dipolar coupling to ¹⁴N, ^• doublet due to residual dipolar coupling to ^{35 7}Cl, " confirmed through Interrupted Decouplmg CP/MAS experiments

7. A process for the preparation of Form III, the anhydrous crystal form of l-(3-Chlorophenyl)-4-[l-(4-cyanobenzyl)-δ- imidazolylme thy 1] -2-piperazinone according to Claim 2, which comprises

33 - a) mixing the monohydrate form of l-(3-chlorophenyl)-4- [l-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone with a solvent for about 20 to about 36 hours at ambient temperature to produce a crystalline solid, and 5 b) isolating the crystalline solid.

8. The process of Claim 7 wherein the solvent comprises ethanol, n-propanol, butanol, acetonitrile, acetone, ethyl acetate or tetrahydrofuran . 0

9. The process of Claim 8 wherein the solvent is acetonitrile.

10. A process for the preparation of Form IV, the 5 isopropanol (IPA) solvate crystal form of l-(3-Chlorophenyl)-4-[l-(4- cyanobenzyl)-δ-imidazolylmethyl] -2-piperazinone according to Claim 4, which comprises: a) mixing the monohydrate form of l-(3-chlorophenyl)-4- [l-(4-cyanobenzyl)-δ-imidazolylmethyl] -2-piperazinone with isopropanol 0 for about 20 to about 36 hours at ambient temperature to produce a crystalline solid, and b) isolating the crystalline solid.

11. A pharmaceutical composition comprising a δ pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of the crystal form of Claim 2.

12. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective 0 amount of the crystal form of Claim 4.

13. A pharmaceutical composition made by combining the crystal form of Claim 2 and a pharmaceutically acceptable carrier.

- 34