WO2004092128A1

WO2004092128A1 - Anhydrous crystalline forms of (2s, 4s)-1-{(2r)-2-amino-3-‘4-methoxybenzyl)sulfonyl!-3-methylbutanoyl}-4-fluoropyrrolindine-2-carbonitrile

Info

Publication number: WO2004092128A1
Application number: PCT/US2004/010600
Authority: WO
Inventors: Martin Howard Osterhout; Steven Michael Reister
Original assignee: Smithkline Beecham Corporation
Priority date: 2003-04-10
Filing date: 2004-04-07
Publication date: 2004-10-28

Abstract

The present invention includes anhydrous crystalline forms of (2S,4S)-1-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile. Formula (I).

Description

ANHYDROUS CRYSTALLINE FORMS OF ( 23 , 4S ) -l- { (2R) -2-AMINO-3- ^λ 4-METHOXYBENZYL) SULFONYL ! -3-METHYLBUTANOYL } -4-FLUOROPYRROLINDINE-2-CARBONITRILE

FIELD OF THE INVENTION The present invention relates to particular forms, for example, anhydrate and hydrated forms of fluoro cyanopyrrolidine compounds. More particularly, the present invention relates to two (2) solid state anhydrous forms of (2S,4S)-l-{(2R)-2-ar no-3-[(4- methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile. These compounds are inhibitors of serine proteases, such as dipeptidyl peptidases, and are useful in the treatment of disorders, for example metabolic disorders, such as hyperglycosemia and/or other conditions ofdiabetes. The particular forms disclosed herein demonstrate unexpectedly beneficial physical properties for use as commercial medicaments.

BACKGROUND OF THE INVENTION International Patent Application PCT/US02/20471 having an international filing date of 26 June 2002, and published as WO 03/02531 on 9 January 2003, discusses serine proteases including Dipeptydyl Peptidase IV (DPP IV) and compounds that demonstrate activity as inhibitors of DPP IV. This published application, which is herein incorporated by reference, discloses bicyclic heterocyclic compounds, including (2S,45)-l-{(2i?)-2- amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2- carbonitrile. As noted in the above-referenced publication, the compound (2(S,4»S)-l-{(2i?)-2- ammo-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2- carbonitrile demonstrates inhibition activity of DPP TV. Problems exist, however, with several salts of this compound due to the absorption of very large amounts of water at the expected exposure humidities if utilized in a medicament (e.g., 20-75% relative humidity (RH)).

Additionally these salts present other problems due to an inability to be isolated as crystalline solids. As a result, suitability of these salts as a commercial medicament would be compromised unless special handling and storage procedures were instituted. The present inventors have now identified novel forms of (2S,4S)-l-{(2R)-2- amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2- carbonitrile which are commercially suitable as serine protease inhibitors, such as dipeptidyl peptidase inhibitors, such as DPP-IV inhibitors.

The compounds of the present invention maybe prepared in crystal form and therefore have enhanced physical stability. More specifically, the amine base of the present invention sorbs much lower amounts of water when exposed to a broad range of humidities and can be prepared in a physically stable crystal form, thus enhancing its suitability as a medicament.

SUMMARY OF THE INVENTION

The present invention includes forms of the compound of Formula (I)

Preferably, the present invention includes two (2) solid state forms, namely anyhydrous form 1 and anhydrous form 2. Most preferably, the present invention includes anhydrous form 2, which demonstrates the physical characteristics desired for commercial development as a medicament. More specifically, anyhdrous form 2 is crystalline, is thermodynamically stable, and provides a development moisture sorption profile.

The present invention includes anhydrous, hydrated, or solvated forms of a compound of Formula I,

including mixtures thereof. Preferably the compound is the anhydrate form. As examples of the preferred compounds of the present invention, anhydrous form 1 maybe characterized by, among other properties, a melting point of about 173°C. Likewise anhydrous form 2 may be characterized by, among other properties, a melting point of about 190°C.

One embodiment of the present invention includes a crystalline form of anhydrous form 1 of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4- fluoropyrrolidine-2-carbonitrile characterized by a powder x-ray diffraction pattern comprising the following peaks:

More particularly, the crystalline form has a powder x-ray diff action pattern that is substantially similar to the pattern in Figure 1.

Another embodiment of the present invention includes a crystalline form of anhydrous form 2 of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile characterized by a powder x-ray diffraction pattern comprising the following peaks:

More particularly the crystalline form has a powder x-ray diffraction pattern that is substantially similar to the pattern in Figure 2.

Another aspect of the present invention includes a pharmaceutical composition comprising a compound as described herein. More particularly, the present invention includes pharmaceutical compositions comprising either anhydrous form 1 of (2S,4S)-1- {(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2- carbonitrile or anhydrous form 2 of (2S,4S)-l-{(2R)-2-amino-3-[(4- methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile, or admixtures thereof.

Furthermore, the present invention should be interpreted to include pharmaceutical compositions that include one or more hydrated form of (2S,4S)-l-{(2R)-2-amino-3-[(4- methoxybenzyl)sulfonyl]-3-methylbutanoyl} -4-fluoropyrrolidine-2-carbonitrile, one or more solvated form of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile, and/or one or more amorphous form of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4- fluoropyrrolidine-2-carbonitrile.

Preferably, as used herein pharmaceutical compositions include one or more pharmaceutically acceptable carrier, diluent, or excipient.

Another aspect of the present invention includes a method for the treatment or prophylaxis of metabolic disorders, gastrointestinal disorders, viral disorders, autoimmune disorders, dermatological or mucous membrane disorders, compliment mediated disorders, inflammatory disorders, and psychosomatic, depressive, and neuropsychiatric disorders, including, without limitation, diabetes, obesity, hyperlipidemia, psoriasis, intestinal distress, constipation, encephalomyelitis, glumerulonepritis, lipodystrophy, tissue damage, HIV infection, allergies, inflammation, arthritis, transplant rejection, high blood pressure, congestive heart failure, tumors, and stress-induced abortions that includes the administration of a compound of the present invention, including anhydrates, hydrates, and solvates thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the powder X-ray diffraction pattern of (2S,4<S)-l-{(2i?)-2-amino- 3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile, form 1, using a conventional powder X-ray diffractometer with Bragg-Brentano geometry and copper K alpha radiation.

Figure 2 depicts the powder X-ray diffraction pattern of (25',45)-l-{(2i?)-2-amino- 3 - [(4-methoxybenzyl)sulfonyl] -3 -methylbutanoyl} -4-fluoropyrrolidine-2-carbonitrile, form 2, using a conventional powder X-ray diffractometer with Bragg-Brentano geometry. Figure 3 depicts the powder X-ray diffraction pattern of (2S,4S)- 1 - {(2i?)-2-amino-

3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile hydrochloride.

Figure 4 illustrates a simulated powder X-ray diffraction pattern for form 1 calculated with copper K-alpha wavelength radiation. Figure 5 illustrates a simulated powder X-ray diffraction pattern calculated for form 2 with copper K-alpha wavelength radiation. Figure 6 illustrates FT Raman spectra of form 1. Figure 7 illustrates FT Raman spectra of form 2.

DETAILED DESCRIPTION OF THE INVENTION As discussed and illustrated throughout, the present invention includes certain solid state crystalline forms. Several methods for characterizing such forms exist, and the invention should not be limited by the methods chosen or the instrumentation used in characterizing the compounds of the present invention. For example, with regard to x-ray diffraction patterns, the diffraction peak intensities in the experimental patterns can vary, as is known in the art, primarily due to preferred orientation (non-random orientation of the crystals) in the prepared sample. As such, the scope of the present invention must be considered in light of the variability of characterization that is appreciated by those skilled in the art.

As used herein, the teπn "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

As used herein, the terms "anhydrous" and "anhydrate" are used interchangably. Likewise the terms "hydrous" and "hydrate" are used intechangeably.

In one embodiment, the compound is the anhydrate form 1 of the compound of formula I. Typically, the anhydrate form 1 has a water content of about 0.1-2.5% w/w water by coulometric KFT preferably below about 1.0% w/w, more preferably below about 0.5% w/w, more preferably about 0.35% w/w.

In another embodiment, the compound is the anhydrate form 2 of the compound of formula I. Typically the anhydrate form 2 has a water content of about 0.1-2.5% w/w water by coulometric KFT preferably below about 1.0% w/w, more preferably below about 0.5% w/w, more preferably about 0.2% w/w. Importantly, although the above-referenced water contents are noted, the water content should not be considered as descriptive of any particular pharmaceutical composition or formulation comprising the forms of the present invention. Rather, when in admixture with other pharmaceutically acceptable carriers, diluents, or excipients, the water content may be higher or lower. The water contents given above should be considered as descriptive of the specific forms, themselves.

In one embodiment, the compound is the anhydrate form 1 of the compound of formula I characterized, in part, by a powder x-ray diffraction pattern as shown in Figure 1. The anhydrate form 1 of the compound of formula I may be characterized by including, but not limited to, the peaks of Table I.

Table I (Form 1)

* Based on Cu Kα radiation. K 2 was removed prior to peak location. Notably, in a mixture of the compound of formula I with another phase, not all the peaks listed in Table I may be apparent in the mixture's powder diffraction pattern.

In another embodiment, the compound is the anhydrate form 2 of the compound of formula I characterized, in part, by a powder x-ray diffraction pattern as shown in Figure 2. The anhydrate form 2 of the compound of formula I maybe characterized by including, but not limited to, the peaks of Table π. Table π (Form 2)

* Based on Cu Kα radiation. Kα2 was removed prior to peak location Notably, in a mixture of the compound of formula I and another phase, not all the peaks listed in Table LI may be apparent in the mixture's powder diffraction pattern.

The present invention includes within the scope substantially pure anhydrous, hydrated, or solvate forms, as well as mixtures thereof. The present invention includes within the scope crystalline or amorphous forms and mixtures of crystalline and amorphous forms.

The free base and HC1 salts of the compound of Formula I may be prepared according to the procedures of the International Patent Application No. PCT/US02/20471, filed 26 June 2002, and published as WO 03/02531 on 9 January 2003, which application is incorporated herein by reference. As illustrated in Schemes A and B, the compound of formula I, namely, (2_JS',4S)-1-

{(2i?)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2- carbonitrile, has been prepared in two distinct forms, an anhydrate designated as Form 1 and an anhydrate form designated as Form 2. The relationship of these forms is illustrated in Scheme B below. Anhydrous Form 1

Form 1 of (2^,45)-l-{(2i?)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile may be prepared by reacting tert-butyl (lR)-l-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-l-yl]carbonyl}-2-[(4- methoxybenzyl)sulfonyl]-2-methylpropylcarbamate in dichloromethane or acetonitrile in the presence of an acid, such as methanesulfonic acid or trifluoroacetic acid, followed by (a) introducing an dilute aqueous base solution, such as a aqueous solution of sodium hydrogen carbonate followed by adding an extraction solvent such as dichloromethane and separating the layers. Concentration of the organic layer followed by purification of the material by chromatography, such as silica gel chromatography, using a non-polar solvent and a polar solvent along with a small amount of ammonium hydroxide as eluent. Likewise, Form 1 can be isolated when the reaction mixture solvent is acetonitrile by adding (b) introducing an dilute aqueous base solution, such as a aqueous solution of sodium hydrogen carbonate and isolating the solids. Anhydrous Form 2 Form 2 of (2S,4»S)-l-{(2i?)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile may be prepared by suspending Form l of(2S,4S)-l-{(2i?)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4- fluoropyrrolidine-2-carbonitrile in an alcohol solvent, such as a lower alkyl alcohol, such as methanol, ethanol or isopropyl alcohol, preferably methanol. The solution may be heated but heat is not necessary.

Form 2 of (2S,4S)-1 - {(2i?)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile may be prepared by reacting tert-butyl (lR)-l-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-l-yl]carbonyl}-2-[(4- methoxybenzyl)sulfonyl]-2-methylpropylcarbamate in acetonitrile or methanol, or other alcohol solvent, in the presence of an acid, such as methanesulfonic acid or trifluoroacetic acid, followed by (a) introducing an dilute aqueous base solution, such as a aqueous solution of sodium hydrogen carbonate, and isolating the solids or (b) concentration of the alcoholic solution followed by the addition of a dilute aqueous base solution, such as a aqueous solution of sodium hydrogen carbonate, and isolating the solids. The solutions may be heated but heat is not necessary.

Consistent polymorph control of drug substance preparation can be achieved with an alcoholic slurry, preferably at elevated temperature but heat is not necessary. Rapid conversions of form 1 to form 2 have been observed in either isopropanol or methanol in the presence of seed crystals at 60 °C. Other polar protic and potentially polar aprotic solvents may be useful. Methanol is preferrred. Such conversion rates have been monitored by Raman spectroscopy. In a typical process, as described below, (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile (either form 1, form 2, or a mixture of polymorphic forms) is suspended in methanol in the presence of 0.5 wt% form 2 seed material and warmed to 60 °C for one hour.

Additional studies have demonstrated that in DSC and hot-stage XRD studies that form 1 can convert to form 2 during melt.

Scheme A

Scheme B

Formula I Formula I (form 1) (form 2)

A series of crystallization experiments were performed to investigate whether compound of Formula I can exist in more than one solid-state form or has a propensity to form solvates. This series of experiments employed 47 solvent systems and four crystallization modes (slow evaporation, fast evaporation, cooling, and ripening). These experiments indicated the existence of at least one additional non-solvated solid-state form in addition to a previously identified non-solvated form. There was no evidence of solvated forms from the experiments conducted.

While it is possible that, for use in therapy, therapeutically effective amounts of a compound of the present invention including anhydrate and/or hydrate forms thereof, may be administered as the raw chemical, it is possible to present the active ingredient as a pharmaceutical composition. Accordingly, the invention further provides pharmaceutical compositions which include therapeutically effective amounts of compounds of the present invention including anhydrate and/or hydrate forms thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compounds of the present invention including anhydrate and/or hydrate forms thereof, are as described above. The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. According to another aspect of the invention there is also provided a process for the preparation of a pharmaceutical formulation including admixing a compound of the present invention including anhydrate and/or hydrate forms thereof, with one or more pharmaceutically acceptable carriers, diluents or excipients.

Compounds of the present invention including anhydrate and/or hydrate forms thereof may be formulated for administration by any route, and the appropriate route will depend on the disease being treated as well as the subjects to be treated. Suitable pharmaceutical formulations include those for oral, rectal, nasal, topical (including buccal, sub-lingual, and transdermal), vaginal or parenteral (including intramuscular, subcutaneous, intravenous, and directly into the affected tissue) administration or in a form suitable for administration by inhalation or insufflation. The formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well know in the pharmacy art. Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing and coloring agent can also be present. Capsules are made by preparing a powder mixture as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like can also be added.

Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like. The compounds of the present invention including anhydrate and/or hydrate forms thereof, can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. The compounds of the present invention including anhydrate and/or hydrate forms thereof may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysme substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels. Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986), incorporated herein by reference with regard to such delivery systems.

Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For treatments of the eye or other external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water- miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.

Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered, dose pressurised aerosols, nebulizers or insufflators. Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions maybe prepared from sterile powders, granules and tablets. hi addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

Also provided in the present invention, is a method for inhibiting a post proline/analine cleaving protease, such as a serine protease, such as a dipeptidyl peptidase, such as DPP-IV, which includes administering a therapeutically effective amount of a compound of the present invention including anhydrate and/or hydrate forms thereof, to the mammal. The compounds of the present invention including anhydrate and/or hydrate forms thereof are as herein described.

The determination of a therapeutically effective amount of a compound of the present invention including anhydrate and/or hydrate forms thereof will depend on a number of factors including, but not limited to, the age and weight of the mammal, the precise disorder requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physcian or veternarian. Typically, the compounds of the present invention including anhydrate and/or hydrate forms thereof will be given for treatment in the range of 0.1 to 100 mg/kg body weight of recipient (mammal) per day and more usually in the range of 1 to 10 mg/kg body weight per day. Acceptable daily dosages, may be from about 0.1 to about 1000 mg/day, and preferably from about 0.1 to about 100 mg/day.

The compounds of the present invention including anhydrate and/or hydrate forms thereof, described above, are useful in therapy and in the preparation of medicaments for treating a disorder in a mammal, which is characterized by the need for inhibition of a post proline/analine cleaving protease, such as a serine protease, such as a dipeptidyl peptidase, such as DPP IV. The compounds of the present invention including anhydrate and/or hydrate forms thereof are useful for treating or preventing metabolic disorders, gastrointestinal disorders, viral disorders, autoimmune disorders, dermatological or mucous membrane disorders, compliment mediated disorders, inflammatory disorders, and psychosomatic, depressive, and neuropsychiatric disorders, including, without limitation, diabetes, obesity, hyperlipidemia, psoriasis, intestinal distress, constipation, encephalomyelitis, glumerulonepritis, lipodystrophy, tissue damage, HIV infection, allergies, inflammation, arthritis, transplant rejection, high blood pressure, congestive heart failure, tumors, and stress-induced abortions.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.

Example 1 Preparation of(2S.4S)-l-{(2R)-2-amino-3- (4-methoxybenzyl)sulfonyl]-3- methylbutanoyiχ4-fluoropyrrolidine-2-carbonitrile. Form 1

To a 1L 3-necked RBF equipped with a stir bar was charged with the 90g (18.1 mmol) of tert-Butyl (lR)-l-{[(2S,4S)-2-cyano-4-fluoroρyrrolidin-l-yl]carbonyl}-2-[(4- methoxybenzyl)thio]-2-methylpropylcarbamate and dichloromethane. To this was slowly added 15.3 ml of trifiuoroacetic acid (19.9 mmol). This solution was allowed to stir at room temperature for 12 hours at which time an additional 60 ml of trifiuoroacetic acid (78 mmol) was added and the reaction stirred at room temperature for an additional 24 hours. The reaction mixture was quenched with sat. aq. sodium hydrogen carbonate and diluted with additional dichloromethane. The organic layer was separated and washed with brine. The organic layer was dried over sodium sulfate, concentrated and purified by running through a plug silica gel column initially using 80%EtOAc/19%Heptane/ l%NH₄OH and then 5%MeOH/94%DCM/l%NH₄OH to flush of the column. The collected fractions were combined and concentrated to give a off-white solid. Yield: 35.7 g

Example 2

Preparation of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl] -3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile. Form 2

a) Preparation of(4S)-l-(tert-butoxycarbonyl)-4-fluoro-L-prolinamide Charged the reactor with (4»S)-l-(tert-butoxycarbonyl)-4-fluoro-L-proline (130 g, lwt, 1 eq.), dichloromethane (520 mL, 4 vol), pyridine (55 mL, 0.4 vol, 1.2 eq), and Boc- anhydride (145 g, 1.1 wt, 1.2 eq.). Stir the reaction solution at approximately 20°C for 2 hours. Charged the reactor with ammonium bicarbonate (62 g, 0.5 wt, 1.44 eq). Stirred the reaction solution at approximately 20°C overnight. Filtered the reaction over a bed of celite (130 g, 1 wt.). Washed the filter cake with dichloromethane (260 mL, 2 vol).

Concentrated the filtrate to a volume of 3 volumes. Added heptane (520 mL, 4 vol), and concentrated to a final volume of 3 volumes. Added heptane (390 mL, 3 vol), and cooled to approx. 5°C. for 30 min. Collected the solid by filtration, washed with heptane (260 mL, 2 vol), and then dried under vacuum at approximately 50°C to constant weight. Yield: 88-90%. b) Preparation of (2S.4S)-4-fluoropyrrolidine-2-carbonitrile para-toluenesulfonic acid Charged the reactor with (4S)-l-(tert-butoxycarbonyl)-4-fluoro-L-prolinamide (116 g, lwt., 1 eq.), isopropyl acetate (578 mL, 5 vol), pyridine (88 mL, 0.8 vol, 2.2 eq). Stirred the resulting slurry at approx. 20°C. Added trifiuoroacetic anhydride (77 mL, 1.0 wt., 1.1 eq.) over at least 30 minutes, maintaining the temperature at approx. 20°C. The reaction solution is stirred at approx. 20°C for 1 hour. Slowly charged the reactor with water (578 mL, 5 vol). Stirred the mixture for 15 minutes, let the layers separate, and discarded the aqueous, lower layer. Concentrated the organic layer under vacuum at a jacket temperature of approximately 50°C to half volume. Diluted the reaction back up to 5 volumes with isopropyl acetate. Cooled reactor contents to 20°C. Charged the reactor with p-toluenesulfonic acid (94 g, 0.8 wt, 1 eq). Stirred the reaction for 2 hours. GC at this point should show complete consumption of (4S)-l-(tert-butoxycarbonyl)-4-fluoro-L- prolinamide. Concentrated the reaction to 3 volumes under full vacuum at a jacket temperature of approximately 50°C and added 2 volumes of isopropyl alcohol and concentrated to a final volume of 4 volumes. Cooled the reaction to 0°C and hold for 30 minutes. Filtered the solids, washed with isopropyl alcohol (1 vol), and then dried under vacuum at approx. 50°C to constant weight. Yield: 68-71%). c) Preparation oftert-Butyl flR)-l-{[(2S.4S)-2-cvano-4-fluoropytrolidin-l-yl]carbonv - 2-[(4-methoxybenzyl)thio J -2-methylpropylcarbamate

Charged the reactor with (2i?)-2-[(tert-butoxycarbonyl)amino]-3-[(4- methoxybenzyl)thio]-3-methylbutanoic acid (100 g, 1 wt, 1 equiv.), (2S,4S)-4- fluoropyrrolidine-2-carbonitrile para-toluenesulfonic acid (81.4 g, 0.81 wt, 1.05 equiv.), 0-(7-Azabenzotriazol-l-yl)-N N N, N-tetramethyluronium hexaflurophosphate [i.e. HATU] (108 g, 1.08 wt, 1.05 equiv.), and DMF (400 mL, 4 vol). Cooled the mixture to approximately 0 °C. Added Hunig's base (126 mL, 1.26 vol, 2.68 equiv.) over at least 30 minutes. Stirred the mixture at approximately 0 °C until the reaction is complete (ca. 5 hours). Added MTBE (400 mL, 4 vol). Added water (400 mL, 4 vol) over at least 45 minutes to quench the reaction. Extracted the aqueous phase with MTBE (2x400 mL, 8 vol). Combined the organic phases, washed with a mixture of saturated ΝaHCO3 (200 mL, 2 vol) and brine (400 mL, 4 vol). Concentrated the organic phase under vacuum to 3 vol. Added methanol (600 mL, 6 vol). Concentrated under vacuum to 3 vol. Added methanol (600 mL, 6 vol). Added the resultant methanol solution to a mixture containing oxone (333 g, 3.33 wt, 2.0 equiv.) in water (1000 mL, 10 vol) at approximately 0 °C under inert atmosphere over at least 1.5 hours. Stirred the mixture at ca. 0 °C for 1 hour. Warmed the reaction mixture in a controlled fashion to 20 °C and stirred until the reaction is complete (overnight). Dissolved sodium hydrogensulfite (118 g, 1.18 wt, 4 equiv.) in process water (300 mL, 3 vol). Added the sodium hydrogensulfite solution over at least 30 minutes. Filtered to collect the solid. Transfered the wet cake back to the reactor. Added ethyl acetate (1200 mL, 12 vol) to dissolve the solid. Filtration of the resulting mixture removed any insoluble inorganic salts. Washed ethyl acetate solution with process water (300 mL, 3 vol). Concentrated the organic layer under vacuum at a jacket temperature of approximately 50C to 3 vol. Added heptane (300 mL, 3 vol). Aged the mixture at approximately 20 °C for 1 hour. Filtered to collect the solid and pull to dryness. Dried in vacuum at approximately 50 °C to constant weight. Yield: 67-71%. d) Preparation of(2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-βuoropyrrolidine-2-carbonitrile Charged the reactor with tert-Butyl (lR)-l-{[(2S,4S)-2-cyano-4-fluoropyrrolidin-l- yl]carbonyl}-2-[(4-methoxybenzyl)thio]-2-methylpropylcarbamate (90 g, 1 wt, 1 equiv.), and acetonitrile (180 mL, 2 vol). Added methanesulfonic acid (35.2 mL, 0.39 vol, 3 equiv.) over at least 30 minutes at approximately 20 °C. Stirred the mixture at approximately 20 °C until the reaction is complete (ca. 1 hour). Dissolved sodium bicarbonate (51.9 g, 0.58 wt, 3.3 equiv.) in water (900 mL, 10 vol). Added the sodium bicarbonate solution to the reaction mixture over at least 30 minutes at approximately 20

°C. Stirred the mixture at approximately 20 °C for 1 hour. Filtered to collect the solid.

Washed the cake with water (200 mL, 2 vol) and pulled to dryness. Dried in vacuum at approximately 55 °C to constant weight. Yield: 81-88%. e) Preparation of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl] -3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile (Form 2)

Charged the reactor with (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile (3.3kg, 8.3 mol) and methanol (3 vol). Stirred the slurry at approximately 60 °C for 1 hour. Cooled the mixture to 20 °C.

Filtered to collect the solid, washed with methanol (2 vol), pulled to dryness, and dried in vacuum at approximately 50 °C to constant weight. Yield: 96%.

Example 3 Powder X-Rav Diffraction of(2S.4S)-X{(2R)-2-amino-3-f(4-methoxybenzyl)sulfonyll-3- methylbutanoyl}-4-flιιoropyrrolidine-2-carbonitrile. Form 1

The anhydrate free amine prepared according to Example 1 was prepared by dusting the sample on to a silicon zero background plate and scanning with a conventional Bragg- Brentano X-ray diffractometer using copper K alpha radiation. The X-ray diffraction pattern obtained is shown in Figure 1.

As noted above, the diffraction peak intensities in the experimental patterns can vary, as is known in the art, primarily due to preferred orientation (non-random orientation of the crystals) in the prepared sample.

Example 4

Powder X-Rav Diffraction of(2S, 4S)-l-{(2R)-2~amino-3-f(4-methoxybenzyl)sulfonyll-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile. Form 2 The anhydrate free amine prepared according to Example 2 was prepared by filling a 0.25-mm well in a silicon plate and smoothing the top surface with a glass microscope slide and scanning with a conventional Bragg-Brentano X-ray diffractometer using copper K alpha radiation. The x-ray diffraction pattern obtained is shown in Figure 2. As noted above, the diffraction peak intensities in the experimental patterns can vary, as is known in the art, primarily due to preferred orientation (non-random orientation of the crystals) in the prepared sample. Example 5 Single Crystal X-ray Diffraction of(2SAS)-l-{(2R)-2-amino-3-f(4- methoxybenzyl)sulfonyl]-3-methylbutanoyli-4-fluoropyrrolidine-2-carbonitrile. Form 1 and Form 2

The crystal structure of Form 1 was determined using copper K-alpha radiation at 150 K. The unit cell parameters and space group were determined to be the orthorhombic crystal system (α = β = γ = 90°), P2₁2₁2₁, with a = 6.7512(3) A, b = 10.0494(4) A, c = 27.9689(12) A at 150 K and a - 6.7767(17) A, b = 10.147(2) A, c = 28.264(8) A at 298 K. Using the atomic coordinates of the single crystal data and room temperature (298K) unit cell parameters, a simulated powder X-ray diffraction pattern was calculated with copper K-alpha wavelength radiation and is presented in Figure 4.

The diffraction peak relative intensities in the simulated pattern differ from those in the experimental powder pattern primarily due to preferred orientation (non random orientation of crystals) in the prepared sample for experimental powder diffraction. The relative peak intensities in the simulated pattern are affected not only by the input single crystal data but also by the peak profile (shape and broadening) used in the calculation of the simulated powder pattern.

The single crystal structure of Form 2 was determined using copper K-alpha radiation at 150 K. The unit cell parameters and space group were determined to be the orthorhombic crystal system (α = β = γ = 90°), P2₁2₁2₁, with a = 9.9579(7) A, b = 10.2175(7) A, c = 19.0760(12) A at 150 K and a = 10.0513(17), b = 10.2960(16), c = 19.210(3) at 295 K.

Using the atomic coordinates of the single crystal data and room temperature (295K) unit cell parameters, a simulated powder X-ray diffraction pattern was calculated with copper K-alpha wavelength radiation and is presented in Figure 5. The diffraction peak relative intensities in the simulated pattern differ from those in the experimental powder pattern primarily due to preferred orientation (non random orientation of crystals) in the prepared sample for experimental powder diffraction. The relative peak intensities in the simulated pattern are affected not only by the input single crystal data but also by the peak profile (shape and broadening) used in the calculation of the simulated powder pattern.

Example 6

Preparation of(2S.4S)-l-{(2R)-2-amino-3-f(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile hydrochloride To a CH C1₂ (60 mL) solution containing trifiuoroacetic acid (8 mL) was added tert-Butyl (1R)- 1 - {[(2S,4S)-2-cyano-4-fluoropyrrolidin- 1 -yl]carbonyl} -2-[(4- methoxybenzyl)thio]-2-methylpropylcarbamate (1.2 g, 2.58 mmol, 1.0 eq). The resulting pale yellow solution was stirred at RT for 2 h at which time the solvent was removed in vacuo. The resulting TFA salt was converted to the free base by addition of a sat. NaHCO₃ solution and extracting the aqueous layer (2x) with EtOAc. The combined organic extracts were dried over Na₂SO₄ and concentrated in vacuo. The resulting off- white foam was purified via flash chromatography using 5% MeOH (with 0.1% NH₃) in CH₂C1₂ as the mobile phase affording 897 mg (2.45 mmol, 95% yield) of a white foam. To form the HC1 salt, the free base was taken up in Et O and acetone added until all solids were in solution. 2.0 M HC1 in ether was added dropwise until no more precipitate formed. The precipitate was then filtered and washed several times with Et₂O. The resulting salt was dried under high vacuum.

1H NMR (D₂O) 400 MHz δ 7.31 (d, 2H, J= 8.6 Hz), 6.88 (d, 2H, J= 8.5 Hz), 5.49 (d, 1H, J= 50.5 Hz), 5.00 (d, 1H, J= 9.8 Hz), 4.02-3.52 (m, 8H), 2.65 (t, 1H, J= 15.7 Hz), 2.40 (m, 1H), 1.44 (s, 3H), 1.31 (s, 3H) ppm. Similar to the powder x-ray diffractiobn methods described in Examples 3 and 4, above, the compound of Example 6 provides the x-ray powder diffraction pattern as shown in Figure 3. As noted above, the diffraction peak intensities in the experimental patterns can vary, as is known in the art, primarily due to preferred orientation (non- random orientation of the crystals) in the prepared sample.

Example 7

Moisture sorption testing of(2S.4S)-l-f(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile

Approximately 20 mg of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile was weighed into a sample pan of a symmetrical integrated gas flow microbalance system (model number SGA-100 , manufactured by VTI). The sample was dried at 60C under a dry nitrogen stream until the rate of weight loss was less than 0.015% in 5 minutes. After drying the sample was equilibrated at 25C and the relative humidity increased stepwise (adsorption) to 5, 15, 25, 35, 45, 55, 65, 75, 85 and 95% - each relative humidity step was held until the sample equilibrated at that condition. Equilibrium was defined as a weight change of less than 0.015% in 5 minutes. The relative humidity was then decreased step wise (desorption) to 90, 80, 70, 60, 50, 40, 30, and 20% - each step held until equilibrium was reached. The equilibrium condition was the same as in the sorption phase. The % w/w increase or decrease in moisture content of the sample is reported for each equilibrated RH condition. The anhydrate form 1 and anhydrate form 2 of the compound of formula I are not hygroscopic; they adsorb very little water at relative humidity between 0% to 95% at 25C.

Typically, the anhydrate form 1 adsorbs less than 1% w/w water, preferably less than 0.5% w/w water, more preferably less than 0.2% w/w water at relative humidity between 0% and 95% at 25C. Water adsorbed by anhydrate form 1 readily desorbs when the relative humidity is decreased.

Typically, the anhydrate form 2 adsorbs less than 1% w/w water, preferably less than 0.5% w/w water, more preferably less than 0.35% w/w water at relative humidity between 0% and 95% at 25C. Water adsorbed by anhydrate form 2 readily desorbs when the relative humidity is decreased.

Example 8 h a typical process, (2<S',45)-l-{(2i-)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile form 1 is suspended in methanol in the presence of 0.5 wt% form 2 seed material and warmed to 60°C for one hour. FT Raman spectra of the undissolved solids collected as a function of time show the disappearance of spectral features associated with form 1 and appearance of spectral features associated with form 2.

The FT Raman spectra of form 1 and form 2 are given in Figures 6 and 7, respectively.

Below are tables summarizing several of the major peaks and their corresponding relative intensities. As noted hereinabove, due to the variability appreciated by those skilled in the art, the existance of peaks and intensities should not necessarily be interpreted too critically in the characterization of a compound within the scope of the present invention. Peak Table for FT Raman Spectrum of Form 1

Peak Table for FT Raman Spectrum of Form 2

Example 9

Stability testing

Upon the collection of data regarding the purity of the compounds of the present invention, the compounds of the present invention show beneficial properties with regard to stability, such as after two (2) weeks in an 40 C/75% RH oven. As such, the forms, and in particular anhydrous form 2 of (2S,4S)-l-{(2R)-2-amino-3-[(4- methoxybenzyl)sulfonyl] -3 -methylbutanoyl} -4-fluoropyrrolidine-2-carbonitrile, presents a beneficial profile as a commercially viable medicament.

BIOLOGICAL DATA Materials:

H-Ala-Pro-pNA«HCl was purchased from BACHEM Bioscience hie. (product no. L- 1115). A 500 mM stock solution was prepared with dimethylsulfoxide and stored at -20 °C. Gly-Pro-AMC was purchased from Enzyme System Products (product no. AMC-39) and stored at -20 °C as a 10 mM stock solution in dimethylsulfoxide. Test compound was dissolved to 10 mM in dimethylsulfoxide and this was used as a stock solution for DPP- IV titration assay. Athens Research and Technology, hie prepared the purified human DPP-IV. The material was isolated from human prostasomes using the method of DeMeester et al., J. Immunol. Methods 189, 99-105. (1996), incorporated herein by reference to the extent of describing such method. DPP-IV Assay:

Two-fold serial dilutions of test compounds in 100 % dimethylsulfoxide were performed in 96-well polystyrene flat bottom plates (Costar, #9017). The average enzymatic activity from wells containing dimethylsulfoxide but lacking test compound was used as a control value for calculating percent inhibition. DPP-IV (20 ng/mL) was mixed in microtiter plates with test compound, substrate and assay buffer to yield 100 μM H-Ala-Pro- pNA«HCl in 25 mM Tris, pH 7.5, 10 mM KC1, 140 mM NaCl. The intact peptide contains a p-nitrophenylanilide which, when hydrolyzed by DPP-IV, releases the absorbant p-nitrophenylaniline. The absorbency was monitored in 20 minutes intervals at a wavelength of 387 nm using a Molecular Devices SpectraMax 250 absorbency plate reader. The enzymatic activity was determined by estimating the best linear fit to the data. Values for enzymatic activity were taken directly from the linear fit determined by the software on the plate reader.

Data Analysis: The enzymatic activity was determined by estimating the best linear fit to the data. Data reduction was performed using the Microsoft Excel RoboSage. Determination of IC₅₀ values: The enzymatic activity was plotted against the concentration of test compound, including [I] = 0, and the IC₅₀ determined from a fit of equation (2) to the data.

RATE = V_max/ (1 + ([I]/ IC₅₀ )) (2) V_max was the best fit estimate of the maximal enzymatic activity.

Determination of Kj values: Ki values were calculated from IC₅₀ values using equation (3) assuming a competitive model.

iT,. = /C *[1 ] (3)

⁵⁰ (s+κ_my

The apparent pKi values for the test compound was > 5.0. All research complied with the principles of laboratory animal care (NTH publication No. 85-23, revised 1985) and GlaxoSmithKline policy on animal use.

Although specific embodiments of the present invention have been illustrated and described in detail, the invention is not limited thereto. The above detailed description of preferred embodiments is provided for example only and should not be construed as constituting any limitation of the invention. Modifications will be obvious to those skilled in the art, and all modifications that do not depart from the spirit of the invention are intended to be included within the scope of the appended claims. Furthermore, when identifying peaks associated with the forms of the present invention, such diffraction patterns should be considered as including such peaks but not limited thereto.

Claims

What is claimed is:

1. Anhydrous, hydrated, or solvated forms of a compound of Formula I,

2. The compound of claim 1, wherein the compound is the anhydrate form. 3. The compound of claim 2, wherein in the compound has a melting point of about

173°C.

The compound of claim 2, wherein the compound has a melting point of about

190°C.

A crystalline form of anhydrous form 1 of (2S,4S)-l-{(2R)-2-amino-3-[(4- methoxybeιιzyl)sulfonyl]-3-methylbutanoyl}-4-fluoiOpyrrolidine-2-carbonitrile characterized by a powder x-ray diffraction pattern comprising the following p©3 -S.

The crystalline form of claim 5 wherein the powder x-ray diffraction pattern is substantially similar to the pattern in Figure 1. A crystalline form of anhydrous form 2 of (2S,4S)-l-{(2R)-2-amino-3-[(4- methoxybenzyl)sulfonyl]-3-methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile characterized by a powder x-ray diffraction pattern comprising the following peaks:

8. The crystalline form of claim 7 wherein the powder x-ray diffraction pattern is substantially similar to the pattern in Figure 2. 9. A pharmaceutical composition comprising a compound as claimed in claims 1-8.

10. A pharmaceutical composition comprising: anhydrous form 1 of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl} -4-fluoropyrrolidine-2-carbonitrile; or anhydrous form 2 of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoiOpyrrolidine-2-carbonitrile; or a mixture thereof.

11. The pharmaceutical composition of claim 10 further comprising one or more hydrated form of (2S,4S)- 1 - {(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile. 12. The pharmaceutical composition of claim 10 further comprising one or more solvated form of (2S,4S)-l-{(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl}-4-fluoropyrrolidine-2-carbonitrile.

13. The pharmaceutical composition of claim 10 further comprising one or more amorphous form of (2S,4S)-1- {(2R)-2-amino-3-[(4-methoxybenzyl)sulfonyl]-3- methylbutanoyl} -4-fluoropyrrolidine-2-carbonitrile.

14. The pharmaceutical composition as claimed in claims 9-13 further comprising one or more pharmaceutically acceptable carrier, diluent, or excipient.

15. A method for the treatment or prophylaxis of metabolic disorders, gastrointestinal disorders, viral disorders, autoimmune disorders, dermatological or mucous membrane disorders, compliment mediated disorders, inflammatory disorders, and psychosomatic, depressive, and neuropsychiatric disorders comprising administering a compound as claimed in any of claims 1 to 8.

16. The method of claim 15 wherein the disorder is diabetes, obesity, hyperlipidemia, psoriasis, intestinal distress, constipation, encephalomyelitis, glumerulonepritis, lipodystrophy, tissue damage, HIV infection, allergies, inflammation, arthritis, transplant rejection, high blood pressure, congestive heart failure, tumors, or stress-induced abortion.

17. A compound as claimed in any one of claims 1 to 8 for use in therapy.