WO2008034781A1

WO2008034781A1 - Cysteine protease inhibitor salt

Info

Publication number: WO2008034781A1
Application number: PCT/EP2007/059752
Authority: WO
Inventors: Craig Grant; Stephen Watt
Original assignee: Medivir Ab
Priority date: 2006-09-18
Filing date: 2007-09-17
Publication date: 2008-03-27
Also published as: GB0618263D0

Abstract

The compound N-[(S)-1 -((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)- 3-methyl-butyl]-4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]-benzamide benzenesulphonate, its use as a medicament and methods of preparation.

Description

Cysteine protease inhibitor salt

The present invention relates to a novel salt of a known cysteine protease inhibitor. In particular it relates to the benzenesulphonate salt of N-[(S)-1-((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro- furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]- benzamide. The present invention also relates to pharmaceutical formulations containing the compound and to therapeutic uses thereof, particularly for the treatment of inflammatory and allergic conditions.

The papain superfamily of cysteine proteases is widely distributed in diverse species including mammals, invertebrates, protozoa, plants and bacteria. A number of mammalian cathepsin enzymes, including cathepsins B, F, H, K, L, O and S, have been ascribed to this superfamily, and inappropriate regulation of their activity has been implicated in a number of metabolic disorders including arthritis, muscular dystrophy, inflammation, glomerulonephritis and tumour invasion. Pathogenic cathepsin like enzymes include the bacterial gingipains, the malarial falcipains I, II, III et seq and cysteine proteases from Pneumocystis carinii, Trypanosoma cruzei and brucei, Crithidia fusiculata, Schistosoma spp.

The inappropriate regulation of cathepsin K has been implicated in a number of disorders including osteoporosis, gingival diseases such as gingivitis and periodontitis, Paget's disease, hypercalcaemia of malignancy and metabolic bone disease. In view of its elevated levels in chondroclasts of osteoarthritic synovium, cathepsin K is implicated in diseases characterised by excessive cartilage or matrix degradation, such as osteoarthritis and rheumatoid arthritis.

Metastatic neoplastic cells typically express high levels of proteolytic enzymes that degrade the surrounding matrix and inhibition of cathepsin K may thus assist in treating neoplasias.

International patent application WO2005/066180 discloses a number of cysteine protease inhibitors including N-[(S)-1 -((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole-4- carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide (corresponding to Example 8.19 therein).

(Compound I) N-[(S)-1-((3aS,6S_!6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]- 4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide (referred to herein as Compound I free base), is specifically disclosed in WO2005/066180 in the form of the free base.

In respect of its use as a pharmaceutical agent, Compound I free base suffers from a number of disadvantages relating to its relatively low water solubility, generally amorphous nature and moisture stability.

There is therefore a need for a form of Compound I which has one or more of the following properties:

(i) increased water solubility (ii) a higher degree of crystallinity

(iii) improved moisture stability

Thus, according to the present invention there is provided N-[(S)-1-((3aS,6S,6aS)-6-Fluoro-3- oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)- thiazol-4-yl]-benzamide benzenesulphonate (referred to herein as Compound I benzenesulphonate).

As will be demonstrated, Compound I benzenesulphonate has an improved physico-chemical profile, especially in terms of crystallinity, moisture stability and adequate water solubility than Compound I free base and other salt forms of Compound I.

The invention is illustrated by reference to the following figures: Figure 1 shows an X-ray powder diffraction pattern of Compound I benzenesulphonate.

Figure 2 shows a comparison of the X-ray powder diffraction patterns of Compound I benzenesulphonate, methanesulphonate and free base.

Figure 3 shows a comparison of the X-ray powder diffraction patterns of Compound I hydrochloride, naphthalene-2-sulphonate, D-gluconate, acetate and free base. Figure 4 shows X-ray powder diffraction patterns of Compound I benzenesulphonate before and after storage for 1 week at 4O⁰C and 75% relative humidity.

Figure 5 shows a gravimetric vapour sorption isotherm for Compound I benzenesulphonate.

Figure 6 shows X-ray powder diffraction patterns of Compound I benzenesulphonate before and after gravimetric vapour sorption testing.

Figure 7 shows X-ray powder diffraction patterns of Compound I benzenesulphonate prepared at different scales.

Figure 8 XRPD of Compound I benzenesulphonate precipitate from different solvents. Figure 9 XRPD of Compound I benzenesulphonate recovered from water.

Benzenesulphonate (also known as 'besylate') is the anion formed by the reaction of benzene sulphonic acid with a base:

Water solubility (i.e. hydrophilicity) is an important physical property of pharmaceutical agents which impacts their pharmacokinetics. In many circumstances an increased water solubility is desirable.

Crystallinity is another important physical property. Highly crystalline solids are generally easier to handle (for example, having more consistent physical properties) compared to amorphous or partially-crystalline solids. Furthermore, the exact crystalline form can affect, for example, dissolution rates and stability (such as moisture stability).

In a further aspect of the present invention there is provided Compound I benzenesulphonate in solid crystalline form. In particular, there is provided the crystalline form in which Compound I benzenesulphonate is obtained when a solution of benzenesulphonic acid in tetrahydrofuran is added to a solution of Compound I free base in dichloromethane. One skilled in the art will recognise that the same crystalline form of Compound I benzenesulphonate may be prepared from other solvents or solvent mixtures.

Additionally, there is provided Compound I benzenesulphonate in the crystalline form having an X-ray powder diffraction pattern with at least one (for example, one, two, three, four, five and preferably all six) a signals at 6.12, 8.92, 9.46, 11.12, 13.25 or 16.86 (± 0.2 degrees, 2-theta values).

Also provided is Compound I benzenesulphonate in the crystalline form having an X-ray powder diffraction pattern with signals at 6.12, 8.92, 9.46, 1 1.12, 13.25 and 16.86 (± 0.2 degrees, 2- theta values), in particular the crystalline form having an X-ray powder diffraction pattern substantially as shown in Figure 1.

Consistency and reliability in pharmaceutical applications are of the utmost importance, both in the context of the initial manufacture of a pharmaceutical product and during the subsequent period prior to administration. Stability of a pharmaceutical agent, for example stability of the physical form to moisture, is therefore also a desirable property. In one embodiment of the invention the Compound I benzenesulphonate is unsolvated. In a second embodiment of the invention the Compound I benzenesulphonate is in the form of a physiologically acceptable solvate (for example a hydrate).

Compound I may be prepared by solution or solid phase synthetic procedures which are known to those skilled in the art, for example, according to the procedure described in WO2005/066180 (summarised in Scheme 1 below).

Scheme 1

\ A \

Compound I

Compound I benzenesulphonate may be prepared by the reaction of Compound I free base with benzenesulphonic acid. Typically, Compound I free base is dissolved in a suitable solvent (for example dichloromethane) before mixing with benzenesulphonic acid in a suitable solvent (for example tetrahydrofuran). In such circumstances, the Compound I benzenesulphonate may form a precipitate and, after allowing sufficient time for reaction to occur, may be separated by filtration.

Additional solvents in which Compound I free-base may be dissolved include acetone, and also mixtures of acetone and with methyl-tert-butyl ether (MTBE), methyl-ethyl-ketone (MEK) or methyl-isobutyl-ketone (MIBK).

Accordingly, in a further aspect of the present invention there is provided a method for the preparation of Compound I benzenesulphonate comprising reacting Compound I free base with benzene sulphonic acid.

Compound I benzenesulphonate may be expected to be of use in the treatment of disorders mediated by cathepsin K.

In another aspect of the invention there is provided the use of Compound I benzenesulphonate as a medicament. Also provided is the use of Compound I benzenesulphonate in the manufacture of a medicament for the treatment of disorders mediated by cathepsin K. Additionally provided is a method for the treatment of a disorder mediated by cathepsin K comprising administering a safe and effective amount of Compound I benzenesulphonate.

Particular disorders which may be mediated by cathepsin K include: osteoporosis; gingival diseases such as gingivitis and periodontitis; Paget's disease; hypercalcaemia of malignancy; metabolic bone disease; diseases characterised by excessive cartilage or matrix degradation, such as osteoarthritis and rheumatoid arthritis; bone cancers including neoplasia; and pain.

While it is possible for Compound I benzenesulphonate to be administered in isolation, it will typically be presented as part of a pharmaceutical composition. Such a composition will comprise Compound I benzenesulphonate together with one or more pharmaceutically acceptable excipients. Said pharmaceutically acceptable excipients will be suitable for administration and will be compatible with the other ingredients of the composition. An additional aspect of the present invention is therefore a pharmaceutical composition comprising Compound I benzenesulphonate and one or more pharmaceutically acceptable diluents or carriers.

The compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. Suitably the pharmaceutical composition is an orally administered formulation. The compositions may conveniently be presented in unit dosage form (e.g. tablets and sustained release capsules) and may be prepared by any method known in the art of pharmacy.

Such methods include the step of bringing into association Compound I benzenesulphonate with the one or more pharmaceutically acceptable diluents or carriers. In general, the compositions are prepared by uniformly and intimately bringing into association the Compound I benzenesulphonate with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

In a further aspect there is provided a method for the preparation of a pharmaceutical composition comprising bringing Compound I benzene sulphonate into association with one or more pharmaceutically acceptable diluents or carriers.

Formulations for oral administration in the present invention may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion and as a bolus etc.

With regard to compositions for oral administration (e.g. tablets and capsules), the term excipient includes: binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the Compound I benzenesulphonate in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the Compound I benzenesulphonate in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the Compound I benzenesulphonate in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the Compound I benzenesulphonate in a suitable liquid carrier.

Appropriate dosage levels for the Compound I benzenesulphonate will depend upon the indication and the individual patient receiving treatment. Suitable dosages may be determined by conventional animal trials. Dosages providing intracellular (for inhibition of physiological proteases of the papain superamily) concentrations of the order 0.01 -100 uM, such as 0.01-10 uM or 0.1-25 uM, are typically desirable and achievable.

When used as a medicament, it may be advantageous for the Compound I benzenesulphonate to be administered in combination with a further pharmaceutically active agent. Such further pharmaceutically active agents will be selected appropriately depending on the disorder to be treated. The Compound I benzenesulphonate and further pharmaceutically active agent may be administered concurrently, sequentially or at different times through the same or different routes.

Where appropriate administration regimes are possible, the Compound I benzenesulphonate and further pharmaceutically active may be formulated together in a pharmaceutical combination.

Compound I benzenesulphonate is also useful in combination with known agents useful for treating or preventing osteoporosis, glucocorticoid induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, atherosclerosis, obesity, parasitic infection, rheumatoid arthritis, osteoarthritis, periprosthetic osteolysis, osteogenesis imperfecta, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma. Combinations Compound I benzenesulphonate with other agents useful in treating or preventing osteoporosis or other bone disorders are therefore considered to fall within the scope of the invention. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the disease involved. Such agents include the following: an organic bisphosphonate; an estrogen receptor modulator; an androgen receptor modulator; an inhibitor of osteoclast proton ATPase; an inhibitor of HMG- CoA reductase; an integrin receptor antagonist; Vitamin D or an analogue thereof, an osteoblast anabolic agent, such as PTH; a selective cyclooxygenase-2 inhibitor (COX-2 inhibitor); an inhibitor of interleukin-1- beta; a LOX/COX inhibitor, a RANKL inhibitor; and pharmaceutically acceptable salts and mixtures thereof.

"Androgen receptor modulators" refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5cc-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

"An inhibitor of osteoclast proton ATPase" refers to an inhibitor of the proton ATPase, which is found on the apical membrane of the osteoclast, and has been reported to play a significant role in the bone resorption process. This proton pump represents an attractive target for the design of inhibitors of bone resorption which are potentially useful for the treatment and prevention of osteoporosis and related metabolic diseases. See C. Farina et al., "Selective inhibitors of the osteoclast vacuolar proton ATPase as novel bone antiresorptive agents," DDT, 4: 163- 172 (1999), which is hereby incorporated by reference in its entirety.

"HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3- methylglutaryl CoA reductase. 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 IG-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 U.S. Patent Nos. 4,231 , 938, 4, 294,926 and 4,319,039), simvastatin - 19 (ZOCOR); see U.S. Patent Nos. 4, 444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL; see U.S. Patent Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180, 589), fluvastatin (LESCOL); see U.S. Patent Nos. 5,354,772, 4,911 ,165, 4, 929, 437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR; see U.S. Patent Nos. 5,273,995, 4,681 ,893, 5,489,691 and 5,342, 952) and cerivastatin (also known as rivastatin and BAYCHOL; see US Patent No. 5,177,080). The structural formulae 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 therefore the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.

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 tetramethylammonium, as well as those salts formed from amines such as ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N, N'- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidine-1 '-yl-methylbenz-imidazole, diethylamine, piperazine, and tris(hydroxymethyl) 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, hexyl resorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, 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.

As used above, "integrin receptor antagonists" refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the alpha-v-beta-3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the alpha-v-beta-5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the alpha-v-beta-3 integrin and the alpha-v-beta-5 integrin, and to compounds 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 alpha-v-beta-6, alpha-v-beta-8, alpha-1-beta-1 , alpha-2-beta-1 , alpha-6-beta-1 and alpha-6- beta-4 integrins. The term also refers to antagonists of any combination of alpha-v-beta-3, alpha-v-beta-5, alpha-v-beta-6, alpha-v-beta-8, alpha-1-beta-1 , alpha-2-beta-1 , alpha-5-beta-1 , alpha-6-beta-1 and alpha-6-beta-4 integrins. H.N. Lode and coworkers in PNAS USA 96: 1591- 1596 (1999) have observed synergistic effects between an antiangiogenic alpha-v integrin antagonist and a tumor-specific antibody-cytokine (interleukin-2) fusion protein in the eradication of spontaneous tumor metastases. Their results suggested this combination as having potential for the treatment of cancer and metastatic tumor growth. Alpha-v-beta-3 integrin receptor antagonists inhibit bone resorption through a new mechanism distinct from that of all currently available drugs. Integrins are heterodimeric transmembrane adhesion receptors that mediate cell-cell and cell matrix interactions. The alpha-v-beta-3 integrin subunits interact non-covalently and bind extracellular matrix ligands in a divalent cation-dependent manner. The most abundant integrin on osteoclasts is alpha-v-beta-3 (>10⁷/ osteoclast), which appears to play a rate-limiting role in cytoskeletal organization important for cell migration and polarization. The alpha-v-beta-3 antagonizing effect is selected from inhibition of bone resorption, inhibition of restenosis, inhibition of macular degeneration, inhibition of arthritis, and inhibition of cancer and metastatic growth.

"An osteoblast anabolic agent" refers to agents that build bone, such as PTH. The intermittent administration of parathyroid hormone (PTH) or its amino-terminal fragments and analogues have been shown to prevent, arrest, partially reverse bone loss and stimulate bone formation in animals and humans. For a discussion refer to D. W. Dempster et al., "Anabolic actions of parathyroid hormone on bone," Endocr Rev 14: 690-709 (1993). Studies have demonstrated the clinical benefits of parathyroid hormone in stimulating bone formation and thereby increasing bone mass and strength. Results were reported by RM Neer et al., in New Eng J Med 344 1434-1441 (2001 ).

"A selective cyclooxygenase-2 inhibitor," or COX-2 inhibitor, refers to a type of nonsteroidal anti- inflammatory drug (NSAID), that inhibit the with the COX-2 coenzyme, which contributes to pain and inflammation in the body. Nonlimiting examples of COX-2 inhibitors include: celecoxib, etoricoxib, parecoxib, rofecoxib, lumaricoxib and valdecoxib.

In addition, parathyroid hormone-related protein fragments or analogues, such as PTHrP (1-36) have demonstrated potent anticalciuric effects; see M.A. Syed et al., "Parathyroid hormone- related protein-(1-36) stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy, " JCEM 86: 1525- 1531 (2001 ) and may also have potential as anabolic agents for treating osteoporosis.

A preferred combination in accordance with the invention comprises codosing the salt of the invention simultaneously or sequentially with parathyroid hormone (PTH) or a fragment thereof, such as PTHrP (1-36).

Those skilled in the art will recognise that the term treatment may also be extended to cover prophylaxis.

EXAMPLES

General Methods

Abbreviations

DCM dichloromethane

MTBE methyl-tert-butyl ether

MEK methyl-ethyl-ketone

MIBK methyl-isobutyl-ketone THF tetrahydrofuran

X-Ray Powder Diffraction (XRPD)

XRPD patterns were collected either on a Bruker AXS C2 GADDS or a Siemens D5000 diffractometer.

X-ray powder diffraction patterns on the Bruker C2 were acquired using Cu Ka radiation (40 kV, 40 mA), automated XYZ stage, laser video microscope for auto-sample positioning and a HiStar 2-dimensional area detector. X-ray optics consist of a single Gobel multilayer mirror coupled with a pinhole collimator of 0.3 mm.

The beam divergence, i.e. the effective size of the X-ray beam on the sample, was approximately 4 mm. A θ-θ continuous scan mode was employed with a sample-detector distance of 20 cm which gives an effective 2Θ range of 3.2-29.8°. Typically the sample would be exposed to the X-ray beam for 120 seconds.

Samples run under ambient conditions were prepared as flat plate specimens using powder as received without grinding. Approximately 1-2 mg of the sample was lightly pressed on a glass slide to obtain a flat surface. X-ray powder diffraction patterns on samples acquired on a Siemens D5000 diffracto meter using Cu Ka radiation (40 kV, 40 mA), θ-θ goniometer, automatic divergence and receiving slits, a graphite secondary monochromator and a scintillation counter. The data were collected over an angular range of 2° to 42° 2Θ in continuous scan mode using a step size of 0.05° 2Θ and a step time of 5 second.

Samples run under ambient conditions were prepared as flat plate specimens using powder as received without grinding. Approximately 25-50 mg of the sample was gently packed into 12 mm diameter, 0.5 mm deep cavities cut into polished, zero-background (510) silicon wafers (The Gem Dugout, 1652 Princeton Drive, Pennsylvania State College, PA 16803, USA).

Example 1 - Synthesis of N-[(S)-1 -((3aS,6S,6aS)-6-Fluoro-3-oxo-hexahydro-furo[3,2- b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]- benzamide free base.

Compound I free base and subsequent reaction of Compound I free base with benzene sulphonic acid provides Compound I benzene sulphonate using the methodology summarised in Scheme 2 below.

Scheme 2

18 19

Reaction steps:

1 ) Trifluoromethanesulfonic anhydride, pyridine, dichloromethane

2) Sodium azide, N,N-dimethylformamide

3) 50% Acetic acid in water

4) p-toluenesulfonyl chloride, pyridine, dichloromethane 5) Palladium on carbon, hydrogen, methanol

6) Benzylchloroformate, methyl-tertbutylether, water, sodium hydrogen carbonate

7) Benzylbromide, sodium hydride, N,N-dimethylformamide

8) Triethylsilane, borontrifluoride diethyl etherate, chloroform 9) Palladium on carbon, hydrogen, di-tert-butyl dicarbonate, ethylacetate

10) Potassium acetate, acetic anhydride, tetrahydrofuran

1 1 ) Pearlman's catalyst, hydrogen, tetrahydrofuran

12) Deoxyfluor, pyridine dichloromethane

13) Sodium methoxide, methanol 14) Hydrochloric acid in dioxane

15) N-carbobenzyloxy-l-leucine, 1-hydroxybenzotriazole hydrate, N-(3-dimethylaminopropyl)- N'-ethylcarbodiimide, 4-methyl morpholine, N,N-dimethylformamide

16) Dess-martin periodinane, dichloromethane

17) Trimethylorthoformate, p-toluenesulphonic acid, methanol 18) Palladium on carbon, hydrogen, methanol

19) 4-[2-(4-Methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acid hydrobromide, 1- hydroxybenzotriazole hydrate, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, 4-methyl morpholine, N,N-dimethylformamide

20) Trifluoroacetic acid 21 ) Benzene sulphonic acid, acetone, 2% water.

A representative synthesis of compound 1 as the free base (i.e. steps 1-20 of Scheme 2) is described immediately below. The salt form can be subsequently prepared as described in Examples 6 and 6A below.

Initial synthetic steps to synthesise the bicyclic moiety - (3R,3aR,6S,6aS)-6-fluoro-hexahydro- furo[3.2-b]pyrrol-3-ol hydrochloride were carried out as described in WO 2005/066180 (Compound 64, page 42).

Synthesis of [1 S-1 -((3R,3aR,6S,6aS)-Fluoro-S-hydroxy-hexahydro-furo[3,2-b]pyrrole-4- carbonyl)-3-methyl-butyl]-carbamic acid benzyl ester,

A 160 L glass-lined steel reactor set-up for cooling to -10 ⁰C and heating to 70 ⁰C is charged with N,N-dimethylformamide (7.9 kg) and (3R,3aR,6S,6aS)-6-fluoro-hexahydro-furo[3,2-b]pyrrol- 3-ol hydrochloride (1.9 kg, 10.3 mol). To the suspension is added 1-hydroxybenzotriazole (12 % H₂O) (1.6 kg, 10.6 mol), Z-L-Leucine (94 % pure, 2.9 kg, 10.3 mol) followed by N-(3- dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (3.6 kg, 1 8.8 mol). The reaction mixture is kept under nitrogen, and the temperature is adjusted to 20-25 ⁰C. Over a period of 1 - 2 hours, 4-methylmorpholine (1.6 kg, 15.8 mol) is added. The resulting solution is allowed to react for 4 to 24 hours. The course of the reaction is followed by TLC until the reaction is deemed complete (typically 10 to 24 hours). The completed reaction mixture is quenched using tap water (79 kg). The product is extracted into dichloromethane (4 x 13 kg). The organic phases are washed with 1 M hydrochloric acid (2 x 10 kg) and 6 % sodium hydrogencarbonate (10.6 kg). The organic extracts are combined, and the solvent is removed by distillation at reduced pressure, finally at 50 - 60 ⁰C and at a pressure < 150 mbar. The residue is dissolved in tert-butyl methyl ether (10 kg), and washed with tap water (2 x 13 kg) and saturated with 25 % sodium chloride (5.2 kg). The aqueous phases are extracted with tert-butyl methyl ether (10 kg). The organic phases are combined, and the solvent is removed by distillation to dryness at reduced pressure, finally at 50 - 60 ⁰C and at a pressure < 150 mbar. Toluene (5 kg) is added, and distillation is continued as above. The distillation residue is re-dissolved in toluene (8 kg), and purified by column chromatography on a column prepared from silica gel 60, 40-63 μ (24 kg) and toluene (53-74 kg). First the column is eluted with a mixture of toluene (251 kg) and ethyl acetate (29 kg), then with a mixture of toluene (168 kg) and ethyl acetate (116 kg). Fractions of approximately 10 L, are taken and analyzed by TLC. Sufficiently pure fractions (purity ≥ 90 %) are pooled, and the solvent is removed by distillation at reduced pressure, finally at 50 - 60 ⁰C and p < 50 mbar. The residue is re-dissolved in dichloromethane (25 kg). A sample of this solution is used for estimating the yield 3.8 kg (94 %).

Synthesis [(S)-I -((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3- methyl-butyl]-carbamic acid benzyl ester

A 63 L glass-lined steel reactor set up for cooling to -10 ⁰C and heating to 70 ⁰C is charged with the dichloromethane solution of [1S-1-((3R,3aR,6S,6aS)-6-fluoro-3-hydroxy-hexahydro- furo[3,2b]pyrrole-4-carbonyl)-3-methyl-butyl]-carbamic acid benzyl ester (neat 3.8 kg, 9.6 mol). Dess-Martin periodinane (4.6 kg, 10.8 mol) is added. The suspension is kept under nitrogen and the temperature is adjusted to 20 - 25 ⁰C. The reaction is stirred for 10 to 24 hours. The course of the reaction is followed by LC-MS. The reaction is stirred for another 4 - 24 hours. The reaction mixture is quenched with a suspension of sodium hydrogen carbonate (2.4 kg, 28.6 mol) and tap water (26 kg). The reaction mixture is stirred for 2 to 4 hours, and celite 545 (2.1 kg) is added. The suspension is filtered on a pad of celite 545 back into the reactor. The lower, organic phase is separated and secured. The filter cake is washed with dichloromethane (26 - 40 kg). The filtrate is used to wash the upper, aqueous phase. The lower, organic phase is separated and secured. The organic phases are washed with 6 % sodium hydrogen carbonate (26 kg), and then washed with 25 % sodium chloride (5.2 kg). The organic phases are combined and dried with magnesium sulfate (3 - 5 kg). The suspension is filtered. The filter cake is washed with dichloromethane (10 - 15 kg). The filtrate and wash are combined and evaporated by distillation at reduced pressure until a final volume of 10 - 15 L, using a jacket temperature of 50 - 60 ⁰C. Methanol (15 - 20 kg) is added to the residue and concentrated to a final volume of 15 - 20 L by distillation at reduced pressure, using a jacket temperature of 50 - 60 ⁰C. A sample of this solution is used for estimating the yield 4.3 kg (113 %).

[(S)-I -((3 aS,6S,6aS)-6-Fluoro-3-3-dimethoxy.-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3- methyl-butyl]-carbarmic acid benzyl ester

A 63 L glass-lined steel reactor set up for cooling to - 10 ⁰C and heating to 70 ⁰C is charged with the methanol solution of [1S-1-((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole- 4-(carbonyl)-3-methyl-butyl]-carbamic acid benzyl ester (neat 4.3 kg, 11.0 mol). Trimethyl orthoformate (6 kg, 56.5 mol) is added. Benzenesulfonic acid (0.2 kg, 1.3 mol) is added. The mixture is kept under nitrogen, and the temperature is adjusted to 40 - 60 ⁰C. The reaction is continued for 4 to 8 hours (acceptance criteria; absent or barely visible, based on TLC), allowing the formed methyl formate to distill off.

The solvent is removed by distillation at reduced pressure, finally at pressure ≤ 50 mBar and at a jacket temperature of 50 - 60 ⁰C. The residue is dissolved in toluene (8 kg). The resulting solution is quenched on 6 % sodium hydrogencarbonate (11 kg). The phases are separated, and the aqueous phase is extracted with additional toluene (4 kg). The organic phases are washed with 25e/e sodium chloride solution (5.2 kg). The organic phases are combined, and the solvent is removed by distillation at reduced pressure, finally at p < 50 mBar and at a jacket temperature of 50 - 60 ⁰C. The evaporation residue is re-dissolved in toluene (4.5 kg). The crude product solution is purified on a column prepared from silica gel 60 (21 kg) and toluene (45 kg). The toluene solution of the crude product is applied to the column. The column is eluted with toluene (95 kg), 2.5 % (v/v) acetone in toluene (230 kg) and then with 5 % (v/v) acetone in toluene (138 kg). Fractions of approximately 10 L are taken. Fractions which look pure by TLC (purity > 90 % by TLC) are pooled, and the solvent is removed by distillation at reduced pressure, finally at p < 50 mBar, and at a jacket temperature of 50 - 60 ⁰C. The residue is re- dissolved in methanol (10 kg). A sample of this solution is used for estimating the yield - 4.3 kg (90 %).

(S)-2-Amino-1 -((3aS,6S,6aS)-6-fluoro-3,3-di-methoxy-hexahydro-furo[3,2-b]pyrrol-4-yl)-4- methyl -pentan-1 -one

A 63 L glass-lined steel reactor set up for cooling to - 10 ⁰C and heating to 70 ⁰C is filled with nitrogen and charged with 10 % palladium on charcoal (0.17 - 0.18 kg). The methanolic solution of [1 S-1 -((3aS,6S,6aS)-6-fluoro-3,3-dimethoxy-hexahydro-furo-[3,2-b]pyrrole-4-carbonyl)-3- methyl-butyl]-carbamic acid benzyl ester (neat 3.5 kg, 8.0 mol) is added. The solution is kept under hydrogen, and the temperature is adjusted to 20 - 3OeC. The hydrogenation is continued in a stream of hydrogen for 4 to 8 hours. The course of the reaction is followed by TLC and when deemed to have reached completion, the reaction mixture is filtered through celite (2 - 3 kg). The filter cake is washed with methanol (5 - 10 kg). The filtrates are combined, and the solvent is removed by distillation at reduced pressure, finally at a pressure < 50 mBar, and at a jacket temperature of 50 - 60 ⁰C. The evaporation residue is dissolved in toluene (3 - 6 kg) and sampled for yield and purity determination. The solvent is removed at reduced pressure, finally at a pressure ≤ 50 mBar and at a jacket temperature of 50 - 60 ⁰C. The evaporation residue is dissolved in N,N-dimethylformamide (25 - 26 kg). Yield of desired product is 2.2 kg (91 %).

N-[(S)-1 -((3aS,6S,6aS)-6-Fluoro-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)- 3-methyl-butyl]-4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]-benzamide

A 160 L glass-lined steel reactor set up for cooling to - 10 ⁰C and heating to 70 ⁰C is filled with nitrogen and charged with the N,N-dimethylformamide solution of 2-amino-[1S-1-((3aS,6S,6aS)- fluoro-3,3-dimethoxy-hexahydro-furo-[3,2-b]pyrrole-4-yl)-4-methyl]-pentan-1-one (~ 50 kg, neat - 3.5 kg, 11 .5 mol). 1-Hydroxybenzotriazole, 12 % water (2.05 kg, 13.5 mol), 4-[2-(4-methyl-1- piperazinyl)-4-thiazolyl]benzoic acid, hydrobromide (4.45 kg, 11.6 mol) and N-(3- dimethylaminopropyl)-N'-ethyl-carbodiimide, hydrochloride (2.6 kg, 13.6 mol) are added. The mixture is stirred at 15 - 20 ⁰C under nitrogen. 1-Methylmorpholine (1.20 kg, 1 1.9 mol) is added over a period of 12 hours. The mixture is then stirred at 15 - 20 ⁰C for a minimum of 4 hours. A sample of the reaction mixture is taken for IPC. Once the reaction is deemed to have reached completion, the reaction mixture is quenched on a mixture of tap water (120 kg) , sodium hydrogencarbonate (7.4 kg) and tert-butyl methyl ether (43 kg). The organic phase is separated. The aqueous phase is further extracted with tert-butyl methyl ether (3 x 21 kg). The organic phases are initially washed with tap water (81 kg), then with a mixture of tap water (41 kg) and sodium hydrogencarbonate (0.4 kg) and finally with 25 % sodium chloride (20 kg). The organic phases are combined, and the solvent is removed by distillation at reduced pressure, finally at 50 - 60 ⁰C and p < 100 mbar. The residue is dissolved in dichloromethane (55 kg) and then stirred with a mixture of tap water (50 kg) and 25 % ammonium hydroxide (0.20 kg) for 5 - 10 minutes. The organic phase is separated. The aqueous phase is further extracted with dichloromethane (2 x 20 kg). The organic phases are washed once more with a mixture of tap water (50 kg) and 25 % ammonium hydroxide (0.20 kg). The organic phases are checked by HPLC to ensure that a maximum of 2 % of (4-[2-(4-methy]-1-piperazinyl)-4-thiazolyl]benzoic acid, hydrobromide remains in the solution. The organic phases are combined and dried with magnesium sulfate (5 kg). The suspension is filtered, and the filter cake is washed with dichloromethane (20 kg). Toluene (15 kg) is added to the filtrate and the wash. The solvent is removed by distillation at reduced pressure, finally at 50 - 60 ⁰C and p < 100 mbar. The residue is dissolved in a mixture of toluene (5.55 kg) and acetone (1.30 kg), and purified on a column prepared from silica gel 60 (63 kg) and a mixture of toluene (104 kg) and acetone (24 kg). The column is eluted with a mixture of toluene (945 kg) and acetone (216 kg), followed by toluene (627 kg) and acetone (380 kg), and finally acetone (400 kg). The pure fractions (purity ≥ 90 %) are selected by HPLC, and the solvent is then removed by distillation at reduced pressure, finally at 50 - 60 ⁰C and p < 100 mbar. The residue is dissolved in dichloromethane (7 kg) Yield: 5.2 kg (76 %) (based on an evaporation residue). N-KSJ-i -USaS.eS.θaSJ-e-Fluoro-S.S-dihydroxy-furolS^-blpyrrole^-carbonylJ-S-methyl- butyl]-4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]-benzamide

A 63 L glass-lined steel reactor set up for cooling to - 10 ⁰C and heating to 30 ⁰C is filled with nitrogen, and charged with trifluoroacetic acid (37.5 kg), and cooled to 9 - 10 ⁰C. Keeping the reaction temperature below 15 ⁰C, the solution of N-[1 S-1-((3aS,6S,6aS)-6-fluoro-3,3- dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1- yl)-thiazol-4-yl]benzamide (2.6 kg, 4.4 mol) in dichloromethane is added. The temperature of the reaction mixture is adjusted to 20 - 22 ⁰C, and the reaction is continued until deemed to have reached completion by HPLC (typically 5 - 6 hours). Heptanes (35 kg) are added. The solvent is evaporated by distillation at reduced pressure, using a jacket temperature of 20 - 22 ⁰C until a residue of approximately 8 L remains. Heptanes (25 kg) are added. The solvent is again evaporated by distillation at reduced pressure, using a jacket temperature of 20 - 22 ⁰C until a residue of approximately 3 L remains. The residue is dissolved in dichloromethane (66 kg). The dichloromethane solution is quenched on a suspension of sodium hydrogencarbonate (3.6 kg) and tap water (60 kg). The organic phase is separated. The aqueous phase is extracted with dichloromethane, and the organic phase is secured. The organic phases are washed with 6 % aqueous sodium hydrogencarbonate (2 x 43.5 kg). The organic phases are washed with 25 % sodium chloride (62 kg). The combined organic phase is applied onto a column prepared from silica gel 60 (11 kg) and dichloromethane. The column is then washed with dichloromethane (20 kg). The product is then washed out with 5 % aqueous acetone (w/w) (123 kg). The fractions containing the desired compound are pooled and evaporated, finally using a rotary evaporator, to dryness at reduced pressure and at a jacket temperature of 15 - 25 ⁰C. Yield: 2.3 kg (96 %) (based on an evaporation residue). The product is depicted above as the hydrate. Example 2 - Crystallinity of a range of N-[(S)-1 -((3aS,6S,6aS)-6-Fluoro-3-oxo-hexahydro- furo[3, 2-b]pyrrole-4-carbonyl)-3 -methyl -butyl]-4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]- benzamide salts.

Method

30 mg of Compound I free base was dissolved in 0.3 ml of dichloromethane (i.e. at a concentration of 100 mg/ml). To the free base solution was added an appropriate quantity of acid solution as summarised in Table 1 below:

Table 1 - Formation of Compound I salts using acid solutions

After mixing the reactions were covered and left overnight at room temperature. Observations were then made before those reactions which did not initially afford a solid were left uncovered and allowed to slowly evaporate to dryness.

Due to poor solubility, certain further acids were added directly to the solution of Compound I free base as solids:

Table 2 - Formation of Compound I salts using solid acid

After mixing the reactions involving solid acids were left stirring for 72 hours before filtration to remove unreacted acid. Evaporation afforded oils in all three cases.

Oils were triturated using diethylether as a means for obtaining solids for further characterisation.

Results

The results of the salt preparation are summarised in Table 3 below.

Table 3 - Result of salt formation

Only two salts of Compound I, the benzenesulphonate and methanesulphonate were found to exhibit significant crystallinity after XRPD analysis. Figure 1 illustrates the diffraction pattern collected for of Compound I benzenesulphonate (using Siemens D5000), six notable peaks were observed at 6.12, 8.92, 9.46, 1 1.12, 13.25 and 16.86 degrees (±0.2, 2-theta values). Figure 2 illustrates the diffraction patterns collected for the benzenesulphonate and methanesulphonate salts compared to the free base control.

Four further salts were found to be weakly or very weakly crystalline, namely the hydrochloride, naphthalene-2-sulphonate, D-gluconate and acetate. Figure 3 illustrates the diffraction patterns collected for these four materials compared to the free base control. It may be noted that the weakly crystalline patterns for the acetate and D-gluconate show some overlap with the free base control, suggesting that salt formation may not have occurred. The remaining salts either did not produce solids or produced solids which were amorphous (XRPD results not shown).

Example 3 - Solubility testing

Method

Aqueous solubility was determined by suspending sufficient test compound in 0.25 ml of water to give a maximum final concentration equivalent to ≥10 mg/ml of the parent free base form of the compound. The suspension was equilibrated at 25⁰C for 24 hours then the pH was measured. The suspension was then filtered through a glass fibre C filter into a 96 well plate. The filtrate was then diluted by a factor of 101.

Quantitation was by HPLC with reference to a control solution of approximately 0.1 mg/ml Compound I free-base in DMSO. Different volumes of the standard solution, and also diluted and undiluted test solution were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard solution injection

Column: Phenomenex Luna C18 (2) 5 uM

Column Dimensions: 50 x 4.6 mm

Column Temperature (⁰C): 25

Injection (ul): 5, 8 and 50

UV Detection Wavelength (nm): 220-300

Flow Rate / ml/min: 2

Mobile Phase A: 0.1 % TFA in water

Mobile Phase B: 0.085% TFA in acetonitrile

Table 5 - HPLC gradient used during solubility testing

Results

The aqueous solubility of a range of salt forms of Compound I are shown in Table 6 below.

Table 6 - Aqueous solubility of Compound I and its salts

The aqueous solubility of Compound I benzenesulphonate was found to be significantly higher than that of Compound I free base.

Example 4 - Moisture stability: Storage at elevated temperature and humidity

Method

Salts prepared in Example 2 were stored for 1 week at 4O⁰C and 75% relative humidity before being analysed again by XRPD.

Results

Compound I salt forms which were amorphous prior to the stability testing remained so. A summary of the results for the crystalline salt forms is provided in Table 7 below.

Table 7 - Result of salt formation

XRPD patterns for Compound I benzenesulphonate before and after moisture stability testing are shown in Figure 4, it being evident that they are essentially the same. As can be seen from Table 7, of the large number of Compound I salts tested, only Compound I benzenesulphonate produced a crystalline product which remained so for a period of 1 week at elevated temperature and humidity.

Example 5 - Moisture stability: Gravimetric Vapour Sorption (GVS)

Method

The stability of Compound I benzenesulphonate was tested by GVS.

Sorption isotherms were obtained using a Hiden IGASorp moisture sorption analyser, controlled by CFRSorp software. Sample temperature was maintained at 25⁰C by the Huber bath re- circulating water through the sample chamber. The humidity was controlled by a mixture of dry and wet nitrogen gas, with a total flow rate of 250 ml/min passed over the sample. The relative humidity (%RH) was measured by a calibrated Vaisala relative humidity probe (dynamic range of 0-95 %RH), located near the sample. The weight change, mass relaxation, of the sample as a function of %RH was constantly monitored by the micro balance (accuracy ±0.001 mg).

The CFRSorp software uses a least squares minimisation together with a model of the mass relaxation, to predict an asymptotic value. The measured mass relaxation value must be within 5% of that predicted by the software, before equilibrium is achieved, before then moving onto the next %RH value is selected. The minimum equilibration time was 1 hour and the maximum 4 hours.

Typically 10-20 mg of sample was added to a mesh stainless steel basket under ambient conditions. All samples were loaded/unloaded at 40 %RH and 25⁰C (typical room humidity and temperature conditions). All samples were re-analysed by XRPD post GVS analysis. A moisture sorption isotherm was performed as outlined below (2 scans giving 1 complete cycle). The standard isotherm was performed at 25⁰C at 10 %RH intervals over a 0-90 %RH range.

Results

Figure 5 shows the GVS results and Figure 6 shows XRPD patterns obtained prior and subsequent to GVS analysis.

As can be seen from the data XRPD testing before and after the sorption isotherm was obtained provided XRPD patterns which were essentially the same.

Example 6 - Preparation of N-[(S)-1 -((3aS,6S,6aS)-6-Fluoro-3-oxo-hexahydro-furo[3,2- b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]- benzamide benzenesulphonate.

Method

To demonstrate the reproducibility of the formation of crystalline Compound I benzenesulphonate its preparation was repeated (i) on a larger scale and (ii) using different solvents

(i) Following the general procedure described in Example 2, Compound I benzenesulphonate was prepared starting with 100 mg of Compound I free base. Compound I free base was dissolved in 1 ml dichloromethane prior to the addition of 1 molar equivalent of 1 M benzenesulphonic acid in tetrahydrofuran. To a solution of 30 mg of Compound I free base in 0.3 ml of the following solvents: tetrahydrofuran; acetone; 1 molar equivalent of 1 M benzene sulphonic acid in THF was added.

To a solution of 30 mg of Compound I free base in 1.2 ml of acetone and 0.3 ml of the following solvents: methyl-isobutyl-ketone; methyl-ethyl-ketone; ethyl acetate; and methyl-tert-butyl ether; 1 molar equivalent of 1 M benzene sulphonic acid in THF was added.

Results

(i) Similar to the initial preparation, a small amount of precipitation was evident on addition of acid. On continuous stirring the precipitate redissolves and after approximately 14 hours precipitation was evident (section 6.4). The solid recovered by filtration using a 5 urn filter was confirmed to be the same crystalline Compound I benzenesulphonate salt reported in Example 2. A comparison of XRPD between the product of Example 2 and of the present example is shown in Figure 7. The two diffraction patterns are essentially the same.

(ii) and (iii) Again, a small amount of precipitation was generally evident on addition of acid. On continuous stirring the precipitate redissolves and after approximately 14 hours precipitation in three solutions was evident (summarised in Table 8 below).

Table 8 - Formation of Compound I benzenesulphonate using alternative solvents

For the cases where precipitation was evident, the resulting solid was recovered by filtration using a 5 um filter and confirmed to be the same crystalline Compound I benzenesulphonate salt reported in Example 2. Figure 8 illustrates the XRPD patterns obtained following preceiptation from aceteone, acetone/Ml BK and acetone/MEK. This provides evidence that the crystalline form of Compound I benzenesulphonate having the XRPD pattern shown in Figure 1 can be reliably produced under a range of conditions.

Example 6a - An optimised procedure for the preparation of N-[(S)-1 -((3aS,6S,6aS)-6- Fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl- piperazin-1 -yl)-thiazol-4-yl]-benzamide benzenesulphonate.

A 63 L glass-lined steel reactor set up for cooling to -10 ⁰C and heating to 30 ⁰C is filled with nitrogen, and charged with N-[(S)-1-((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole- 4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]-benzamide (where the ketone existed as a mixture of ketone and hydrate) (5.0 kg, 8.9 mol) and acetone (16 kg). Purified water (0.5 kg) is added. A pH electrode is installed for measuring pH in the range of 1 to 7. The temperature of the suspension is adjusted to 15-20 ⁰C. Keeping the reaction temperature below 20 ⁰C, benzenesulphonic acid (approximately 1.5 kg, 9.5 mol) is added in order to obtain a final pH of 3.5 to 4.5. The resulting solution is seeded. The crystallizing suspension is cooled to 5 - 8 ⁰C. Stirring at this temperature is continued for 20-72 hours. The suspension is filtered, and the filter cake is washed with acetone (6-10 kg). The wet filter cake is dried in a vacuum dryer, finally at 20 - 25 ⁰C and pressure ≤ 5 mBar for 3-5 days, or until the level of acetone is below an acceptable level. Yield 5.5 kg (86 %). Example 7 - Recovery of undissolved N-[(S)-1 -((3aS,6S,6aS)-6-Fluoro-3-oxo-hexahydro- furo[3, 2-b]pyrrole-4-carbonyl)-3 -methyl -butyl]-4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]- benzamide benzenesulphonate from water.

Method

Following Example 3, wherein the aqueous solubility of N-[(S)-1-((3aS,6S,6aS)-6-Fluoro-3-oxo- hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4- yl]-benzamide benzenesulphonate was tested, the undissolved material was recovered by filtration and analysed by XRPD.

Results

Figure 9 illustrates the XRPD patterns for crystalline Compound I benzenesulphonate and for the residue recovered from aqueous solution. Although the diffraction pattern for the recovered material is weak due to the quantity of material obtained, the pattern is essentially the same as that for crystalline Compound I benzenesulphonate which had not been contacted with water, thereby further demonstrating the stability of crystalline Compound I benzenesulphonate.

All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:

Claims

1. A compound which is N-[(S)-1-((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2- b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]- benzamide benzenesulphonate.

2. A compound according to claim 1 in solid crystalline form.

3. A compound according to claim 2 in the crystalline form in which it is obtained when a solution of benzenesulphonic acid in tetrahydrofuran is added to a solution of N-[(S)-1- ((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]- 4-[2-(4-methyl-piperazin-1 -yl)-thiazol-4-yl]-benzamide in dichloromethane.

4. A compound according to claim 3 in the crystalline form having an X-ray powder diffraction pattern with signals at 6.12, 8.92, 9.46, 1 1.12, 13.25 and 16.86 (± 0.2 degrees, 2-theta values).

5. A compound according to claim 4 in the crystalline form having the X-ray powder diffraction pattern substantially as shown in Figure 1.

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 and one or more pharmaceutically acceptable diluents or carriers.

7. A pharmaceutical combination comprising a compound according to any one of claims 1 to 5 together with a further pharmaceutically active agent.

8. Use of a compound according to any one of claims 1 to 5 as a medicament.

9. Use of a compound according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of disorders mediated by cathepsin K.

10. Use according to claim 9, characterised in that the disorder is selected from the list consisting of: osteoporosis; gingival diseases such as gingivitis and periodontitis;

Paget's disease; hypercalcaemia of malignancy; metabolic bone disease; diseases characterised by excessive cartilage or matrix degradation, such as osteoarthritis and rheumatoid arthritis; bone cancers including neoplasia; and pain.

1 1. Use according to any one of claims 7 to 9, characterised in that the compound according any one of claims 1 to 5 is administered in combination with a further pharmaceutically active agent.

12. A method for the treatment of a disorder mediated by cathepsin K comprising administering a safe and effective amount of a compound according to any one of claims 1 to 5.

13. A method according to claim 12, characterised in that the disorder is selected from the list consisting of: osteoporosis; gingival diseases such as gingivitis and periodontitis;

14. A method according to either claim 12 or 13, characterised in that the compound according to any one of claims 1 to 5 is administered in combination with a further pharmaceutically active agent.

15. A method for the preparation of a compound according to any one of claims 1 to 5 comprising reacting N-[(S)-1 -((3aS,6S,6aS)-6-fluoro-3-oxo-hexahydro-furo[3,2-b]pyrrole- 4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide free base with benzene sulphonic acid.

16. A method according to claim 15, wherein the reaction takes place in a solvent comprising acetone.

17. A method for the preparation of a pharmaceutical composition according to claim 6 comprising bringing a compound according to any one of claims 1 to 5 into association with one or more pharmaceutically acceptable diluents or carriers.