CHEMICAL COMPOUNDS 313

This invention relates to (lR,5S,6r)-3-[5-(adamantan-2-ylcarbamoyl)-6- (methylthio)pyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-carbox ylic acid and pharmaceutically-acceptable salts thereof. The compound possesses human

11-β-hydroxysteroid dehydrogenase type 1 enzyme (1 lβHSDl) inhibitory activity and accordingly have value in the treatment of disease states including metabolic syndrome and are useful in methods of treatment of a warm-blooded animal, such as man. The invention also relates to processes for the manufacture of said compound, to pharmaceutical compositions containing it and to its use in the manufacture of medicaments to inhibit 1 lβHSDl in a warm-blooded animal, such as man.

Glucocorticoids (Cortisol in man, corticosterone in rodents) are counter regulatory hormones i.e. they oppose the actions of insulin (Dallman MF, Strack AM, Akana SF et al. 1993; Front Neuroendocrinol 14, 303-347). They regulate the expression of hepatic enzymes involved in gluconeogenesis and increase substrate supply by releasing glycerol from adipose tissue (increased lipolysis) and amino acids from muscle (decreased protein synthesis and increased protein degradation). Glucocorticoids are also important in the differentiation of pre-adipocytes into mature adipocytes which are able to store triglycerides (Bujalska IJ et al. 1999; Endocrinology 140, 3188-3196). This may be critical in disease states where glucocorticoids induced by "stress" are associated with central obesity which itself is a strong risk factor for type 2 diabetes, hypertension and cardiovascular disease (Bjorntorp P & Rosmond R 2000; Int. J. Obesity 24, S80-S85)

It is now well established that glucocorticoid activity is controlled not simply by secretion of Cortisol but also at the tissue level by intracellular interconversion of active Cortisol and inactive cortisone by the 11 -beta hydroxysteroid dehydrogenases, 1 lβHSDl (which activates cortisone) and 11 βHSD2 (which inactivates Cortisol) (Sandeep TC & Walker BR 2001 Trends in Endocrinol & Metab. 12, 446-453). That this mechanism may be important in man was initially shown using carbenoxolone (an anti-ulcer drug which inhibits both 1 lβHSDl and 2) treatment which (Walker BR et al. 1995; J. Clin. Endocrinol. Metab. 80, 3155-3159) leads to increased insulin sensitivity indicating that

1 lβHSDl may well be regulating the effects of insulin by decreasing tissue levels of active glucocorticoids (Walker BR et al. 1995; J. Clin. Endocrinol. Metab. 80, 3155-3159).

Clinically, Cushing's syndrome is associated with Cortisol excess which in turn is associated with glucose intolerance, central obesity (caused by stimulation of pre-adipocyte differentiation in this depot), dyslipidaemia and hypertension. Cushing's syndrome shows a number of clear parallels with metabolic syndrome. Even though the metabolic syndrome is not generally associated with excess circulating Cortisol levels (Jessop DS et al. 2001; J. Clin. Endocrinol. Metab. 86, 4109-4114) abnormally high 1 lβHSDl activity within tissues would be expected to have the same effect. In obese men it was shown that despite having similar or lower plasma Cortisol levels than lean controls, 1 lβHSDl activity in subcutaneous fat was greatly enhanced (Rask E et al. 2001; J. Clin. Endocrinol. Metab. 1418-1421). Furthermore, the central fat, associated with the metabolic syndrome expresses much higher levels of 1 lβHSDl activity than subcutaneous fat (Bujalska IJ et al. 1997; Lancet 349, 1210-1213). Thus there appears to be a link between glucocorticoids, 1 lβHSDl and the metabolic syndrome.

1 lβHSDl knock-out mice show attenuated glucocorticoid- induced activation of gluconeogenic enzymes in response to fasting and lower plasma glucose levels in response to stress or obesity (Kotelevtsev Y et al. 1997; Proc. Natl. Acad. Sci USA 94, 14924-14929) indicating the utility of inhibition of 1 lβHSDl in lowering of plasma glucose and hepatic glucose output in type 2 diabetes. Furthermore, these mice express an anti-atherogenic lipoprotein profile, having low triglycerides, increased HDL cholesterol and increased apo-lipoprotein AI levels. (Morton NM et al. 2001; J. Biol. Chem. 276,

41293-41300). This phenotype is due to an increased hepatic expression of enzymes of fat catabolism and PP ARa. Again this indicates the utility of 1 lβHSDl inhibition in treatment of the dyslipidaemia of the metabolic syndrome.

The most convincing demonstration of a link between the metabolic syndrome and 1 lβHSDl comes from recent studies of transgenic mice over-expressing 1 lβHSDl

(Masuzaki H et al. 2001; Science 294, 2166-2170). When expressed under the control of an adipose specific promoter, 1 lβHSDl transgenic mice have high adipose levels of corticosterone, central obesity, insulin resistant diabetes, hyperlipidaemia and hyperphagia. Most importantly, the increased levels of 1 lβHSDl activity in the fat of these mice are similar to those seen in obese subjects. Hepatic 1 lβHSDl activity and plasma corticosterone levels were normal, however, hepatic portal vein levels of corticosterone

were increased 3 fold and it is thought that this is the cause of the metabolic effects in liver.

Overall it is now clear that the complete metabolic syndrome can be mimicked in mice simply by overexpressing 1 lβHSDl in fat alone at levels similar to those in obese man.

1 lβHSDl tissue distribution is widespread and overlapping with that of the glucocorticoid receptor. Thus, 1 lβHSDl inhibition could potentially oppose the effects of glucocorticoids in a number of physiological/pathological roles. 1 lβHSDl is present in human skeletal muscle and glucocorticoid opposition to the anabolic effects of insulin on protein turnover and glucose metabolism are well documented (Whorwood CB et al. 2001; J. Clin. Endocrinol. Metab. 86, 2296-2308). Skeletal muscle must therefore be an important target for 1 lβHSDl based therapy.

Glucocorticoids also decrease insulin secretion and this could exacerbate the effects of glucocorticoid induced insulin resistance. Pancreatic islets express 1 lβHSDl and carbenoxolone can inhibit the effects of 11-dehydocorticosterone on insulin release

(Davani B et al. 2000; J. Biol. Chem. 275, 34841-34844). Thus in treatment of diabetes 1 lβHSDl inhibitors may not only act at the tissue level on insulin resistance but also increase insulin secretion itself.

Skeletal development and bone function is also regulated by glucocorticoid action. 1 lβHSDl is present in human bone osteoclasts and osteoblasts and treatment of healthy volunteers with carbenoxolone showed a decrease in bone resorption markers with no change in bone formation markers (Cooper MS et al 2000; Bone 27, 375-381). Inhibition of 1 lβHSDl activity in bone could be used as a protective mechanism in treatment of osteoporosis. Glucocorticoids may also be involved in diseases of the eye such as glaucoma.

1 lβHSDl has been shown to affect intraocular pressure in man and inhibition of 1 lβHSDl may be expected to alleviate the increased intraocular pressure associated with glaucoma (Rauz S et al. 2001; Investigative Opthalmology & Visual Science 42, 2037-2042). There appears to be a convincing link between 1 lβHSDl and the metabolic syndrome both in rodents and in humans. Evidence suggests that a drug which specifically inhibits 1 lβHSDl in type 2 obese diabetic patients will lower blood glucose by reducing

hepatic gluconeogenesis, reduce central obesity, improve the atherogenic lipoprotein phenotype, lower blood pressure and reduce insulin resistance. Insulin effects in muscle will be enhanced and insulin secretion from the beta cells of the islet may also be increased. Currently there are two main recognised definitions of metabolic syndrome.

1) The Adult Treatment Panel (ATP III 2001 JMA) definition of metabolic syndrome indicates that it is present if the patient has three or more of the following symptoms: Waist measuring at least 40 inches (102 cm) for men, 35 inches (88 cm) for women; Serum triglyceride levels of at least 150 mg/dl (1.69 mmol/1); HDL cholesterol levels of less than 40 mg/dl (1.04 mmol/1) in men, less than 50 mg/dl (1.29 mmol/1) in women;

Blood pressure of at least 135/80 mm Hg; and / or Blood sugar (serum glucose) of at least 110 mg/dl (6.1 mmol/1).

2) The WHO consultation has recommended the following definition which does not imply causal relationships and is suggested as a working definition to be improved upon in due course:

The patient has at least one of the following conditions: glucose intolerance, impaired glucose tolerance (IGT) or diabetes mellitus and/or insulin resistance; together with two or more of the following: Raised Arterial Pressure;

Raised plasma triglycerides

Central Obesity

Microalbuminuria

We have found that (lR,5S,6r)-3-[5-(adamantan-2-ylcarbamoyl)-6- (methylthio)pyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-carbox ylic acid or a pharmaceutically-accep table salt thereof, is an effective 1 lβHSDl inhibitors, and accordingly has value in the treatment of disease states associated with metabolic syndrome. We have also found that the compound of the invention has improved properties, which would make it better candidates for use as a pharmaceutical. Accordingly there is provided a (lR,5S,6r)-3-[5-(adamantan-2-ylcarbamoyl)-6-

(methylthio)pyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-car boxylic acid or a pharmaceutically-acceptable salt thereof.

(lR,5S,6r)-3-[5-(Adamantan-2-ylcarbamoyl)-6-(methylthio)p yridin-2-yl]-3- azabicyclo[3.1.0]hexane-6-carboxylic acid has the following structure:

(lR,5S,6r)-3-[5-(Adamantan-2-ylcarbamoyl)-6-(methylthio)p yridin-2-yl]-3- azabicyclo[3.1.0]hexane-6-carboxylic acid may hereinafter be referred to as 'the Agent' . In another aspect there is provided (lR,5S,6r)-3-[5-(adamantan-2-ylcarbamoyl)-6- (methylthio)pyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-carbox ylic acid; or a pharmaceutically-acceptable salt thereof.

A suitable pharmaceutically-acceptable salt of a compound of the invention is, for example, an acid-addition salt of a compound of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulphuric, phosphoric, trifiuoroacetic, citric or maleic acid. In addition a suitable pharmaceutically-acceptable salt of a compound of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, tert- butylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

The invention also relates to in- vivo hydro lysable esters of the Agent. A pharmaceutically-acceptable ester is hydrolysed in the human or animal body to produce the parent acid. Possible pharmaceutically-acceptable esters for the carboxy in the Agent include Ci to C _ό alkoxymethyl esters for example methoxymethyl, Ci- ₆ alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C ₃ -scycloalkoxycarbonyloxyCi- _ό alkyl esters for example 1-cyclohexylcarbonyloxyethyl; l,3-dioxolen-2-onylmethyl esters, for example 5-methyl-l,3-dioxolen-2-onylmethyl; and Ci- _ό alkoxycarbonyloxyethyl esters.

The invention relates to any and all tautomeric forms of the compound that possess 1 lβHSDl inhibitory activity.

It is also to be understood that the compound may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms, which possess 1 lβHSDl inhibitory activity.

In one embodiment of the invention is provided (lR,5S,6r)-3-[5-(adamantan-2- ylcarbamoyl)-6-(methylthio)pyridin-2-yl]-3-azabicyclo[3.1.0] hexane-6-carboxylic acid. In an alternative embodiment are provided pharmaceutically-acceptable salts of (lR,5S,6r)-3- [5-(adamantan-2-ylcarbamoyl)-6-(methylthio)pyridin-2-yl]-3-a zabicyclo[3.1.0]hexane-6- carboxylic acid.

In another aspect the invention relates to a compound: (lR,5S,6r)-3-[5-(adamantan-2-ylcarbamoyl)-6-(methylthio)pyri din-2-yl]-3- azabicyclo[3.1.0]hexane-6-carboxylic acid; and pharmaceutically-acceptable salts thereof.

Another aspect of the present invention provides a process for preparing the Agent or a pharmaceutically acceptable salt thereof which process comprises: a) reacting a compound of the formula:

with (lR,5S,6r)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid wherein the carboxy group is optionally protected; and thereafter if necessary or desirable: i) removing any protecting groups; ii) resolving enantiomers; iii) forming a pharmaceutically-acceptable salt thereof.

The protecting groups may be removed at any convenient stage in the synthesis using conventional techniques well known in the chemical art.

As stated hereinbefore the compounds defined in the present invention possess 1 lβHSDl inhibitory activity. These properties may be assessed using the following assay.

Assays

The conversion of cortisone to the active steroid Cortisol by 1 lβHSDl oxo- reductase activity, can be measured using a competitive homogeneous time resolved fluorescence assay (HTRF) (CisBio International, R&D, Administration and Europe Office, In Vitro Technologies — HTRF® / Bioassays BP 84175, 30204 Bagnols/Ceze Cedex, France. Cortisol bulk HTRF kit: Cat No. 62CORPEC).

The evaluation of compounds described herein was carried out using a baculovirus expressed N terminal 6-His tagged full length human l lβHSDl enzyme(*l). The enzyme was purified from a detergent solublised cell lysate, using a copper chelate column. Inhibitors of 1 lβHSDl reduce the conversion of cortisone to Cortisol, which is identified by an increase in signal, in the above assay.

Compounds to be tested were dissolved in dimethyl sulphoxide (DMSO) to 1OmM and diluted further in assay buffer containing 1% DMSO to 10 fold the final assay concentration. Diluted compounds were then plated into black 384 well plates (Matrix, Hudson NH, USA).

The assay was carried out in a total volume of 20μl consisting of cortisone (Sigma, Poole, Dorset, UK, 16OnM), glucose-6-phosphate (Roche Diagnostics, ImM), NADPH (Sigma, Poole, Dorset, lOOμM), glucose-6-phosphate dehydrogenase (Roche Diagnostics, 12.5μg/ml), EDTA (Sigma, Poole, Dorset, UK, ImM), assay buffer (K ₂ HPO ₄ ZKH ₂ PO ₄ , 10OmM) pH 7.5, recombinant 1 lβHSDl [using an appropriate dilution to give a viable assay window - an example of a suitable dilution may be 1 in 1000 dilution of stock enzyme] plus test compound. The assay plates were incubated for 25 minutes at 37°C after which time the reaction was stopped by the addition of lOμl of 0.5mM glycerrhetinic acid plus conjugated cortisol(XL665 or D2). lOμl of anti-cortisol Cryptate was then added and the plates sealed and incubated for 6 hours at room temperature. Fluorescence at 665nm and 620nm was measured and the 665nm:620nm ratio calculated using an Envision plate reader.

These data was then used to calculate IC50 values for each compound (Origin 7.5, Microcal software, Northampton MA, USA) and/or the % inhibition at 30μM of compound. * 1 The Journal of Biological Chemistry, Vol. 26, No 25, ppl6653 - 16658 Example 1 has an IC50 of 0.009 μM in this assay. Another assay that may be used to determine the IC50 is described as follows:

The conversion of cortisone to the active steroid Cortisol can be measured using mature human adipocytes isolated from adipose tissue taken during subcutaneous needle biopsies from healthy volunteers. Following biopsy, adipose tissue was washed with Phosphate buffered saline (PBS) and digest buffer (30ml) (0.6 mg/ml collagenase (Roche) in digestion medium (Medium M 199 + 1% penicillin and streptomycin + 4% BSA) was added for 1 hour. Mature adipocytes were isolated following filtration using 250 μM gauze and washed four times with 1% wash medium (Medium M 199 + 1% penicillin and streptomycin + 1% BSA). Cells were resuspended in assay medium Dulbecco's Modified Eagles Medium (DMEM) (6% glucose) + 1% penicillin and streptomycin + 10% FCS) prior to assay.

2 ml isolated adipocytes were plated into 6 well plates prior to assay. Cells were incubated with 20 μl 3H-cortisone (1 μCi/ml, GE Healthcare) + 100 nM cortisone (Sigma), in the presence of compound (in 1% DMSO/assay medium) for 6 hours. 800 μl media was removed to eppendorf tubes and stored at -80 ⁰ C prior to analysis. Samples were thawed at room temperature and transferred to clean 5 x 5/8 glass tubes. Ethyl acetate (2.5ml) was added to each sample. Following a 10 second vortex the top solvent layer of each of the samples, containing the tritiated steroids, was transferred to a 4 x 4/8 glass tube. Ethyl acetate was evaporated off at 50 ⁰ C, under nitrogen. Samples were resuspended in 50:50 methanokwater (120 μl). Samples were run on a HPLC linked scintillation counter (Agilent 1100 or 1200 system and Perkin Elmer Flo-Scintillation analyser (500 TR)).

% conversion of 3H-cortisone to 3H-cortisol was calculated using the AUC of the cortisone and Cortisol peaks.

% conversion = [AUC cortisol/(AUC Cortisol + AUC cortisone)] * 100

these data were then used to calculate the IC50 value for the compound (Origin 7.5, Microcal software, Northampton MA, USA). Example 1 was tested twice in this assay giving IC50S of 4.3nM, and 3.OnM.

The oral bioavailability of the compound of the invention may be tested as follows:

Determination of Bioavailability in PK Studies

Compounds are dosed intravenously at 2mg/kg (2ml/kg) and orally at 5mg/kg (5ml/kg) in a 25% HPBCD in sorrensons buffer pH 5.5 formulation. Blood samples (20OuI) are taken Predose, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8 and 24 h post dose for both routes and plasma prepared by centrifugation. Plasma samples are analysed as below. PK parameters (clearance, volume of distribution, bioavailability, fraction absorbed etc.) are calculated by standard

PK methods using suitable PK software (WinNon-Lin).

Bioanalvsis of plasma samples The guidelines described are for the manual preparation of plasma samples following single compound or cassette dosing of project compounds to all PK species used within discovery DMPK. Analysis by open access (LC-MS/MS) or manual approaches (LC-MS) is described.

Contents 1. Materials

2. Generic Extraction Method

3. Example Sample List Using Generic Plate Layout

4. Open Access Batch Submission and System Checks

5. Acceptance Criteria for Batch Pass 1. Materials

Solvents: Methanol, acetonitrile and DMSO

Water: Purified or HPLC grade

ImI shallow 96-well plates OR eppendorf tubes

2ml deep well 96-well plates plus lids Blank (control) plasma

2. Generic Extraction Method

Solubilise compound(s) to lmg/ml using DMSO taking into account salt factors if any.

The DMSO stock(s) may be used to make all calibration & quality control (QC) samples:

2.i Single compound analysis 2. La Preparation of calibration and QC samples: 1. Prepare standard solutions as follows:

2. Transfer 50ul blank plasma to a well of a ImI 96-well plate (shallow well)

3. Transfer 5ul of each of the standard solutions to further wells of the plate

4. Add 50ul blank plasma to each of these wells.

5. To generate the QC samples, add three aliquots of 5ul of the 100ng/ml, 1000ng/ml and 10,000ng/ml standard solutions to the plate (3 QCs at each concentration).

6. Add 50ul blank plasma to each of these.

7. Transfer 50ul of each PK sample to the ImI 96-well plate

8. Add 5ul methanol (- compound) to each of the PK samples

9. Ensure all dose formulations are well mixed by vortex mixing.

10. Dilute intravenous (IV) and oral dose (PO) formulations of expected concentration to lOug/ml in methanol. (For example, a formulation made to an expected concentration of 2 mg/ml would be diluted 1:200 to give 10ug/ml solution).

11. Add 6x 50 ul aliquots of plasma to the plate. Add 5 ul of diluted IV formulation to three of the wells, repeat with PO formulation and remaining 3 wells.

12. Precipitate proteins by adding lOOul acetonitrile containing a project related internal standard (at lug/ml) to all calibration, QC, PK and formulation samples.

13. Vortex mix the plate before centrifugation at 4,00Og for 10 minutes.

14. Transfer lOOul of the supernatant to the wells of a 2ml 96-well plate (see following plate map). Care should be taken not to disturb the pellet.

15. Add ~1.5ml of 50:50 Methanol: Water into the last well.

16. For analysis on triple quad systems: add 400ul water (HPLC grade) to each sample. Gently mix.

17. Add lOOul of the 100,000ng/ml stock of each of the standard solutions to the 2ml plate and add 900ul water. Add a sample of internal standard to a further well (see plate map). These are for compound tuning (denoted on the plate map as tune solutions) 18. For analysis on platform systems: add lOOul water (HPLC grade) to each sample.

Gently mix.

19. Manually tune all compounds using compound solutions prepared to 5,000ng/ml (add lOOul of the 50,000ng/ml standard solutions to 900ul water) 2.ii Cassette dose analysis 2.iia Preparation of calibration and QC samples:

Note: For cassette dosing, the amount of methanol required to dilute the lmg/ml stock will be adjusted according to the number of compounds present.

1. Add 1 OOul of each lmg/ml stock required to a vial.

2. Add the required volume of methanol to yield a total volume of ImI. 3. Perform all further steps as for single compound analysis (steps 2 -16 above).

2.iii In cases where PK samples exceed the Upper limit of Quantification (ULOQ).

1. Prepare a further calibration curve and QC samples as above (steps 1 - 6).

2. Transfer <50ul (e.g. 25ul) of the PK samples that exceed the ULOQ.

3. Add enough control plasma to these samples to yield a final plasma volume of 50ul. Make a note of the dilution made.

4. Transfer 50ul of all remaining PK samples.

5. Prepare all formulation samples and extract all samples as described above, (steps 8 - 16)

Note: Upper concentrations used to generate the calibration curve may be reviewed, however, care must be taken to avoid saturation of the HPLC column or MS equipment. It is for this reason that dilution of PK samples is recommended. 2.iv In cases of poor sensitivity (high Lower Limit of Quantification).

Note: High LLOQ is taken as when most of the plasma concentrations lie below the lower limit of quantification or where the LLOQ is greater the 10ng/ml. The following methods should be applied when either of these scenarios is encountered.

According to a further aspect of the invention there is provided a pharmaceutical composition, which comprises the Agent, or a pharmaceutically-acceptable salt thereof, as defined hereinbefore in association with a pharmaceutically-acceptable diluent or carrier.

The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing). In general, compositions in a form suitable for oral use are preferred.

The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents. Suitable pharmaceutically-acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p_-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.

Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p_-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present. The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these.

Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent. The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic parenterally-accep table diluent or solvent, for example a solution in 1,3-butanediol.

Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.

For further information on formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred

to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

We have found that the Agent, or a pharmaceutically-acceptable salt thereof, is an effective 1 lβHSDl inhibitor, and accordingly have value in the treatment of disease states associated with metabolic syndrome.

It is to be understood that where the term "metabolic syndrome" is used herein, this relates to metabolic syndrome as defined in 1) and/or 2) or any other recognised definition of this syndrome. Synonyms for "metabolic syndrome" used in the art include Reaven's Syndrome, Insulin Resistance Syndrome and Syndrome X. It is to be understood that where the term "metabolic syndrome" is used herein it also refers to Reaven's Syndrome, Insulin Resistance Syndrome and Syndrome X.

According to a further aspect of the present invention there is provided the Agent, or a pharmaceutically-acceptable salt thereof, as defined hereinbefore for use in a method of prophylactic or therapeutic treatment of a warm-blooded animal, such as man. Thus according to this aspect of the invention there is provided the Agent, or a pharmaceutically-acceptable salt thereof, as defined hereinbefore for use as a medicament.

In another aspect of the invention there is provided the Agent, or a pharmaceutically-acceptable salt thereof, as defined hereinbefore for use in the treatment of type II diabetes. In another aspect of the invention there is provided the Agent, or a pharmaceutically-acceptable salt thereof, as defined hereinbefore for use in the treatment of obesity.

In another aspect there is provided the Agent, or a pharmaceutically-acceptable salt thereof, as defined hereinbefore for use in the production of an inhibitory effect in a warm- blooded animal, such as man.

According to another feature of the invention there is provided the use of the Agent, or a pharmaceutically-acceptable salt thereof, as defined hereinbefore in the manufacture of a medicament for use in the production of an 1 lβHSDl inhibitory effect in a warm-blooded animal, such as man. Where production of or producing an 1 lβHSDl inhibitory effect is referred to suitably this refers to the treatment of metabolic syndrome. Alternatively, where production of an 1 lβHSDl inhibitory effect is referred to this refers to the treatment of

diabetes, obesity, hyperlipidaemia, hyperglycaemia, hyperinsulinemia or hypertension, particularly diabetes and obesity. Alternatively, where production of an 1 lβHSDl inhibitory effect is referred to this refers to the treatment of glaucoma, osteoporosis, tuberculosis, dementia, cognitive disorders or depression. Alternatively, where production of an 1 lβHSDl inhibitory effect is referred to this refers to the treatment of cognitive disorders, such as improving the cognitive ability of an individual, for example by improvement of verbal fluency, verbal memory or logical memory, or for treatment of mild cognitive disorders. See for example WO03/086410 and references contained therein, and Proceedings of National Academy of Sciences (PNAS), 2001, 98(8), 4717-4721.

Alternatively, where production of an 1 lβHSDl inhibitory effect is referred to this refers to the treatment of, delaying the onset of and/or reducing the risk of atherosclerosis - see for example J. Experimental Medicine, 2005, 202(4), 517-527.

Alternatively, where production of an 1 lβHSDl inhibitory effect is referred to this refers to the treatment of Alzheimers and/or neurodegenerative disorders.

According to a further feature of this aspect of the invention there is provided a method for producing an 1 lβHSDl inhibitory effect in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of the Agent, or a pharmaceutically-acceptable salt thereof. In addition to their use in therapeutic medicine, the Agent, or a pharmaceutically- salt thereof, is also useful as pharmacological tool in the development and standardisation of in vitro and in vivo test systems for the evaluation of the effects of inhibitors of 1 lβHSDl in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents. The inhibition of 1 lβHSDl described herein may be applied as a sole therapy or may involve, in addition to the subject of the present invention, one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment. Simultaneous treatment may be in a single tablet or in separate tablets. For example agents than might be co-administered with 1 lβHSDl inhibitors, particularly those of the present invention, may include the following main categories of treatment:

1) Insulin and insulin analogues;

2) Insulin secretagogues including sulphonylureas (for example glibenclamide, glipizide), prandial glucose regulators (for example repaglinide, nateglinide), glucagon- like peptide 1 agonist (GLPl agonist) (for example exenatide, liraglutide) and dipeptidyl peptidase IV inhibitors (DPP-IV inhibitors);

3) Insulin sensitising agents including PPARγ agonists (for example pioglitazone and rosiglitazone);

4) Agents that suppress hepatic glucose output (for example metformin);

5) Agents designed to reduce the absorption of glucose from the intestine (for example acarbose);

6) Agents designed to treat the complications of prolonged hyperglycaemia; e.g. aldose reductase inhibitors

7) Other anti-diabetic agents including phosotyrosine phosphatase inhibitors, glucose 6 - phosphatase inhibitors, glucagon receptor antagonists, glucokinase activators, glycogen phosphorylase inhibitors, fructose 1,6 bisphosphastase inhibitors, glutamine:fructose -6-phosphate amidotransferase inhibitors

8) Anti-obesity agents (for example sibutramine and orlistat);

9) Anti- dyslipidaemia agents such as, HMG-CoA reductase inhibitors (statins, eg pravastatin); PPARα agonists (fibrates, eg gemfibrozil); bile acid sequestrants (cholestyramine); cholesterol absorption inhibitors (plant stanols, synthetic inhibitors); ileal bile acid absorption inhibitors (IBATi), cholesterol ester transfer protein inhibitors and nicotinic acid and analogues (niacin and slow release formulations);

10) Antihypertensive agents such as, β blockers (eg atenolol, inderal); ACE inhibitors (eg lisinopril); calcium antagonists (eg. nifedipine); angiotensin receptor antagonists (eg candesartan), α antagonists and diuretic agents (eg. furosemide, benzthiazide);

11) Haemostasis modulators such as, antithrombotics, activators of fibrinolysis and antiplatelet agents; thrombin antagonists; factor Xa inhibitors; factor Vila inhibitors; antiplatelet agents (eg. aspirin, clopidogrel); anticoagulants (heparin and Low molecular weight analogues, hirudin) and warfarin; 12) Anti-inflammatory agents, such as non-steroidal anti-inflammatory drugs (eg. aspirin) and steroidal anti-inflammatory agents (eg. cortisone); and 13) Agents that prevent the reabsorption of glucose by the kidney (SGLT inhibitors).

In the above other pharmaceutical composition, process, method, use and medicament manufacture features, the alternative and preferred embodiments of the compounds of the invention described herein also apply.

Example The invention will now be illustrated by the following Example in which, unless stated otherwise:

(i) temperatures are given in degrees Celsius ( ⁰ C); operations were carried out at room or ambient temperature, that is, at a temperature in the range of 18-25 ⁰ C and under an atmosphere of an inert gas such as argon; (ii) evaporation of solvent was carried out using a rotary evaporator under reduced pressure (600-4000 Pa; 4.5-30 mmHg) with a bath temperature of up to 60 ⁰ C;

(iii) chromatography means flash chromatography on silica gel;

(iv) in general, the course of reactions was followed by TLC and reaction times are given for illustration only; (v) yields are given for illustration only and are not necessarily those which can be obtained by diligent process development; preparations were repeated if more material was required;

(vi) where given, NMR data ( ¹ H) is in the form of delta values for major diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane (TMS), determined at 300 or 400 MHz (unless otherwise stated) using perdeuterio dimethyl sulfoxide

(DMSO-dβ) as solvent, unless otherwise stated; peak multiplicities are shown thus: s, singlet; d, doublet; dd, doublet of doublets; dt, doublet of triplets; dm, doublet of multiplets; t, triplet, m, multiplet; br, broad;

(vii) chemical symbols have their usual meanings; SI units and symbols are used; (viii) solvent ratios are given in volume : volume (v/v) terms;

(xi) mass spectra (MS) were run with an electron energy of 70 electron volts in the chemical ionisation (CI) mode using a direct exposure probe; where indicated ionisation was effected by electron impact (EI), fast atom bombardment (FAB) or electrospray (ESP); values for m/z are given; generally, only ions which indicate the parent mass are reported;

(x) relative volume (rel vol) is the relative volume compared to the amount of the key intermediate. Relative volume is usually used to refer to the amount of solvent.

For example if the key intermediate is lOOg and 1000ml of solvent is used, then this is referred to as 10 rel vol of solvent;

(xi) The following abbreviations may be used below or in the process section hereinbefore:

DMF N,N-dimethylformamide

DCM dichloromethane

DME 1 ,2-dimethoxyethane

THF tetrahydrofuran

EtOH ethyl acetate

DMA N,N-dimethylacetamide

Example 1

QR,5S.,6r)-3-[5-(Adamantan-2-ylcarbamoyl)-6-(methylthio)p yridin-2-yll-3- azabicyclo[3.1.01hexane-6-carboxylic acid

Lithium hydroxide monohydrate (0.735 g, 17.51 mmol) was added in one portion to ethyl (lR,5S)-3-[5-(2-adamantylcarbamoyl)-6-methylsulfanylpyridin- 2-yl]-3- azabicyclo[3.1.0]hexane-6-carboxylate (Intermediate 3, 3.99 g, 8.76 mmol) in THF (45 mL) and methanol (15 mL). Water (9 mL) was then added dropwise with vigorous stirring. The resulting suspension was stirred at room-temperature for 16 hours. The reaction mixture was evaporated to dryness and redissolved in water (50 mL) and acidified with 2M HCl. The precipitate was collected by filtration, washed with water (50 mL) and dried under vacuum to afford (lR,5S,6r)-3-[5-(adamantan-2-ylcarbamoyl)-6- (methylthio)pyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-carbox ylic acid (3.46 g, 92 %) as a solid, which was used without further purification. The reaction may also be carried out

using potassium hydroxide in place of lithium hydroxide monohydrate and ethanol in place of THF.

IH NMR (300.072 MHz, CDCB) 1.53 - 2.10 (m, 15H), 2.34 (s, 2H), 2.56 (s, 3H), 3.59 (d, 2H), 3.90 (d, 2H), 4.18 - 4.31 (m, IH), 6.05 (d, IH), 6.90 (d, IH), 7.84 (d, IH) m/z (ESI+) (M+H)+ = 428; HPLC tR = 2.66 min.

Alternatively Example 1 may be prepared as follows:

Aqueous potassium hydroxide solution (2M) (5 mol eq.) was added to intermediate 3 (1 mol eq.) in ethanol (20 relative vol.) and stirred at ambient temperature overnight. The mixture was carefully brought to pH5 with aqueous HCl and diluted with water and stirred for 1 hour. The resulting solid was filtered off, washed with water and dried under vacuum at 5O ⁰ C for 24 hours.

Intermediate 1 N-(2- Adamantyl)-2,6-dichlor o-pyridine-3-carboxamide

Oxalyl chloride (8.72 ml, 100.00 mmol) was added dropwise to 2,6-dichloronicotinic acid ([Helvetica Chim. Acta, 1976, 59(1), 222], 60 g, 50 mmol) and N,N-dimethylformamide (0.039 ml, 0.50 mmol) in DCM at 20 ⁰ C over a period of 10 minutes under nitrogen. The resulting suspension was stirred at 20 ⁰ C for 2 hours. The resulting mixture was evaporated to dryness and the residue was azeotroped with toluene to afford the crude acid chloride, which was dissolved in DCM (25mL) and added portionwise to a stirred solution of 2- adamantanamine hydrochloride (9.39 g, 50.00 mmol) and N-ethyldiisopropylamine (26.1 ml, 150.0 mmol) in DCM cooled to O ⁰ C, over a period of 15 minutes under nitrogen. The resulting suspension was stirred at 20 ⁰ C for 2 hours. The reaction mixture was evaporated to dryness, stirred with water (5OmL) for 10 mins and the precipitate was collected by filtration, washed with water (2x25 mL) and dried under vacuum to afford N-(2- adamantyl)-2,6-dichloro-pyridine-3-carboxamide (16.01 g, 98 %) as a solid, which was used without further purification

IH NMR (400.132 MHz, CDCB) δ 1.69 - 1.76 (2H, m), 1.79 (2H, s), 1.82 - 1.96 (8H, m), 2.07 (2H, s), 4.27 (IH, d), 6.92 - 7.01 (IH, m), 7.39 (IH, d), 8.19 (IH, d)

m/z (ESI+) (M+H)+ = 325; HPLC t _R = 2.66 min.

Alternatively, Intermediate 1 may be prepared as follows:

Oxalyl chloride (2 mol eq.) was added dropwise to 2,6-dichloronicotinic acid (1 mol eq.) and DMF (0.01 mol eq.) in DCM (23 mol eq) at 2O ⁰ C over 10 minutes under nitrogen. The resulting suspension was stirred at 2O ⁰ C for 2 hours to form a clear solution and then evaporated to dryness. The residue was azeotroped with toluene (2X) to afford the crude acid chloride as an oil. The oil was dissolved in DCM and added portion-wise to a stirred solution of 2-adamantyl hydrochloride (1 mol eq.) and N-ethyldiisopropylamine (3 mol eq.) in DCM (23 relative vol.), cooled to O ⁰ C over 15 minutes under nitrogen. The resulting suspension was stirred at 2O ⁰ C for 2 hours to give a clear solution and then evaporated to a low volume. Water (6.6 relative vol.) was added as a solid formed and the mixture put on a rotavapor to remove residual organics. The solid was collected by filtration, washed twice with water and dried under vacuum to give intermediate 1 as a solid which was used without further purification.

Intermediate 2 N-^-AdamantylJ-ό-chloro-l-methylsulfanyl-pyridine-S-carboxa mide

Sodium thiomethoxide (0.409 g, 5.84 mmol) was added to N-(2-adamantyl)-2,6-dichloro- pyridine-3-carboxamide (Intermediate, 1, 2.0 g, 6.15 mmol) in DMA (10 mL) at 2O ⁰ C under nitrogen. The resulting suspension was stirred at 60 ⁰ C for 3 hours. The reaction mixture was diluted with EtOAc (10OmL) and washed with water (3x20mL), and saturated brine (25 mL). The organic layer was dried over MgSO4, filtered and evaporated to afford crude product which was purified by crystallisation from EtOAc/isohexane to afford N-(2-

adamantyl)-6-chloro-2-methylsulfanyl-pyridine-3-carboxamide (1.150 g, 55.5 %) as a solid. The reaction may also be carried out in DMF at 18 ⁰ C instead of in DMA at 6O ⁰ C. 1U NMR (300.072 MHz, CDCB) δ 1.65 - 1.82 (m, 4H), 1.85 - 1.98 (m, 8H), 2.01 - 2.12 (m, 2H), 2.61 (s, 3H), 4.22 - 4.33 (m, IH), 6.73 - 6.82 (m, IH), 7.07 (d, IH), 7.87 (d, IH)

m/z (ESI+) (M+H)+ = 337; HPLC t _R = 2.97 min.

Alternatively, intermediate 2 may be prepared as follows:

Sodium carbonate (3.5 mol eq.) was added to Intermediate 1 (1 mol eq.) in DMF (10 relative vol.) to form a suspension. Sodium thiomethoxide (1.15 mol eq.) was added over 5 minutes and stirred at ambient temperature overnight. The mixture was then diluted with water 50 relative vol.), stirred for 1 hour and the resulting white solid was filtered off, washed with water and dried under vacuum. Ethyl acetate was added to the solid and heated at reflux to give a cloudy solution. This was filtered whilst hot and the product left to crystallize overnight. The crystals were washed with ethyl acetate.

Intermediate 3

Ethyl (lR,5S)-3-[5-(2-adamantylcarbamoyl)-6-methylsulfanylpyridin- 2-yl]-3- azabicyclo[3.1.0]hexane-6-carboxylate

Potassium carbonate (4.93 g, 35.69 mmol) was added to N-(2-adamantyl)-6-chloro-2- methylsulfanyl-pyridine-3-carboxamide (Intermediate 2, 4.01 g, 11.90 mmol) and (lR,5S,6r)-ethyl 3-azabicyclo[3.1.0]hexane-6-carboxylate hydrochloride (Commercially available from Tyger Scientific Inc., literature reference US5475116, 2.28 g, 11.90 mmol) in butyronitrile (60 mL) at 20 ⁰ C under nitrogen. The resulting suspension was stirred at 120 ⁰ C for 70 hours. The reaction mixture was diluted with EtOAc (25 mL), and washed

sequentially with water (10 mL) and saturated brine (10 mL). The organic layer was dried over MgSO4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% EtOAc in DCM. Pure fractions were evaporated to dryness to afford ethyl (lR,5S)-3-[5-(2-adamantylcarbamoyl)- 6-methylsulfanylpyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-ca rboxylate (3.33 g, 61%) as a white solid. Mixed fractions were combined to give 1.65g of less pure product which was repurified by flash silica chromatography, elution gradient 0 to 10% EtOAc in 1:1 DCM:isohexane. Pure fractions were evaporated to dryness to afford ethyl (lR,5S)-3-[5- (2-adamantylcarbamoyl)-6-methylsulfanylpyridin-2-yl]-3-azabi cyclo[3.1.0]hexane-6- carboxylate (0.660 g, 12%) as a solid giving a total yield of 3.99g, 73%

IH NMR (400.132 MHz, CDC13) δ 1.20 (3H, t), 1.48 - 1.52 (IH, m), 1.61 (2H, d), 1.70 (2H, s), 1.81 (6H, s), 1.88 (2H, d), 1.95 - 1.99 (2H, m), 2.17 - 2.21 (2H, m), 2.50 (3H, s), 3.50 (2H, d), 3.81 (2H, d), 4.08 (2H, q), 4.17 - 4.21 (IH, m), 5.98 (IH, d), 6.78 - 6.83 (IH, m), 7.77 (IH, d) m/z (ESI+) (M+H)+ = 456; HPLC tR = 3.3 min.

Alternatively, intermediate 3 may be prepared as follows: Intermediate 2 (1 mol eq.), (lR,5S,6r)-ethyl 3-azabicyclo[3.1.0]hexane-6-carboxylate hydrochloride (1.2 mol eq.) and potassium carbonate (3 mol eq.) were suspended in butyronitrile (10 relative vol.) and sealed in a Parr pressure bomb. The reaction was heated to 16O ⁰ C for 37 hours. The crude reaction mixture was filtered and resulting solid washed butyronitrile. The solvent was evaporated off to give a solid, which was stirred in DCM for 15minutes. The solid was then filtered off and the filtrate was evaporated to give a solid. The solid was purified on a column (Redisep 75Og silica cartridge) eluting with DCM to 10% EtOAc.