AU2003274373A1 - Inhibitors of 11-beta-hydroxy steroid dehydrogenase type 1 and type 2 - Google Patents

Description

WO 2004/037251 PCT/GB2003/004590 INHIBITORS OF 11-BETA-HYDROXY STEROID DEHYDROGENASE TYPE 1 AND TYPE 2 FIELD OF INVENTION 5 The present invention relates to a compound. In particular the present invention provides compounds capable of inhibiting 1103-hydroxysteroid dehydrogenase (113 HSD). Introduction 10 The role of glucocorticoids Glucocorticoids are synthesised in the adrenal cortex from cholesterol. The principle glucocorticoid in the human body is cortisol, this hormone is synthesised and secreted in response to the adrenocortictrophic hormone (ACTH) from the pituitary gland in a 15 circadian, episodic manner, but the secretion of this hormone can also be stimulated by stress, exercise and infection. Cortisol circulates mainly bound to transcortin (cortisol binding protein) or albumin and only a small fraction is free (5-10%) for biological processes [1]. 20 Cortisol has a wide range of physiological effects, including regulation of carbohydrate, protein and lipid metabolism, regulation of normal growth and development, influence on cognitive function, resistance to stress and mineralocorticoid activity. Cortisol works in the opposite direction compared to insulin meaning a stimulation of hepatic gluconeogenesis, inhibition of peripheral glucose uptake and increased blood glucose 25 concentration. Glucocorticoids are also essential in the regulation of the immune response. When circulating at higher concentrations glucocorticoids are immunosuppressive and are used pharmacologically as anti-inflammatory agents. Glucocorticoids like other steroid hormones are lipophilic and penetrate the cell 30 membrane freely. Cortisol binds, primarily, to the intracellular glucocorticoid receptor (GR) that then acts as a transcription factor to induce the expression of glucocorticoid responsive genes, and as a result of that protein synthesis.

WO 2004/037251 PCT/GB2003/004590 2 The role of the 11 -HSD enzyme The conversion of cortisol (F) to its inactive metabolite cortisone (E) by 11p-HSD was first described in the 1950's, however it was not until later that the biological importance 5 for this conversion was suggested [2]. In 1983 Krozowski et al. showed that the mineralocorticoid receptor (MR) has equal binding affinities for glucocorticoids and mineralocorticoids [3]. Because the circulating concentration of cortisol is a 100 times higher than that of aldosterone and during times of stress or high activity even more, it was not clear how the MR remained mineralocorticoid specific and was not constantly 10 occupied by glucocorticoids. Earlier Ulick et al. [4] had described the hypertensive condition known as, "apparent mineralocorticoid excess" (AME), and observed that whilst secretion of aldosterone from the adrenals was in fact low the peripheral metabolism of cortisol was disrupted. These discoveries lead to the suggestion of a protective role for the enzymes. By converting cortisol to cortisone in mineralocorticoid 15 dependent tissues 11P-HSD enzymes protects the MR from occupation by glucocorticoids and allows it to be mineralcorticoid specific. Aldosterone itself is protected from the enzyme by the presence of an aldehyde group at the C-18 position. Congenital defects in the 11P3-HSD enzyme results in over occupation of the MR by 20 cortisol and hypertensive and hypokalemic symptoms seen in AME. Localisation of the 11 1-HSD showed that the enzyme and its activity is highly present in the MR dependent tissues, kidney and parotid. However in tissues where the MR is not mineralocorticoid specific and is normally occupied by glucocorticoids, 11 P3-HSD is not 25 present in these tissues, for example in the heart and hippocampus [5]. This research also showed that inhibition of 11 3-HSD caused a loss of the aldosterone specificity of the MR in these mineralocorticoid dependent tissues. It has been shown that two iso-enzymes of 11 P1-HSD exist. Both are members of the 30 short chain alcohol dehydrogenase (SCAD) superfamily which have been widely conserved throughout evolution. 11 3-HSD type 2 acts as a dehydrogenase to convert the secondary alcohol group at the C-1 position of cortisol to a secondary ketone, so producing the less active metabolite cortisone. 11 P3-HSD type 1 is thought to act mainly in vivo as a reductase, that is in the opposite direction to type 2 [6] [see below]. 11 P3 35 HSD type 1 and type 2 have only a 30% amino acid homology.

WO 2004/037251 PCT/GB2003/004590 3 Cortisol Cortisone 11 B HSD Tvvel 11 -HSD enzyme activity The intracellular activity of cortisol is dependent on the concentration of glucocorticoids 5 and can be modified and independently controlled without involving the overall secretion and synthesis of the hormone. The role of 11 p-HSD Type 1 10 The direction of 11 -HSD type 1 reaction in vivo is generally accepted to be opposite to the dehydrogenation of type 2. In vivo homozygous mice with a disrupted type 1 gene are unable to convert cortisone to cortisol, giving further evidence for the reductive activity of the enzyme [7]. 11 P3-HSD type 1 is expressed in many key glucocorticoid regulated tissues like the liver, pituitary, gonad, brain, adipose and adrenals ,however, 15 the function of the enzyme in many of these tissues is poorly understood [8]. The concentration of cortisone in the body is higher than that of cortisol , cortisone also binds poorly to binding globulins, making cortisone many times more biologically available. Although cortisol is secreted by the adrenal cortex, there is a growing amount 20 of evidence that the intracellular conversion of E to F may be an important mechanism in regulating the action of glucocorticoids [9]. It may be that 11 13-HSD type 1 allows certain tissues to convert cortisone to cortisol to increase local glucocorticoid activity and potentiate adaptive response and counteracting 25 the type 2 activity that could result in a fall in active glucocorticoids [10]. Potentiation of the stress response would be especially important in the brain and high levels of 11 13 HSD type 1 are found around the hippocampus, further proving the role of the enzyme. 11 1-HSD type 1 also seems to play an important role in hepatocyte maturation [8]. Another emerging role of the 11 P-HSD type 1 enzyme is in the detoxification process of WO 2004/037251 PCT/GB2003/004590 4 many non-steroidal carbonyl compounds, reduction of the carbonyl group of many toxic compounds is a common way to increase solubility and therefore increase their excretion. The 11 p-HSD typel enzyme has recently been shown to be active in lung tissue [11]. Type 1 activity is not seen until after birth, therefore mothers who smoke 5 during pregnancy expose their children to the harmful effects of tobacco before the child is able to metabolically detoxify this compound. The role of 11 p-HSD Type 2 10 As already stated earlier the 11 P-HSD type 2 converts cortisol to cortisone, thus protecting the MR in many key regulatory tissues of the body. The importance of protecting the MR from occupation by glucocorticoids is seen in patients with AME or liquorice intoxification. Defects or inactivity of the type 2 enzyme results in hyperterisive syndromes and research has shown that patients with an hypertensive syndrome have 15 an increased urinary excretion ratio of cortisol : cortisone. This along with a reported increase in the half life of radiolabelled cortisol suggests a reduction of 11 p-HSD type 2 activity [12]. Rationale for the development of 11 p-HSD inhibitors 20 As said earlier cortisol opposes the action of insulin meaning a stimulation of hepatic gluconeogenesis, inhibition of peripheral glucose uptake and increased blood glucose concentration. The effects of cortisol appear to be enhanced in patients suffering from glucose intolerance or diabetes mellitus. Inhibition of the enzyme 11 p-HSD type 1 25 would increase glucose uptake and inhibit hepatic gluconeogenesis, giving a reduction in circulatory glucose levels. The development of a potent 11 p-HSD type 1 inhibitor could therefore have considerable therapeutic potential for conditions associated with elevated blood glucose levels. 30 An excess in glucocorticoids can result in neuronal dysfunctions and also impair cognitive functions. A specific 11 P3-HSD type 1 inhibitor might be of some importance by reducing neuronal dysfunctions and the loss of cognitive functions associated with ageing, by blocking the conversion of cortisone to cortisol. 35 Glucocorticoids also have an important role in regulating part of the immune response WO 2004/037251 PCT/GB2003/004590 5 [13]. Glucocorticoids can suppress the production of cytokines and regulate the receptor levels. They are also involved in determining whether T-helper (Th) lymphocytes progress into either Thl or Th2 phenotype. These two different types of Th cells secrete a different profile of cytokines, Th2 is predominant in a glucocorticoid environment. By 5 inhibiting 11 B-HSD type 1, Thl cytokine response would be favoured. It is also possible to inhibit 11 3-HSD type 2 , thus by inhibiting the inactivation of cortisol, it may be possible to potentiate the anti-inflammatory effects of glucocorticoids. Aspects of the invention are defined in the appended claims. 10 SUMMARY ASPECTS OF THE PRESENT INVENTION In one aspect the present invention provides a compound having Formula I R, N Formula I >R3 N R2 wherein one of R 1 and R 2 is a group of the formula O 01 R5-S L o 15 R4 wherein R 4 is selected from H and hydrocarbyl, R5 is a hydrocarbyl group and L is an optional linker group, or R 1 and R 2 together form a ring substituted with the group O R5- S L R4 20 wherein R 3 is H or a substituent, and wherein X is selected from S, O, NR 6 and

C(R

7

)(R

8 ), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R7 and R 8 are independently selected from H and hydrocarbyl groups.

WO 2004/037251 PCT/GB2003/004590 6 In one aspect the present invention provides a pharmaceutical composition comprising (i) a compound having Formula I R, N Formula I

R

3 X R2 wherein one of R 1 and R 2 is a group of the formula O R-S L N of R4 5 wherein R 4 is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is an optional linker group, or R 1 and R 2 together form a ring substituted with the group O II of R4 wherein R 3 is H or a substituent, and wherein X is selected from S, O, NR 6 and

C(R

7

)(R

8 ), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 10 and R 8 are independently selected from H and hydrocarbyl groups. (ii) optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. In one aspect the present invention provides a compound having Formula I R, -- N Formula I R3 X R2 15 wherein one of R, and R2 is a group of the formula WO 2004/037251 PCT/GB2003/004590 7 O II

R

5 -S L N R4 wherein R 4 is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is an optional linker group, or R 1 and R 2 together form a ring substituted with the group O R5- S L fT R4 5 wherein R 3 is H or a substituent, and wherein X is selected from S, O, NR, and

C(R

7 )(R.), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 and R 8 are independently selected from H and hydrocarbyl groups, for use in medicine. In one aspect the present invention provides a use of a compound in the manufacture of 10 a medicament for use in the therapy of a condition or disease associated with 11 13-HSD, wherein the compound has Formula I

R

1 , N Formula I R2 x> R wherein R4one of RSe acted from His and hydrocarbyl, R is a hydrocarbyl group andof the formulas an 15 optional linker group, or R and R together form a ring substituted with the group fl N wherein R 4 is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is an 15 optional linker group, or R, and R 2 together form a ring substituted with the group WO 2004/037251 PCT/GB2003/004590 8 0 R --- S L o R4 wherein R 3 is H or a substituent, and wherein X is selected from S, O, NR 6 and

C(R

7

)(R

8 ), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 and Re are independently selected from H and hydrocarbyl groups. 5 SOME ADVANTAGES One key advantage of the present invention is that the compounds of the present invention can act as 11 -HSD inhibitors. The compounds may inhibit the interconversion 10 of inactive 1 1-keto steroids with their active hydroxy equivalents. Thus present invention provides methods by which the conversion of the inactive to the active form may be controlled, and to useful therapeutic effects which may be obtained as a result of such control. More specifically, but not exclusively, the invention is concerned with interconversion between cortisone and cortisol in humans. 15 Another advantage of the compounds of the present invention is that they may be potent 11 -HSD inhibitors in vivo. Some of the compounds of the present invention are also advantageous in that they may 20 be orally active. The present invention may provide for a medicament for one or more of (i) regulation of carbohydrate metabolism, (ii) regulation of protein metabolism, (iii) regulation of lipid metabolism, (iv) regulation of normal growth and/or development, (v) influence on 25 cognitive function, (vi) resistance to stress and mineralocorticoid activity. Some of the compounds of the present invention may also be useful for inhibiting hepatic gluconeogenesis. The present invention may also provide a medicament to relieve the effects of endogenous glucocorticoids in diabetes mellitus, obesity (including centripetal 30 obesity), neuronal loss and/or the cognitive impairment of old age. Thus, in a further aspect, the invention provides the use of an inhibitor of 11 P-HSD in the manufacture of a medicament for producing one or more therapeutic effects in a patient to whom the WO 2004/037251 PCT/GB2003/004590 9 medicament is administered, said therapeutic effects selected from inhibition of hepatic gluconeogenesis, an increase in insulin sensitivity in adipose tissue and muscle, and the prevention of or reduction in neuronal loss/cognitive impairment due to glucocorticoid potentiated neurotoxicity or neural dysfunction or damage. 5 From an alternative point of view, the invention provides a method of treatment of a human or animal patient suffering from a condition selected from the group consisting of: hepatic insulin resistance, adipose tissue insulin resistance, muscle insulin resistance, neuronal loss or dysfunction due to glucocorticoid potentiated neurotoxicity, and any 10 combination of the aforementioned conditions, the method comprising the step of administering to said patient a medicament comprising a pharmaceutically active amount of a compound in accordance with the present invention. Some of the compounds of the present invention may be useful for the treatment of 15 cancer, such as breast cancer, as well as (or in the alternative) non-malignant conditions, such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age. DETAILED ASPECTS OF THE PRESENT INVENTION 20 In one aspect the present invention provides a compound having Formula I R, N Formula I R3 x R2 wherein one of R 1 and R 2 is a group of the formula O II Re- S L N o I R4 wherein R 4 is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is an 25 optional linker group, or R 1 and R 2 together form a ring substituted with the group WO 2004/037251 PCT/GB2003/004590 10 O o II R4 wherein R 3 is H or a substituent, and wherein X is selected from S, O, NR, and

C(RT)(R

8 ), wherein R 6 is selected from H and hydrocarbyl groups, wherein each of R 7 and R 8 are independently selected from H and hydrocarbyl groups. 5 In one aspect the present invention provides a pharmaceutical composition comprising (i) a compound having Formula I defined above (ii) optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. 10 In one aspect the present invention provides a compound having Formula I defined above, for use in medicine. In one aspect the present invention provides a use of a compound having Formula I 15 defined above in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 -HSD. In one aspect the present invention provides a use of a compound having Formula I defined above in the manufacture of a medicament for use in the therapy of a condition 20 or disease associated with adverse 11 P-HSD levels. In one aspect the present invention provides a use of a compound having Formula I defined above in the manufacture of a pharmaceutical for inhibiting 11 -HSD activity. 25 In one aspect the present invention provides a use of a compound having Formula I defined above in the manufacture of a pharmaceutical for inhibiting 11 -HSD activity. In one aspect the present invention provides a method comprising (a) performing a 110 HSD assay with one or more candidate compounds having Formula I defined above; (b) 30 determining whether one or more of said candidate compounds is/are capable of modulating 11p-HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of modulating 1 p-HSD activity.

WO 2004/037251 PCT/GB2003/004590 11 In one aspect the present invention provides a method comprising (a) performing a 11j3 HSD assay with one or more candidate compounds having Formula I defined above; (b) determining whether one or more of said candidate compounds is/are capable of 5 inhibiting 11 3-HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of inhibiting 11 -HSD activity. In one aspect the present invention provides * a compound identified by the above method, 10 * the use of the said compound in medicine, * a pharmaceutical composition comprising the said compound, optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, * use of the said compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with 1 I0-HSD, and 15 * use of the said compound in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 -HSD levels. For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each 20 section are not necessarily limited to each particular section. PREFERABLE ASPECTS In one preferred aspect the compound of the present invention has Formula 1I

R

1 N R Formula II

KR

3 S R2 25 In one preferred aspect L is not present. In this aspect the present invention provides a compound having Formula I WO 2004/037251 PCT/GB2003/004590 12 R N Formula I R2R wherein one of R 1 and R 2 is a group of the formula O N ~I R4 wherein R 4 is selected from H and hydrocarbyl, and R, is a hydrocarbyl group; or R, and

R

2 together form a ring substituted with the group O 11 Re5-S 5 R4 wherein R 3 is H or a substituent In one preferred aspect the compound of the present invention R, and R 2 together form a ring substituted with the group R5-S L 100 10 R4 In one preferred aspect the compound of the present invention R, and R 2 together form a carbocyclic ring. 15 In one preferred aspect the compound of the present invention R, and R 2 together form a six membered ring. In one preferred aspect the compound of the present invention R, and R 2 together form a six membered carbocyclic ring.

WO 2004/037251 PCT/GB2003/004590 13 In one preferred aspect the compound of the present invention wherein R 1 and R 2 together form an aryl ring. 5 Preferred compounds of the present invention are those having one of the following formulae. N Formula Ill Rs .S L R 3 R4 O N Formula IV

R

5 -s, N L R3 o I R4 O Formula V R5 | N KR3 o s R4 O Formula VI I IN R - S -NR3 O R 4 R3 O Formula Via I I R O-S-N---L O R4 R3 S

S

WO 2004/037251 PCT/GB2003/004590 14 o R Formula VII

R

5 -S-N II I O R 4 Formula Vila

R

5

-S-N-L--

O R 4 In preferred aspects of the present invention R 3 is selected from H, hydrocarbyl, -S hydrocarbyl, -S-H, halogen and N(R,)(R 10 ), wherein each of R 9 and Rio are independently selected from H and hydrocarbyl groups. 5 In preferred aspects of the present invention R 3 is selected from H, hydroxy, alkyl especially C01-010Co alkyl groups, C1-C6 alkyl, e.g. Cl-C3 alkyl group, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially C,-Cl0 alkoxy groups, C1-C,6 alkoxy, e.g. C-C3 10 alkoxy group, methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents. When R3 is -S-hydrocarbyl, preferably R3 is selected from -S-alkyl, -S-carboxylic acid, S-ether, and -S-amide, preferably selected from -S-Cl- 1 0 alkyl, -S- Cl-locarboxcylic acid, 15 -S- C-o 0 ether, and -S- Cl- 10 amide. In preferred aspects of the present invention R 3 is -CH 3 . Further preferred compounds of the present invention are those having one of the 20 following formulae. R N Formula la x R2 WO 2004/037251 PCT/GB2003/004590 15 0 Formula VIII "N

R

5 - S L R 3 o N N S R4 0 Formula IX Re- N 11 N R 3 01' O S R4 O Formula X I I

R

5 -S-N N o Formula Xa II

R

5 -S-N-L N O R 4

R

3 S N Formula XI 0

R

3 Re-S-N II I O R4 N Formula Xla 0 R3

R

5

-S-N

II I O R 4 In further preferred aspects of the present invention, such as when the compound has Formula la, Formula VIII, Formula IX, Formula X, Formula Xa, Formula XI, or Formula WO 2004/037251 PCT/GB2003/004590 16 Xla, R 3 is selected from O, hydrocarbyl, and N(R,) wherein R 9 is selected from H and hydrocarbyl groups. More preferably R 3 is selected from O, C 1 -Co 1 0 alkenyl groups, such as C 1

-C

6 alkenyl group, and C 1

-C

3 alkenyl group, NH and N-C 1 -Cjo alkyl groups, such as

N-C-C

6 alkyl group, and N-C 1

-C

3 alkyl groups. 5 In further preferred aspects of the present invention R 4 is selected from H and C 1 -Co 0 alkyl groups, such as Cl-C 6 alkyl group, and Cl-C 3 alkyl group. Preferably R 4 is H. In further preferred aspects of the present invention R 4 is a group of the formula. T O-S-O I 10

R

5 In these aspects the group shown above as O

R

5 - S L II N R4 may be of the formula O R-S L s-s o I O0 = 15

R

5 wherein each R 5 ' is independently selected from hydrocarbyl groups. Each R 5 may be the same of different to the other Rs. In one aspect the two R 5 groups are the same. In some preferred aspects of the invention R 5 is a cyclic hydrocarbyl group. Preferably R 5 20 is a cyclic hydrocarbyl group comprising a hydrocarbon ring.

R

5 may be a substituted ring or an unsubstituted ring. In some preferred aspects of the invention Rs is substituted ring.

WO 2004/037251 PCT/GB2003/004590 17 Preferably R 5 is a carbocyclic ring. Preferably R 5 is a six membered ring. 5 Preferably R, is a six membered carbocyclic ring. More preferably R 5 is a substituted six membered carbocyclic ring. In some preferred aspects of the invention R 5 is an aryl ring. Preferably R, is a 10 substituted aryl ring. In one highly preferred aspect R 5 is a group having the formula

R

1 4 R13 R 15 R12 R11 15 wherein each of Rl 1 , R 1 2 , R 1 3 , R 1 4 and R 15 are independently selected from H, halogen, and hydrocarbyl groups. Preferably each of R,1, R 12 , R 13 , R 1 4 and R 1 5 are independently selected from H, halogen, alkyl, such as Cl.,6 alkyl, phenyl, O-alkyl, O-phenyl, nitrile, haloalkyl, such as CF 3 , CC3 20 and CBr 3 , carboxyalkyl, -CO 2 H, CO 2 alkyl, and NH-acetyl groups.. Two or more of R 11 , R 12 , R 1 3 , R 14 and R 1 5 may join to form a ring. The two or more of R 11 ,

R

1 2 , R 1 3 , R 4 and R 15 may or may not be adjacent. The ring may be carbocyclic or heterocyclic ring. The ring may be optionally substituted by any of the R,,, R 1 2 , R 1 3 , R 14 25 and R 1 5 substituents listed above. When two or more of R 1 1 , R 12 , R 13 , R 14 and R 15 may join to form a ring the group WO 2004/037251 PCT/GB2003/004590 18 R14

R

13

R

1 5 R12 R11 may provide a naphthyl, quinolyl, tetrahydroquinolyl, or benzothtrahydropyranyl, each of which may be substituted or unsubstituted. 5 SUBSTITUENTS The compound of the present invention may have substituents other than those of the ring systems show herein. Furthermore the ring systems herein are given as general formulae and should be interpreted as such. The absence of any specifically shown 10 substituents on a given ring member indicates that the ring member may substituted with any moiety of which H is only one example. The ring system may contain one or more degrees of unsaturation, for example is some aspects one or more rings of the ring system is aromatic. The ring system may be carbocyclic or may contain one or more hetero atoms. 15 The compound of the invention, in particular the ring system compound of the invention of the present invention may contain substituents other than those show herein. By way of example, these other substituents may be one or more of: one or more halo groups, one or more O groups, one or more hydroxy groups, one or more amino groups, one or 20 more sulphur containing group(s), one or more hydrocarbyl group(s) - such as an oxyhydrocarbyl group. In general terms the ring system of the present compounds may contain a variety of non interfering substituents. In particular, the ring system may contain one or more hydroxy, 25 alkyl especially lower (Cl-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (Cl-C 6 ) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.

WO 2004/037251 PCT/GB2003/004590 19 For some compounds of the present invention, the compound may be substituted with a hydrocarbylsulphanyl group. The term "hydrocarbylsulphanyl" means a group that comprises at least hydrocarbyl group (as herein defined) and sulphur, preferably -S hydrocarbyl, more preferably -S-hydrocarbon. That sulphur group may be optionally 5 oxidised. Preferably the hydrocarbylsulphanyl group is -S-Cl 1 0 alkyl, more preferably -S-C1.5 alkyl, more preferably -S-C 1 3 alkyl, more preferably -S-CH 2

CH

2

CH

3 , -S-CH 2

CH

3 or -SCH 3 10 FURTHER ASPECTS For some applications, preferably the compounds have a reversible action. For some applications, preferably the compounds have an irreversible action. 15 In one embodiment, the compounds of the present invention are useful for the treatment of breast cancer. The compounds of the present invention may be in the form of a salt. 20 The present invention also covers novel intermediates that are useful to prepare the compounds of the present invention. For example, the present invention covers novel alcohol precursors for the compounds. By way of further example, the present invention covers bis protected precursors for the compounds. Examples of each of these 25 precursors are presented herein. The present invention also encompasses a process comprising each or both of those precursors for the synthesis of the compounds of the present invention. STEROID DEHYDROGENASE 30 111 Steroid dehydrogenase may be referred to as "11 I-HSD" or "HD" for short In some aspects of the invention 11 P-HSD is preferably 11 3-HSD Type 1. 35 In some aspects of the invention 11 -HSD is preferably 11 3-HSD Type 2.

WO 2004/037251 PCT/GB2003/004590 20 STEROID DEHYDROGENASE INHIBITION It is believed that some disease conditions associated with HD activity are due to conversion of a inactive, cortisone to an active, cortisol. In disease conditions 5 associated with HD activity, it would be desirable to inhibit HD activity. Here, the term "inhibit" includes reduce and/or eliminate and/or mask and/or prevent the detrimental action of HD. 10 HD INHIBITOR In accordance with the present invention, the compound of the present invention is capable of acting as an HD inhibitor. 15 Here, the term "inhibitor" as used herein with respect to the compound of the present invention means a compound that can inhibit HD activity - such as reduce and/or eliminate and/or mask and/or prevent the detrimental action of HD. The HD inhibitor may act as an antagonist. 20 The ability of compounds to inhibit steroid dehydrogenase activity can be assessed using the suitable Assay Protocol presented in the Examples section. It is to be noted that the compound of the present invention may have other beneficial properties in addition to or in the alternative to its ability to inhibit HD activity. 25 HYDROCARBYL The term "hydrocarbyl group" as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of 30 such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the 35 hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non- WO 2004/037251 PCT/GB2003/004590 21 limiting example of a hydrocarbyl group is an acyl group. A typical hydrocarbyl group is a hydrocarbon group. Here the term "hydrocarbon" means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may 5 be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the. hydrocarbon backbone and on the branch. 10 In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from optionally substituted alkyl group, optionally substituted haloalkyl group, aryl group, alkylaryl group, alkylarylakyl group, and an alkene group. 15 In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from C1-Co0 alkyl group, such as Cl-Ce alkyl group, and C,-C3 alkyl group. Typical alkyl groups include C, alkyl, C2 alkyl, C3 alkyl, C4 alkyl, Co alkyl, C7 alkyl, and C8 alkyl. 20 In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from Cj-Clo haloalkyl group, C-C6 haloalkyl group, Cl-C. haloalkyl group, C-Clo bromoalkyl group, C,-C6 bromoalkyl group, and C,-Cs bromoalkyl group. Typical haloalkyl groups include C, haloalkyl, C2 haloalkyl, C3 haloalkyl, C4 haloalkyl, C haloalkyl, C7 haloalkyl, C8 haloalkyl, C, bromoalkyl, C2 bromoalkyl, C3 25 bromoalkyl, C4 bromoalkyl, C, bromoalkyl, C7 bromoalkyl, and C. bromoalkyl. In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from aryl groups, alkylaryl groups, alkylarylakyl groups, -(CH 2

)

1 10 -aryl, -(CH 2 )1-1 0 -Ph, (CH 2 )1-1 0 -Ph-C 1 1 0 alkyl, -(CH 2 )1- 5 -Ph, (CH 2

),

5 -Ph-C-5 alkyl, -(CH 2 )1- 3 30 Ph, (CH 2 )1-3-Ph-C 1 .z alkyl, -CH 2 -Ph, and -CH 2 -Ph-C(CH 3

)

3 . The aryl groups may contain a hetero atom. Thus the aryl group or one or more of the aryl groups may be carbocyclic or more may heterocyclic. Typical hetero atoms include O, N and S, in particular N. In some aspects of the present invention, one or more hydrocarbyl groups is 35 independently selected from -(CH 2 )1- 10 -cycloalkyl, -(CH 2

)

1

.

1 0

-C

3 .- locycloalkyl, -(CH 2 )1- 7 -C3 7 cycloalkyl, -(CH 2

)

1 5

-C

3

.

5 cycloalkyl, -(CH 2

)

1

.

3 "C3- 5 cycloalkyl, and -CH 2 - C 3 cycloalkyl.

WO 2004/037251 PCT/GB2003/004590 22 In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from alkene groups. Typical alkene groups include C-C1o alkene group, C1-C6 alkene group, C-C3 alkene group, such as C1, C 2 , C3, C4, C5, C6, or 07 5 alkene group. In a preferred aspect the alkene group contains 1, 2 or 3 C=C bonds. In a preferred aspect the alkene group contains 1 C=C bond. In some preferred aspect at least one C=C bond or the only C=C bond is to the terminal C of the alkene chain, that is the bond is at the distal end of the chain to the ring system. 10 In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from oxyhydrocarbyl groups. OXYHYDROCARBYL 15 The term "oxyhydrocarbyl" group as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than 20 one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen. 25 In one embodiment of the present invention, the oxyhydrocarbyl group is a oxyhydrocarbon group. Here the term "oxyhydrocarbon" means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an 30 oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch. 35 Typically, the oxyhydrocarbyl group is of the formula C,6,O (such as a C,30).

WO 2004/037251 PCT/GB2003/004590 23 ANIMAL ASSAY MODEL FOR DETERMINING OESTROGENIC ACTIVITY (PROTOCOL 1) 5 Lack of in vivo oestrogenicity The compounds of the present invention may be studied using an animal model, in particular in ovariectomised rats. In this model, compounds which are oestrogenic stimulate uterine growth. 10 The compound (10 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol). A further group received the estrogenic compound EMATE subcutaneously in an amount of 10pg/day for five days. At the end of the study uteri were obtained and weighed with the results being expressed 15 as uterine weight/whole body weight x 100. Compounds having no significant effect on uterine growth are not oestrogenic. REPORTERS 20 A wide variety of reporters may be used in the assay methods (as well as screens) of the present invention with preferred reporters providing conveniently detectable signals (e.g. by spectroscopy). By way of example, a reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties. 25 Other protocols include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non interfering epitopes may even be used. These and other assays are described, among 30 other places, in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8:121 1). Examples of reporter molecules include but are not limited to (P-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, ( 35 glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabelled or fluorescent tag-labelled nucleotides can be incorporated into nascent transcripts which WO 2004/037251 PCT/GB2003/004590 24 are then identified when bound to oligonucleotide probes. By way of further examples, a number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH) 5 supply commercial kits and protocols for assay procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241. 10 HOST CELLS The term "host cell" - in relation to the present invention includes any cell that could comprise the target for the agent of the present invention. 15 Thus, a further embodiment of the present invention provides host cells transformed or transfected with a polynucleotide that is or expresses the target of the present invention. Preferably said polynucleotide is carried in a vector for the replication and expression of polynucleotides that are to be the target or are to express the target. The cells will be 20 chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells. The gram negative bacterium E. coli is widely used as a host for heterologous gene expression. However, large amounts of heterologous protein terid to accumulate inside 25 the cell. Subsequent purification of the desired protein from the bulk of E.coli intracellular proteins can sometimes be difficult. In contrast to E.coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other 30 bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas. Depending on the nature of the polynucleotide encoding the polypeptide of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells 35 are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not WO 2004/037251 PCT/GB2003/004590 25 processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected. Examples of suitable expression hosts within the scope of the present invention are fungi 5 such as Aspergillus species (such as those described in EP-A-0184438 and EP-A 0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. By way 10 of example, typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae. 15 The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention. 20 ORGANISM The term "organism" in relation to the present invention includes any organism that could comprise the target according to the present invention and/or products obtained therefrom. Examples of organisms may include a fungus, yeast or a plant. 25 The term "transgenic organism" in relation to the present invention includes any organism that comprises the target according to the present invention and/or products obtained. 30 TRANSFORMATION OF HOST CELLS/HOST ORGANISMS As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see 35 Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology WO 2004/037251 PCT/GB2003/004590 26 (1995), John Wiley & Sons, Inc. If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns. 5 In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces 10 cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow). 15 For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a 20 wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae. A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a 25 vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.). Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating 30 plasmid vectors. In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. 35 The constructs contain a promoter active in yeast fused to the nucleotide sequence, usually a promoter of yeast origin, such as the GALl promoter, is used. Usually a signal WO 2004/037251 PCT/GB2003/004590 27 sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system. For the transformation of yeast several transformation protocols have been developed. 5 For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168). 10 The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, e.g. G418. 15 Another host organism is a plant. The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material. Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. 20 A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP A-0449375. 25 Thus, the present invention also provides a method of transforming a host cell with a nucleotide sequence that is to be the target or is to express the target. Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression of the encoded protein. The protein produced by a recombinant cell may be displayed on the surface of the cell. If desired, and as will be understood by those of 30 skill in the art, expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).

WO 2004/037251 PCT/GB2003/004590 28 VARIANTS/HOMOLOGUES/DERIVATIVES In addition to the specific amino acid sequences and nucleotide sequences mentioned 5 herein, the present invention also encompasses the use of variants, homologue and derivatives thereof. Here, the term "homology" can be equated with "identity". In the present context, an homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% 10 identical. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. Homology comparisons can be conducted by eye, or more usually, with the aid of readily 15 available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly 20 compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration 25 that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without 30 penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology. However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence 35 alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap WO 2004/037251 PCT/GB2003/004590 29 costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be 5 modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. 10 Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, 15 the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program. 20 A further useful reference is that found in FEMS Microbiol Lett 1999 May 15;174(2):247 50 (and a published erratum appears in FEMS Microbiol Lett 1999 Aug 1;177(1):187-8). Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a 25 scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is 30 preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of 35 the sequence comparison and generates a numerical result.

WO 2004/037251 PCT/GB2003/004590 30 The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the 5 residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, 10 phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: 15 Table 1 ALIPHATIC Non-polar G A P ILV Polar - uncharged C S T M NQ Polar - charged D E KR AROMATIC HF W Y EXPRESSION VECTORS 20 The nucleotide sequence for use as the target or for expressing the target can be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in 25 eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above. The protein produced by a host recombinant cell by expression of the nucleotide WO 2004/037251 PCT/GB2003/004590 31 sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane. 5 FUSION PROTEINS The target amino acid sequence may be produced as a fusion protein, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S 10 transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target. 15 The fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The 20 antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance. In another embodiment of the invention, the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of 25 peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognised by a commercially available antibody. THERAPY 30 The compounds of the present invention may be used as therapeutic agents - i.e. in therapy applications. The term "therapy" includes curative effects, alleviation effects, and prophylactic effects. 35 The therapy may be on humans or animals, preferably female animals.

WO 2004/037251 PCT/GB2003/004590 32 PHARMACEUTICAL COMPOSITIONS In one aspect, the present invention provides a pharmaceutical composition, which 5 comprises a compound according to the present invention and optionally a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). The pharmaceutical compositions may be for human or animal usage in human and 10 veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with 15 regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). 20 Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. 25 There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, 30 for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes. Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for 35 example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.

WO 2004/037251 PCT/GB2003/004590 33 Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets 5 containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may 10 contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner. 15 COMBINATION PHARMACEUTICAL The compound of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutically active agents. 20 By way of example, the compounds of the present invention may be used in combination with other 11 P-HSD inhibitors and/or other inhibitors such as an aromatase inhibitor (such as for example, 4hydroxyandrostenedione (4-OHA)), and/or a steroid sulphatase inhibitors such as EMATE and/or steroids - such as the naturally occurring sterneurosteroids dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate 25 (PS) and/or other structurally similar organic compounds. In addition, or in the alternative, the compound of the present invention may be used in combination with a biological response modifier. 30 The term biological response modifier ("BRM") includes cytokines, immune modulators, growth factors, haematopoiesis regulating factors, colony stimulating factors, chemotactic, haemolytic and thrombolytic factors, cell surface receptors, ligands, leukocyte adhesion molecules, monoclonal antibodies, preventative and therapeutic vaccines, hormones, extracellular matrix components, fibronectin, etc. For some 35 applications, preferably, the biological response modifier is a cytokine. Examples of cytokines include: interleukins (IL) - such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- WO 2004/037251 PCT/GB2003/004590 34 9, IL-10, IL-11, IL-12, IL-19; Tumour Necrosis Factor (TNF) - such as TNF-a; Interferon alpha, beta and gamma; TGF-P3. For some applications, preferably the cytokine is tumour necrosis factor (TNF). For some applications, the TNF may be any type of TNF such as TNF-ca, TNF-P3, including derivatives or mixtures thereof. More preferably the 5 cytokine is TNF-a. Teachings on TNF may be found in the art - such as WO-A-98/08870 and WO-A-98/13348. ADMINISTRATION 10 Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited. 15 The compositions of the present invention may be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight. 20 By way of further example, the agents of the present invention may be administered in accordance with a regimen of 1 to 4 times per day, preferably once or twice per day. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound 25 employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. Aside from the typical modes of delivery - indicated above - the term "administered" 30 also includes delivery by techniques such as lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes. 35 The term "administered" includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a WO 2004/037251 PCT/GB2003/004590 35 parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route. Thus, for pharmaceutical administration, the compounds of the present invention can be 5 formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc. and usually for parenteral administration. Approximate effective dose rates may be in the range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or even from 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of 10 average (70Kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day. They may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days. For oral administration they may be formulated in tablets, capsules, solution or suspension 15 containing from 100 to 500 mg of compound per unit dose. Alternatively and preferably the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the 20 bodyweight of the patient, such variations being within the skill and judgement of the physician. CELL CYCLING 25 The compounds of the present invention may be useful in the method of treatment of a cell cycling disorder. As discussed in "Molecular Cell Biology" 3rd Ed. Lodish et al. pages 177-181 different eukaryotic cells can grow and divide at quite different rates. Yeast cells, for example, 30 can divide every 120 min., and the first divisions of fertilised eggs in the embryonic cells of sea urchins and insects take only 1530 min. because one large pre-existing cell is subdivided. However, most growing plant and animal cells take 10-20 hours to double in number, and some duplicate at a much slower rate. Many cells in adults, such as nerve cells and striated muscle cells, do not divide at all; others, like the fibroblasts that assist 35 in healing wounds, grow on demand but are otherwise quiescent.

WO 2004/037251 PCT/GB2003/004590 36 Still, every eukaryotic cell that divides must be ready to donate equal genetic material to two daughter cells. DNA synthesis in eukaryotes does not occur throughout the cell division cycle but is restricted to a part of it before cell division. 5 The relationship between eukaryotic DNA synthesis and cell division has been thoroughly analysed in cultures of mammalian cells that were all capable of growth and division. In contrast to bacteria, it was found, eukaryotic cells spend only a part of their time in DNA synthesis, and it is completed hours before cell division (mitosis). Thus a gap of time occurs after DNA synthesis and before cell division; another gap was found 10 to occur after division and before the next round of DNA synthesis. This analysis led to the conclusion that the eukaryotic cell cycle consists of an M (mitotic) phase, a G 1 phase (the first gap), the S (DNA synthesis) phase, a G 2 phase (the second gap), and back to M. The phases between mitoses (G 1 , S, and G 2 ) are known collectively as the interphase. 15 Many nondividing cells in tissues (for example, all quiescent fibroblasts) suspend the cycle after mitosis and just prior to DNA synthesis; such "resting" cells are said to have exited from the cell cycle and to be in the Go state. 20 It is possible to identify cells when they are in one of the three interphase stages of the cell cycle, by using a fluorescence-activated cell sorter (FACS) to measure their relative DNA content: a cell that is in G 1 (before DNA synthesis) has a defined amount x of DNA; during S (DNA replication), it has between x and 2x; and when in G 2 (or M), it has 2x of DNA. 25 The stages of mitosis and cytokinesis in an animal cell are as follows (a) Interphase. The G 2 stage of interphase immediately precedes the beginning of mitosis. Chromosomal DNA has been replicated and bound to protein during the S 30 phase, but chromosomes are not yet seen as distinct structures. The nucleolus is the only nuclear substructure that is visible under light microscope. In a diploid cell before DNA replication there are two morphologic chromosomes of each type, and the cell is said to be 2n. In G 2 , after DNA replication, the cell is 4n. There are four copies of each chromosomal DNA. Since the sister chromosomes have not yet separated from each 35 other, they are called sister chromatids.

WO 2004/037251 PCT/GB2003/004590 37 b) Early prophase. Centrioles, each with a newly formed daughter centriole, begin moving toward opposite poles of the cell; the chromosomes can be seen as long threads. The nuclear membrane begins to disaggregate into small vesicles. 5 (c) Middle and late prophase. Chromosome condensation is completed; each visible chromosome structure is composed of two chromatids held together at their centromeres. Each chromatid contains one of the two newly replicated daughter DNA molecules. The microtubular spindle begins to radiate from the regions just adjacent to the centrioles, which are moving closer to their poles. Some spindle fibres reach from 10 pole to pole; most go to chromatids and attach at kinetochores. (d) Metaphase. The chromosomes move toward the equator of the cell, where they become aligned in the equatorial plane. The sister chromatids have not yet separated. 15 (e) Anaphase. The two sister chromatids separate into independent chromosomes. Each contains a centromere that is linked by a spindle fibre to one pole, to which it moves. Thus one copy of each chromosome is donated to each daughter cell. Simultaneously, the cell elongates, as do the pole-to-pole spindles. Cytokinesis begins as the cleavage furrow starts to form. 20 (f) Telophase. New membranes form around the daughter nuclei; the chromosomes uncoil and become less distinct, the nucleolus becomes visible again, and the nuclear membrane forms around each daughter nucleus. Cytokinesis is nearly complete, and the spindle disappears as the microtubules and other fibres depolymerise. Throughout 25 mitosis the "daughter" centriole at each pole grows until it is full-length. At telophase the duplication of each of the original centrioles is completed, and new daughter centrioles will be generated during the next interphase. (g) Interphase. Upon the completion of cytokinesis, the cell enters the G 1 phase of 30 the cell cycle and proceeds again around the cycle. It will be appreciated that cell cycling is an extremely important cell process. Deviations from normal cell cycling can result in a number of medical disorders. Increased and/or unrestricted cell cycling may result in cancer. Reduced cell cycling may result in 35 degenerative conditions. Use of the compound of the present invention may provide a means to treat such disorders and conditions.

WO 2004/037251 PCT/GB2003/004590 38 Thus, the compound of the present invention may be suitable for use in the treatment of cell cycling disorders such as cancers, including hormone dependent and hormone independent cancers. 5 In addition, the compound of the present invention may be suitable for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcomas, melanomas, prostate cancer, pancreatic cancer etc. and other solid tumours. 10 For some applications, cell cycling is inhibited and/or prevented and/or arrested, preferably wherein cell cycling is prevented and/or arrested. In one aspect cell cycling may be inhibited and/or prevented and/or arrested in the G 2 /M phase. In one aspect cell cycling may be irreversibly prevented and/or inhibited and/or arrested, preferably wherein cell cycling is irreversibly prevented and/or arrested. 15 By the term "irreversibly prevented and/or inhibited and/or arrested" it is meant after application of a compound of the present invention, on removal of the compound the effects of the compound, namely prevention and/or inhibition and/or arrest of cell cycling, are still observable. More particularly by the term "irreversibly prevented and/or inhibited 20 and/or arrested" it is meant that when assayed in accordance with the cell cycling assay protocol presented herein, cells treated with a compound of interest show less growth after Stage 2 of the protocol I than control cells. Details on this protocol are presented below. Thus, the present invention provides compounds which: cause inhibition of growth of 25 oestrogen receptor positive (ER+) and ER negative (ER-) breast cancer cells in vitro by preventing and/or inhibiting and/or arresting cell cycling; and/or cause regression of nitroso-methyl urea (NMU)-induced mammary tumours in intact animals (i.e. not ovariectomised), and/or prevent and/or inhibit and/or arrest cell cycling in cancer cells; and/or act in vivo by preventing and/or inhibiting and/or arresting cell cycling and/or act as 30 a cell cycling agonist. CELL CYCLING ASSAY (PROTOCOL 2) Procedure 35 Stage 1 WO 2004/037251 PCT/GB2003/004590 39 MCF-7 breast cancer cells are seeded into multi-well culture plates at a density of 105 cells/well. Cells were allowed to attach and grown until about 30% confluent when they are treated as follows: 5 Control - no treatment Compound of Interest (COl) 20p.M Cells are grown for 6 days in growth medium containing the COI with changes of medium/COl every 3 days. At the end of this period cell numbers were counted using a 10 Coulter cell counter. Stage 2 After treatment of cells for a 6-day period with the COI cells are re-seeded at a density of 15 104 cells/well. No further treatments are added. Cells are allowed to continue to grow for a further 6 days in the presence of growth medium. At the end of this period cell numbers are again counted. CANCER 20 As indicated, the compounds of the present invention may be useful in the treatment of a cell cycling disorder. A particular cell cycling disorder is cancer. Cancer remains a major cause of mortality in most Western countries. Cancer therapies 25 developed so far have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumrnours. However, more aggressive chemotherapy is currently employed for the treatment of hormone-independent tumours. Hence, the development of a pharmaceutical for anti-cancer treatment of hormone 30 dependent and/or hormone independent tumours, yet lacking some or all of the side effects associated with chemotherapy, would represent a major therapeutic advance. We believe that the compound of the present invention provides a means for the treatment of cancers and, especially, breast cancer. 35 In addition or in the alternative the compound of the present invention may be useful in WO 2004/037251 PCT/GB2003/004590 40 the blocking the growth of cancers including leukaemias and solid tumours such as breast, endometrium, prostate, ovary and pancreatic tumours. OTHER THERAPIES 5 It is also to be understood that the compound/composition of the present invention may have other important medical implications. For example, the compound or composition of the present invention may be useful in the 10 treatment of the disorders listed in WO-A-99/52890 - viz: In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635. For ease of reference, part of that list is now provided: diabetes including Type 11 diabetes, obesity, 15 cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, 20 malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulceration, retinopathy and surgical wound 25 healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis. In addition, or in the alternative, the compound or composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859. For ease of 30 reference, part of that list is now provided: cytokine and cell proliferation/differentiation activity; immunosuppressant or immunostimulant activity (e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity); regulation of haematopoiesis, e.g. 35 treatment of myeloid or lymphoid diseases; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e.g. for healing wounds, treatment of burns, ulcers and WO 2004/037251 PCT/GB2003/004590 41 periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiinflammatory activity (for treating e.g. 5 septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine. In addition, or in the alternative, the composition of the present invention may be useful 10 in the treatment of disorders listed in WO-A-98/09985. For ease of reference, part of that list is now provided: macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity; anti-immune activity, i.e. inhibitory effects against a cellular and/or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular 15 matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic 20 heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic 25 diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, 30 intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic 35 neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and WO 2004/037251 PCT/GB2003/004590 42 inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's 5 disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, 10 chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral 15 nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular 20 immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue. 25 As previously mentioned, in one aspect the present invention provides use of a compound as described herein in the manufacture of a medicament for use in the therapy of a condition or disease associated with 11 -HSD. 30 Conditions and diseases associated with 11 3-HSD have been reviewed in Walker, E. A,; Stewart, P. M.; Trends in Endocrinology and Metabolism, 2003, 14 (7), 334-339. In a preferred aspect, the condition or disease is selected from the list consisting of: * metabolic disorders, such as diabetes and obesity 35 * cardiovascular disorders, such as hypertension * glaucoma WO 2004/037251 PCT/GB2003/004590 43 * inflammatory disorders, such as arthritis or asthma * immune disorders * bone disorders, such as osteoporosis * cancer 5 * intra-uterine growth retardation * apparent mineralocorticoid excess syndrome (AME) * polycystic ovary syndrome (PCOS) * hirsutism * acne 10 * oligo- or amenorrhea * adrenal cortical adenoma and carcinoma * Cushing's syndrome * pituitary tumours * invasive carcinomas 15 * breast cancer; and * endometrial cancer. SUMMARY 20 In summation, the present invention provides compounds for use as steroid dehydrogenase inhibitors, and pharmaceutical compositions for the same. BRIEF DESCRIPTION OF THE FIGURES 25 The present invention will be described in further detail by way of example only with reference to the accompanying figures in which: Figure 1 is of graph 1 which shows the amount of protein per pL of rat liver and rat kidney. 30 Figure 2 is of graph 2 which shows the enzyme concentration and time-dependency course, E to F, in rat liver, 11 p-HSD type 1 activity. Figure 3 is of graph 3 which shows the enzyme concentration and time-dependency course, F to E, in rat kidney, 11 1-HSD type 2 activity. Figure 4 is a graph showing extraction efficiencies obtained with four extraction 35 methods.

WO 2004/037251 PCT/GB2003/004590 44 Figure 5 is a graph showing a comparison of 11 1-HSD1 activity in rat and human hepatic microsomes. Figure 6 is a series of graphs showing the effect of incubation time on human microsomal 11 P-HSD1 activity. 5 Figure 7 is a series of graphs showing the effect of microsomal protein concentration on human microsomal 11 P3-HSD1 activity. Figure 8 is a graph showing the substrate (cortisone) saturation curve for human hepatic microsomal 1113 HSDI. Figure 9 is a Lineweaver-Burke plot of substrate saturation data for human hepatic 10 microsomal 113 HSDI. Figure 10 is a graph showing the IC50 determination for glycyrrhetinic acid. Figure 11 is a graph showing the IC50 determination for carbenoxolone. Figures 12(A), 12(B) and 12(C) are graphs showing the 11 P-HSD1 activity measured by Immunoassay. Figure 12(A) shows the effect of protein; Figure 12(B) shows the 15 effect of cortisone; and Figure 12(C) shows the effect of Tween-80. Figure 13 is a graph showing the evaluation of the Assay Designs Cortisol Immunoassay. Figure 14 is a graph showing the effect of increasing microsomal protein on measurement of 11 3 HSD1 activity detected by Assay Designs Immunoassay. 20 Figure 15 is a graph showing the detection of 1113 HSD1 activity by RIA using the Immunotech anti-cortisol antibody. Figure 16 is a graph showing the effect of lowering the Immunotech antibody concentration on the signal to noise (microsome group compared to GA blank group). Figure 17 is a graph showing the Immunotech antibody saturation curve for detection of 25 1113 HSD1 activity by RIA. Figure 18 is a graph showing the linearity of human hepatic microsomal 1113 HSD1 activity detected by RIA. Figure 19 is a graph showing the effect of Tween 80 on detection of human hepatic microsomal 1113 HSD1 activity by RIA. 30 Figure 20 is a graph showing the effect of buffer systems on detection of human hepatic microsomal 1113 HDS1 activity by RIA. Figure 21 is a graph showing the linearity of human hepatic microsomal 1113 HSD1 activity with incubation time detected by RIA. Figure 22 is a graph showing the substrate saturation curve for human hepatic 35 microsomal 11p HDS1 activity detected by RIA.

WO 2004/037251 PCT/GB2003/004590 45 Figure 23 is a Lineweaver-Burke plot of substrate saturation data for human hepatic microsomal 11 3 HDS1 activity detected by RIA. Figure 24 is a graph showing the DMSO tolerance of human hepatic microsomal 11P3 HSD1 activity. 5 Figure 25 is an IC, 0 curve for inhibition of human hepatic microsomal 11P HSD1 activity by glycyrrhetinic acid. EXAMPLES 10 The present invention will now be described only by way of example. MATERIALS AND METHODS Materials 15 Enzymes - Rat livers and rat kidneys were obtained from normal Wistar rats (Harlan Olac, Bicester, Oxon,UK). Both the kidneys and livers were homogenised on ice in PBS sucrose buffer (1g/ 10 ml) using an Ultra-Turrax. After the livers and kidneys were homogenised the homogenate was centrifuged for five minutes at 4000 rpm. The 20 supernatant obtained was removed and stored in glass vials at -20 0 C. The amount of protein per pl of rat liver and kidney cytosol was determined using the Bradford method [14]. Apparatus 25 * Incubator: mechanically shaken water bath, SW 20, Germany. * Evaporator, Techne Driblock DB 3A, UK * TLC aluminium sheets 20 x 20 cm silica gel 60 F 254 , Merck, Germany. * Scintillation vials: 20 ml polypropylene vials with caps, SARSTEDT, Germany. * Scintillation counter: Beckman LS 6000 SC, Beckman Instruments Inc., USA. 30 Solutions * Assay medium: PBS-sucrose buffer, Dulbecco's Phosphate Buffered Saline, 1 tablet/100 ml with 0,25 M sucrose, pH 7,4 BDH Laboratory supplies, UK. * Scintillation fluid: Ecoscint A (National Diagnostics, USA). 35 * Radioactive compound solutions: [1,2,6,7- 3 H]-cortisol (Sp. Ac. 84 Ci/mmol) NEN Germany, [4- 14 C]-cortisol (Sp. Ac. 53 mCi/mmol) NEN Germany.

WO 2004/037251 PCT/GB2003/004590 46 * CrO 3 and Acetic acid (Sigma Chemical Co., UK). * Extraction fluid: Di-ethylether, Fischer Chemicals, UK. * Bradford Reagent solution: Coomassie Brilliant Blue G-250, 100 mg in 95% ethanol with 100 ml of phosphoric acid (85% w/v) diluted to 1 litre. 5 Compounds * Inhibitors: compounds were synthesised in accordance with the synthetic routes below. * Cofactor: NADPH and NADP, Sigma Chemical Co., UK. 10 Methods Synthesis of radio labelled cortisone 15 Labelled cortisol (F) ( 3 H-F and " 4 C-F) was oxidised at the C-1l position with Cr0 3 in order to synthesize to the corresponding labelled cortisone ( 3 H-E and 1 4 C-E). For this reaction F was oxidised in a 0,25% CrO3 (w/v) dissolved in a 50% acetic acid/distilled water (v/v) solution. The labelled F was then added to 1 ml of the CrO3 20 solution, vortex mixed and put in an incubator for 20 minutes at 37 0 C. The aqueous reaction mixture was extracted twice with 4 ml of di-ethylether, the di-ethylether was then evaporated and the residue transferred to a TLC-plate, which was developed in the following system, chloroform : methanol 9:1 (v/v). Unlabelled cortisone (E) was also run on the TLC-plate to locate the position of the labelled steroids. After locating the spot of 25 the labelled steroids this area is cut out from the TLC-plate and eluted with 0,5 ml of methanol. The amount of protein per gL of rat liver and rat kidney 30 The amount of protein in rat liver and rat kidney needed to be determined. The experiment was done according to the Bradford method [14]. The following method was used: first a BSA (protein) solution was prepared (1 mg/ml). Protein solutions containing 10 to 100 pg protein were pipetted into tubes and volumes adjusted with distilled water. Then 5 ml of protein reagent was added to the tubes and vortex mixed. The absorbance 35 was measured at 595 nm after 15 minutes and before 1 hour in 3 ml cuvettes against a reagent blank. The weight of the protein was plotted against the corresponding WO 2004/037251 PCT/GB2003/004590 47 absorbance resulting in a standard curve used to determine the protein concentration in rat liver and rat kidney cytosols. Assay validation - Enzyme concentration and time-dependency of 11 13-HSD 5 activity Before carrying out 11 P-HSD assays to examine the conversion E to F and F to E and the influence that different inhibitors have on these conversions the amount of rat liver homogenate and rat kidney homogenate and their incubation time need to be 10 determined. 11 1-HSD type 1 is the enzyme responsible for the conversion E to F and this type of enzyme is present in rat liver. The substrate solution used in this assay contained 70,000 cpm/ml 3 H-E in PBS-sucrose and 0.5 pM of unlabelled E and co-factor NADPH (9 15 mg/10 ml of substrate solution). 1 ml of the substrate solution and the different amounts of rat liver homogenate was added to all tubes. The amount of rat liver homogenate needed for an assay was determined by incubating the substrate solution with 25, 50, 100 and 150 ptl for 30, 60, 90 and 120 minutes at 370C 20 in a water bath with the tubes being mechanically shaken. After the incubation 50 pL of recovery solution was added, containing about 8,000 cpm/ 50 gL of 14 C-F and 50 ptg/50 VL of unlabelled F for visualising the spot on the TLC-plate, to correct for the losses made in the next two steps. F was then extracted from the aqueous phase with 4 ml of ether (2 x 30 sec cycle, vortex mix). The aqueous phase was then frozen using dry-ice 25 and the organic layer was decanted and poured into smaller tubes and evaporated. 6 drops of ether were then added to the small tubes to re-dissolve the residue which was transferred to an aluminium thin layer chromatography plate (TLC-plate). The TLC-plate was developed in a TLC tank under saturated conditions. The solvent system used was chloroform : methanol 9:1 (v/v). The F spots on the TLC-plate were visualised under 30 UV- light and cut out from the TLC-plate (Rf=0.45). The spots from the TLC- plate were then put into scintillation vials and 0.5 ml of methanol was added to all vials to elute the radioactivity from the TLC-plate for 5 minutes. 10 ml of Ecoscint was added to the scintillation vials and they were put into the scintillation counter to count amount of product formed. 35 WO 2004/037251 PCT/GB2003/004590 48 The same procedure was used for the 11 P3-HSD type 2 assay, the conversion F to E, to determine the amount of rat kidney to be used and the incubation time. Except this time the substrate solution contained 3 H-F and unlabelled F and the recovery contained 14 C-E and unlabelled E and cortisone has a Rf value of 0.65 on the TLC-plate. 5 Assay procedure - The 11 1-HSD inhibitors In these assays the influence of different inhibitors on the 11 P3-HSD activity both in reductive (type 1) and oxidative (type 2) directions were assessed. In the reductive 10 direction E is the substrate and F the product and visa versa in the case of oxidation. The method described here is for the oxidative direction. The substrate solution contained about 50,000 cpm/ml 3 H-F in PBS-sucrose and 0.5 pM F. I ml of the substrate solution was added to each tube, the inhibitors were also added, 15 at a 10 ptM concentration, to each tube except to the "control" and "blank" tubes. 150 [IL was added to all tubes except to the blanks, this was done to correct for the amount of 3 H-F spontaneously formed. The tubes were incubated for 60 minutes in a mechanically shaken water bath at 370C. The amount of kidney liver homogenate and incubation time used resulted from the enzyme- and time-dependency assay. After incubation 50 pL of 20 recovery was added to correct for the losses made in the next steps, containing 5000 cpm/50 plL of 14 C-E and 50pig/50 pL of unlabelled E (to visualise the spot on the TLC plate). The aqueous mixture was then extracted with 4 ml of ether (2 x 30 sec cycle, vortex mix). After freezing the aqueous phase, the ether (upper) layer was decanted into smaller tubes and evaporated at 450C until completely dry. The residue was then re 25 dissolved in 6 drops of ether and transferred to a TLC-plate. The TLC-plate was developed in chloroform : methanol (9:1 v/v) solvent system, the TLC-plate ran for about 90 minutes until the solvent front had moved about 18 cm. The position of the product E was visualised under UV-light and cut out from the TLC-plate and put into scintillation vials. Radioactivity was eluted over 5 minutes with 0.5 ml methanol. 0.5 ml of PBS 30 sucrose and 10 ml of Ecoscint were then added and vortex mixed before counting in the scintillation counter. Before counting the samples, two total activity vials were prepared. These contained 0.5 ml of the substrate solution, 50 [iL of the recovery, 0.5 ml of methanol and 10 ml of Ecoscint. These two total activity vials were needed to determine the amount of 14 C-E and 3 H-F added in the beginning to make the calculations. 35 WO 2004/037251 PCT/GB2003/004590 49 In case of the reductive direction, E to F ,the same method was used. Only the substrate solution containing 3 H-E and unlabelled E and the recovery containing 14 C-F and unlabelled F are different to the method used in the oxidative direction. 5 After testing all the inhibitors at 10 pM a dose-response experiment was done for the most potent 11 P3-HSD type 1 and type 2 inhibitors. To look at the percentage of inhibition four different concentrations, 1, 5, 10 and 20 pM, were used. The method for both the rat liver, type 1 the reductive, and rat kidney, type 2 the oxidative, stay the same throughout the entire experiment. 10 RESULTS The amount of protein per gL of rat liver and rat kidney 15 An initial experiment was carried out to determine the amount of protein in rat liver cytosol and rat kidney cytosol, to be added to each tube. Graph 1 in figure 1 shows the standard curve from which the amount of protein used in both experiments was calculated. The amount of protein added to each tube in the rat liver experiment was 75.5 lg (per 25 pL). In the rat kidney experiment the amount of protein added to each 20 tube was 135.6 p.g (per 150jL). Enzyme concentration and time-dependency of 11 P-HSD activity In this experiment the amount of rat liver homogenate and rat kidney homogenate added 25 to each tube and the incubation time was determined. Graph 2 in figure 2 shows the enzyme concentration and time-dependency course of the rat liver experiment E to F, 11 3-HSD type 1 activity. Graph 3 in figure 3 shows the enzyme concentration and time dependency course F to E, 11 13-HSD type 2 activity. After drawing the graphs the optimal amount of rat liver cytosol and rat kidney cytosol and both their incubation times 30 were selected. One important rule when selecting both variables, to select an amount of rat liver and rat kidney and incubation time on a linear part of the graph. This is done to avoid fluctuations in enzyme activity. The amount of rat liver cytosol selected was 25 lL and 90 minutes of incubation time, the amount of rat kidney cytosol selected was 150 gL and 60 minutes of incubation time. 35 WO 2004/037251 PCT/GB2003/004590 50 The 11 P3-HSD inhibitors In this experiment the influence of different inhibitors on the conversion E to F and F to E was determined. The reason why inhibition in both directions was examined was to 5 make a comparison between the inhibitors and which type of 11 p-HSD they inhibit more. Compounds were screened for their ability to inhibit 11 P-HSD type 1 (E to F) and type 2 (F to E). All the inhibitors were initially tested at a 10 pM concentration. The percent of inhibition was calculated as the percentage of decrease in radio labelled 3 H-E and 3 H-F of product formed, compared with the control activity (the tubes without an 10 inhibitor in it). All the results calculated are means, n=2. Table 2: Inhibitory Effect STX Structure % inhibition of 11 % inhibition of 113I No. HSD1 @ 10 pM HSD2 @ 10 pM typical typical sd ± 5% sd ± 5% 412 0 27 3 S 413 53 n=2 0.2 4/ UIC 5 0 = 6.6 pM 421 60 n=2 0.9 \/~NH 1C 50 = 10 PM 0os 0 7 1 O 424 24 0.7 0 425 40 0.0 0 \ / 469 ci 63 29 ~ 0/ WO 2004/037251 PCT/GB2003/004590 51 STX Structure % inhibition of 113 % inhibition of 11 3 No. HSD1 @ 10 pM HSD2 @ 10 pM typical typical sd ± 5% sd ± 5% 470 ci 39 30 4 0001o 519 Br H48 8 0 Is--N 521 /\ 0.5 5 o 522 37 6 ci ' C'- / S'-NH 523 21 8 Br 0 N NH 0 INH ci 0/ NH cl 552 --- 18 24 553 O 0.7 18 \/

S-NH

WO 2004/037251 PCT/GB2003/004590 52 STX Structure % inhibition of 11P % inhibition of 11 No. HSD1 @ 10 pM HSD2 @ 10 pM typical typical sd _ 5% sd ± 5% 554 s 69 43 /N- o N 575 o 62 1.6 OH - 0 N cl No 580 c 75 1.4 \-NH ci 0/ N 581 c- 77 32

N

oN.. o S!\X_ 0 582 0- 40 0.7 / NH 583 N 29 0.4 /\ /,2N S N N S 584 o0- 48 10 o 585 48 1.6 o 0 WO 2004/037251 PCT/GB2003/004590 53 STX Structure % inhibition of 113 % inhibition of 111 No. HSD1 @ 10 pM HSD2 @ 10 pM typical typical sd _ 5% sd ± 5% 701 o 34 36 \ / s-ONH 703 35 4 0 \ / s-NH 704 0 38 4 o 705 o0 6 6 os NH 0 706 29 7 '9 \/ soNH 707 -o 21 11 oo //,O o 708 39 11 0 709 10 13 \ /s-NH o 710 55 10 0 \-- 8 -NH / \ o~ \'%_ WO 2004/037251 PCT/GB2003/004590 54 STX Structure % inhibition of 113 % inhibition of 113 No. HSD1 @ 10 pM HSD2 @ 10 pM typical typical sd _ 5% sd ±+ 5% 711 o 37 6 sH H-"\ / -N 712 F F 24 3 F 0, \ / SONH F F 713 26 3 0 730 32 9 00 o \ /to/ o S N 0 o 731 N 45 12 /- \ N S 750 CI4 10 SS 751 CI10 5 z - \N N0 WO 2004/037251 PCT/GB2003/004590 55 STX Structure % inhibition of 113 % inhibition of 113 No. HSD1 @ 10 pM HSD2 @ 10 pM typical typical sd + 5% sd + 5% 752 c 5 1 0 N O o 753 8 2 OH o o 754 ci 20 6 0 ,o 755 21 8 NS BIOLOGICAL ASSAY DEVELOPMENT USING HUMAN 113-HYDROXYSTEROID DEHYDROGENASE TYPE 1. 5 Standard Operating Procedure for the 11p-Hydroxysteroid Dehydrogenase Type 1 cortisol Radioimmunoassay. 11p HSD1 cortisol RIA 10 Reagents: Cortisone, Cortisol (Hydrocortisone), NADPH, Glucose-6-phosphate, Glycyrrhetinic acid (GA), Dextran coated charcoal (C6197) and DMSO were obtained from Sigma Aldrich, Carbenoxolone was obtained from ICN Biomedicals, Product 215493001, 3 H-cortisone was obtained from American Radiolabelled Componds Inc, 15 Product ART-743, 3 H-cortisol was obtained from NEN, Product NET 396, 14 C-cortisol WO 2004/037251 PCT/GB2003/004590 56 was obtained from NEN, Product NEC 163, human hepatic microsomes were obtained from XenoTech, product H0610 / Lot 0210078, rat hepatic microsomes were obtained from XenoTech, SPA beads were obtained from Amersham, Product RPNQ0017, the Immunoassay kit was obtained from Assay Designs, Product 900-071, the 5 Immunologicals Direct anti-cortisol antibody was Product OBT 0646, the Sigma anti cortisol antibody was Product C8409 and the Immunotech antibody was supplied by Beckman, Product IMBULK3 6D6. Buffer Solutions 10 Buffer 1, from Barf [15]: 30 mM Tris-HCL, pH 7.2, containing 1 mM EDTA Buffer 2, from the Sterix protocol: PBS (pH 7.4) containing 0.25M sucrose Buffer 3, from the Sigma RIA protocol: 50 mM Tris-HCL, pH 8, containing 0.1 M NaCL and 0.1 % gelatin 15 Stop solution, from Barf [15]: 1 mM glycyrrhetinic acid in 100 % DMSO Enzyme assays were carried out in the presence of 181 pM NADPH, 1 mM Glucose-6 Phosphate and cortisone concentrations indicated for each experiment. Enzyme assay buffer: 30 mM Tris-HCL, pH 7.2 containing 1 mM EDTA 20 Antibody binding buffer: 50 mM Tris-HCL, pH 8, containing 0.1 M NaCI and 0.1 % gelatin Compound preparation: Prepare 10 mM stock solutions in 100% DMSO at 100 times the required assay concentration. Dilute into assay buffer 1 in 25. Also dilute neat 25 DMSO 1 in 25 into assay buffer for controls. Substrate preparation: Prepare a solution of cortisone in ethanol 600 times the required assay concentration (175 nM). Dilute this 1 in 50 into assay buffer. Prepare NADPH as a 1.8 mg/ml solution in assay buffer. 30 Prepare G-6-P as a 3.65 mg/ml solution in assay buffer. Mix these 3 solutions 1:1:1 to make a solution of sufficient volume for 25 pl additions to each sample. Add 0.5 pCi tritiated cortisone per 25 .l and mix the solution well. Microsome preparation: Dilute stock 20 mg/ml solution 1 in 100 with assay buffer. 35 WO 2004/037251 PCT/GB2003/004590 57 Antibody preparation: Dilute stock antibody solution to 17 fg/ml in antibody binding buffer. Dextran coated charcoal preparation: Make a 20 mg/ml solution in antibody binding 5 buffer and chill on ice. Enzyme assay: To a u-bottom polypropylene 96 well plate add: 25 pI compound dilution or diluted DMSO to controls, NSB's and blanks 10 gl 1 mM GA in DMSO (enzyme stop solution) to blanks 10 25 pl substrate mixture to all samples 50 pil diluted microsomes to all samples Incubate plate for 30 min at 370C shaking Add 10 pl enzyme stop solution to all wells except the blanks Add 100 pI antibody solution to all wells except the NSB's, add antibody binding buffer to 15 these wells Incubate at 37 0 C for 1 h Chill plate on ice for 15 min Add 50 pl / well charcoal solution and mix with an 8-channel pipette (4 - 5 aspirations) Chill the plate on ice 20 Centrifuge at 4oC, 2000 x g for 15 min Transfer 100 pl supernatant into an Optiplate, also add 25 0l substrate mixture to 2 empty wells to indicate counting efficiency Add 200 pI Microscint-40 to all wells and count on a Topcount 25 Radioimmunoassay The 1113 HSD1 enzyme assay was carried out following the standard operating procedure described above in u-bottom polypropylene 96 well plates or 1.5 ml Eppendorf tubes as indicated for each experiment. Subsequent to stopping the enzyme reaction, 30 100 pl antibody prepared in buffer 3 unless otherwise indicated was added to test samples and 100 gl buffer 3 was added to the NSB samples. The samples were incubated for 1 hour at 370C and the chilled on ice for 15 mins. Dextran coated charcoal (50 pl / sample) prepared to the indicated concentration in buffer 3 was added and the samples were mixed (vortex for tubes and aspiration 5 times with an 8-channel pipette WO 2004/037251 PCT/GB2003/004590 58 for 96 well plates) and chilled for a further 10 min. The samples were centrifuged at 2000 x g for 15 min at 40C to pellet the charcoal. Aliquots of the supernatant (100 pl) were transferred to an Optiplate and counted on the Topcount in 150-200 pA Microscint 40. In some experiments, aliquots of supernatant were transferred to scintillation vials 5 and counted on the Tricarb LSC in 5 ml Ultima Gold scintillant. 11p HSD1 ASSAY DEVELOPMENT 11 HSD1 TLC format assay 10 Separation of Cortisone and Cortisol Prior to performing an enzyme assay, solvent systems reported in the literature for separation of cortisone from cortisol were investigated [16, 17]. Solutions of cortisone 15 and cortisol at 10 mg/ml were prepared in methanol, and aliquots spotted onto a silica gel TLC plate. The plate was run in CH 2

CI

2 : IMS 92 : 8 v/v (2). The plate was then air dried and sprayed with 0.1% Rhodamine B in methanol to visualise the spots. The table below describes the separation obtained. 20 Table 3: Separation of cortisone from cortisol by TLC Steroid Distance run from origin Solvent front migration / (cm) steroid migration (cm) Cortisone 7.5 2.3 Cortisol 4.5 3.8 This separation was considered adequate for use in an enzyme assay. 25 The literature details several methods of extracting cortisol from aqueous solution [16, 17]. In order to select a method for use, [ 1 4 C-labelled cortisol was obtained from NEN. A stock was prepared in phosphate buffered saline (PBS) containing 4000 DPM in 50p1 with cold cortisol (1g) added as a carrier. The final ethanol concentration was 0.4%. Aliquots of this solution were added to glass tubes (100.l) and the following extractions 30 were carried out: 1. 1 ml CH 2 CI2,vortex and pass through phase separating filter paper (Whatman, IPS) 2. 1ml ethyl acetate, vortex and pass through phase separating filter WO 2004/037251 PCT/GB2003/004590 59 paper 3. 1 ml CH 2

CI

2 and 200pI 0.05% CaCI 2 , vortex, centrifuge (500g for 5 min) and remove upper aqueous phase 4. 1 ml ethyl acetate and 200pl 0.05 % CaC 2 , vortex, centrifuge (500g for 5 min) and collect upper organic phase. The organic phases were dried and the residues were taken up in 100tl IMS. An aliquot of this was spotted onto a 5 TLC plate and the plate run as before. Following visualisation with Rhodamine B, the spots were scraped into scintillation vials and counted on a liquid scintillation counter (Packard TriCarb) in 5ml Ultima gold scintillant. Extraction efficiencies were calculated and are given in Figure 4. 10 From these results it appears that 90 % of the cortisol is lost by phase separating filtration. Ethyl acetate appears to extract cortisol more efficiently than CH 2

CI

2 , possibly because the organic phase is easier to collect. Ethyl acetate appears to be a suitable method of extraction. 15 Human and Rat Hepatic Microsomal 1113-HSD1 Activity. 11 3-HSD1 activity in rat and human hepatic microsomes was evaluated, to determine the minimum microsomal protein concentrations required for measurement of enzyme activity. The experiment was done according to the Bradford Method [14]. The assay 20 was performed in Buffer 2 and the cortisone concentration used was 2M containing 0.5pCi [ 3 H]-cortisone per incubation. Microsomes were tested at concentrations ranging from 50pg to 400gg protein per incubation in a final incubation volume of 100jI in glass tubes. Samples were incubated for 1 h in a shaking water bath at 370C and the assay was stopped by addition of I ml ethyl acetate. To correct for recovery, 50pl [14C]-cortisol 25 was added to the samples followed by 200pl 0.05 % CaCl 2 . The samples were vortex mixed and centrifuged as described above. The upper organic phase was removed and dried down, and the residue dissolved in 100l methanol and 50p aliquots were spotted onto TLC plates, which were run as described above. Samples were counted on a TriCarb liquid scintillation counter using a dual label programme. Recovery efficiency 30 was determined from the DPM obtained in 50p l [ 14 C]-cortisol solution, which was counted with the samples. Results are shown in Figure 5. The 11 -HSD1 activities in rat and human microsomes were similar, 0.7pmol/mg/min and 0.5pmol/mg/min for rat and human microsomes respectively. The activity in human WO 2004/037251 PCT/GB2003/004590 60 microsomes is apparently not related to microsomal protein concentration, which may suggest that that the protein concentration range examined is too high. Lower human microsome protein concentrations were evaluated; 3.7pg to 100 Ig per 5 sample. The time course of activity was also determined, from 0 to 60 minutes at 37 0 C. The extraction conditions were as described above. The results from these experiments are shown in figures 6 and 7. The results shown in figures 6 and 7 demonstrate that enzyme activity is linear at 10 incubation times up to 30 min at all the microsomal protein concentrations tested, and that enzyme activity is linear at microsomal protein concentrations below 30pg per sample. The influence of substrate concentration on activity was examined. The [ 3 H]-cortisone 15 concentration was kept constant at 0.5tCilsample, and unlabelled cortisone varied from 44nM to 2pM. The assay was carried out with 10pg microsomal protein per sample with an incubation time of 30 minutes at 37oC. The results are shown in figure 8. A double reciprocal plot (Lineweaver-Burke) of these data gives an apparent Km for cortisone of 660nM, figure 9. 20 The standard compounds glycyrrhetinic acid and carbenoxolone were examined in this assay system, as part of the validation process. The assay was performed using 175nM cortisone substrate, with 10tg microsomal protein and a 30 minute incubation at 370C, as described by Barf [15]. Although the data in figures 8 and 9 above suggest that this 25 substrate concentration is not saturating under these assay conditions. Glycyrrhetinic acid and carbenoxolone were tested at concentrations from 0.012pM to 3pM, the DMSO concentration was 1% in all samples. The results are shown in figures 10 and 11. Glycyrrhetinic acid and carbenoxolone give IC0 values of 40nM and 119nM respectively. 30 The IC50 reported for carbenoxolone by Barf et aL. using the SPA format and recombinant 1113-HSD is 330nM [15], approximately three-fold less potent. The difference in potency in the two assay systems is probably due to the different assay conditions, SPA compared to tic end point, and also the enzyme source, native hepatic enzyme compared to recombinant enzyme. 35 WO 2004/037251 PCT/GB2003/004590 61 The assay conditions described above support good enzyme activity however, which should be transferable to a 96 well plate format. Development of High Throughput 1113 HSD1 Assays 5 Supply of the antibody used by Barf [15] in the Scintillation Proximity assay (SPA) proved problematic. A sample batch of the antibody (from Immunotech) was tested for suitability and a second order was placed for a larger quantity. A robust 96 well plate assay using Radioimmunoassay (RIA) format was developed using the Immunotech 10 antibody available, this is described below. Immunoassay Format An Assay Designs enzyme immunoassay system was evaluated as a potential assay 15 format. The basis of the assay is competition for antibody binding between sample cortisol, generated by 11 P-HSD1, and labelled cortisol binding. The anti-cortisol detection antibody provided in the kit is a mouse monoclonal, reported to cross react less than 0.1% with cortisone. The kit is designed for the analysis of cortisol levels in saliva, urine, serum and plasma and also in tissue culture media, rather than for 20 determining enzyme activity however. The 11 p-HSD1 enzyme assay conditions described by Barf et al [15] were used; human hepatic microsomes in Buffer 1 at protein concentrations from 25Vg to 200kg, cortisone at concentrations from 44nM to 700nM incubated for 60 minutes at 370C. The effect of 25 0.9% Tween 80 was also investigated, as this detergent is reported to improve the activity of enzymes involved in steroid metabolism. Results are shown in Figure 12. Figure 12(A) shows the effect of protein. Data taken from the 700pM cortisone group tested in the presence of Tween-80. 30 Figure 12(B) shows the effect of cortisone. Data taken from the 25pg microsomal protein group tested in the presence of Tween-80. Figure 12(C) shows the effect of Tween-80. Data taken from the 25Ag microsomal 35 protein group tested in the presence of 700 gM cortisone.

WO 2004/037251 PCT/GB2003/004590 62 The assay detected cortisol in the standard curve (313 pg/ml to 10,000 pg/ml) as expected but the signal obtained from the enzyme assay samples decreased with increasing microsomal protein concentration, suggesting that the microsomal protein 5 may interfere with the immunoassay, figure 12(A). Addition of exogenous cortisone had no effect on levels of cortisol detected in the enzyme assay samples, suggesting the antibody does not cross react with cortisone, figure 12(B). Inclusion of detergent in the enzyme assay buffer had little effect, figure 12(C). 10 The assay conditions were varied to determine if it was feasible to use the immunoassay system to detect 1113-HSD1 activity; 24gg microsomal protein per sample and 2pM cortisone substrate in Buffer 2. Enzyme activity was also measured in samples following the addition of steroid displacement reagent; a kit component which releases cortisol from cortisol binding protein, if present in the sample. The assay detected the cortisol in 15 the standard curve (313pg/ml to 10,000pg/ml). Figure 13 shows the absorbance at 405m obtained for the different groups: The lowest and highest concentrations of the cortisol standard have been included in Figure 13 as 313 pg/ml and 1000 pg/ml together with the NSB absorbance to show the 20 dynamic range obtained in the assay. Absorbance obtained in the presence of reaction mixture taken from samples incubated with microsomal protein ("Enzyme") are lower than those in the presence of reaction mixture not containing microsomal protein ("No enzyme") indicating increases in levels of 25 cortisol. In the presence of the kit steroid displacement reagent ("DR") these two reaction mixtures show the same pattern but the signal is depressed. 30 Glycyrrhetinic acid (GA) in the presence of the top concentration of cortisol standard has no effect on the ability of the kit to measure cortisol concentrations. Although the signal to background ratio of 2.5 for the assay is rather poor, these data demonstrate that the antibody can bind the cortisol:AP conjugate and that this can be 35 displaced by cortisol. An experiment was carried out to examine the effect of increasing microsomal protein concentration, in an attempt to improve the signal to noise obtained.

WO 2004/037251 PCT/GB2003/004590 63 Microsomal protein was tested from 100pg/incubation down to 51ig/incubation using 2tM cortisone in Buffer 2. All other conditions were identical to those detailed above. The results are shown in figure 14. 5 Decreasing microsomal protein from 10pg/incubation to 5pg/incubation results in a corresponding decrease in enzyme activity. Increasing microsomal protein above 10g/incubation results in a quenching of signal which may be due to the colour of the microsomes. Therefore the dynamic range of this assay cannot be improved by increasing the microsomal protein concentration. 10 RIA development using Immunotech Antibody The 113 HSD1 assay was carried out using 10.g/well human hepatic microsomal protein. The Immunotech antibody was used in the RIA at concentrations from 15 6.25pg/well to 25pg/well, the results are shown in figure 15. The Immunotech antibody worked well in the assay and gave good signal to background at all the concentrations tested. The signal to noise with 12.5 and 6.1 ig antibody per well was similar suggesting it may be possible to reduce the antibody concentration. 20 The antibody titre, at concentrations from 0.67pg/well to 6.7pg/well, was examined. The 1113 HSD1 assay was carried out using human microsomal protein at 20g/well, to generate the optimum signal to background. Each antibody concentration was tested against a "no enzyme" blank (buffer substituted for microsomes), a "GA blank" (10 pil 25 stop solution added prior to microsomes) and a control group. The results are shown in figures 16 and 17. The saturation curve indicates that there is no difference in the detection of enzyme activity above 1.68 gg/well. The signal to background ratio with this antibody 30 concentration is good, (6 fold).Consequently the antibody will be used at 1.7 gg/well in future assays. Linearity of enzyme activity with human hepatic microsomal protein concentration using RIA detection was examined. The 1113 HSD1 assay was carried with microsomal protein 35 concentrations varying from 1 pg/well to 40 [Lg/well. 1113 HSD1 activity was linear with WO 2004/037251 PCT/GB2003/004590 64 protein up to concentrations of 20 pig/well, figure 18, confirming the results obtained with the classical enzyme assay (figure 7). The optimal concentration of human microsomal protein to use in the assay appears to 5 be 10Otg/well. The effect of including Tween 80 in the enzyme assay buffer was also investigated. This assay was carried out in parallel with the assay above and under the same conditions except that the enzyme assay buffer (Buffer 2) contained 0.05 % Tween 80. Microsomal 10 protein was tested at four concentrations. Tween 80 was found to increase the blank CPM, reducing the signal to noise of the assay. Representative data, from the group tested 10pg/well microsomal protein, are shown in figure 19. Similar results were obtained with all the microsome protein concentrations examined, consequently Tween will not be used in future studies.. 15 To simplify the protocol such that both enzyme assay and RIA stages are carried out in the same buffer, both phases were carried out in either enzyme assay buffer (Buffer 2) or Buffer 3 (RIA buffer). The microsomal protein concentration used was 10 ptg/well and the cortisone concentration was 175 nM. Performing both enzyme assay and RIA in 20 Buffer 3 appears to improve the data slightly, figure 20. Linearity of enzyme activity with incubation time was investigated. The enzyme assay was carried out with 10pg/well microsomal protein and with 175nM cortisone, and stopped at varying time points, the results are shown in figure 21. 25 With microsome protein concentrations of 10jag/well and 175nM substrate, the reaction is linear at time points up to 30 minutes. These results indicate that a substrate concentration of 175nM is too low. The apparent Km observed in the classical 11 p HSD1 assay was 660 nM (figures 8 and 9), although these assays are end-point 30 measurement, hence it is not certain that initial rates were measured in the low substrate groups with a 30 minute incubation time. However, published Km values for cortisone in human hepatic microsomal 113 HSD1 assays are in the micromolar range [18, 19]. Although 175nM substrate is well below the apparent Km, it may not be possible to increase the concentration significantly for two reasons: 35 WO 2004/037251 PCT/GB2003/004590 65 (i) If the compounds are competitive with cortisone, the measured inhibition will fall if the substrate is increased above the concentration used in Reference 1. (ii) Increasing the substrate concentration will reduce the specific activity of the label, reducing the sensitivity of the assay. This could be overcome by adding 5 higher concentrations of [ 3 H]-cortisone, but the protocol uses 0.5pxCi/well and there is a cost implication if higher levels of radioactivity are used. Substrate saturation was examined. The enzyme assay was carried out exactly described in the methods section, in Buffer 3 with 10pg/well microsomal protein and with 10 [cold cortisone] as indicated. [3H]-cortisone was 0.5pxCi/sample throughout. The reaction was stopped after 30 min by the addition of 10gl stop solution. The RIA was carried out exactly as indicated in the methods section. The results are shown in figures 22 and 23. 15 The apparent Km (700 nM), determined from the Lineweaver-Burke plot of these data shown in figure 23 is very similar to that determined in the tic format 11 3 HSD1 assay (Figure 9, apparent Km ~660 nM). The data suggests that at 10pg microsomal protein, the enzyme is not saturated at 175nM cortisone, over an incubation period of 30 minutes. 20 Lowering the microsomal protein concentration or the incubation time to bring the reaction within the linear range would partly overcome the problem. However either of these adjustment adjustments would decrease the assay sensitivity, and decrease the apparent potency of inhibitors. Consequently the initial experiments were performed with 25 175nM cortisone. 11 D-HSD1 Assay Validation Prior to compound testing, the tolerance of the enzyme assay to DMSO was determined. 30 Inclusion of DMSO at 1 % in the enzyme assay does not affect total or blank values, but slightly increases enzyme activity and the signal to noise ratio (Table 4). The experiment was repeated over a range of DMSO concentrations from 0.3 to 10%, figure 24.

WO 2004/037251 PCT/GB2003/004590 66 Table 4:Control and blank CPM obtained in the Glycyrrhetinic acid IC 50 assay showing effect of 1% DMSO and signal to noise ratio obtained. Group 1 % DMSO No DMSO NSB 670 661 GA blank 640 660 Control 3515 2583 Signal to noise 5 fold 4 fold 5 There is a slight increase in microsomal enzyme activity in the presence of 0.3% and 1% DMSO. At DMSO concentrations above 1%, there is a linear reduction in enzyme activity. It is reported that DMSO can both increase and reduce microsomal enzyme activity, depending on the concentration, presumably due to effects on the microsomal 10 membranes. On the basis of these data, it is intended that compounds be screened in the presence of 1% DMSO. An IC50 value was generated for the standard inhibitor glycyrrhetinic acid, the compound was tested at concentrations between 0.012pM and 3 1 M, with a final DMSO 15 concentration of 1%, figure 25. Glycyrrhetinic acid gives a concentration-related inhibition of the enzyme with an IC50 of 41nM, with good curve fit values (r 2 = 0.962) and Hillslope. This is similar to the value of 40nM generated using the tic format assay, (see figure 10). An IC50 value of 30nM has 20 been reported for glycyrrhetinic acid inhibition of 11P3 HSD1 in human hepatic microsomes, using dehydro-dexamethasone as the substrate [19]. However, these values are lower than the value reported by Barf et al. [15]. Table 5: Inhibition Data 25 STX No. Structure % inhibition of Human 113 HSD1 @ 10 pM typical sd ± 5% N=2 976 110 C.

N

WO 2004/037251 PCT/GB2003/004590 67 STX No. Structure % inhibition of Human 113 HSD1 @ 10 pM typical sd ± 5% N=2 993 89 CI N N 01< I S CC 994 83 CI N ~NNJ 01 1029 78 CI 984 73 0 995 68 00 WO 2004/037251 PCT/GB2003/004590 68 STX No. Structure % inhibition of Human 1113 HSD1 @ 10 pM typical sd ± 5% N=2 469 66 1018 66 / CI H /NH 986 65 S9 CI 00 1020 64 NA 996 63 N HN

K

O SO WO 2004/037251 PCT/GB2003/004590 69 STX No. Structure % inhibition of Human 1113 HSD1 @ 10 pM typical sd ± 5% N=2 987 62 NHC 1030 61 H 992 • 61 Cl 9CI 985 61 N

BH

WO 2004/037251 PCT/GB2003/004590 70 STX No. Structure % inhibition of Human 11P HSD1 @ 10 pM typical sd - 5% N=2 977 61 N 1019 61 CI H Cl 521 60 S N 978 60 10 6Cl 1017 60 Cl

NN

WO 2004/037251 PCT/GB2003/004590 71 STX No. Structure % inhibition of Human 11 3 HSD1 @ 10 pM typical sd - 5% N=2 998 58 Cl 585 58 H

N

999 55 Cl C 1021 54 CI 470 54 554 53

-I

WO 2004/037251 PCT/GB2003/004590 72 STX No. Structure % inhibition of Human 1 1p HSD1 @ 10 pM typical sd ± 5% N=2 982 53 0 H c o 991 51 CI c 997 51 M 575 50 Cl 0 OH 709 49 0 VS N WO 2004/037251 PCT/GB2003/004590 73 STX No. Structure % inhibition of Human 1113 HSD1 @ 10 pM typical sd + 5% N=2 553 48 519 48 Br N 424 47 H0 oo 522 47 Ci Hq Cl \ o S N 552 45 C /N N

.

0

N

WO 2004/037251 PCT/GB2003/004590 74 STX No. Structure % inhibition of Human 1113 HSD1 @ 10 pM typical sd ± 5% N=2 975 45 O / CI 'J N 989 45 Cl cii N OHS 704 44 H 0 Io 524 44 N N Cl WO 2004/037251 PCT/GB2003/004590 75 STX No. Structure % inhibition of Human 113 HSD1 @ 10 pM typical sd _ 5% N=2 421 42 H NI 425 41 ;l H 701 41 HO 981 40 NN 703 37 0 WO 2004/037251 PCT/GB2003/004590 76 STX No. Structure % inhibition of Human 113 HSD1 @ 10 pIM typical sd + 5% N=2 412 36 Cl 0 Cl \ N 710 35 0/ H 582 34 H -o o/ - S 580 30 413 CI 413 29 CI %K 0S WO 2004/037251 PCT/GB2003/004590 77 STX No. Structure % inhibition of Human 11 3 HSD1 @ 10 pM typical sd + 5% N=2 581 29 0H F583 28 N 583 28 'r / 705 28 0 0 0 H 831 28 SO C 0 751 22 \H Cl WO 2004/037251 PCT/GB2003/004590 78 STX No. Structure % inhibition of Human 1113 HSD1 @ 10 pM typical sd + 5% N=2 708 21 H 11 / 0 584 20 S O0 SULPHONAMIDE SYNTHESIS Method A 5 To the amine (1 eq.) dissolved in pyridine (3 eq.) was added the corresponding sulphonyl chloride (1.2 eq.) and the reaction mixture was stirred at RT under N 2 overnight. The resulting mixture was poured into aq. HCI and the organic layer was extracted with ethyl acetate, dried (MgSO 4 ), filtered and concentrated under reduced pressure to give the desired sulphonamide as crystalline solid or as a thick syrup. The 10 crude compound was then purified by flash chromatography using EtOAc/hexane (3:2) or CH 2

CI

2 /EtOAc (4:1) as eluent to give crystalline solid. Method B To the amine (1 eq.) dissolved in Et 3 N (5 eq.) was added the corresponding sulphonyl 15 chloride (1.2 eq.) and the reaction mixture was stirred at RT under N 2 overnight. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO 4 ), filtered and concentrated under reduced pressure to give the desired sulphonamide as crystalline solid or as a thick syrup. The crude compound was then purified by flash chromatography using EtOAc/hexane (3:2) or CH2CI 2 /EtOAc (4:1) 20 as eluent to give crystalline solid.

WO 2004/037251 PCT/GB2003/004590 79 Note : Insoluble amines and sulphonyl chlorides were dissolved in minimum amount of

CH

2

CI

2 , THF or DMF. Method C 5 To a solution Arylsulphonyl chloride (1.1 eq.) in DCM were added Pyridine (2.2 eq.) and catalytic amount of DMAP. The solution was stirred at room temperature under nitrogen for 10 minutes. Then the amine (1 eq.) was added and the reaction mixture was stirred at room temperature under nitrogen for 4-16 hrs. The resulting mixture was partitioned between DCM and 5% sodium bicarbonate. The organic layer was washed with brine, 10 dried over MgSO 4 , and concentrated to give a solid or a thick syrup. The crude compound was then purified by flash chromatography to give desired arylsulphonamide as crystalline solid. DGSO3020A (STX412) 15 Synthesised by method A. Off-white crystals of DGSO3020A (186 mg; 55%). mp 189 190 'C; TLC Rf: 0.68 EtOAc/Hexane (3:2); 1 H NMR (CDCl 3 ) 6 2.80 (s, 3H, CH 3 ), 7.13 (s, 1H, N-H, exchanged with D 2 0), 7.212 (dd,IH, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.27 (dd, 1H, Ar-H, J= 1.95 Hz and 8.59 Hz), 7.51 (d, 1H, Ar-H, J= 1.95 Hz), 7.65 (d, 1H, Ar-H, J = 1.95 Hz), 7.69 (d, 1H, Ar-H, J = 8.59 Hz), 7.91 (d, 1H, Ar-H, J = 8.59 Hz); MS (FAB+) 20 372.9 [100, (M+H)*]; HRMS m/z (FAB+) 372.9627, C 1 4

H

1 0 35

CI

2

N

2 0 2

S

2 requires 372.9639, 376.9574, C 14

H

10 37

C

2

N

2 0 2

S

2 requires 376.9580; HPLC tr 3.65 min (92 : 08 = MeOH :

H

2 0). DGSO3022A (STX413) 25 Synthesised by method A. Off-white crystals of DGSO3022A (233 mg; 72%). mp 178 OC; TLC Rf: 0.71 EtOAc/Hexane (3:2); 'H NMR (CDCl 3 ) 6 2.75 (s, 3H, CH 3 ), 2.80 (s, 3H,

CH

3 ), 6.75 (s, 1H, N-H, exchanged with D 2 0), 7.11 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 7.17 - 7.21 (m, 1H, Ar-H), 7.53 (d, 1H, Ar-H, J = 1.17 Hz), 7.55 (d, 1H, Ar-H, J = 1.95 Hz), 7.68 (d, 1H, Ar-H, J = 8.20 Hz), 7.92 (dd, 1H, Ar-H, J = 1.17 Hz and 7.81 Hz); 30 MS (FAB+) 164.1 [35, (5-Amino-2-methyl benzothiazole)*], 353.0 [100, (M+H)*]; HRMS m/z (FAB+) 353.0176, C 15 H1 4 35

CIN

2 0 2

S

2 requires 353.0185, 355.0155, C 1 5

H

14 37

CIN

2 0 2

S

2 requires 355.0156; HPLC tr 3.78 min (92: 08 = MeOH : H 2 0). DGSO3024A (STX421) 35 Synthesised by method A. White crystals of DGSO3024A (240 mg; 76%). mp 133-134 OC; TLC Rf : 0.7 EtOAc/Hexane (3:2); 'H NMR (CDCI 3 ) 6 0.90 (t, 3H, C-H 3

CH

2

CH

2 , J = WO 2004/037251 PCT/GB2003/004590 80 7.42 Hz), 1.56 - 1.66 (m, 2H, CH 3 CH2CH 2 ), 2.59 (t, 2H, CH 3

H

2

CH

2 , J = 7.42 Hz), 2.80 (s, 3H, CH 3 ), 6.71 (s, 1H, N-H, exchanged with D 2 0), 7.17 (d 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.201 - 7.214 (m, 1H, Ar-H), 7.218- 7.223 (m, 1H, Ar-H), 7.57 (d, 1H, Ar-H, J = 2.34 Hz), 7.76-7.69 (m, 3H, Ar-H); MS (FAB+) 347.1 [100, (M+H)+]; HRMS m/z (FAB+) 5 347.0881, C 1 7

H

1 9

N

2 0 2

S

2 requires 347.0887; HPLC t, 3.69 min (92 : 08 = MeOH : H 2 0). DGSO3034A (STX424) Synthesised by method A. White crystals of DGSO3034A (262 mg; 86%). mp 152 OC; TLC Rf: 0.48 EtOAclHexane (3:2); 'H NMR (CDCl 3 ) 6 10.31 (s, 1H, NH, Ex. With D 2 0), 10 7.85 (d, 1H, Ar-H, J = 8.59 Hz), 7.66 - 7.69 (m, 2H, Ar-H), 7.57 (d, 1H, Ar-H, J = 1.95 Hz), 7.11 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.02 - 7.05 (m, 2H, Ar-H), 3.76 (s, 3H, OCH 3 ), 2.73 (s, 3H, CH 3 ); MS (FAB+) 164.0[25 (Amine SM*)], 335.0 [100, (M+H)+]; HRMS m/z (FAB+) 335.0519, C, 3

H

15

N

2 0 3

S

2 requires 335.0524; HPLC tr 1.94 min (80 : 20 = MeOH: H 2 0). 15 DGSO3036A (STX425) Synthesised by method A. White crystals of DGSO3036A (136 mg; 42%). mp 295-296 OC; TLC Rf: 0.56 EtOAc/Hexane (3:2); 1 H NMR (DMSO-d 6 ) 65 10.66 (s, 1H, NH, Ex. With

D

2 0), 7.87 (d, 1H, Ar-H, J = 8.59 Hz), 7.52 (d, 1H, Ar-H, J = 1.95 Hz), 7.32 - 7.44 (m, 20 3H, Ar-H), 7.12 (dd, 1H, Ar-H, J = 2.3 Hz and 8.59 Hz), 2.73 (s, 3H, CH 3 ), 2.64 (s, 3H,

CH

3 ); MS (FAB+) 164.0 [40, (Starting amine)'], 353.0 [100, (M+H)+]; HRMS m/z (FAB+) 353.0187, C 15

H

14 3 1CIN 2 0 2

S

2 requires 353.0185, 355.0165, C, 1 5

H

14 37

CIN

2 0 2

S

2 requires 355.0155; HPLC t, 1.94 min (80: 20 = MeOH : H 2 0z). 25 DGS03058A (STX519) Synthesised by method A. White crystals of DGS03058A (199 mg; 57%). mp 172 'C; TLC Rf : 0.56 EtOAc/Hexane (3:2); 'H NMR (DMSO-d 6 ) 65 10.53 (s, 1H, NH, Ex. With

D

2 0), 7.88 (d, 1H, Ar-H, J = 8.59 Hz), 7.74 - 7.77 (m, 2H, Ar-H), 7.64 - 7.68 (m, 2H, Ar H), 7.58 (d, 1H, Ar-H, J = 1.95 Hz), 7.11 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 2.74 30 (s, 3H, CH 3 ); MS (FAB+) 384.9 [100, (M+H)+]; HRMS m/z (FAB+) 384.9494, C1 4

H

2 81 BrN 2 0 2

S

2 requires 384.9503, 382.9501, C14H12 79 BrN20 2

S

2 requires 382.9523; HPLC tr 2.64 min (90: 10 = MeOH : H 2 0). DGSO3062B (STX469) 35 To a stirred solution of DGSO3022A (50 mg, 0.14 mmol, 1 eq.) in anhy. DMF (5 ml) and NaH (7 mg, 0.16 mmol, 1.1 eq.) was added Mel (3 ml, 0.21 mmol, 1.5 eq.) and the WO 2004/037251 PCT/GB2003/004590 81 mixture was stirred for lh. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO 4 ), filtered and concentrated under reduced pressure to give a yellow suspension. The crude compound (70 mg) was purified by flash chromatography using EtOAc/hexane (3:2) as eluent to give white 5 crystals of DGSO3062A (36 mg; 69%). mp 97-98 OC; TLC Rf: 0.61 EtOAc/Hexane (3:2); 1 H NMR (CDC1 3 ) 6 7.73 (dd, 1H, Ar-H, J = 1.17 Hz and 7.81 Hz), 7.37 (d, 1H, Ar-H, J = 8.59 Hz), 7.59 (d, 1H, Ar-H, J= 1.95 Hz), 7.49 (dd, 1H, Ar-H, J = 1.17 Hz and 8.2 Hz), 7.24 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.11 - 7.15 (m, 1H, Ar-H), 3.24 (s, 3H,

CH

3 ), 2.76 (s, 3H, CH 3 ), 2.35 (s, 3H, CH 3 ); MS (FAB+) 366.9 [100, (M+H)*]; HRMS m/z 10 (FAB+) 366.0262, C 1 6 H 1 35

CIN

2 0 2

S

2 requires 366.0262, 368.0300, C 1 6 H3 15 7

CIN

2 0 2

S

2 requires 368.0234; HPLC t, 1.93 min (96 : 04 = MeOH : H 2 0). DGSO3072A (STX470) To a stirred solution of DGSO3022A (50 mg, 0.14 mmol, 1 eq.) in anhy. DMF (5 ml) and 15 NaH (10 mg, 0.16 mmol, 1.1 eq.) was added Etl (23 mg, 0.21 mmol, 1.5 eq.) and the mixture was stirred for 1h. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO 4 ), filtered and concentrated under reduced pressure to give a yellow suspension. The crude compound (75 mg) was purified by flash chromatography using EtOAc/hexane (3:2) as eluent to give a pale 20 yellow thick syrup of DGSO3072A (16 mg; 30%). TLC Rf : 0.71 EtOAc/Hexane (3:2); 'H NMR (CDCI3) 6 7.76 - 7.78 (m, 2H, Ar-H), 7.66 (m, 1H, Ar-H), 7.53 - 7.55 (m, 1 H, Ar-H), 7.27 - 7.28 (m, 1H, Ar-H), 7.14 - 7.18 (m, 1H, Ar-H), 7.11 - 7.18 (m, 1H, Ar-H), 5.30 (s, 1H, NH, Ex. with D20), 3.74 (q, 2H, Ar-H, J = 7.42 Hz and 7.03 Hz), 2.83 (s, 3H, CH 3 ), 2.53 (s, 3H, CH 3 ), 1.12 (t, 3H, CH 3 , J= 7.03 Hz), MS (FAB+) 381.1 [100, (M+H)i]; HRMS 25 m/z (FAB+) 381.1062, C 1 7

H

1 7 35

CIN

2 0 2

S

2 requires 381.1058, 385.0952, C1 7

H

1 7 3 7

CIN

2 0 2

S

2 requires 385.0949. DGSO3082A (STX521) Synthesised by method A. White crystals of DGSO3082A (230 mg; 67%). mp 85-86 OC; 30 TLC Rf: 0.64 EtOAc/Hexane (3:2); 1 H NMR (CDCl3) 5 10.54 (s, 1H, NH, Ex. With D 2 0), 7.87 (d, 1H, Ar-H, J= 8.59 Hz), 7.84 (broad s, 4H, Ar-H), 7.67 - 7.69 (m, 2H, Ar-H), 7.62 (d, 1H, Ar-H, J = 1.95 Hz), 7.39 - 7.49 (m, 3H, Ar-H), 7.17 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 2.73 (s, 3H, CH 3 ); MS (FAB+) 381.2 [100, (M+H)*]; HRMS m/z (FAB+) 381.0730, C 20

HI

7

N

2 0 2

S

2 requires 381.0731; HPLC t, 1.36 min (96: 04 = MeOH : H 2 0).

WO 2004/037251 PCT/GB2003/004590 82 DGSO3084A (STX522) Synthesised by method A. Yellow crystals of DGSO3084A (46 mg; 10%). mp 253-254 OC; TLC Rf : 0.74 EtOAc/Hexane (3:2); 1H NMR (DMSO-d 6 ) 5 11.09 (s, 1H, NH, Ex. with 5 D 2 0), 7.91 (d, 1H, Ar-H, J= 8.59 Hz), 7.86 (s, 2H, Ar-H), 7.59 (d, 1H, Ar-H, J= 2.34 Hz), 7.15 (dd, 1H, Ar-H, J= 1.95 Hz and 8.59 Hz), 2.74 (s, 3H, CH 3 ); MS (FAB+) 409.1 [100, (M+H)*]; MS (FAB-) 407.0 [100, (M-H) ]; HRMS m/z (FAB+) 406.9176, C1 4 H19 35

C

3

N

2 0 2

S

2 requires 406.9167, 408.9136, C14H1 9 3 7 C1 3

N

2 0 2

S

2 requires 408.9140. 10 DGSO3086A (STX523) Synthesised by method A. Pale yellow crystals of DGSO3086A (101 mg; 57%). mp 219 OC; TLC Rf: 0.71 EtOAc/Hexane (3:2); 'H NMR (DMSO-d 6 ) 6 10.68 (s, 1H, NH, Ex. With

D

2 0), 7.87 (d, 1H, Ar-H, J = 8.59 Hz), 7.79 (d, 1H, Ar-H, J = 8.59 Hz), 7.64 (d, 1H, Ar-H, J = 1.95 Hz), 7.54 - 7.57 (m, 2H, Ar-H), 7.11 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 15 2.73 (s, 3H, CH 3 ), 2.59 (s, 3H, CH 3 ); MS (FAB+) 399.0 [100, (M+H)], 164.1 [50, (Starting amine)*]; HRMS m/z (FAB+) 398.9663, C,5H1 3 81 BrN20 2

S

2 requires 398.9569, 396.9684, C1 5 H13 79 BrN20 2

S

2 requires 396.9680; HPLC tr 1.39 min (96 : 04 = MeOH :

H

2 0). 20 DGS03064 2,4-Dichloro benzoic acid (10 g, 0.0523 mol, 1 eq.) was heated to 115 OC with excess chlorosulphonic acid (10.5 mL, 0.1571 mol, 3 eq.) under N 2 for 18 h. The resulting mixture was cooled and consciously poured into ice-water. The resulted white precipitate was filtered out, washed with plenty of water and dried under vacuum over night. The 25 crude DGS03064 (11.5 g, 76 %) was used for the subsequent reaction without further purification. mp 173-174 OC; TLC Rf; 0.48 (4:1, CH 2

CI

2 /EtOAc); 1H NMR (CDC3) 6 8.28 (1H, s, Ar-H), 7.65 (1H, s, Ar-H); MS m/z (FAB+) 286.9 [100, (M+H)1; HRMS m/z (FAB+) 287.8798, C 7

H

3 35 C1 3 0 4 S requires 287.8818, 291.8755, C 7

H

3 37

C

3 0 4 S requires 291.8759. 30 DGSO3088A (STX524) Synthesised by method B. Two compounds were isolated - DGSO3088A and DGS03088A. White crystals of DGSO3088A (48 mg; 13%). mp 153-155 OC; TLC Rf : 0.79 EtOAc/Hexane (3:2); 'H NMR (CDC3) 6 8.31 (s, 1H, NH, Ex. With D20), 8.07 (s, 35 1H, NH, Ex. With D 2 0), 8.07 (s, 1H, Ar-H), 7.71 - 7.79 (m, 4H, Ar-H), 7.67 (d, 1H, Ar-H, J = 1.95 Hz), 7.58 (s, 1H, Ar-H), 7.27 (dd, 1H, Ar-H, J = 2.72 Hz and 8.59 Hz), 2.83 (s, WO 2004/037251 PCT/GB2003/004590 83 3H, CHa), 2.79 (s, 3H, CH 3 ); MS (FAB+) 562.9 [100, (M+H)+]; HRMS m/z (FAB+) 562.9825, C 23

H

17 3 Cl 2 N40,S requires 562.9839, 566.9778, C 23

H

1 7 37 1C 2

N

4 0 3 SS requires 566.9781; HPLC t, 1.33 min (96 : 04 = MeOH : H 2 0). 5 DGS03088-1 (STX575) White crystals of DGS03088-1 (31 mg; 12%). mp 147-148 OC; TLC Rf : 0.45 EtOAc/Hexane (3:2); 1 H NMR (CDC3) 6 8.45 (s, 1H, NH, Ex. With D 2 0), 8.17 (d, 1H, Ar H, J = 8.09 Hz), 8.04 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.50 (d, 1H, Ar-H, J = 1.83 Hz), 7.35 (dd, 1H, Ar-H, J = 1.83 Hz and 8.05 Hz), 2.85 (s, 3H, CH 3 ); LC-MS 418.1 [100, 10 (M')]; HPLC tr 1.97 min (96: 04 = MeOH : H 2 0). DGS03100A (STX552) Synthesised by method B. White crystals of DGS03100A (224 mg; 69%). mp 222-223 OC; TLC Rf: 0.56 CH 2 CI2/EtOAc (4:1); 'H NMR (DMSO-d 6 ) 5 10.27 (s, 1H, NH, Ex. With 15 D20), 9.16 - 9.17 (m, 1H, Ar-H), 8.48 - 8.51 (m, 2H, Ar-H), 8.36 - 8.38 (m, 2H, Ar-H), 8.23 - 8.25 (m, 1H, Ar-H), 7.67 - 7.34 (m, 3H, Ar-H), 7.51 - 7.12 (m, 1H, Ar-H), 7.09 7.12 (m, 1H, Ar-H), 2.67 (s, 3H, CH 3 ); LC-MS 355.7 [(M)+]; MS (FAB+) 356.0 [100, (M+H)*]; HRMS m/z (FAB+) 356.0531, C 17

H

14

N

3 0 2

S

2 requires 356.0527; HPLC t, 1.86 min (96: 04 = MeOH: H 2 0). 20 DGSO3102A (STX553) Synthesised by method B. Pale yellow crystals of DGSO3102A (170 mg; 52%). mp 89-90 'C; TLC Rf: 0.55 CH 2 Cl 2 /EtOAc (4:1); 1 H NMR (DMSO-d 6 ) 6 10.87 (s, 1H, NH, Ex. With

D

2 0), 8.28 - 8.24 (m, 1H, Ar-H), 8.06 - 8.22 (m, 2H, Ar-H), 8.05 (d, 1H, Ar-H, J = 8.20 25 Hz), 7.60 -7.77 (m, 2H, Ar-H), 7.47 (d, 1H, Ar-H, J = 1.95 Hz), 7.04 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 2.69 (s, 3H, CH 3 ); MS (FAB+) 355.0 [100, (M+H)*]; HRMS m/z (FAB+) 355.0576, C, 8 Hj 5

N

2 0 2

S

2 requires 355.0575; HPLC tr 1.93 min (96: 04 = MeOH:

H

2 0). 30 DGS03104A (STX554) Synthesised by method B. Yellow crystals of DGSO3104A (230 mg; 63%). mp 85-86 OC; TLC Rf: 0.65 CH 2

CI

2 /EtOAc (4:1); 1 H NMR (DMSO-d 6 ) 5 10.84 (s, 1H, NH, Ex. With D20), 8.40 - 8.42 (m, 2H, Ar-H), 8.22 - 8.23 (m, 1H, Ar-H), 7.76 - 7.78 (m, 1H, Ar-H), 7.58 - 7.65 (m, 2H, Ar-H), 7.51 - 7.56 (m, 1H, Ar-H), 7.23 - 7.25 (m, 1H, Ar-H), 7.05 35 7.07 (m, 1H, Ar-H), 2.79 (s, 6H, 2xCH 3 ), 2.69 (s, 3H, CH 3 ); MS (FAB+) 398.1 [100, WO 2004/037251 PCT/GB2003/004590 84 (M+H)]; HRMS m/z (FAB+) 398.0978, C 20

H

20

N

3 0 2

S

2 requires 398.0997; HPLC t, 2.01 min (96: 04 = MeOH: H 2 0). DGSO3116A (STX580) 5 Synthesised by method B. Pale yellow crystals of DGSO3116A (151 mg; 44%). mp 153 'C; TLC Rf: 0.55 CH 2 CI2/EtOAc (4:1); 'H NMR (DMSO-d 6 ) 6 10.96 (s, 1H, NH, Ex. With

D

2 0), 8.00 (d, 1H, Ar-H, J = 2.34 Hz), 7.90 (d, 1H, Ar-H, J = 8.59 Hz), 7.66 - 7.73 (m, 2H, Ar-H), 7.58 (d, 1H, Ar-H, J = 2.34 Hz), 7.17 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 2.74 (s, 3H, CH 3 ); MS (FAB+) 372.8 [100, (M+H)+]; HRMS m/z (FAB+) 375.9599, 10 0 14

H

1 1 37 0 2

N

2 0 2

S

2 requires 375.9502, 372.9606, C 14 H 1 3 5C1 2

N

2 0 2

S

2 requires 372.9639; HPLC tr 2.98 min (90: 10 = MeOH : H 2 0). DGSO3118A (STX581) Synthesised by method B. White crystals of DGSO3118A (416 mg; 42%). mp 88-89 OC; 15 TLC Rf: 0.49 CH 2

C

2 I/EtOAc (4:1); 1 H NMR (DMSO-d 6 ) 6 10.47 (s, 1H, NH, Ex. With

D

2 0), 7.74 (d, 1H, Ar-H, J = 8.59 Hz), 7.58 - 7.61 (m, 2H, Ar-H), 7.35 - 7.38 (m, 1 H, Ar H), 7.13 - 7.17 (m, 1H, Ar-H), 4.76 - 4.78 (m, 2H, CH 2 ), 3.75 - 3.79 (m, 2H, CH 2 ), 2.90 2.93 (m, 2H, CH 2 ), 2.73 (s, 3H, CHA); MS (FAB+) 456.0 [100, (M+H)+]; HRMS m/z (FAB+) 456.0663, C 19

H

17

F

3

N

3 0 3

S

2 requires 456.0663; HPLC tr 1.63 min (96: 04 = MeOH 20 : H 2 0). DGSO3120A (STX582) Synthesised by method B. Pale yellow crystals of DGSO3120A (185 mg; 55%). mp 91-92 OC; TLC Rf: 0.51 CH 2 Cl2/EtOAc (4:1); 'H NMR (DMSO-de) 6 10.35 (s, 1H, NH, Ex. With 25 D 2 0), 7.85 (d, 1H, Ar-H, J = 8.98 Hz), 7.69 (d, 1H, Ar-H, J = 2.34 Hz), 7.61 (dd, 1H, Ar H, J = 2.73 Hz and 8.98 Hz), 7.55 (d, 1H, Ar-H, J = 2.73 Hz), 7.20 (d, 1 H, Ar-H, J = 8.98 Hz), 7.15 (dd, 1H, Ar-H, J = 2.3 Hz and 8.59 Hz), 3.89 (s, 3H, OCH 3 ), 2.73 (s, 3H, CH 3 ); MS (FAB+) 369.0 [100, (M+H)]; HRMS m/z (FAB+) 371.0114, C 1 5

H

14 37

CIN

2 0 3

S

2 requires 371.0105, 369.0135, C 1 5

H

14 3 5

CN

2 0 3

S

2 requires 369.0134; HPLC tr 1.68 min (96 : 04 = 30 MeOH : H 2 0). DGSO3122A (STX731) Synthesised by method B. Two compounds were isolated - DGSO3122A and DGSO3122B. Yellow crystals of DGS03122A (67 mg; 22%). mp 272-273 0C; TLC Rf: 35 0.59 CH 2

CI

2 /EtOAc (4:1); 'H NMR (DMSO-d 6 ) 5 8.15 (m, 5H,, Ar-H), 8.02 - 8.09 (m, 4H, WO 2004/037251 PCT/GB2003/004590 85 Ar-H), 7.74 (d, 1H, Ar-H, J = 2.3 Hz), 7.14 (dd, 1H, Ar-H, J= 1.95 Hz and 8.59 Hz), 2.84 (s, 3H, CH 3 ); MS (FAB+) 495.0 [100, (M+H)*]; HPLC t, 1.79 min (90: 10 = MeOH: H 2 0). DGS03122B (STX583) 5 Yellow crystals of DGSO3122B (47 mg; 16%). mp 204-206 0C; TLC Rf : 0.48

CH

2

CI

2 /EtOAc (4:1); 'H NMR (DMSO-d 6 ) 5 10.95 (s, 1H, NH, Ex. With D 2 0), 8.03 - 8.07 (m, 2H, Ar-H), 7.86 - 7.91 (m, 2H, Ar-H), 7.77 - 7.81 (m, 1H, Ar-H), 7.55 (d, 1H, Ar-H, J = 1.95 Hz), 7.12 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 2.74 (s, 3H, CH 3 ); MS (FAB+) 330.0 [100, (M+H)]; HRMS m/z (FAB+) 330.0370, CjH1 2

N

3 0 2

S

2 requires 330.0371; 10 HPLC t, 1.84 min (90: 10 = MeOH : H 2 0). DGSO3124A (STX584) Synthesised by method B. Pale yellow crystals of DGSO3124A (125 mg; 55%). mp 188 189 OC; TLC Rf : 0.37 CH 2 Cl 2 /EtOAc (4:1); 'H NMR (DMSO-de) 6 10.09 (s, 1H, NH, Ex. 15 With D 2 0), 7.81 (d, 1H, Ar-H, J= 8.59 Hz), 7.63 (d, 1H, Ar-H, J= 8.20 Hz), 7.56 (d, 1H, Ar-H, J = 1.95 Hz), 7.14 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 6.96 (s, 1H, Ar-H), 6.81 (d, 1H, Ar-H, J = 8.59 Hz), 3.87 (s, 3H, OCH 3 ), 2.72 (s, 3H, CH 3 ), 2.28 (s, 3H, CH 3 ); MS (FAB+) 219.1 [20, (sulphonyl chloride-H)*], 349.0 [100, (M+H)*]; HRMS m/z (FAB+) 349.0678, C 16

H,

7

N

2 0 3

S

2 requires 349.0681; HPLC tr 1.80 min (96: 04 = MeOH : H 2 0). 20 DGSO3126A (STX585) Synthesised by method B. Pale yellow crystals of DGSO3126A (145 mg; 40%). mp 84-86 OC; TLC Rf: 0.71 CH 2 CI2/EtOAc (4:1); 1 H NMR (DMSO-d 6 ) 6 10.42 (s, 1H, NH, Ex. With

D

2 0), 7.88 (d, 1H, Ar-H, J = 8.59 Hz), 7.73 - 7.77 (m, 2H, Ar-H), 7.59 (d, 1H, Ar-H, J = 25 1.95 Hz), 7.41 - 7.46 (m, 2H, Ar-H), 7.22 - 7.26 (m, 2H, Ar-H), 7.13 (dd, 1H, Ar-H, J = 8.59 Hz and 2.34 Hz), 7.02 - 7.10 (m, 4H, Ar-H), 2.75 (s, 3H, CH 3 ); MS (FAB+) 397.0 [100, (M+H)*]; HRMS m/z (FAB+) 397.0671, C 20

H,

7

N

2 0 3

S

2 requires 397.0681; HPLC t, 1.93 min (96 : 04 = MeOH: H 2 0). 30 DGSO3130A (STX730) Synthesised by method B. Two compounds were isolated - DGSO3130A and DGSO3130B. Synthesised by method B. Pale yellow crystals of DGSO3130A (105 mg; 33%). mp 125-126 'C; TLC Rf: 0.55 CH 2

CI

2 /EtOAc (4:1); 'H NMR (DMSO-d 6 ) 5 8.21 8.24 (m, 4H, Ar-H), 8.13 (d, 1H, Ar-H, J = 8.59 Hz), 7.99 - 8.03 (m, 4H, Ar-H), 7.57 (d, 35 1H, Ar-H, J = 1.95 Hz), 7.03 (dd, 1 H, Ar-H, J = 8.59 Hz and 1.95 Hz), 2.82 (s, 3H, CH 3

);

WO 2004/037251 PCT/GB2003/004590 86 2.69 (s, 6H, 2xCH 3 ); MS (FAB+) 529.0 [100, (M+H)*]; MS (FAB-) 527.1 [70, (M-H)+], 345.0 [100, (M-2-Acetyl sulphonyl chloride)']; HPLC tr 1.81 min (96: 04 = MeOH : H 2 0). DGSO3130B (STX701) 5 Pale yellow crystals of DGSO3130A (45 mg; 14%). mp 169 OC; TLC Rf : 0.42

CH

2 Cl 2 /EtOAc (4:1); 1 H NMR (DMSO-d 6 ) 6 10.63 (s, 1H, NH, Ex. With D20), 8.04 - 8.07 (m, 2H, Ar-H), 7.86 -7.89 (m, 3H, Ar-H), 7.59 (d, 1H, Ar-H, J = 1.95 Hz), 7.13 (dd, 1H, Ar-H, J = 8.9 Hz and 2.3 Hz), 3.73 (s, 3H, CH 3 ); 2.56 (s, 3H, CH 3 ); MS (FAB+) 347.0 [100, (M+H)+], 219.1 [10, (sulphonyl chloride+H)+]; HRMS m/z (FAB+) 347.0522, 10 0 16

H,

15

N

2 0 3

S

2 requires 347.0524; HPLC t, 1.77 min (96 : 04 = MeOH : H 2 0). DGSO3134A (STX7O3) Synthesised by method B. Pale yellow crystals of DGSO3134A (91 mg; 23%). mp 206 207 'C; TLC Rf: 0.81 CH 2

CI

2 /EtOAc (4:1); 1 H NMR (DMSO-d 6 ) 65 10.37 (s, 1H, NH, Ex. 15 With D 2 0), 7.87 (d, 1H, Ar-H, J= 8.59 Hz), 7.46 (d, 1H, Ar-H, J = 1.95 Hz), 7.19 (s, 2H, Ar-H), 7.07 (dd, 1H, Ar-H, J = 8.59 Hz and 1.95 Hz), 4.13 - 4.20 (m, 2H, 2x(CHz) 2 H), 2.83 - 2.89 (m, 1H, (CH 3 )2H), 2.72 (s, 3H, CH 3 ), 1.15 (d, 12H, 4x(CH 3

)

2 , J = 7.03 Hz), 1.11 (d, 9H, 2x(CH 3

)

2 , J = 6.64 Hz); LC-MS 429.72 (M) ; HPLC tr 2.84 min (90 : 10 = MeOH : H 2 0). 20 DGSO3136A (STX704) Synthesised by method B. Pale yellow crystals of DGSO3136A (225 mng; 71%). mp 54-55 OC; TLC Rf: 0.50 CH 2 CIJ2EtOAc (4:1); 1 H NMR (CDCl3) 5 7.65 (m, 3H, Ar-H), 7.58 (d, 1H, Ar-H, J = 2.34 Hz), 7.18 (dd, 1H, Ar-H, J = 8.6 Hz and 1.95 Hz), 6.84- 6.85 (m, 2H, Ar 25 H), 6.82 (s, 1H, NH, Ex. With D20), 4.51 - 4.60 (m, 1H, (CH 3

)

2 H), 2.80 (s, 3H, CH 3 ), 1.31 (s, 6H, (CH 3

)

2 ); LC-MS 347.6 (M)'; HRMS m/z (FAB+) 347.0847, C 1 7 HjN 2 0 2

S

2 requires 347.0837; HPLC tr 2.39 min (90: 10 = MeOH : H 2 0). DGSO3138B (STX705) 30 Synthesised by method B. Pale yellow crystals of DGSO3138B (24 mg; 7%). mp 248 OC; TLC Rf: 0.52 CH 2 CI2/EtOAc (4:1); 1 H NMR (CDC3) 6 8.18 (d, 1H, Ar-H, J = 8.59 Hz), 8.15 (d, 1H, Ar-H, J = 1.95 Hz), 7.89 - 8.04 (m, 4H, Ar-H), 7.51 (dd, 1 H, Ar-H, J = 8.20 Hz and 1.95 Hz), 7.27 (s, 1H, NH, Ex. With D20), 2.89 (s, 3H, CH 3 ), 1.59 (s, 3H, CH 3 ); LC-MS 372.90 (M+CH3CN)*; HRMS m/z (FAB+) 371.2281, C 16

H

1 5N 2 0 4

S

2 requires 35 371.2278; HPLC tr 2.22 min (90: 10 = MeOH : H 2 0).

WO 2004/037251 PCT/GB2003/004590 87 DGSO3140A (STX711) Synthesised by method B. Brown crystals of DGSO3140A (85 mg; 26%). mp 73-75 OC; TLC Rf: 0.59 CH 2

CI

2 /EtOAc (4:1); 1 H NMR (CDC3) 5 7.85 (d, 1H, Ar-H, J = 8.59 Hz), 7.80 (d, 1H, Ar-H, J = 8.59 Hz), 7.68 (s, 1H, NH, Ex. With D20), 7.57 (d, 1H, Ar-H, J = 5 1.95 Hz), 7.54 (s, 1H, NH, Ex. With D 2 0), 7.24 (d, 1H, Ar-H, J= 2.34 Hz), 7.18 (dd, 1H, Ar-H, J = 8.20 Hz and 1.95 Hz), 7.03 (dd, 1H, Ar-H, J = 8.59 Hz and 2.34 Hz), 6.77 (dd, 1H, Ar-H, J = 8.59 Hz and 2.34 Hz), 2.78 (s, 3H, CH 3 ), 2.24 (s, 3H, CH 3 ); LC-MS 362.32 (M)*; HRMS m/z (FAB+) 361.0587, C 1 6

H

16

N

3 0 3

S

2 requires 361.0636; HPLC t, 2.09 min (90: 10 = MeOH : H 2 0). 10 DGSO3142A (STX706) Synthesised by method B. Pale yellow crystals of DGSO3142A (79 mg; 24%). mp 89-91 oC; TLC Rf: 0.65 CH 2

C

2 /EtOAc (4:1); 1 H NMR (CDCI3) 6 7.78 (d, 1H, Ar-H, J = 8.20 Hz), 7.61 (d, 1H, Ar-H, J = 1.56 Hz), 6.98 (dd, 1H, Ar-H, J = 1.95 Hz and 8.20 Hz), 6.93 (s, 15 1H, Ar-H), 6.92 (s, 1H, NH, Ex. With D20), 3.99 (s, 6H, 2xCH 3 ), 3.93 (s, 6H, 2xCH 3 ), 2.85 (s, 3H, CH 3 ); LC-MS 361.48 (M)*; HRMS m/z (FAB+) 361.1605, C 18

H

21

N

2 0 2

S

2 requires 361.1606; HPLC t, 2.26 min (90 : 10 = MeOH : H 2 0). DGSO3144A (STX707) 20 Synthesised by method B. Pale yellow crystals of DGSO3144A (79 mg; 24%). mp 89-91 OC; TLC Rf: 0.69 CH 2

CI

2 /EtOAc (4:1); 'H NMR (CDC13) 5 7.70 (d, 1 H, Ar-H, J= 8.59 Hz), 7.58 (d, 1H, Ar-H, J = 2.34 Hz), 7.39 (dd, 1H, Ar-H, J = 2.34 Hz and 8.59 Hz), 7.19 (d, 1H, Ar-H, J = 1.95 Hz), 7.17 (t, 1 H, Ar-H, J = 1.95 Hz), 6.83 (d, 1H, Ar-H, J = 8.59 Hz), 6.59 (s, 1H, NH, Ex. With D20), 3.89 (s, 3H, OCH 3 ), 3.76 (s, 3H, OCH 3 ), 2.81 (s, 3H, 25 CH 3 ); LC-MS 363.02 (M)'; HRMS m/z (FAB+) 365.0642, Cj6H 1 7

N

2 0 4

S

2 requires 365.0585; HPLC t, 2.15 min (90: 10 = MeOH : H 2 0). DGS03146A (STX708) Synthesised by method B. Pale yellow crystals of DGSO3146A (181 mg; 51%). mp 175 30 OC; TLC Rf: 0.57 CH 2 Cl 2 /EtOAc (4:1); 'H NMR (CDC13) 5 7.71 (dd, 1H, Ar-H, J = 2.3 Hz and 8.98 Hz), 7.59 (d, 1H, Ar-H, J = 1.95 Hz), 7.43 (d, 1H, Ar-H, J = 8.98 Hz), 7.21 (dd, 1H, Ar-H, J= 1.95 Hz and 8.59 Hz), 6.67 (s, 1H, NH, Ex. With D 2 0), 2.81 (s, 3H, OCH 3 ), 1.59 (s, 6H, 2xCH 3 ), 1.29 (s, 6H, 2xCH 3 ); LC-MS 377.01 (M)+; HRMS m/z (FAB+) 377.0988, C 18

H

2 1 N 2 0 3

S

2 requires 377.0994; HPLC tr 2.53 min (90: 10 = MeOH : H 2 0).

WO 2004/037251 PCT/GB2003/004590 88 DGSO3148A (STX709) Synthesised by method B. Off-white crystals of DGSO3148A (102 mg; 31%). mp 214 215 OC; TLC Rf: 0.62 CH2CI 2 /EtOAc (4:1); 1 H NMR (CDC 3 ) 5 7.71 - 7.73 (m, 2H, Ar-H), 5 7.60 - 7.61 (m, 1H, Ar-H), 7.44 - 7.46 (m, 2H, Ar-H), 7.21 - 7.24 (m, 2H, Ar-H), 6.61 (s, 1H, NH, Ex. With D 2 0), 2.83 (s, 3H, CH 3 ), 1.31 (s, 9H, (CH 3

)

3 ); LC-MS 360.12 (M)'; HRMS m/z (FAB+) 361.1057, C 18

H

21

N

2 0 3

S

2 requires 361.1044; HPLC t, 2.67 min (90 : 10 = MeOH : H 2 0). 10 DGSO3150A (STX710) Synthesised by method B. Pale yellow crystals of DGSO3150A (101 mg; 30%). mp 200 201 OC; TLC Rf: 0.50 CH 2

CI

2 /EtOAc (4:1); 1 H NMR (CDCI 3 ) 5 7.65 (d, 1H, Ar-H, J= 8.59 Hz), 7.44 (d, 1H, Ar-H, J = 1.95 Hz), 7.09 (dd, 1H, Ar-H, J = 1.95 Hz and 8.59 Hz), 6.75 (s, 1H, NH, Ex. With D 2 0), 2.79 (s, 3H, CH 3 ), 2.57 (s, 6H, 2xCH 3 ), 2.24 (s, 3H, CH 3 ), 15 2.19 (s, 6H, 2xCH 3 ); LC-MS 374.10 (M)*; HRMS m/z (FAB+) 375.1195, C 19

H

23

N

2 0 2

S

2 requires 375.1201; HPLC t 3.15 min (80: 20 = MeOH : H 2 0). DGSO3152A (STX712) Synthesised by method B. Pale yellow crystals of DGS03152A (120 mg; 33%). mp 181 20 182 OC; TLC Rf: 0.65 CH 2

CI

2 /EtOAc (4:1); 'H NMR (CDC1 3 ) 6 7.63 (d, 1H, Ar-H, J= 8.59 Hz), 7.59 (d, 1H, Ar-H, J = 2.3 Hz), 7.22 (dd, 1H, Ar-H, J= 2.3 Hz and 8.59 Hz), 4.91 (s, 1H, NH, Ex. With D 2 0), 3.82 (s, 3H, CH 3 ); LC-MS 392.96 (M)*; HRMS m/z (FAB+) 394.9941, C 14

H

8

F

5

N

2 0 2

S

2 requires 394.9947; HPLC tr 2.49 min (90: 10 = MeOH : H 2 0). 25 DGS03158A (STX713) Synthesised by method B. Yellow crystals of DGSO3158A (158 mg; 40%). mp 334-335 OC; TLC Rf: 0.47 CH 2 CIJ2/EtOAc (4:1); 1 H NMR (CDCI 3 ) 5 7.65 (d, 1H, Ar-H, J = 8.59 Hz), 7.47 (d, 1H, Ar-H, J= 2.3 Hz), 7.10 (dd, 1H, Ar-H, J = 2.3 Hz and 8.59 Hz), 6.69 (s, 1H, NH, Ex. With D 2 0), 2.79 (s, 3H, CH 3 ), 2.61 (t, 2H, CH 2 , J = 6.64 Hz), 2.55 (s, 3H, CH 3 ), 30 2.51 (s, 3H, CH 3 ), 2.08 (s, 3H, CH 3 ), 1.79 (t, 2H, CH 2 , J = 7.03 Hz), 1.29 (s, 6H, 2xCH 3 ); LC-MS 431.11 (M)+; HPLC tr 3.24 min (90: 10 = MeOH : H 2 0).

WO 2004/037251 PCT/GB20031004590 89 Synthesis of Benzothiazole Arylsulphonamide Derivatives SH N b 0 'N R

H

2 N C s H 2 Na Nl "SN IC R = - 2

H

5 R =-C 2 H1 R = -O 2

H

4 00H 3 R =-C 2

H

4 0CH 3 R = -CH 2 0000 2

H

5 R = -CH 2

COOC

2

H

5 NS~N 0 H N H S N NN H H C N \ IC Ir' s HH R =-H R = -OH 3 Ar =3-CI-2-CH 3 -pheny), R = -H Ar =3-CI-2-CH 3 -phenyl, R =-OH 3 , Ar =4-n-propylphenyl, R = -OH 3 , Ar =2.5-dichiorophenyl, R -OH 3 , HH -R N H >= b 0! NS >

H

2 N S 01- 6'' -- S s" H R R = -OH 3 R = -CH 2

COOC

2 HS a) RX, NaH, THF rt. b) ArSO 3 O, DOMPyridine or ArSO 3 OI, DOM,PyridineIDMAP c) Diethylamine, DOM, AIC1 3 d) RX, K 2 00 3 , Acetone, reflux WO 2004/037251 PCTIGB2003/004590 90 Na 0 2 N ~NN 0 2 N

H

2 N N j N I >-ci + \>I SS H 2 S 0 H c c \\ N S- 0( CNN '0 S sH Nl 0 ci H R =-NHCH 3 R =-NHCH 3 R =-N(C 2

H

5 9) 2 R = -N(C 2

H

5

)

2 ,a N a N b ~ N N Ra c R

NO

2

NH

2 Q.NH R = -OH 3 R -H R = -OCH 3 R R= -CH 3 ci ci

NO

2

NH

2 b\ 0 ~ ~ N c - 'NH ~N a S/ b I -C rN -~ N N 0 2 N H 2 N s'P' S H Ar = 3-CI-2-CH 3 -phenyl Ar = 2,5-dichiorophenyl a) HNO 3 , H 2 S0 4 -5 - OC b) H 2 , 5% Pd/C, C 2

H

5 OH, c) ArSO 3 CI, DCM,Pyridine or ArSO 3 CI, DGM,Pyridine/DMAP d) amine, THF, reflux WO 2004/037251 PCT/GB20031004590 91

H

2 N N b N

H

2 N~~~~ b~ I~ C sS

H

2 N N b 0' I - Ar-~ Ar 3-CI-2-CH 3 -phenyl Ar = 2,5-dichiorophenyl Ar =4-n-propylphenyl 0 0 N C Br cN -SS 0 I N N a) N-chlorosuccinimide, IPA b) ArS0 3 CI, DCM,Pyridine or ArSO 3 CI, DCM,Pyridine/DMAP c) N-bromosucinimide, 0014, benzoyl peroxide d) 3 -chloro-2methylbenzenesulphomamide, K 2 00 3 , CH 3

CN

WO 2004/037251 PCT/GB20031004590 92 0N 0 HN ONO 0 SN K l s c H H STX755, XDSO1 141A TX73, XDSOI 4 14 5B ,XS0 4 I- N HN C" &~ H STX75, XDS1 13945A86 X SOI 63 XDS8145 J1 a' NN" N' N Nl /S 0> CIa 'ON S' H H I STX888, XDS01 139 c - STX889OOI18B c clSTX889,XSlI8 DS ,8 o N a--- N 'I >=O ~ N S O= S CS H 01 STX753, XDS01I188 ST3, XS~B STX76, STX889 XDSO 188AXDS1 19 0 N H Na 0, NicN N CH I -, Y O SX5,XS113STX86B, XDS01I163B STX4, S16 aCI STX767, XDSO 1151A WO 2004/037251 PCT/GB20031004590 93 0 H H 0\N N \\ NS\ I - HH N STX8, XISII7ST85 DS 176 ST83,XDOI :N c N s H c STX87, XDSO16 SX8, XDS00176 STX6 X02117 N~ N \>- \l S NH 0 ~ sN 5/ s\\NH S 61 H SX7,XS11STX996, XDS02038 CI XSO214 S1X997XDSD020O49 / -c NS_ NH 0 / NH \ ::S = cl/ STX99, XS02047 SXDS020S48B2 I~ cl STX997 XDS02048 N HH CI CI c sT95 XDSO2O44 XDO24 'N - S l H-- cl-- s STXOI7, XDS2045B STX 2, XDS22B STs00 XDS202A WO 2004/037251 PCT/GB2003/004590 94 General Method for the Preparation of N-Benzothiazole Benzenesulphonamide derivatives: To a solution arylsulphonyl chloride (1.1 eq.) in DCM (5-10 mL) were added pyridine (2.2 5 eq.) and catalytic amount of DMAP. The solution was stirred at room temperature under nitrogen for 10 minutes. Then the amine (1 eq.) was added and the reaction mixture was stirred at room temperature under nitrogen for 4-16 hrs. The resulting mixture was partitioned between DCM and 5% sodium bicarbonate. The organic layer was washed with brine, dried over MgSO 4 , and concentrated to give a yellow residue. The crude 10 compound was then purified by flash chromatography to give desired benzenesulphonamide as crystalline solid. (Yield 40-90%). Synthesis of 2-Alkylsulfanyl-benzothiazol-6-yl-amine To a solution of 6-amino-2-merceptobenzothiazole (273mg, 1.5 mmol) in anhydrous THF 15 (10 mL) was added NaH (60% dispersion, 1.5 mmol), followed by alkyl halide (1.5 mmol). The mixture was stirred at rt for 24h, partitioned between ethyl acetate and 5% sodium bicarbonate. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to a yellow solid, which was purified with recrystallization or flash chromatography. (Yield 60-90%). 20 The following amines were synthesized with the method described above: 2-Ethylsulfanylbenzothiazol-6-ylamine Yellow crystalline solid. mp 77-780C (lit.77 0 C). TLC single spot at Rf 0.78 (8% 25 methanol/DCM); 1 H NMR (270 MHz, DMSO): 6 7.50 (1H, d, J= 8.5 Hz, 4-H), 6.98 (1H, d, J= 2.2 Hz, 7-H), 6.69 (1H, dd, J= 8.5, 2.2 Hz, 5-H), 5.33 (2H, broad, NH 2 ), 3.23 (2H, q, J= 7.3 Hz, SCH 2 ), 1.35 (3H, t, J= 7.3 Hz, CH 3 ). (Francolor, S.A.; US 2500093; 1945) 30 2-(2-Methoxyethylsulfanyl)-benzothiazol-6-ylamine Yellow thick syrup. TLC single spot at Rf 0.65 (30% ethyl acetate/DCM); 'H NMR (270 MHz, DMSO): 6 7.49 (1H, d, J = 8.8 Hz, 4-H), 6.97 (1H, d, J = 2.2 Hz, 7-H), 6.69 (1H, dd, J = 8.8, 2.2 Hz, 5-H), 5.34 (2H, broad, NH 2 ), 3.63 (2H, t, J = 6.3 Hz, CH 2 ), 3.43 (2H, t, J = 6.3 Hz, CH 2 ), 3.26 (3H, s, CH 3

).

WO 2004/037251 PCT/GB2003/004590 95 (6-aminobenzothiazol-2-ylmercapto)-acetic acid ethyl ester Off white solid. mp 87-890C (lit.92 0 C, [20]); TLC single spot at Rf 0.72 (8% methanol/DCM); 1 H NMR (270 MHz, DMSO): 5 7.48 (1H, d, J = 8.7 Hz, 4-H), 7.01 (1H, 5 d, J= 1.8 Hz, 7-H), 6.71 (1H, dd, J= 8.7, 1.8 Hz, 5-H), 5.58 (2H, broad, NH 2 ), 4.17 (2H, s, SCH 2 ), 4.12 (2H, t, J= 7.3 Hz, CH 2 ), 1.19 (3H, t, J= 7.3 Hz, CH 3 ). The following compounds were synthesized with the general method for N benzothiazole benzenesulphonamide: 10 3-Chloro-N-(2-ethylsulfanylbenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX751, XDS01141) Off-white solid (220 mg; 55%). TLC single spot at Rf: 0.83 (17% EtOAc/DCM); HPLC purity 96% (tR 1.9 min in methanol); 1 HNMR (400MHz, DMSO-d6) 5 10.8 (1H, s, NH), 15 7.89 (1H, dd, J = 8.0, 1.0 Hz, 6'-H of benzene), 7.71 (1H, d, J = 8 Hz, 4-H of benzothiazole), 7.70 (1H, d, J= 2 Hz, 7-H of benzothiazole), 7.70 (1H, dd, J = 8.0, 1.0 Hz, 4'-H of benzene), 7.36 (1H, t, J = 8 Hz, 5'-H of benzene), 7.15 (1H, dd, J = 8.0, 2.0 Hz, 5-H of benzothiazole), 3.30 (2H, q, J = 7.0 Hz, SCH 2 ), 2.66 (3H, s, CH 3 ), 1.38 (3H, t, J = 7.0 Hz, CH 3 ); APCI-MS 397.99 (M)+; FAB-HRMS calcd for C16H16CIN202S3 (MH') 20 399.0062, found 399.0048.

[

6

-(

3 -Chloro-2-methylbenzenesulphonylamino)-benzothiazol-2-ylsulfanyl]-.acetic acid ethyl ester (STX752, XDS01142) White crystalline solid (210 mg; 46%). TLC single spot at Rf: 0.69 (17% EtOAc/DCM); 25 HPLC purity 99% (tR 2.9 min in 10% water-methanol); 1 HNMR (400MHz, DMSO-d6) 6 10.8 (1 H, s, SO 2 NH), 7.88 (1 H, dd, J= 8.0, 1.0 Hz, 6'-H of benzene), 7.72 (1 H, d, J = 2.0 Hz, 7-H of benzothiazole), 7.69 (1H, dd, J = 8.0, 1.0 Hz, 4'-H of benzene), 7.68 (1H, d, J = 8.0 Hz, 4-H of benzothiazole), 7.36 (1H, t, J= 8.0 Hz, 5'-H of benzene), 7.15(1H, dd, J = 8.0, 2.0 Hz, 5-H of benzothiazole), 4.25 (2H, s, 2-SCH 2 -), 4.13 (2H, q, J = 7.1 Hz, 30 COOCH 2 ), 2.64 (3H, s, CH 3 ), 1.17 (3H, t, J = 7.1 Hz, 2-COOCH 2

CH

3 ); APCI-MS 456.0 (M)'; FAB-HRMS calcd for C18H18CIN20 4

S

3 (MH) 457.0117, found 457.0109. 3-Chloro-N-[2-(2-methoxyethylsulfanyl)-benzothiazol-6-yl]-2 methylbenzenesulphonamide (STX754, XDS01144) 35 Off-White solid (150 mg; 77%). TLC single spot at Rf 0.60 (17% EtOAc/DCM); HPLC purity 94% (tR 3.1 min in 10% water-methanol); 1 HNMR (400MHz, DMSO-d6) 6 10.8 WO 2004/037251 PCT/GB2003/004590 96 (1 H, s, SO 2 NH), 7.88 (1H, dd, J = 8, 1 Hz, 6'-H of benzene), 7.71 (1H, d, J = 8 Hz, 4-H of benzothiazole), 7.70 (1H, dd, J= 8, 1 Hz, 4'-H of benzene), 7.69 (1H, d, J= 2 Hz, 7-H of benzothiazole), 7.36 (1H, t, J = 8 Hz, 5'-H of benzene), 7.15(1H, dd, J = 8, 2 Hz, 5-H of benzothiazole), 3.64 (2H, t, J = 6 Hz, CH 2 ), 3.50 (2H, t, J = 6 Hz, SCH 2 ), 3.27 (3H, s, 5 CH 3 ), 2.65 (3H, s, CH 3 ); APCI-MS 428.0 (M)'; FAB-HRMS calcd for C17H18CIN203S 3 (MH) 429.0168, found 429.0159. 2-[6-(3-Chloro-2-methylbenzenesulphonylamino)-benzothiazol-2-ylsulfanyl]-N,N. diethylacetamide (STX755, XDS01145) and 2-[6-(3-chloro-2-methyl 10 benzenesulphonylamino)-benzothiazol-2-yl]-N,N-diethylacetamide (STX763, XDS01145B) To a suspension of AIC1 3 (50 mg) in DCM (5 ml) was added diethylamine ( 0.4 ml). The solution was stirred under nitrogen at room temperature for 10 minutes. [6-(3-Chloro-2 methyl-benzenesulphonylamino)-benzothiazol-2-ylsulfanyl]-acetic acid ethyl ester 15 (STX752, 100 mg) was added and the mixture was kept stirring at room temperature for 30 minutes. The reaction was quenched with water, partitioned between DCM and 5% NaHCO 3 . The organic phase was washed with water, dried over MgSO 4 and evaporated in vacuo to give a yellow residue, which was purified with flash column chromatography using 20-30% ethyl acetate-DCM as eluting solvent. STX755 (50 mg, 47%) was 20 obtained as white solid. TLC single spot at Rf 0.60 (25% EtOAc/DCM); HPLC purity 89% (tR 2.7 min in 10% water-methanol); 1 HNMR (270MHz, DMSO-d6) 5 10.7 (1H, s,

SO

2 NH), 7.86 (1H, d, J = 8 Hz, 6'-H of benzene), 7.64-7.68 (3H, m, 4'-H of benzene and 4,7-H of benzothiazole), 7.34 (1H, t, J = 8 Hz, 5'-H of benzene), 7.12 (1H, dd, J = 8, 2 Hz, 5-H of benzothiazole), 4.42 (2H, s, 2-SCH 2 -), 3.26-3.38 (4H, m, -N(CH 2

)

2 -), 2.50 (3H, 25 s, 1'-CH 3 ), 1.17 (3H, t, J = 7 Hz, -NCH2CH 3 ), 1.00 (3H, t, J = 7 Hz, -NCHCQH 3 ); APCI-MS 484.0 (M)'; FAB-HRMS calcd for C20H23CIN30 3

S

3 (MH) 484.0590, found 484.0584. STX763 (25 mg, 25%) was obtained as white solid. TLC single spot at Rf 0.39 (25% EtOAc/DCM); LCMS purity 98% (tR 6.9 min in 10% water-CH 3 CN); 'HNMR (400 MHz, DMSO-d6) 6 10.8 (1H, s, SO 2 NH), 7.88 (1H, dd, J = 8.1, 1.2 Hz, 6'-H of benzene), 7.79 30 (1H, d, J = 8.6 Hz, 4-H), 7.72 (1H, d, J = 2.0 Hz, 7-H), 7.68 (1H, dd, J = 8.1, 1.2 Hz, 4'-H of benzene), 7.35 (1H, t, J= 8.1 Hz, 5'-H of benzene), 7.17 (1H, dd, J= 8.6, 2 Hz, 5-H of benzothiazole), 4.2 (2H, s, 2-SCH 2 -), 3.26-3.38 (4H, m, -N(CH 2

)

2 -), 2.65 (3H, s, CH 3 ), 1.10 (3H, t, J = 7 Hz, -NCH2QH 3 ), 1.02 (3H, t, J = 7 Hz, -NCH 2

CH

3 ); APCl-MS 451.0 (M)+; FAB-HRMS calcd for C20H23CIN303S 2

(MH

+

) 452.0869, found 452.0870. 35 3-Chloro-N-benzothiazol-6-yl-2-methylbenzenesulphonamide (STX750, XDS01139) WO 2004/037251 PCT/GB2003/004590 97 Light pink needles (260 mg; 77%). TLC single spot at Rf 0.46 (17% EtOAc/DCM); HPLC purity 99% (tR 2.5 min in 10% water-methanol); 1 HNMR (400MHz, DMSO-d6) 6 10.9 (1H, S, SO 2 NH), 9.25 (1H, s, 2-H of benzothiazole), 7.95 (1H, d, J = 9 Hz, 4-H of benzothiazole), 7.92 (1H, dd, J = 8.0, 1.0 Hz, 6'-H of benzene), 7.84 (1H, d, J = 2 Hz, 7 5 H of benzothiazole), 7.70 (1H, dd, J = 8.0, 1.0 Hz, 4'-H of benzene), 7.37(1H, t, J = 8 Hz, 5'-H of benzene), 7.25 (1H, dd, J = 9.0, 2.0 Hz, 5-H of benzothiazole), 2.66 (3H, s, CH 3 ); APCI-MS 337.9 (M)-; FAB-HRMS calcd for C20H23CIN303S 2 (MH*) 452.0869, found 452.0870. 10 3-Chloro-N-(2-methylbenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX886, XDS01187B) Off-white solid. TLC single spot at Rf 0.65 (10% methanol/DCM); HPLC purity >99% (tR 2.4 min in 10% water-methanol); 'HNMR (270MHz, DMSO-d6) 6 10.7 (1H, s, NH), 7.87 (1H, dd, J = 7.8, 1.9 Hz, ArH), 7.75 (1H, d, J = 8.8 Hz, ArH), 7.69 (1H, d, J= 2.2 15 Hz, ArH), 7.68 (1H, dd, J = 7.8, 1.9 Hz, ArH), 7.34 (1 H, t, J = 7.8 Hz, ArH), 7.15 (1H, dd, J = 8.8, 2.2 Hz, ArH), 2.71 (3H, s, CH 3 ), 2.63 (3H, s, CH 3 ); APCI-MS 351 (M-H)*; FAB HRMS calcd for C15H14CIN202S2 (MH

+

) 353.0185, found 353.0197. N-(2-methylbenzothiazol-6-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3.-chloro-.2.

20 methylbenzenesulphonamide (STX887, XDS01187A) Off-white powder. TLC single spot at Rf 0.89 (10% methanol/DCM); HPLC purity 91% (tR 3.1 min in 10% water-methanol); 1 HNMR (270MHz, DMSO-d6) 65 8.09 (1H, d, J= 2.2 Hz, ArH), 7.92 (1H, d, J = 8.6 Hz, ArH), 7.85-7.90 (4H, m, ArH), 7.46 (2H, t, J = 8.0 Hz, ArH), 7.35 (1H, dd, J= 8.8, 2.2 Hz, ArH), 2.81 (3H, s, CH 3 ), 2.33 (6H, s, 2 x CH 3 ); APCl 25 MS 539 (M-H)*; FAB-HRMS calcd for C22H19CI2N204S3 (MH

+

) 540.9884, found 540.9897. 2,5-Dichloro-N-(2-methylbenzothiazol.6-yl)-benzenesulphonamide (STX888, XDS01188B) 30 White crystalline solid. TLC single spot at Rr 0.68 (10% methanol/DCM); HPLC purity >99% (tR 2.3 min in 10% water-methanol); 1 HNMR (270MHz, DMSO-d6) 5 10.9 (1H, s, NH), 7.98 (1H, d, J= 2.3 Hz, ArH), 7.76 (1H, d, J = 8.9 Hz, ArH), 7.74 (1H, d, J = 2.3 Hz, ArH), 7.69 (1H, dd, J= 8.6, 2.3 Hz, ArH), 7.66 (1H, d, J = 8.6 Hz, ArH), 7.18 (1H, dd, J = 8.9, 2.3 Hz, ArH), 2.71 (3H, s, CH 3 ); APCI-MS 371 (M-H) t ; FAB-HRMS calcd for 35 C14H11CI2N202S2 (MH*) 372.9639, found 372.9651.

WO 2004/037251 PCT/GB2003/004590 98 N-(2-Methylbenzothiazol-6-yl)-N-(2,5-dichlorophenylsulphonyl)-2,5-dichloro benzenesulphonamide (STX889, XDS01 188A) Yellow solid. TLC single spot at Rf 0.72 (10% methanol/DCM); HPLC purity 94% (tR 2.9 5 min in 10% water-methanol); 1 HNMR (270MHz, DMSO-d6) 6 8.13 (1H, d, J 2.2 Hz, ArH),, 7.99 (2H, d, J = 2.4 Hz, ArH), 7.90-7.94 (3H, m, ArH), 7.77 (2H, d, J = 8.4 Hz, ArH), 7.29 (1 H, dd, J = 8.6, 2.2 Hz, ArH), 2.86 (3H, s, CH 3 ); APCI-MS 581 (M)'; FAB HRMS calcd for C20H13CI4N204S3 (MH) 580.8792, found 580.8777. 10 N-(2-Methylbenzothiazol-6-yl)-4-propylbenzenesulphonamide (STX890, XDS01189) Off-white solid. TLC single spot at Rf 0.72 (10% methanol/DCM); HPLC purity 99% (tR 2.3 min in 10% water-methanol); 1 HNMR (270MHz, DMSO-d6) 6 10.3 (1H, s, NH), 7.67 (1H, d, J= 8.7 Hz, ArH) 7.64 (1H, d, J= 1.5 Hz, ArH), 7.59 (2H, d, J= 7.7 Hz, ArH), 7.27 (2H, d, J= 7.7 Hz, ArH), 7.09 (1H, dd, J= 8.7, 1.5 Hz, ArH), 2.65 (3H, s, CH 3 ), 2.49 15 (2H, t, J= 7.9 Hz, CH 2 ), 1.47 (2H, sextet, J= 7.9 Hz, CH 2 ), 0.58 (3H, t, J= 7.9 Hz, CH 3 ); APCI-MS 345 (M-H)+; FAB-HRMS calcd for C17H19N202S2 (MH) 347.0888, found 347.0904. 3-Chloro-2-methyl-N-(2-oxo-2,3-dihydro-benzothiazol-6-yl)-benzenesulphonamide 20 (STX753, XDS01143) White crystalline solid (160 mg; 45%). TLC single spot at Rf 0.42 (17% EtOAc/DCM); HPLC purity 98% (tR 2.3 min in 10% water-methanol); 1 HNMR (400MHz, DMSO-d6) 6 11.8 (1H, s, 3-NH), 10.5 (1H, s, SO 2 NH), 7.81 (1H, dd, J = 8, 1 Hz, 6'-H of benzene), 7.71 (1H, dd, J= 8, 1 Hz, 4'-H of benzene), 7.36 (1H, t, J = 8 Hz, 5'-H of benzene), 7.28 25 (1H, d, J = 2 Hz, 7-H of benzothiazole), 6.93-6.98 (2H, m, 4,5-H of benzothiazole), 2.63 (3H, s, CH 3 ); APCI-MS 353.7 (M)'; FAB-HRMS calcd for C14H12CIN203S2 (MH*) 354.9978, found 354.9980. 3-Chloro-N-methyI-N-(3-methyl-2-oxo-2,3-dihydro-benzothiazol-6-yl)-2 30 methylbenzenesulphonamide (STX831, XDS01163) To a solution of STX753 (66 mg, 0.19 mmol) in acetone (3 mL) was added potassium carbonate (66 mg), followed by methyl iodide (66 mg). The mixture was stirred at rt for 2 h, extracted into DCM and washed with brine. After drying over sodium sulphate, the solvent was removed in vacuo to give an oily residue that was purified with flash 35 chromatography. Off white solid (59 mg, 80%) was obtained. TLC single spot at Rf 0.37 (100% DCM); HPLC purity 99% (tR 2.0 min in 10% water-methanol); 1 HNMR (400MHz, WO 2004/037251 PCT/GB2003/004590 99 DMSO-d6) 6 7.73-7.80 (2H, m, ArH), 7.61 (1H, d, J = 2.1 Hz, ArH), 7.42 (1H, t, J = 8.0 Hz, ArH), 7.29 (1H, d, J = 8.8 Hz, ArH), 7.21 (1H, dd, J = 8.1, 2.1 Hz, ArH), 3.38 (3H, s,

CH

3 ), 3.32 (3H, s, CH 3 ), 2.33 (3H, s, CH 3 ); APCI-MS 383 (MH) 4 ; FAB-HRMS calcd for C16H16CIN 2 0 3

S

2 (MH) 383.0291, found 383.0273. 5 [(3-Chloro-2-methylbenzenesulphonyl)-(3-ethoxycarbonylmethyl-2-oxo-2,3 dihydro-benzothiazol-6-yl)-amino]-acetic acid ethyl ester (STX764, XDS01149) To a solution of STX753 (20 mg, 0.056 mmol) in acetone (3 mL) was added potassium carbonate (20 mg), followed by methyl 2-bromoethyl acetate (50 pl). The mixture was 10 stirred at rt for 4 h, extracted into EtOAc and washed with brine. After drying over sodium sulphate, the solvent was removed in vacuo to give an oily residue that was purified with flash chromatography. White crystalline solid (20 mg, 68%) was obtained. TLC single spot at Rf 0.51 (30% ethyl acetate/hexane); HPLC purity 98% (tR 2.6 min in 10% water-methanol); 1 HNMR (400MHz, DMSO-d6) 5 7.75-7.78 (2H, m, ArH), 7.70 15 (1H, d, J= 2.3 Hz, ArH), 7.37 (1H, t, J= 8.2 Hz, ArH), 7.30 (1H, d, J= 8.6 Hz, ArH), 7.24 (1H, dd, J = 8.6, 2.3 Hz, ArH), 4.81 (2H, s, CH 2 ), 4.56 (2H, s, CH 2 ), 4.14 (2H, q, J = 7.0 Hz, CH 2 ), 4.06 (2H, q, J= 7.0 Hz, CH 2 ), 2.44 (3H, s, CH 3 ), 1.19 (3H, t, J = 7.0 Hz, CH 3 ), 1.13 (3H, t, J = 7.0 Hz, CH 3 ); FAB-MS 527 (MH)-; FAB-HRMS calcd for C22H24CIN207S2 (MWH) 527.0713, found 527.0694. 20 Synthesis of 2-chlorobenzothiazol-6-yl-amine and 2-chloro-benzothiazol-5-yl amine To a solution of 2-chlorobenzothiazole (12.0 g, 70.7 mmol) in concentrated H 2

SO

4 (60 mL) was added HNO 3 (69% solution, 6 mL) dropwise at 0oC for 20 min. The mixture 25 was stirred at 5oC for 3h, poured into ice-water (150 mL). The precipitate was collected and washed with 5% sodium bicarbonate and water, dried in vacuo. 'H NMR analysis showed the mixture contained 78% 6-nitro-2-chlorobenzothiazole and 8% 5-nitro-2 chlorobenzothiazole. Recrystallization from ethanol gave 6-nitro-2-chlorobenzothiazole as white crystalline solid (11 g, 72%). 3.5 g of the solid was dissolved in refluxing 30 ethanol-acetic acid (150 : 15 mL), Iron powder was added in one portion.. The mixture was refluxed for 1.5h, filtered. The filtrate was concentrated in vacuo to half volume and neutralized with 10% NaOH to pH 7.5, extracted with ethyl acetate. The organic phase was washed with brine, dried over magnesium sulphate and evaporated to give a residue, which was recrystallized from ethanol. Light purple crystals (2.5 g, 83%) were 35 obtained. Mp 160-164°C; TLC single spot at Rf 0.27 (30% EtOAc/hexane); 1

HNMR

WO 2004/037251 PCT/GB2003/004590 100 (270 MHz, DMSO-d6) 65 7.58 (1H, d, J = 9.0 Hz, 4-H), 7.03 (1H, d, J = 2.0 Hz, 7-H), 6.77 (1H, dd, J = 9.0, 2.0 Hz, 5-H), 5.55 (2H, s, NH 2 ). The mother liquor from the recrystallization of nitration product was evaporated and subjected to iron powder reduction as described above. The crude product was purified 5 with flash chromatography (ethyl acetate-DCM gradient elution) to give 2-chloro benzothiazol-5-yl-amine as yellow solid. Mp 146-149°C; TLC single spot at Rf 0.52 (10% EtOAc/DCM); 1 HNMR (270 MHz, DMSO-d6) 6 7.63 (1H, d, J= 8.6 Hz, 7-H), 7.05 (1H, d, J= 2.3 Hz, 4-H), 6.78 (1H, dd, J= 8.6, 2.3 Hz, 6-H), 5.40 (2H, s, NH 2 ). 10 The following compounds were synthesized with the general method for N benzothiazole benzenesulphonamide: N-(2-chlorobenzothiazol-6-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3-chloro-2 methylbenzenesulphonamide (STX767, XDS01151A) 15 White crystalline solid. TLC single spot at Rf 0.78 (33% EtOAc/DCM); HPLC purity 95% (tR 6.4 min in 10% water-methanol); 1 HNMR (270MHz, DMSO-d6) 65 8.21 (1H, d, J = 1.3 Hz, ArH), 8.00 (1H, d, J = 8.8 Hz, ArH), 7.83-7.90 (4H, m, ArH), 7.46 (2H, t, J = 8.0 Hz, ArH), 7.37 (1H, dd, J= 8.8, 1.8 Hz, ArH), 2.33 (6H, s, 2 x CH 3 ); APCI-MS 560 (M)'; FAB-HRMS calcd for C21H16C13N 2 0 4

S

3 (MH) 560.9338, found 560.9344. 20 3-Chloro-N-(2-chlorobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX768, XDS01151B) Off-white crystalline solid. TLC single spot at Rf 0.68 (33% EtOAc/DCM); HPLC purity 99% (tR 1.7 min in methanol); 1 HNMR (270MHz, DMSO-d6) 5 10.9 (1H, s, NH), 7.91 25 (1H, d, J = 8.1 Hz, ArH), 7.82 (1H, d, J = 8.8 Hz, ArH), 7.80 (1H, d, J = 3.1 Hz, ArH), 7.70 (1H, d, J= 8.1 Hz, ArH), 7.36 (1H, t, J= 8.1 Hz, ArH), 7.23 (1H, dd, J= 8.8, 3.0 Hz, ArH), 2.63 (3H, s, CH 3 ); APCI-MS 372 (M)'; FAB-HRMS calcd for C14H11C12N202S2 (MH) 372.9639, found 372.9651. 30 3-Chloro-N-(2-chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834, XDS01168) White crystalline solid. TLC single spot at Rf 0.52 (30% EtOAc/hexane); HPLC purity 99% (tR 1.7 min in methanol); 1 HNMR (270MHz, DMSO-d6) 5 10.9 (1H, s, NH), 7.91 7.96 (2H, m, ArH), 7.71 (1H, d, J= 8.1 Hz, ArH), 7.58 (1H, d, J= 2.2 Hz, ArH), 7.39 (1H, 35 d, J= 8.1 Hz, ArH), 7.22 (1H, dd, J= 8.1, 2.2 Hz, ArH), 2.64 (3H, s, CH 3 ); APCI-MS 371 (M-H)*; FAB-HRMS calcd for C14H11 C12N20 2

S

2 (MH*) 372.9639, found 372.9656.

WO 2004/037251 PCT/GB2003/004590 101 3-Chloro-2-methyl-N-(2-methylaminobenzothiazol-6-yl)-benzenesulphonamide (STX833, XDS01167) The solution of 3-chloro-N-(2-chlorobenzothiazol-6-yl)-2-methylbenzenesulphonamide 5 (STX768, 150 mg, 0.40 mmol) in CH 3 NH-THF (2M, 3 mL) was stirred at 820C in a sealed tube for 24h, extracted with ethyl acetate. The organic phase was washed brine, dried over sodium sulphate and concentrated in vacuo to give a residue that was purified with flash chromatography (ethyl acetate/DCM gradient elution). White crystals (100 mg, 68%) were obtained. TLC single spot at Rf 0.27 (30% EtOAc/DCM); HPLC purity 99% 10 (tR 1.8 min in 4% water-methanol); 1 HNMR (270MHz, DMSO-d6) 6 10.2 (1H, s, NH), 7.82 (1 H, q, J = 4.8 Hz, NH), 7.72 (1H, d, J= 7.7 Hz, ArH), 7.61 (1 H, d, J= 7.7 Hz, ArH), 7.29 (1H, d, J= 2.2 Hz, ArH), 7.26 (1H, t, J= 8.0 Hz, ArH), 7.15 (1H, d, J= 8.7 Hz, ArH), 6.79 (1H, dd, J = 8.7, 2.2 Hz, ArH), 2.80 (3H, d, J = 4.8 Hz, NCH 3 ), 2.54 (3H, s, CH 3 ); APCI-MS 366 (M-H)-; FAB-HRMS calcd for C1 5 H1 5 CIN302S2 (MH') 368.0294, found 15 368.0292. 3-Chloro-2-methyl-N-(2-methylaminobenzothiazol-5-yl)-benzenesulphonamide (STX835, XDS01176) The compound was prepared as described for STX833 using 3-chloro-N-(2 20 chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834, 80 mg, 0.21 mmol) as starting material. White crystals (60 mg, 78%) were obtained. TLC single spot at Rf 0.25 (30% EtOAc/DCM); HPLC purity 99% (tR 2.3 min in 10% water-methanol); 1 HNMR (270MHz, DMSO-d6) 6 10.5 (1H, s, NH), 7.96 (1H, q, J= 4.7 Hz, NH), 7.86 (1H, d, J= 8.1 Hz, ArH), 7.69 (1H, d, J= 8.1 Hz, ArH), 7.47 (1H, d, J= 8.0 Hz, ArH), 7.37 (1H, t, J= 25 8.1 Hz, ArH), 7.05 (1H, d, J= 1.9 Hz, ArH), 6.73 (1H, dd, J= 8.0, 1.9 Hz, ArH), 2.88 (3H, d, J = 4.7 Hz, NCH 3 ), 2.64 (3H, s, CH 3 ); APCI-MS 368 (MH)*; FAB-HRMS calcd for C15H15CIN302S2 (MH') 368.0294, found 368.0292. 3-Chloro-N-(2-diethylaminobenzothiazol-5-yl)-2-methylbenzenesulphonamide 30 (STX836, XDS01177) The compound was prepared as described for STX833 using 3-chloro-N-(2 chlorobenzothiazol-5-yl)-2-methylbenzenesulphonamide (STX834, 70 mg, 0.18 mmol) and diethylamine-THF (3 mL) as starting material. White crystals (50 mg, 68%) were obtained. TLC single spot at Rf 0.60 (30% EtOAc/DCM); HPLC purity 97% (tR 3.0 min in 35 10% water-methanol)-i'HNMR (270MHz, DMSO-d6) 6 10.5 (1H, s, NH), 7.85 (1H, d, J = 7.9 Hz, ArH), 7.68 (1H, d, J= 8.0 Hz, ArH), 7.52 (1H, d, J= 8.4 Hz, ArH), 7.36 (1H, t, J WO 2004/037251 PCT/GB2003/004590 102 = 8.0 Hz, ArH), 7.06 (1H, d, J = 2.2 Hz, ArH), 6.75 (1H, dd, J = 8.3, 2.2 Hz, ArH), 3.45 (4H, q, J = 7.0 Hz, N(CH 2

)

2 ), 2.63 (3H, s, CH 3 ), 1.15 (6H, t, J = 7.0 Hz, 2 x CH 3 ); APCI MS 410 (MH)*; FAB-HRMS calcd for C18H21CIN30 2 S2 (MH*) 410.0764, found 410.0753. 5 3-Chloro-N-(2-diethylaminobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX878, XDS01164) The compound was prepared as described for STX833 using 3-chloro-N-(2 chlorobenzothiazol-6-yl)-2-methylbenzenesulphonamide (STX768, 240 mg, 0.64 mmol) 10 and diethylamine-IPA (3 mL) as starting material. Off-white crystalline solid (128 mg, 49%) were obtained. TLC single spot at Rf 0.33 (30% EtOAc/hexane); HPLC purity 96% (tR 2.2 min in 4% water-methanol); 1 HNMR (270MHz, DMSO-d6) 5 7.56-7.62 (3H, m, ArH), 7.18-7.27 (2H, m, ArH), 7.00 (1H, dd, J= 8.5, 1.7 Hz, ArH), 5.52 (1H, s, NH), 3.54 (4H, q, J = 7.0 Hz, N(CH 2

)

2 ), 2.25 (3H, s, CH 3 ), 1.22 (6H, t, J = 7.0 Hz, 2 x CH 3 ); APCI 15 MS 409 (M)'; FAB-HRMS calcd for C1 8 H21CIN302S2 (MH') 410.0764, found 410.0698. Synthesis of 2,6-Dimethylbenzothiazol-7-ylamine To a solution of 2,6-dimethylbenzothiazol (350 mg, 2.15 mmol) in Conc. H 2

SO

4 (4 mL) was added HNO 3 (69%, 0.3 mmol) at 0oC. After stirred at 00C for 0.5h, the mixture was 20 poured over ice-water. The precipitate was collected and washed with 5% sodium bicarbonate and water, recrystallized from ethanol to give 7-nitro-2,6 dimethylbenzothiazol as yellow solid (160 mg). The product (150 mg) was hydrogenated over 5% Pd/C in ethanol-THF (10 : 2 mL) at atmosphere pressure to give 2,6-dimethyl benzothiazol-7-ylamine as yellow solid (120 mg). TLC single spot at Rf 0.55 (10% 25 EtOAc/DCM); 1 H NMR (270 MHz, DMSO): 6 7.07 (2H, s, ArH), 5.23 (2H, s, NH 2 ), 2.73 (3H, s, CH 3 ), 2.20 (3H, s, CH 3 ). Synthesis of 6-Methoxy-2-methylbenzothiazol-7-ylamine The compound was prepared as described above starting from 6-methoxy-2 30 methylbenzothiazol. Yellow solid was obtained. mp 117-1190C (lit.121-122°C); TLC single spot at Rf 0.55 (40% EtOAc/DCM); 1 H NMR (270 MHz, DMSO): 6 7.14 (1H, d, J = 8.7 Hz, ArH), 7.05 (1H, d, J = 8.7 Hz, ArH), 5.10 (2H, broad, NH 2 ), 3.83 (3H, s, OCH 3 ), 2.71 (3H, s, CH 3 ). (Friedman, S.G. J Gen Chem USSR 31, 1961, 3162-3167) 35 WO 2004/037251 PCT/GB2003/004590 103 Synthesis of 2,5-Dimethylbenzothiazol-4-ylamine and 2,5-Dimethylbenzothiazol-6 ylamine To a solution of 2,5-dimethylbenzothiazol (1.63 g, 10 mmol) in Conc. H 2

SO

4 (12 mL) was added HNO 3 (69%, 1 mmol) at -5 0 C. After stirred at -5 - 00C for 2h, the mixture was 5 poured over ice-water (150 mL). The precipitate was collected and washed with 5% sodium bicarbonate, water and 70% ethanol. The product (1.98 g) was a mixture of 4 nitro-2,5-dimethylbenzothiazol and 6-nitro-2,5-dimethylbenzothiazol in 1 : 1 ratio judged by NMR. The product (998 mg) was hydrogenated over 5% Pd/C (600 mg) in ethanol THF (50 : 20 mL) at atmosphere pressure to give a yellow solid (880 mg). Separation 10 with flash chromatography (EtOAc/DCM gradient elution) yielded 2,5 dimethylbenzothiazol-4-ylamine as yellow crystals (400 mg). TLC single spot at Rf 0.60 (15% EtOAc/DCM); 1 H NMR (270 MHz, DMSO): 6 7.05 (1H, d, J = 8.0 Hz, ArH), 6.99 (1H, d, J = 8.0 Hz, ArH), 5.26 (2H, s, NH 2 ), 2.74 (3H, s, CH 3 ), 2.18 (3H, s, CH 3 ); APCI MS 177 (M-H)*. 15 2,5-Dimethylbenzothiazol-6-ylamine was obtained as yellow solid (320 mg). TLC single spot at Rf 0.55 (15% EtOAc/DCM); 1 H NMR (270 MHz, DMSO): 5 7.47 (1H, s, ArH), 7.05 (1H, s, ArH), 5.05 (2H, s, NH 2 ), 2.65 (3H, s, CH 3 ), 2.16 (3H, s, CH 3 ); APCI-MS 177 (M H)*. 20 Synthesis of 4-chloro-2-methylbenzothiazol-5-ylamine and 4,6-dichloro-2 methylbenzothiazol-5-ylamine To a solution of 5-amino-2-methylbenzothiazole (818 mg, 4.99 mmol) in isopropanol (12 mL) was added N-chlorosuccinimide (732 mg, 5.48 mmol). The mixture was stirred at 600C for 15 min., partitioned between DCM and 5% sodium bicarbonate. The organic 25 phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a residue that was purified with flash chromatography (EtOAc/DCM gradient elution). 4-Chloro-2-methylbenzothiazol-5-ylamine was obtained as off-white crystalline solid (510 mg, 51%). mp 121-122oC (lit.124 0 C); TLC single spot at Rf 0.51 (20% EtOAc/DCM); 1 H NMR (270 MHz, DMSO): 6 7.61 (1H, d, J= 8.6 Hz, ArH), 6.91 (1H, d, J 30 = 8.6 Hz, ArH), 5.49 (2H, s, NH 2 ), 2.75 (3H, s, CH 3 ); APCI-MS 199 (MH)*. 4,6-Dichloro-2-methylbenzothiazol-5-ylamine was obtained as yellow solid (60 mg, 5%). TLC single spot at Rf 0.57 (20% EtOAc/DCM); 1 H NMR (270 MHz, DMSO): 5 7.89 (1H, s, ArH), 5.26 (2H, s, NH 2 ), 2.77 (3H, s, CH 3 ); APCI-MS 233 (MH)*.

WO 2004/037251 PCT/GB2003/004590 104 The following compounds were synthesized with the general method for N benzothiazole benzenesulphonamide. 5 3-Chloro-N-(6-methoxy-2-methylbenzothiazol-7-yl)-2-methylbenzenesulphonamide (STX989, XDS02038) White crystalline solid. TLC single spot at Rf 0.71 (30% EtOAc/DCM); HPLC purity 99% (tR 2.3 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 6 10.1 (1H, s, NH), j 7.76 (1H, d, J = 8.9 Hz, ArH), 7.72 (1H, d, J = 8.2 Hz, ArH), 7.53 (1H, d, J = 8.2 Hz, 10 ArH), 7.23 (1H, t, J = 8.2 Hz, ArH), 7.05 (1H, d, J = 8.9 Hz, ArH), 3.29 (3H, s, OCH 3 ), 2.74 (3H, s, CH 3 ), 2.70 (3H, s, CH 3 ); APCI-MS 381 (M-H)+; FAB-HRMS calcd for C16H16CIN203S2 (MH) 383.0291, found 383.0284. 3-Chloro-N-(2,6-dimethyl-benzothiazol-7-yl)-2-methyl-benzenesulphonamide 15 (STX1021, XDS02069) Off-white crystalline solid. TLC single spot at Rf 0.49 (10% EtOAc/DCM); HPLC purity 98% (tR 2.0 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 5 10.3 (1H, s, NH), 7.78 (1H, d, J= 7.9 Hz, ArH), 7.74 (1H, d, J= 8.4 Hz, ArH), 7.64 (1H, d, J= 7.9 Hz, ArH), 7.34 (1H, t, J = 7.9 Hz, ArH), 7.30 (1H, d, J= 7.9 Hz, ArH), 2.68 (3H, s, CH 3 ), 2.61 20 (3H, s, CH 3 ), 2.03 (3H, s, CH 3 ); FAB-MS 367 (MH)*; FAB-HRMS calcd for C16H16CIN202S2 (MH) 367.0342, found 367.0347. 3-Chloro-N-(2,5-dimethyl-benzothiazol-4-yl)-2-methyl-benzenesulphonamide (STX996, XDS02047) 25 White crystalline solid. TLC single spot at Rf 0.76 (10% EtOAc/DCM); HPLC purity >99% (t 2.9 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 6 9.98 (1H, s, NH), 7.81 (1H, d, J = 8.3 Hz, ArH), 7.64 (1H, d, J = 7.9 Hz, ArH), 7.45 (1H, d, J = 7.9 Hz, ArH), 7.31 (1H, d, J = 8.3 Hz, ArH), 7.12 (1H, t, J = 7.9 Hz, ArH), 2.73 (3H, s, CH 3 ), 2.47 (3H, s, CH 3 ), 2.44 (3H, s, CH 3 ); APCI-MS 367 (MH)*; FAB-HRMS calcd for 30 C1 6

H

16 CIN202S2 (MH*) 367.0342, found 367.0342. N-(2,5-dimethylbenzothiazol-6-yi)-N-(3-chloro-2-methylphenylsulphonyl)-3-chloro 2-methylbenzenesulphonamide (STX997, XDSO2048A) Off-white syrup. TLC single spot at Rf 0.78 (10% EtOAc/DCM); HPLC purity 85% (tR 4.2 35 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 5 8.06 (1H, s, ArH), 7.86 7.95 (5H, m, ArH), 7.68 (1H, s, ArH), 7.52 (2H, t, J = 8.2 Hz, ArH), 2.83 (3H, s, CH 3

),

WO 2004/037251 PCT/GB2003/004590 105 2.29 (6H, s, 2 x CH 3 ), 2.05 (3H, s, CH 3 ); APCI-MS 555 (MH)*; FAB-HRMS calcd for C23H21Cl2N204S3 (MH) 555.0040, found 555.0041. 3-Chloro-N-(2,5-dimethylbenzothiazol-6-yl)-2-methylbenzenesulphonamide 5 (STX998, XDSO2048B) White crystalline solid. TLC single spot at Rf 0.39 (10% EtOAc/DCM); HPLC purity 96% (tR 2.1 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 5 10.0 (1H, s, NH), D 7.74 (1H, d, J= 7.9 Hz, ArH), 7.70 (1H, s, ArH), 7.67 (1H, d, J= 7.9 Hz, ArH), 7.63 (1H, s, ArH), 7.33 (1H, t, J = 7.9 Hz, ArH), 2.75 (3H, s, CH 3 ), 2.60 (3H, s, CH 3 ), 2.13 (3H, s, 10 CH 3 ); APCI-MS 367 (MH) ; FAB-HRMS calcd for C16H16CIN202S2 (MH*) 367.0342, found 367.0350. 2,5-Dichloro-N-(2,5-dimethylbenzothiazol-6-yl)-benzenesulphonamide (STX999, XDS02049) 15 White crystalline solid. TLC single spot at Rf 0.43 (10% EtOAc/DCM); HPLC purity 98% (tR 2.0 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 6 10.3 (1H, s, NH), 1 7.76 (3H, s, ArH), 7.72 (1H, s, ArH), 7.67 (1H, s, ArH), 2.75 (3H, s, CH 3 ), 2.23 (3H, s,

CH

3 ); APCI-MS 387 (MH)+; FAB-HRMS calcd for C15H13CI2N202S2 (MH) 386.9795, found 386.9806. 20 N-(4-Chloro-2-methyl-benzothiazol-5-yl)-N-(3-chloro-2-methylphenylsulphonyl)-3 chloro-2-methyl-benzenesulphonamide (STX991, XDSO2042A) White powder. TLC single spot at Rf 0.75 (8% EtOAc/DCM); HPLC purity >99% (tR 4.4 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 6 8.19 (1H, d, J= 8.7 Hz, 2 25 ArH), 7.93 (4H, d, J = 8.2 Hz,, ArH), 7.59 (1H, d, J = 8.7 Hz, ArH), 7.50 (2H, d, J = 8.2 Hz, ArH), 2.85 (3H, s, CH 3 ), 2.41 (6H, s, 2 x CH 3 ); APCI-MS 575 (MH)*; FAB-HRMS calcd for C22H18Cl3N204S3 (MH 4 ) 574.9494, found 574.9492. 3-Chloro-N-(4-chloro-2-methylbenzothiazol-5-yl)-2-methylbenzenesulphonamide 30 (STX992, XDSO2042B) White crystalline solid. TLC single spot at Rf 0.69 (8% EtOAc/DCM); HPLC purity 99% (tR 2.5 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 5 10.5 (1H, s, NH), 3 7.96 (1H, d, J = 8.7 Hz, ArH), 7.73 (1H, d, J = 7.9 Hz,, ArH), 7.64 (1H, d, J = 7.9 Hz, ArH), 7.30 (1H, d, J

=

8.7 Hz, ArH), 7.29 (1 H, t, J = 7.9 Hz,, ArH), 2.79 (3H, s, CH 3 ), 2.70 WO 2004/037251 PCT/GB2003/004590 106 (3H, s, CHa); APCI-MS 385 (M-H)'; FAB-HRMS calcd for C15H13CI2N202S2 (MH*) 386.9795, found 386.9790. 2,5-Dichloro-N-(4-chloro-2-methylbenzothiazol-5-yl)-benzenesulphonamide 5 (STX993, XDSO2043B) White crystalline solid. TLC single spot at Rf 0.71 (8% EtOAc/DCM); HPLC purity 99% (tR 5.0 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 6 10.7 (1H, s, NH), j 7.97 (1H, d, J = 8.6 Hz, ArH), 7.71-7.78 (3H, m,, ArH), 7.28 (1H, d, J = 8.6 Hz, ArH), 2.81 (3H, s, CH 3 ); APCI-MS 407 (MH)*; FAB-HRMS calcd for C14H10CI3N202S2 (MH +) 10 406.9249, found 406.9234. N-(4-Chloro-2-methylbenzothiazol-5-yl)-4-propylbenzenesulphonamide (STX994, XDSO2044B) White crystalline solid. TLC single spot at Rf 0.70 (8% EtOAc/DCM); HPLC purity 99% 15 (tR 2.7 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 6 10.1 (1H, s, NH), 7.93 (1H, d, J = 8.6 Hz, ArH), 7.60 (2H, d, J = 8.2 Hz, ArH), 7.35 (2H, d, J = 8.2 Hz, ArH), 7.28 (1H, d, J= 8.4 Hz, ArH), 2.79 (3H, s, CH 3 ), 2.69 (2H, t, J= 7.2 Hz, CH 2 ), 1.59 (2H, m, CH 2 ), 0.86 (3H, t, J = 7.2 Hz, CH 3 ); APCI-MS 381 (MH)*; FAB-HRMS calcd for C17H18CIN202S2 (MH) 381.0498, found 381.0484. 20 N-(4-Chloro-2-methylbenzothiazol-5-yl)-N-(4-propylphenylsulphonyl)-4 propylbenzenesulphonamide (STX995, XDSO2044A) White powder. TLC single spot at Rf 0.70 (8% EtOAc/DCM); HPLC purity 99% (tR 3.8 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO-d6) 6 8.10 (1H, d, J = 8.4 25 Hz, ArH), 7.74 (4H, d, J = 8.1 Hz, ArH), 7.50 (4H, d, J = 8.1 Hz, ArH), 7.08 (1H, d, J= 8.4 Hz, ArH), 2.91 (3H, s, CH 3 ), 2.71 (4H, t, J = 7.1 Hz, 2 x CH 2 ), 1.59 (4H, m, CH 2 ), 0.86 (6H, t, J = 7.1 Hz, 2 x CH 3 ); APCI-MS 561 (M-H)*; FAB-HRMS calcd for C26H28CIN204S 3 (MH*) 563.0900, found 563.0886. 30 Synthesis of 3-Chloro-2-methyl-N-(2-methyl-benzothiazol-5-ylmethyl) benzenesulphonamide (STX1029, XDSO2070A) and 3-Chloro-2-methyl-N,N-bis-(2 methyl-benzothiazol-5-ylmethyl)-benzenesulphonamide (STX1030, XDSO2070B) To a solution of 3-chloro-2-methylbenzenesulphonamide (103 mg, 0.5 mmol) in CH 3 CN was added potassium carbonate (100 mg), followed 5-bromomethyl-2 35 methylbenzothiazole (121mg, 0.5 mmol). The mixture was refluxed under N 2 for 6h, WO 2004/037251 PCT/GB2003/004590 107 partitioned between ethyl acetate and water. The organic phase was washed brine, dried over sodium sulphate and concentrated in vacuo to give a yellow residue, which was separated with flash chromatography (ethyl acetate/DCM, gradient elution). STX1029 was obtained as white solid. TLC single spot at Rf 0.55 (10% EtOAc/DCM); 5 HPLC purity >99% (tR 2.0 min in 10% water-methanol); 1 HNMR (270 MHz, CDCI, 3 ) 5 7.89 (1H, d, J = 7.9 Hz, ArH), 7.66 (1H, d, J = 7.9 Hz, ArH), 7.65 (1H, d, J = 1.3 Hz, ArH), 7.49 (1H, d, J= 7.9 Hz, ArH), 7.17 (1H, t, J= 7.9 Hz, ArH), 7.13 (1H, dd, J = 7.9, 1.5 Hz, ArH), 5.35 (1H, t, J = 5.9 Hz, NH), 4.24 (2H, d, J = 5.9 Hz, CH 2 ), 2.79 (3H, s,

CH

3 ), 2.62 (3H, s, CH 3 ); APCI-MS 367 (MH)-; FAB-HRMS calcd for C16H16CIN202S 2 10 (MH ) 367.0342, found 367.0330. STX1030 was obtained as white solid. TLC single spot at Rf 0.50 (10% EtOAc/DCM); HPLC purity 99% ( t R 6.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO-d6) 6 7.87 (3H, d, J = 8.1 Hz, ArH), 7.75 (1H, d, J = 8.0 Hz, ArH), 7.59 (2H, broad wl/ 2 = 1.1 Hz, ArH), 7.38 (1H, t, J= 8.0 Hz, ArH), 7.11 (2H, dd, J= 8.1, 1.1 Hz, ArH), 4.56 (4H, s, 2 15 x NCH 2 ), 2.78 (6H, s, 2 x CH 3 ), 2.58 (3H, s, CH 3 ); APCI-MS 528 (MH)+; FAB-HRMS calcd for C25H23CIN302S3 (MH ) 528.0641, found 528.0630. Synthesis of N-Indole or N-Indoline Arysulfonamide Derivatives rO H O. H ) H H 0 H H H H Cl Cl CIl STX832, XDS01165 STX981, XDS02019 STX982, XDS02020 0 H \N 0 H \ O H c H N S Cl CIH STX986, XDS02030 STX1 018, XDS02061 STX1019, XDS02062 NN, N N qn.-.N -O:N HCI CI CI 20 STX1020, XDS02063 STX984, XDS02025 STX987, XDS02031 WO 2004/037251 PCT/GB2003/004590 108 General method for synthesis N-indole or N-indoline arylsulphonamide derivatives (STX832, STX981-982, STX984, STX986-987, STX1018-1020): To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (2.2 eq.) and catalytic amount of DMAP, followed by the corresponding amine (1 eq.). The reaction 5 mixture was stirred at rt under nitrogen for 4-6 h, then partitioned between ethyl acetate and 5% sodium bicarbonate after TLC showed completion of the reaction. The organic layer was washed with brine, dried over sodium sulphate, and concentrated in vacuo to give crude product as solid or thick syrup. The compound was then purified by flash chromatography (methanol-DCM gradient elution) to give desired arylsulphonamide as 10 crystalline solid. Yield ranges from 50-85%. 3-Chloro-2-methyl-N-(2-methyl-IH-indol-5-yl)-benzenesulphonamide (STX832, XDS01165) White crystalline solid. TLC single spot at Rf 0.68 (30% ethyl acetate/hexane); HPLC 15 purity > 99 % (tR 1.8 min in 4 % water-methanol); 'H NMR (270 MHz, DMSO): 6 10.9 (1H, s, NH), 9.98 (1H, s, NH), 7.72 (1H, d, J = 8 Hz, ArH), 7.63 (1H, d, J= 8 Hz, ArH), 7.27 (1H, t, J= 8 Hz, ArH), 7.07 (1H, d, J= 8 Hz, ArH), 7.05 (1H, d, J= 2 Hz, ArH), 6.67 (1H, dd, J=8, 2 Hz, ArH), 5.99 (1H, s, 3-H), 2.60 (3H, s, CH 3 ), 2.29 (3H, s, CH 3 ); APCI MS 334 (M+); FAB-HRMS calcd for C16H16CIN202S (MH+) 335.0621, found 335.0609 20 3-Chloro-2-methyl-N-(1 H-indol-5-yl)-benzenesulphonamide (STX981, XDSO2019) White crystalline solid. TLC single spot at Rf 0.72 (6% methanol/DCM); HPLC purity 98 % (tR 2.1 min in 10 % water-methanol); 'H NMR (270 MHz, DMSO): 5 11.1 (1H, s, NH), 10.1 (1H, s, NH), 7.76 (1H, d, J= 7.9 Hz, ArH), 7.66 (1H, d, J= 7.9 Hz, ArH), 7.22-7.32 25 (4H, m, ArH), 6.81 (1H, dd, J=7.9, 1.2 Hz, ArH), 6.33 (1H, broad, 3-H), 2.64 (3H, s, CH 3 ); APCI-MS 319 (M-H-); FAB-HRMS calcd for C15H14CIN202S (MH ) 321.0465, found 321.0453. 3-Chloro-2-methyl-N-(1H-indol-6-yl)-benzenesulphonamide (STX982, XDSO2020) 30 White crystalline solid. TLC single spot at Rf 0.88 (10% methanol/DCM); HPLC purity 98 % (tR 2.5 min in 20 % water-methanol); 'H NMR (270 MHz, DMSO): 6 11.0 (1H, s, NH), 10.3 (1H, s, NH), 7.80 (1H, d, J= 7.9 Hz, ArH), 7.67 (1H, d, J= 7.9 Hz, ArH), 7.31-7.38 (2H, m, ArH), 7.26 (1H, m, ArH), 7.80 (1H, d, J = 1.2 Hz, ArH), 6.75 (1H, dd, J=7.9, 1.2 Hz, ArH), 6.31 (1H, broad, 3-H), 2.65 (3H, s, CH 3 ); APCI-MS 319 (M-H ); FAB-HRMS 35 calcd for C15H14ClN202S (MH

+

) 321.0465, found 321.0446.

WO 2004/037251 PCT/GB2003/004590 109 5-(3-Chloro-2-methylbenzenesulfonylamino)-1 H-indole-2-carboxylic acid ethyl ester (STX986, XDSO2030) White crystalline solid. TLC single spot at Rf 0.82 (8% methanol/DCM); HPLC purity > 99 % (t 2.3 min in 10% water-methanol); 'H NMR (270 MHz, DMSO): 5 11.9 (1H, s, 5 NH), 10.3 (1H, s, NH), 7.78 (1H, d, J = 7.9 Hz, ArH), 7.67 (1H, d, J = 7.9 Hz, ArH), 7.28 7.33 (3H, m, ArH), 7.06 (1H, d, J= 2.2 Hz, ArH), 7.00 (1H, dd, J= 8.2, 2.2 Hz, ArH), 4.32 (2H, q, J = 6.9 Hz, OCH 2 ), 2.63 (3H, s, CH 3 ), 1.31 (3H, t, J = 6.9 Hz, CH 3 ); APCI-MS 391 (M-H*); FAB-HRMS calcd for C18H18CIN204S (MH) 393.0676, found 393.0659 10 3-Chloro-2-methyl-N-(2,3-dimethyl-1H-indol-5-yl)-benzenesulphonamide (STX1018, XDSO2061) White crystalline solid. TLC single spot at Rf 0.83 (30% ethyl acetate/hexane); HPLC purity 97% (tR 2.9 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 5 10.6 (1H, s, NH), 10.0 (1H, s, NH), 7.74 (1H, d, J = 7.5 Hz, ArH), 7.65 (1H, d, J = 7.5 Hz, ArH), 15 7.29 (1H, t, J= 8.0 Hz, ArH), 7.05 (1H, d, J= 8.6 Hz, ArH), 6.99 (1H, d, J= 1.7 Hz, ArH), 6.66 (1H, dd, J= 8.6, 1.7 Hz, ArH), 2.62 (3H, s, CH 3 ), 2.25 (3H, s, CH 3 ), 2.03 (3H, s,

CH

3 ); APCI-MS 349 (MH*); FAB-HRMS calcd for C17H18CIN202S (MH

+

) 349.0778, found 349.0737. 20 2,5-Dichloro-N-(2,3-dimethyl-1H-indol-5-yl)-benzenesulphonamide (STX1019, XDS02062) White amorphous powder. TLC single spot at Rf 0.82 (10% ethyl acetate/hexane); HPLC purity 98 % (tR 3.0 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO): 5 10.7 (1H, s, NH), 10.2 (1H, s, NH), 7.80 (1H, m, ArH), 7.67-7.70 (2H, m, ArH), 7.07 (1H, 25 d, J = 8.5 Hz, ArH), 7.05 (1H, d, J = 1.7 Hz, ArH), 6.73 (1H, dd, J= 8.5, 1.7 Hz, ArH), 2.25 (3H, s, CH 3 ), 2.04 (3H, s, CH 3 ); APCI-MS 367 (M-H'); FAB-HRMS calcd for C16H14Cl2N202S (M') 368.0153, found 368.0146 4-n-Propyl-N-(2,3-dimethyl-1 H-indol-5-yl)-benzenesulphonamide (STX1020, 30 XDS02063) Off-white crystalline solid. TLC single spot at Rf 0.82 (10% ethyl acetate/hexane); HPLC purity 97 % (tR 2.9 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 6 10.6 (1H, s, NH), 9.6 (1H, s, NH), 7.56 (2H, d, J= 8.3 Hz, ArH), 7.30 (2H, d, J= 8.3 Hz, ArH), 7.03 (1H, d, J = 8.3 Hz, ArH), 6.95 (1H, d, J = 1.7 Hz, ArH), 6.68 (1H, dd, J= 8.3, 1.7 Hz, 35 ArH), 2.56 (2H, t, J= 7.3 Hz, CH 2 ), 2.24 (3H, s, CH 3 ), 2.01 (3H, s, CH 3 ), 1.55 (2H, sextet, WO 2004/037251 PCT/GB2003/004590 110 J = 7.3 Hz, CH 2 ), 0.85 (3H, t, J = 7.3 Hz, CH 3 ),; APCI-MS 343 (MH+); FAB-HRMS calcd for C19H22N202S (M') 342.1402, found 342.1403 3-Chloro-2-methyl-N-(1-acetyl-2,3-dihydro-1 H-indol-5-yl)-benzenesulphonamide 5 (STX984, XDS02025) White crystalline solid. TLC single spot at Rf 0.58 (5% methanol/DCM); HPLC purity 95 % (tR 2.2 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO): 5 10.4 (1H, s, NH), 7.80-7.86 (2H, m, ArH), 7.71 (1H, d, J= 7.8 Hz, ArH), 7.36 (1H, t, J= 8.0 Hz, ArH), 6.93 (1H, d, J = 1.8 Hz, ArH), 6.83 (1H, dd, J = 8.2, 1.8 Hz, ArH), 4.01 (2H, t, J = 8.3 Hz, 10 CH 2 ), 3.03 (2H, t, J = 8.4 Hz, CH 2 ), 2.62 (3H, s, CH 3 ), 2.09 (3H, s, CH 3 ); APCI-MS 363 (M-H*); FAB-HRMS calcd for C17H18CIN203S (MH

+

) 365.0727, found 365.0796. 5-(3-Chloro-2-methyl-benzenesulfonylamino)-1-ethyl-2,3-dihydro-1 H-indolium chloride (STX987, XDSO2031) 15 The free base of STX987 was synthesized as above. A purple amorphous powder was obtained; TLC single spot at Rf 0.79 (8% methanol/DCM); 'H NMR (270 MHz, CDC3): 6 7.79 (1H, d, J= 7.9 Hz, ArH), 7.53 (1H, d, J= 7.9 Hz, ArH), 7.15 (1H, t, J= 8.0 Hz, ArH), 6.75 (1H, d, J= 1.8 Hz, ArH), 6.58 (1H, dd, J= 8.1, 1.8 Hz, ArH), 6.35 (1H, s, NH), 6.22 (1H, d, J= 8.1 Hz, ArH), 3.30 (2H, t, J= 8.4 Hz, CH 2 ), 3.05 (2H, q, J= 7.2 Hz, CH 2 ), 2.84 20 (2H, t, J= 8.3 Hz, CH 2 ), 2.67 (3H, s, CH 3 ), 1.11 (3H, t, J= 7.2 Hz, CH.). The free base was treated with HCI-ether solution to give STX987 as light pink crystalline solid. HPLC purity 93% (tR 3.6 min in 20 % water-methanol); 'H NMR (270 MHz, DMSO): 6 10.3 (1H, s, NH), 7.82 (1H, d, J= 8.1 Hz, ArH), 7.72 (1H, d, J= 8.1 Hz, ArH), 7.38 (1H, t, J= 8.1 Hz, ArH), 6.79-6.89 (3H, m, broad, ArH), 3.42 (2H, t, J = 8.4 Hz, CH 2 ), 3.16 (2H, q, J = 25 7.0 Hz, CH 2 ), 2.91 (2H, t, J = 8.4 Hz, CH 2 ), 2.62 (3H, s, CH 3 ), 1.10 (3H, t, J = 7.0 Hz,

CH

3 ); APCI-MS 349 (M-HCl-H*); FAB-HRMS calcd for C,1 7

H

20

CIN

2 0 2 S (M-HCl+H*) 351.0934, found 351.0941. 1-Acetyl-5-aminoindoline 30 The solution of 1-acetyl-5-nitroindoline (1.0 g, 4.85 mmol) in ethanol-THF (100 mL : 30 mL) was hydrogenated over 5% Pd/C (600 mg) at atmosphere pressure for 2h, filtered through Celite and concentrated in vacuo to give a white solid which was recrystalilized from ethanol. White crystalline solid (580 mg, 68%) was obtained. Mp 185-186.50C (lit 184-185oC, [21]); 'H NMR (270 MHz, DMSO): 6 7.73 (1H, d, J = 8.6 Hz, ArH), 6.45 (1H, 35 s broad, wl/2= 1.8 Hz, ArH), 6.33 (1H, dd, J= 8.6, 1.8 Hz, ArH), 4.82 (2H, s, NH 2 ), 3.97 WO 2004/037251 PCT/GB2003/004590 111 (2H, t, J = 8.4 Hz, CH 2 ), 2.99 (2H, t, J = 8.4 Hz, CH 2 ), 2.07 (3H, s, CH 3 ); APCI-MS 175 (M-H+). 1-Ethyl-5-aminoindoline 5 To a suspension of 1-acetyl-5-aminoindoline (130 mg, 0.74 mmol) in anhydrous THF (10 mL) was added LiAIH 4 (42 mg, 1.11 mmol). The mixture was stirred at rt for 6h, quenched with saturated NH 4 CI and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a purple residue (80 mg, 67%) that was used without further purification. 1 H NMR (270 10 MHz, DMSO): 6 6.56 (1H, s, ArH), 6.47 (1H, d broad, J = 8.1 Hz, ArH), 6.37 (1H, d, J= 8.0 Hz, ArH), 3.29 (2H, s, NH 2 ), 3.20 (2H, t, J = 7.6 Hz, CH 2 ), 3.02 (2H, q, J = 6.9 Hz,

CH

2 ), 2.86 (2H, t, J= 7.6 Hz, CH 2 ), 1.17 (3H, t,J= 6.9 Hz, CH 3 ). Synthesis of 5-( 3 -chloro-2-methyl-benzenesulfonamino)-lH-indole-3-carboxylic 15 acid methyl ester, STX 1050 (KRB01132): C 0 O 5-amino-.lH-indole-3-carboxylic acid methyl ester (KRB01131): To a solution of 5 20 nitro-1H-indole-3-carboxylic acid methyl ester (206 mg, 0.940 mmol) in methanol (40 mL) was added 5% palladium on carbon (40 mg) and the mixture was stirred under 1 atm H 2 for 5h. The mixture was filtered through celite and the filtrate evaporated to yield a brown solid that was used without further purification (173 mg, 97%), single spot at Rf 0.64 (ethyl acetate). 1 H NMR (d 6 -DMSO): 6 11.50 (1H, s, N-H), 7.83 (1H, d, J=3.2 Hz), 25 7.17 (1H, d, J=2.0 Hz), 7.14 (1H, d, J=8.4 Hz), 6.56 (1H, dd, J=8.6, 2.2 Hz), 4.77 (2H, s,

N-H

2 ), 3.76 (3H, s). To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (124 mg, 0.552 mmol) in dichloromethane (4 mL) was added pyridine (100 pL, 1.3 mmol) and the mixture was 30 stirred under N 2 for 5 min, after which time 5-amino-lH-indole-carboxylic acid methyl ester (100 mg, 0.526 mmol) was added. The resulting mixture was stirred for 1.5 h at room temperature, then saturated NaHCO 3 solution (15 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, WO 2004/037251 PCT/GB2003/004590 112 dried (Na 2 SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a white solid (129 mg, 65%), single spot at Rf 0.84 (ethyl acetate). mp 216.8-219.3oC, [22] , HPLC purity 99+% (tR 2.07 min in 10% water acetonitrile). 'H NMR (de-DMSO): 6 11.91 (1H, s), 10.32 (1H, s), 8.03 (1H, d, J=3.0 Hz), 5 7.82 (1H, d, J=7.9 Hz), 7.70-7.67 (2H, m), 7.37-7.31 (2H, m), 6.95 (1H, dd, J=8.6, 2.0 Hz), 3.77 (3H, s), 2.65 (3H, s). LCMS: 377.09. FAB-MS (MH+, C, 7

H

1 5CIN 2 0 4 S): calcd 378.0441, found 378.0439.

WO 2004/037251 PCT/GB2003/004590 113 Synthesis of Benzimidazole Arysulphonamide Derivatives 0 2 N N a 0 2 N N 2N H R R 0 2 N N N b H

H

2 N - N b cN

H

2 N c N V-2J ' I I 0 S c Ar e SR H R OANHR=

-OH

3 d~ CI H Ar = 2-Me-3-OCI-phenyl R = -CH2COOC 2

H

5 R = -CH 2

CH(CH

3

)

2 Ar = 2-Me-3-CI-phenyl R = -CH 2

C

6

H

5 R = -CH 2

COOC

2

H

5 Ar = 4-n-propylphenyl R = -CH 3 R = -CH 2

C

6

H

5 Ar = 2,5-dichlorophenyl R = -CH 3 Ar = 2,4-dichlorophenyl R = -CH 3 Ar = 2-Me-4-Br-phenyl R = -CH 3 Ar = 4-biphenyl R = -CH 3

H

2 N N e H N O =CF -) N 1-' H H 10 H Cl

H

2 N - N H 2 N N N H H H a) RX, K 2

CO

3 , Acetone ,rt or reflux b)H 2 / 5%Pd-C, Ethanol-THF r.t. or Fe, AcOH-ethanol c) ArSO 3 CI, DCM,Pyridine or ArSO 3 CI, DCM,Pyridine/DMAP d)HOBt, THF, r.t. e)N-chlorosuccimide, IPA 5 WO 2004/037251 PCT/GB2003/004590 114

S

0 HH CI 1 N N \\NN N N q'b N I 'N N NN ~I 0> CI CI Cl STX975, XDS02001 STX976, XDS02003 STX1121, XDS02102 ON H N H -'N N N N C 1o STX1112, XDS02088 STX 113, XDS02089 STX1 114, XDS02090 0 HH 0 H N I- 0 "I N N C 1 -0 N N Br \ C STX1115, XDS02091 STX1116, XDS02092 STX1110, XDS02084 HH N H 0N N 0 N1 I .

N c, 6 "-, CI CI STX1I 11, XDS02085 STX1 119, XDS02100 STX1 120, XDS02101 O I 0 HH H 0 N NN 0 s N Cl STX977, XDS02015 STX978, XDS02017 STX117, XDS02098 H 9KN H N H P r N oCF 3 N 5-' N -N -~N - N CI H H STX1 118, XDS02099 STX879, XDS01 173 STX985, XDS02026 Preparation of I -AlkyI-5-am ino-2-methylbenzimidazole and I -alkyl-6-amino-2 methylbenzimidazole To a solution of 5-nitrobenzimidazole (1.0 g, 5.6 mmol) in acetone (50 mL) was added 5 potassium carbonate (1.0 g), followed by alkyl halide (1.2 - 1.5 equivalents). The mixture was stirred under nitrogen at rt, then partitioned between ethyl acetate and water WO 2004/037251 PCT/GB2003/004590 115 after TLC showed completion of the reaction. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a mixture of 1-alkyl 5-nitro-2-methylbenzimidazole and 1-alkyl-6-nitro-2-methylbenzimidazole, which were dissolved in ethanol-THF (100 mL, 2 : 1) and hydrogenated over 5% Pd-C under 5 atmosphere pressure for 8h. After filtration through celite®, the filtrate was evaporated to give a yellow solid that was separated with flash chromatography (Methanol-DCM gradient elution). 1-Alkyl-5-aminobenzimidazole and 1-alkyl-6-aminobenzimidazole were obtained as yellow solid or thick syrup. 10 5-Amino-1,2-dimethylbenzimidazole (XDS01191B, XDSO2082B): Yellow solid, mp 126-1270C (lit.128 0 C, [23]). TLC single spot at Rf 0.30 (5%methanol/DCM); 'H NMR (270 MHz, DMSO): 5 7.08 (1H, d, J = 8.7 Hz, 7-H), 7.65 (1H, d, J = 1.5 Hz, 4-H), 6.50 (1H, dd, J = 8.7, 1.5 Hz, 6-H), 4.63 (2H, broad, NH 2 ), 3.58(3H, s, NCH 3 ), 2.39 (3H, s, CH 3 ). 15 6-Amino-1,2-dimethylbenzimidazole (XDS01191A, XDSO2082A): Yellow solid. TLC single spot at Rf 0.33 (5%methanol/DCM); 'H NMR (270 MHz, DMSO): 5 7.13 (1H, d, J= 8.4 Hz, 4-H), 6.48 (1H, d, J= 2.0 Hz, 7-H), 6.43 (1H, dd, J= 8.4, 2.0 Hz, 5-H), 4.83 (2H, broad, NH 2 ), 3.53(3H, s, NCH 3 ), 2.39 (3H, s, CH 3 ). 20 5-Amino-l-ethyl-2-methylbenzimidazole (XDSO2079B): Yellow syrup. TLC single spot at Rf 0.27 (5%methanol/DCM); 1H NMR (270 MHz, DMSO): 6 7.12 (1H, d, J = 8.3 Hz, 7-H), 6.68 (1H, d, J = 2.0 Hz, 4-H), 6.51 (1H, dd, J = 8.3, 2.0 Hz, 6-H), 4.68 (2H, broad, NH 2 ), 4.08 (2H, q, J = 7.2 Hz, NCH 2 ), 2.43 (3H, s, 25 CH 3 ), 1.24(3H, t, J = 7.2 Hz, CH 3 ); APCI-MS 175 (M'). 6-Amino-l-ethyl-2-methylbenzimidazole (XDSO2079A): Yellow solid. TLC single spot at Rf 0.30 (5%methanol/DCM); 1H NMR (270 MHz, DMSO): 6 7.16 (1H, d, J = 8.4 Hz, 4-H), 6.68 (1H, d, J = 1.7 Hz, 7-H), 6.46 (1H, dd, J= 30 8.4, 1.7 Hz, 5-H), 4.85 (2H, broad, NH 2 ), 4.02 (2H, q, J = 7.9 Hz, NCH 2 ), 2.42 (3H, s,

CH

3 ), 1.24(3H, t, J= 7.9 Hz, CH 3 ); APCI-MS 175 (M'). 5-Amino-I-i-butyl-2-methylbenzimidazole (XDSO2093B): Yellow syrup. TLC single spot at Rf 0.42 (10%methanol/DCM); 1H NMR (400 MHz, 35 DMSO): 5 7.08 (1H, d, J 8.5 Hz, 7-H), 6.65 (1H, d, J= 1.9 Hz, 4-H), 6.48 (1H, dd, J= WO 2004/037251 PCT/GB2003/004590 116 8.5, 1.9 Hz, 6-H), 4.63 (2H, broad, NH 2 ), 3.82 (2H, d, J = 7.4 Hz, NCH 2 ), 2.41 (3H, s,

CH

3 ), 2.07 (1H, m, CH), 0.84 (6H, d, J = 7.0 Hz, 2 x CH 3 ); APCI-MS 204 (MH+) 6-Amino-l-i-butyl-2-methylbenzimidazole (XDSO2093A): 5 Yellow solid. TLC single spot at Rf 0.45 (10%methanol/DCM); 1 H NMR (270 MHz, DMSO): 6 7.15 (1H, d, J = 8.2 Hz, 4-H), 6.62 (1H, d, J = 1.6 Hz, 7-H), 6.44 (1H, dd, J = 8.2, 1.8 Hz, 5-H), 4.83 (2H, broad, NH 2 ), 3.79 (2H, d, J = 7.7 Hz, NCH 2 ), 2.41 (3H, s,

CH

3 ), 2.10 (1H, m, CH), 0.87 (6H, d, J = 6.6 Hz, 2 x CH 3 ); APCI-MS 204 (MH ' ) 10 (5-Aminobenzoimidazol-1-yl)-acetic acid ethyl ester (XDSO2012B) Yellow solid. TLC single spot at Rf 0.36 (5%methanol/DCM); 'H NMR (270 MHz, DMSO): 6 7.08 (1H, d, J = 8.4 Hz, 7-H), 6.69 (1H, d, J = 2.2 Hz, 4-H), 6.49 (1H, dd, J = 8.4, 2.2 Hz, 6-H), 5.02 (2H, s, NCH 2 ), 4.68 (2H, s, NH 2 ), 4.16 (2H, q, J = 7.2 Hz, CH 2 ), 2.36 (3H, s, CH 3 ), 1.21 (3H, t, J= 7.2 Hz, CH 3 ); APCI-MS 234 (MH'). 15 (6-Aminobenzoimidazol-1-yl)-acetic acid ethyl ester (XDSO2012A) Yellow solid. TLC single spot at Rf 0.40 (5%methanol/DCM); 'H NMR (270 MHz, DMSO): 5 7.17 (1H, d, J = 9.0 Hz, 4-H), 6.45-6.48 (2H, m, 5 and 7-H), 4.95 (2H, s,

NCH

2 ), 4.87 (2H, s, NH 2 ), 4.17 (2H, q, J = 7.1 Hz, CH 2 ), 2.36 (3H, s, CH 3 ), 1.22 (3H, t, J 20 = 7.1 Hz, CH 3 ); APCI-MS 234 (MH*). 5-Amino-l-benzyl-2-methylbenzimidazole (XDSO2086B): Yellow syrup. TLC single spot at Rf 0.27 (5%methanol/DCM); 1H NMR (400 MHz, DMSO): 6 7.26-7.32 (2H, m, ArH), 7.23 (1H, tt, J = 7.5, 2.3 Hz, ArH), 7.05-7.09 (3H, m, 25 ArH), 6.69 (1H, d, J = 2.3 Hz, 4-H), 6.46 (1H, dd, J = 8.2, 2.3 Hz, 6-H), 5.30 (2H, s, CH 2 ), 4.68 (2H, broad, NH 2 ), 2.40 (3H, s, CH 3 ); APCI-MS 238 (MH'). 6-Amino-l-benzyl-2-methylbenzimidazole (XDSO2086A): Yellow solid. TLC single spot at Rf 0.30 (5%methanol/DCM); 'H NMR (400 MHz, 30 DMSO): 6 7.28-7.32 (2H, m, ArH), 7.23 (1H, tt, J = 7.5, 2.3 Hz, ArH), 7.17 (1H, d, J = 8.2, Hz, ArH), 7.06 (2H, m ArH), 6.43-6.46 (2H, m, ArH), 5.26 (2H, s, CH 2 ), 4.63 (2H, s,

NH

2 ), 2.40 (3H, s, CH 3 ); APCI-MS 238 (MH). Preparation of 5-amino-4-chloro-1,2-dimethylbenzimidazole (XDSO2096A) 35 To a solution of 5-amino-1,2-dimethylbenzimidazole (600 mg, 3.73 mmol) in IPA (15 mL) was added N-chlorosuccinimide (548 mg, 4.10 mmol). The mixture was stirred at rt for WO 2004/037251 PCT/GB2003/004590 117 20 min, diluted with DCM (80 mL) and washed with 5% sodium bicarbonate and brine. The dark brown solution was dried over sodium sulphate and concentrated in vacuo to give a brown residue, which was subjected to flash chromatography (methanol-DCM gradient elution). Yellow solid (220 mg, 33%) was obtained. TLC single spot at Rf 0.69 5 (10%methanol/DCM); 1 H NMR (270 MHz, DMSO): 5 7.16 (1H, d, J = 7.9 Hz, ArH), 6.72 (1H, d, J= 7.9, Hz, ArH), 4.90 (2H, s, NH 2 ), 3.64 (3H, s, NCH 3 ), 2.40 (3H, s, CH 3 ); APCI MS 196 (MH) General method for synthesis of N-benzimidazole arylsulphonamide derivatives: 10 To a solution arylsulphonyl chloride (1.1 eq.) in DCM were added pyridine (2.2 eq.) and catalytic amount of DMAP, followed by the corresponding amine (1 eq.). The reaction mixture was stirred at rt under nitrogen for 4-16 h, then partitioned between ethyl acetate and 5% sodium bicarbonate after TLC showed completion of the reaction. The organic layer was washed with brine, dried over sodium sulphate, and concentrated in vacuo to 15 give crude product as solid or thick syrup. The compound was then purified by flash chromatography (Methanol-DCM gradient elution) to give desired arylsulphonamide as crystalline solid. Yield ranges from 50-80%. 3-Chloro-N-(1,2-dimethyl-1H-benzoimidazol-6-yl)-2-methylbenzenesulphonamide 20 (STX975, XDSO2001) White crystalline solid. Mp 265-266°C; TLC single spot at Rf 0.43 (5% methanol/DCM); HPLC purity > 99% (tR 2. min in 10% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.4 (1H, s, NH), 7.84 (1H, d, J= 7.9 Hz, ArH), 7.67 (1H, d, J = 7.9 Hz, ArH), 7.34 (1H, d, J= 8.2 Hz, ArH), 7.32 (1H, t, J= 7.9 Hz, ArH), 7.14(1H, d, J= 2 Hz, ArH ), 6.80 (1H, 25 dd, J= 8.2, 2.0 Hz, ArH), 3.61 (3H, s, NCH 3 ), 2.64 (3H, s, CH 3 ), 2.46 (3H, s, CH); APCI MS 348 (M-H+); FAB-HRMS calcd for C16H17CIN302S (MH) 350.0730, found 350.0749. 3-Chloro-N-(1,2-dimethyl-1 H-benzoimidazol-5-yl)-2-methylbenzenesulphonamide 30 (STX976, XDSO2003) White crystalline solid. Mp 283-283.5'C; TLC single spot at Rf 0.38 (5% methanol/DCM); HPLC purity >99% (tR 2.0 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.3 (1H, s, NH), 7.77 (1H, d, J = 7.6 Hz, ArH), 7.66 (1H, d, J= 7.6 Hz, ArH), 7.32 (1H, d, J= 8.4 Hz, ArH), 7.30 (1H, t, J= 7.6 Hz, ArH), 7.16(1H, d, J = 2 Hz, 35 ArH ), 6.90 (1H, dd, J= 8.4, 2.0 Hz, ArH), 3.64 (3H, s, NCH 3 ), 2.64 (3H, s, OH 3 ), 2.44 WO 2004/037251 PCT/GB2003/004590 118 (3H, s, CH 3 ); APCI-MS 348 (M-H ); FAB-HRMS calcd for C16H17CIN302S (MH) 350.0730, found 350.0747. 3-Chloro-N-(4-chloro-1,2-dimethyl-1H-benzoimidazol-5-yl)-2 5 methylbenzenesulphonamide (STX1121, XDSO2102B) Off-white crystalline solid. TLC single spot at Rf 0.50 (10% methanol/DCM); HPLC purity 95% (tR 2.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 6 10.1 (1H, s, NH), 7.70 (1H, dd, J= 7.7, 1.7 Hz, ArH), 7.56 (1H, dd, J= 7.8, 1.7 Hz, ArH), 7.39 (1H, d, J = 8.2 Hz, ArH), 7.25 (1H, t, J = 7.7 Hz, ArH), 7.04 (1H, d, J = 8.2 Hz, ArH), 3.69 (3H, s, 10 NCH 3 ), 2.67 (3H, s, CH 3 ), 2.51 (3H, s, CH 3 ); APCI-MS 384 (MH). N-(1,2-Dimethyl-1H-benzoimidazol-5-yl).4-propylbenzenesulphonamide (STX1112, XDS02088) White crystalline solid. TLC single spot at Rf 0.38 (5% methanol/DCM); HPLC purity 15 >99% (tR 2.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 6 9.90 (1H; s, NH), 7.58 (2H, d, J= 8.3 Hz, ArH), 7.29-7.32 (3H, m, ArH), 7.17 (1H, d, J= 1.5 Hz, ArH), 6.91 (1H, dd, J = 8.6, 2.0 Hz, ArH), 3.64 (3H, s, NCH 3 ), 2.55 (2H, m, CH 2 ), 2.50 (3H, s,

CH

3 ), 1.55 (2H, sextet, J = 7.6 Hz, CH 2 ), 0.84 (3H, t, J = 7.6 Hz, CH 3 ); APCI-MS 344 (MH*). 20 2,5-Dichloro-N-(1,2-dimethyl-1 H-benzoimidazol-5-yl)-benzenesulphonamide (STXI 113, XDS02089) White crystalline solid. TLC single spot at Rf 0.67 (10% methanol/DCM); HPLC purity 99% (tR 2.0 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 5 10.5 (1H, s, 25 NH), 7.84 (1 H, t, J = 1.4 Hz, ArH), 7.68 (2H, d, J = 2.0 Hz, ArH), 7.35 (1H, d, J= 8.5 Hz, ArH), 7.21 (1H, d, J = 2.0 Hz, ArH), 6.97 (1H, dd, J = 8.2, 2.0 Hz, ArH ), 3.64 (3H, s,

NCH

3 ), 2.45 (3H, s, CH 3 ); APCI-MS 370 (MH). 2,4-Dichloro-N-(1,2-dimethyl-1 H-benzoimidazol-5-yl)-benzenesulphonamide 30 (STX1114, XDSO2090) Off-white crystalline solid. TLC single spot at Rf 0.59 (10% methanol/DCM); HPLC purity >99% (tR 2.0 min in 20% water-methanol); 1H NMR (270 MHz, DMSO): 5 10.4 (1H, s, NH), 7.88 (1H, d, J = 8.7 Hz, ArH), 7.84 (2H, d, J = 1.9 Hz, ArH), 7.52 (1H, dd, J = 8.7, 1.9 Hz, ArH), 7.33 (1H, d, J = 8.5 Hz, ArH), 7.20 (1H, d, J = 1.7 Hz, ArH ), 6.95 (1H, dd, 35 J= 8.7, 1.7 Hz, ArH ), 3.63 (3H, s, NCH 3 ), 2.45 (3H, s, CH 3 ); APCI-MS 370 (MH).

WO 2004/037251 PCT/GB2003/004590 119 4-Bromo-N-(1,2-dimethyl-1 H-benzoimidazol-5-yl)-2-methylbenzenesulphonamide (STX1115, XDSO2091) White crystalline solid. TLC single spot at Rf 0.67 (10% methanol/DCM); HPLC purity >99% (tR 2.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 5 10.1 (1H, s, 5 NH), 7.62-7.69 (2H, m, ArH), 7.50 (1H, dd, J= 8.5, 2.2 Hz, ArH), 7.32 (1H, d, J= 8.5 Hz, ArH), 7.14 (1H, d, J = 1.9 Hz, ArH), 6.89 (1H, dd, J = 8.5, 1.9 Hz, ArH), 3.63 (3H, s,

NCH

3 ), 2.55 (3H, s, CH 3 ), 2.45 (3H, s, CH 3 ); APCI-MS 394 (MH 4 ). N-(1,2-Dimethyl-1H-benzoimidazol-5-yl)-4-phenylbenzenesulphonamide (STX1 116, 10 XDS02092) White crystalline solid. TLC single spot at Rf 0.72 (5% methanol/DCM); HPLC purity >99% (tR 2.1 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 5 10.0 (1H, s, NH), 7.67-7.82 (6H, m, ArH), 7.41-7.50 (3H, m, ArH), 7.33 (1H, d, J = 8.5 Hz, ArH), 7.22 (1H, d, J = 1.9 Hz, ArH), 6.95 (1H, dd, J = 8.5, 1.9 Hz, ArH), 3.64 (3H, s, NCH), 2.44 15 (3H, s, CH 3 ); APCI-MS 378 (MH). 3-Chloro-N-(1-ethyl-2-methyl-1H-benzoimidazol-6-yl)-2 methylbenzenesulphonamide (STX1 110, XDS02084) Off-white solid. TLC single spot at Rf 0.45 (8% methanol/DCM); HPLC purity >99% (tR 20 2.2 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 6 10.4 (1H, s, NH), 7.84 (1H, d, J = 7.9 Hz, ArH), 7.67 (1H, d, J= 7.8 Hz, ArH), 7.35 (1H, d, J= 8.5 Hz, ArH), 7.32 (1 H, t, J = 7.9 Hz, ArH), 7.10 (1H, d, J = 2.2 Hz, ArH ), 6.82 (1H, dd, J= 8.5, 2.1 Hz, ArH), 4.09 (2H, q, J =7.1 Hz, CH 2 ), 2.61 (3H, s, CH 3 ), 2.46 (3H, s, CH 3 ), 1.18 (3H, t, J = 7.1 Hz, CH 3 ); APCI-MS 364 (MH). 25 3-Chloro-N-(1-ethyl-2-methyl-1 H-benzoimidazol-5-yl)-2 methylbenzenesulphonamide (STX1111, XDS02085) Off-white solid. TLC single spot at Rf 0.42 (8% methanol/DCM); HPLC purity >99% (tR 2.2 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.3 (1H, s, NH), 7.78 30 (1H, d, J = 7.9 Hz, ArH), 7.66 (1H, d, J = 7.9 Hz, ArH), 7.36 (1H, d, J = 8.2 Hz, ArH), 7.32 (1H, t, J= 7.9 Hz, ArH), 7.16 (1H, d, J= 1.9 Hz, ArH ), 6.90 (1H, dd, J= 7.9, 2.0 Hz, ArH), 4.12 (2H, q, J =7.1 Hz, CH 2 ), 2.64 (3H, s, CH 3 ), 2.46 (3H, s, CH 3 ), 1.23 (3H, t, J = 7.1 Hz, CH 3 ); APCI-MS 364 (MH*).

WO 2004/037251 PCT/GB2003/004590 120 3-Chloro-N-(1-isobutyl-2-methyl-1lH-benzoimidazol-6-yl)-2 methylbenzenesulphonamide (STX1 119, XDSO2100) Off-white solid. TLC single spot at Rf 0.57 (8% methanol/DCM); HPLC purity 99% (tR 2.2 5 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.4 (1H, s, NH), 7.80 (1H, d, J = 8.9 Hz, ArH), 7.66 (1H, d, J= 8.8 Hz, ArH), 7.36 (1H, d, J= 8.5 Hz, ArH), 7.29 (1H, t, J = 7.9 Hz, ArH), 7.03 (1H, d, J = 1.9 Hz, ArH ), 6.84 (1H, dd, J= 8.5, 1.8 Hz, ArH), 3.85 (2H, d, J =7.2 Hz, NCH 2 ), 2.61 (3H, s, CH 3 ), 2.45 (3H, s, CH 3 ), 1.91 (1H, m, CH), 0.81 (6H, d, J = 7.0 Hz, 2 x CH 3 ); APCI-MS 392 (MH+). 10 3-Chloro-N-(1-isobutyl-2-methyl-1H-benzoimidazol-5-yl)-2 methylbenzenesulphonamide (STX1120, XDSO2101) Off-white solid. TLC single spot at Rf 0.52 (8% methanol/DCM); HPLC purity 99% (tR 2.3 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.3 (1H, s, NH), 7.80 (1H, 15 d, J= 7.9 Hz, ArH), 7.68 (1H, d, J= 7.9 Hz, ArH), 7.38 (1H, d, J= 8.8 Hz, ArH), 7.33 (1H, t, J = 7.9 Hz, ArH), 7.16 (1H, d, J = 1.9 Hz, ArH ), 6.90 (1H, dd, J= 8.7, 1.9 Hz, ArH), 3.91 (2H, d, J =7.3 Hz, NCH 2 ), 2.62 (3H, s, CH 3 ), 2.47 (3H, s, CH 3 ), 2.05 (1H, m, CH), 0.83 (6H, d, J = 7.0 Hz, 2 x CH 3 ); APCl-MS 392 (MH+). 20 [6-(3-Chloro-2-methylbenzenesulphonylamino)-2-methylbenzoimidazol-.1-yl]-acetic acid ethyl ester (STX977, XDSO2015) Off-white solid. TLC single spot at Rf 0.46 (6% methanol/DCM); HPLC purity >99% (tR 2.0 min in 10% water-methanol); 'H NMR (270 MHz, DMSO): 6 10.4 (1H, s, NH), 7.82 (1H, d, J = 8.0 Hz, ArH), 7.67 (1H, d, J = 8.0 Hz, ArH), 7.37 (1H, d, J = 8.5 Hz, ArH), 25 7.30 (1H, t, J= 8.0 Hz, ArH), 7.11 (1H, d, J= 2.0 Hz, ArH ), 6.83 (1H, dd, J= 8.5,2.0 Hz, ArH), 5.09 (2H, s, NCH 2 ), 4.16 (2H, q, J = 7.1 Hz, CH 2 ), 2.62 (3H, s, CH 3 ), 2.45 (3H, s,

CH

3 ), 1.21 (3H, t, J = 7.1 Hz, CH 3 ); APCI-MS 420 (M-H+); FAB-HRMS calcd for C1 9H21 CIN304S (MH) 422.0941, found 422.0942. 30 [5-(3-Chloro-2-methylbenzenesulphonylamino)-2-methylbenzoimidazol-1-yl]-acetic acid ethyl ester (STX978, XDSO2017) Off-white solid. TLC single spot at Rf 0.40 (6% methanol/DCM); HPLC purity 99% (tR 2.0 min in 10% water-methanol); 'H NMR (270 MHz, DMSO): 6 10.3 (1H, s, NH), 7.80 (1H, d, J= 7.9 Hz, ArH), 7.67 (1H, d, J = 7.9 Hz, ArH), 7.29-7.34 (2H, m, ArH), 7.18 (1H, d, J 35 = 1.7 Hz, ArH ), 6.90 (1H, dd, J= 8.6, 1.7 Hz, ArH), 5.11 (2H, s, NCH 2 ), 4.15 (2H, q, J = 7.1 Hz, CH 2 ), 2.64 (3H, s, CH 3 ), 2.40 (3H, s, CH 3 ), 1.19 (3H, t, J = 7.1 Hz, CH 3 ); APCI- WO 2004/037251 PCT/GB2003/004590 121 MS 420 (M-H+); FAB-HRMS calcd for C19H21CIN304S (MH) 422.0941, found 422.0944. 3-Chloro-N-(1-benzyl-2-methyl-1lH-benzoimidazol-6-yl)-2 5 methylbenzenesulphonamide (STX1117, XDSO2098) Off-white solid. TLC single spot at Rf 0.70 (10% methanol/DCM); HPLC purity 99% (t 2.2 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.4 (1H, s, NH), 7.66 (1H, d, J= 7.9 Hz, ArH), 7.64 (1H, d, J= 7.9 Hz, ArH), 7.30-7.39 (4H, m, ArH), 7.21 (1H, t, J = 7.9 Hz, ArH), 7.11 (1H, d, J = 2.0 Hz, ArH), 7.02-7.06 (2H, m, ArH ), 6.83 (1H, dd, 10 J= 7.9, 2.0 Hz, ArH), 5.34 (2H, s, NCH 2 ), 2.58 (3H, s, CH 3 ), 2.45 (3H, s, CH 3 ); APCI-MS 426 (MH). 3-Chloro-N-(1-benzyl-2-methyl-1lH-benzoimidazol-5-yl)-2 methylbenzenesulphonamide (STXI 118, XDS02099) 15 Off-white solid. TLC single spot at R, 0.65 (10% methanol/DCM); HPLC purity 99% (tR 2.2 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.3 (1H, s, NH), 7.80 (1H, d, J= 7.9 Hz, ArH), 7.67 (1H, d, J= 7.9 Hz, ArH), 7.25-7.35 (5H, m, ArH), 7.19 (1H, d, J = 1.9 Hz, ArH), 7.06-7.09 (2H, m, ArH ), 6.87 (1H, dd, J= 8.5, 1.9 Hz, ArH), 5.38 (2H, s, NCH 2 ), 2.62 (3H, s, CH 3 ), 2.45 (3H, s, CH 3 ); APCI-MS 426 (MH). 20 3-Chloro-N-(2-trifluromethyl-1 H-benzoimidazol-5-yl)-2 methylbenzenesulphonamide (STX879, XDSO1173) White crystalline solid. TLC single spot at Rf 0.58 (20% ethyl acetate/DCM); HPLC purity 99% (tR 2.4 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 6 13.9 (1H, s, 25 NH), 10.7 (1H, s, NH), 7.85 (1H, d, J = 8.0 Hz, ArH), 7.68 (1H, d, J= 8.0 Hz, ArH), 7.60 (1H, d broad, J = 8.1 Hz, ArH), 7.34 (2H, m, ArH), 7.10(1H, d, J= 8.3 Hz, ArH ), 2.64 (3H, s, CH 3 ); APCI-MS 388 (M-H*); FAB-HRMS calcd for C15H12CIF3N302S (MH) 390.0291, found 390.0291. 30 Preparation of 3-chloro-N-(2-methyl-1 H-benzoimidazol-5-yl)-2 methylbenzenesulphonamide (STX985, XDS02026) The coupling reaction of 3-chloro-2-methylbenzenesulphonyl chloride (2 eq.) with 2 methylbenzimidazoel (1 eq.) under the condition described above yielded a mixture of 3 chloro-N-[1-(3-chloro-2-methylbenzenesulphonyl)-2-methyl-1H-benzoimidazol-5-yl]-2 35 methyl-benzenesulphonamide and 3-chloro-N-[1-(3-chloro-2-methylbenzenesulphonyl) 2-methyl-lH-benzoimidazol-6-yl]-2-methyl-benzenesulphonamide in 1 : 1 ratio as judged WO 2004/037251 PCT/GB2003/004590 122 by HNMR. 1 H NMR (270 MHz, DMSO): 5 10.7 (2H, s, 2 x NH), 7.86-7.96 (3H, m, ArH), 7.65-7.78 (5H, m, ArH), 7.52-7.58 (5H, m, ArH), 7.25-7.40 (3H, m, ArH), 7.07 (2H, t, J = 8.2 Hz, ArH), 2.61 (3H, s, CH 3 ), 2.58 (3H, s, CH 3 ), 2.54 (6H, s, 2 x CH 3 ), 2.39 (3H, s,

CH

3 ), 2.33 (3H, s, CH 3 ). The mixture (200 mg) was dissolved in THF (15 mL), N 5 hydroxybenzotriazole (200 mg) was added. After stirred at rt for 48h, the mixture was partitioned between ethyl acetate and 5% sodium bicarbonate. The organic phase was washed with brine, dried over sodium sulphate and concentrate in vacuo to give a yellow residue, which was purified with flash chromatography (methanol/DCM gradient elution). Off white amorphous powder was obtained. TLC single spot at Rf 0.38 (10% 10 methanol/DCM); HPLC purity 99% (tR 2.0 min in 10% water-methanol); 1 H NMR (270 MHz, DMSO): 6 12.1 (1H, s, NH), 10.3 (1H, s, NH), 7.78 (1H, d, J = 7.9 Hz, ArH), 7.68 (1H, d, J= 7.9 Hz, ArH), 7.29-7.35 (2H, m, ArH), 7.12(1H, s, ArH ), 6.83 (1H, dd, J= 8.4, 1.8 Hz, ArH), 2.63 (3H, s, CH 3 ), 2.41 (3H, s, CH 3 ); APCI-MS 334 (M-H'); FAB-HRMS calcd for C15H15CIN302S (MH') 336.0573, found 336.0583. 15 Synthesis of N-Benzimidazole Arvilsulphonamide Derivatives 0 H H S-N N 0 2 N NO 2 a H 2 ,.aNH2 'NH2N SN )CNH NH N11 'CN I Cl STX1140, XDS02110 c O CI C H _ N NN eN- _ e-S N 20 N jPhenyl-benzene-1,2,4-triamine: To a solution of 2,4-dinitrophenylamine (1.5 g, 5.8 mmol) in ethanol-THF (150 : 50 mL) were added hydrazine hydrate (2 mL, 65 mmol) and Raney Nickel (2.0 g). The reaction mixture was stirred at rt for 20 min, filtered through Celite. Evaporation of the solvent gave a black residue, which was purified by flash chromatography (methanol-DCM 25 gradient elution). A black crystalline solid (1.0 g, 87%) was obtained. Mp 128-1290C; TLC single spot at Rf 0.46 (8% methanol/DCM); H NMR (270 MHz, DMSO): 5 7.25 (2H, WO 2004/037251 PCT/GB2003/004590 123 t, J = 7.5 Hz, ArH), 6.73 (1H, s, NH), 6.61 (1H, d, J = 8.3 Hz, ArH), 6.49-6.55 (3H, m, ArH), 5.99 (1H, d, J = 2.5 Hz, ArH), 5.83 (1H, dd, J = 8.2, 2.5 Hz, ArH), 4.66 (2H, s,

NH

2 ), 4.44 (2H, s, NH 2 ); APCI-MS 198 (M-H'). 5 N-(2-Methyl-1l-phenyl-1 H-benzimidazol-5-yl)-acetamide: N'-Phenyl-benzene-1,2,4-triamine (800 mg, 4 mmol) was dissolved in acetic acid (10 mL), acetic anhydride (1.0 mL) was added to the solution. The mixture was stirred at 800C for 6h, cooled to rt and neutralized with 5% sodium carbonate, then extracted with ethyl acetate. The organic phase was washed with brine, dried over magnesium 10 sulphate and concentrated to give a residue, which was crystallized from ethanol. A brown crystalline solid (0.85 g, 80%) was obtained. Mp 231-232°C; TLC single spot at Rf 0.39 (10% methanol/DCM); 'H NMR (270 MHz, DMSO): 6 9.92 (1H, s, NH), 7.97 (1H, d, J = 1.6 Hz, ArH), 7.51-7.67 (5H, m, ArH), 7.31 (1H, dd, J = 8.3, 1.9 Hz, ArH), 7.04 (1H, d, J = 8.3 Hz, ArH),, 2.41 (3H, s, CH 3 ), 2.05 (3H, s, CH 3 ); APCI-MS 264 (M-H'). 15 2-Methyl-1-phenyl-1 H-benzoimidazol-5-ylamine: The solution of N-(2-methyl-1-phenyl-1H-benzimidazol-5-yl)-acetamide (800 mg, 3 mmol) in 6N HCI (5 mL) was stirred at 750C for 3h, cooled to rt and neutralized with sodium carbonate to pH 7, then extracted with ethyl acetate. The organic phase was 20 washed with brine, dried over magnesium sulphate and concentrated to give a dark brown solid (600 mg, 90%). Mp 145-146oC; TLC single spot at Rf 0.47 (10% methanol/DCM); 'H NMR (270 MHz, DMSO): 6 7.58-7.64 (2H, m, ArH), 7.46-7.53 (3H, m, ArH), 6.82 (1H, d, J = 8.5 Hz, ArH), 6.76 (1H, d, J = 1.9 Hz, ArH), 6.51 (1H, dd, J= 8.5,1.9 Hz, ArH), 4.78 (2H, s, NH 2 ), 2.36 (3H, s, CH 3 ); APCI-MS 223 (M'). 25 3-Chloro-2-methyl-N-(2-methyl-1l-phenyl-1 H-benzoimidazol-5-yl) benzenesulphonamide (STX1 140, XDSO2110) The compound was prepared with general method of benzenesulphonamide formation. Light pink crystalline solid was obtained. Mp 254-256°C; TLC single spot at Rf 0.62 (8% 30 methanol/DCM); HPLC purity > 99% (tR 2.6 min in 20% water-methanol); 'H NMR (270 MHz, DMSO): 6 10.4 (1H, s, NH), 7.83 (1H, dd, J = 8.6, 1.8 Hz, ArH), 7.69 (1H, dd, J = 8.6, 1.7 Hz, ArH), 7.58-7.63 (5H, m, ArH), 7.34 (1H, t, J = 8.0 Hz, ArH), 7.28 (1H, d, J= 1.9 Hz, ArH), 6.90-7.00 (2H, m, ArH), 2.66 (3H, s, CH 3 ), 2.36 (3H, s, CH 3 ); APCI-MS 412 (MH ). 35 WO 2004/037251 PCT/GB2003/004590 124 3-Chloro-N-[1-(2-hydroxyethyl)-2-methyl-1 H-benzoimidazol-6-yl]-2-methyl benzenesulfonamide (STX1141, XDSO2115) To a solution of [6-(3-Chloro-2-methyl-benzenesulfonylamino)-2-methyl-benzoimidazol 1-yl]-acetic acid ethyl ester (100 mg, 0.237 mmol) in anhydrous THF (10 mL) was added 5 LiAIH 4 (54 mg, 1.42 mmol) at 00C. The mixture was stirred at 00C for 0.5h, quenched with saturated ammonium chloride solution, neutralized with 6N HCI and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulphate and concentrated in vacuo to give a light pink crystalline solid (82 mg, 91%). Mp 213 214.50C; TLC single spot at Rf 0.39 (12% methanol/DCM); HPLC purity > 99% (tR 2.0 10 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.4 (1H, s, NH), 7.84 (1H, d, J= 7.7 Hz, ArH), 7.67 (1H, d, J= 7.9 Hz, ArH), 7.28-7.35 (2H, m, ArH), 7.15 (1H, d, J = 1.9 Hz, ArH), 6.80 (1H, dd, J = 8.5, 1.9 Hz, ArH), 4.94 (1H, t, J = 5.2 Hz, OH), 4.10 (2H, t, J = 5.0 Hz, NCH 2 ), 3.59 (2H, q, J = 5.2 Hz, CH 2 ), 2.51 (3H, s, CH 3 ), 2.47 (3H, s,

CH

3 ); APCI-MS 380 (MH). 15 3-Chloro-N-[1-(2-hydroxy-ethyl)-2-methyl-lH-benzoimidazol-5-yl]-2-methyl benzenesulfonamide (STX1142, XDSO2116) The compound was prepared as above from [5-(3-Chloro-2-methyl benzenesulfonylamino)-2-methyl-benzoimidazol-1-yl]-acetic acid ethyl ester (35 mg, 20 0.083 mmol). White crystalline solid (22 mg, 89%) was obtained. Mp 245-247oC; TLC single spot at Rf 0.38 (12% methanol/DCM); HPLC purity > 99% (tR 2.0 min in 20% water-methanol); 1 H NMR (270 MHz, DMSO): 6 10.3 (1H, s, NH), 7.79 (1H, d, J = 8.0 Hz, ArH), 7.67 (1H, d, J = 8.0 Hz, ArH), 7.29-7.35 (2H, m, ArH), 7.16 (1H, s, ArH), 6.89 (1H, d, J= 8.5, ArH), 4.90 (1H, t, J = 5.0 Hz, OH), 4.14 (2H, t, J = 5.0 Hz, NCH 2 ), 3.63 25 (2H, q, J= 5.2 Hz, CH 2 ), 2.65 (3H, s, CH 3 ), 2.48 (3H, s, CH 3 ); APCI-MS 380 (MH'). Synthesis of Benzoxazole Derivatives R*

R

1 R2N R2 0 RS 'O - R 3 '~~ N R40 H

R

4 H STX 839: R 1

=R

2 =H, R 3 =CI, R 4 =Me STX 842: RI=R 2 =H, R 3 = C I , R 4

=

Me STX 840: RI=R 3

=R

4 =H, R 2 =n-propyl STX 843: Ri=R 3

=R

4 =H, R 2 =n-propyl STX 841: R=R 4 =CI, R 2

=R

3 =H STX 846: R 1

=R

4 =CI, R 2

=R

3 = H 30 WO 2004/037251 PCT/GB2003/004590 125 Synthesis of 3-chloro-2-methyl-N-(2-methyl-benzooxazol-6-yl) benzenesulfonamide, STX 839 (KRB01009): C0 N 0 H 5 To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (163 mg, 0.723 mmol) in dichloromethane (3 mL) was added pyridine (140 pL, 1.72 mmol) and the mixture was stirred under N 2 for 5 min, after which time 6-amino-2-methylbenzoxazole (102 mg, 0.688 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, 10 then saturated NaHCO 3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na 2

SO

4 ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a white solid (151 mg, 65%), single spot at Rf 0.50 (60:40 hexane:ethyl acetate). mp 127.1-127.50C, HPLC purity 97% (tR 2.05 min in 10% water-acetonitrile). 1 H NMR 15 (CDC 3 ): 6 7.85 (1H, dd, J=8.1, 1.1 Hz), 7.53 (1H, dd, J=8.1, 1.3 Hz), 7.45 (1H, d, J=8.4 Hz), 7.27 (1H, d, J=2.2 Hz), 7.17 (1H, t, J=7.9 Hz), 6.93 (1H, s, N-H), 6.86 (1H, dd, J=8.4, 2.2 Hz), 2.71 (3H, s), 2.58 (3H, s). LCMS: 335.14 (M-). FAB-MS (MH+,

C

15

H

13

CIN

2 0 3 S): calcd 337.0413, found 337.0406. 20 Synthesis of N-(2-methyl-benzooxazol-6-yl)-4-propyl-benzenesulfonamide, STX 840 (KRB01010): 0 O H 25 To a solution of 4n-propylbenzenesulphonyl chloride (163 mg, 0.744 mmol) in dichloromethane (3 mL) was added pyridine (140 pL, 1.72 mmol) and the mixture was stirred under N 2 for 5 min, after which time 6-amino-2-methylbenzoxazole (105 mg, 0.709 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO 3 solution was added (8 mL) and the mixture was extracted into 30 ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na 2

SO

4 ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale pink solid (164 mg, 70%), single spot at Rf 0.49 (60:40 hexane:ethyl WO 2004/037251 PCT/GB2003/004590 126 acetate). mp 101.7-102.3'C, HPLC purity 99% (tR 2.02 min in 10% water-acetonitrile). 1 H NMR (CDC 3 ): 6 7.61 (2H, m), 7.43 (1H, d, J=8.4 Hz), 7.37 (1H, d, J=1.8 Hz), 7.19 (2H, m), 6.83 (2H, m), 2.57 (5H, m), 1.58 (2H, sextet, J=7.3 Hz), 0.88 (3H, t, J=7.3 Hz). LCMS: 329.21 (M-). FAB-MS (MH+, C1 7

H

1 8

N

2 0 3 S): calcd 331.1116, found 331.1107. 5 Synthesis of 2,5-dichloro-(2-methyl-benzooxazol-6-yl)-benzenesulfonamide, STX 841 (KRB01011): Cl N C 0 10 To a solution of 2,5-dichlorobenzenesulphonyl chloride (174 mg, 0.709 mmol) in dichloromethane (3 mL) was added pyridine (140 pL, 1.72 mmol) and the mixture was stirred under N 2 for 5 min, after which time 6-amino-2-methylbenzoxazole (100 mg, 0.675 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, 15 then saturated NaHCO, solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na 2

SO

4 ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a white solid (154 mg, 64%), single spot at Rf 0.50 (60:40 hexane:ethyl acetate). mp 167.0-167.3oC, HPLC purity 97% (tR 1.97 min in 10% water-acetonitrile). 1 H NMR 20 (CDCI 3 ): 6 7.93 (1H, d, J=2.3 Hz), 7.47 (1H, d, J=8.6 Hz), 7.46-7.40 (4H, m), 7.00 (1H, dd, J=8.6, 2.0 Hz), 2.61 (3H, s). LCMS: 355.07 (M-). FAB-MS (MH+, C 14

H

1 0 C1 2

N

2 0 3 S): calcd 356.9867, found 356.9875. Synthesis of 3-chloro-2-methyl-N-(2-methyl-benzooxazol.5-yl). 25 benzenesulfonamide, STX 842 (KRB01014): Sd N " N To a solution of 3-chloro-2-methylbenzenesulphonyl chloride (96 mg, 0.43 mmol) in 30 dichloromethane (2 mL) was added pyridine (80 pL, 1.0 mmol) and the mixture was stirred under N 2 for 5 min, after which time 5-amino-2-methylbenzoxazole (60 mg, 0.40 WO 2004/037251 PCT/GB2003/004590 127 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO 3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na 2

SO

4 ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a 5 white solid (89 mg, 64%), single spot at Rf 0.52 (1:1 hexane:ethyl acetate). mp 180.2 180.5 0 C, HPLC purity 99% (tR 2.32 min in 10% water-acetonitrile). 'H NMR (CDCl 3 ): 6 7.82 (1H, dd, J=8.1, 1.1 Hz), 7.52 (1H, dd, J=7.7, 1.1 Hz), 7.32 (1H, d, J=8.4 Hz), 7.25 (1H, d, J-2.9 Hz (overlap with CHCI 3 )), 7.14 (1H, t, J=8.1 Hz), 6.99 (1H, dd, J=8.4, 2.2 Hz), 6.67 (1H, s, N-H), 2.71 (3H, s), 2.58 (3H, s). LCMS: 335.01 (M-). FAB-MS (MH+, 10 C 15

H

13

CIN

2 0 3 S): calcd 337.0413, found 337.0420. Synthesis of N-( 2 -methyl-benzooxazol-5-yl)-4-propyl-benzenesulfonamide, STX 843 (KRB01015):

N-.,N

15 d H To a solution of 4 n-propylbenzenesulphonyl chloride (93 mg, 0.43 mmol) in dichloromethane (2 mL) was added pyridine (80 pL, 1.0 mmol) and the mixture was stirred under N 2 for 5 min, after which time 5-amino-2-methylbenzoxazole (60 mg, 0.40 20 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO 3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na 2 SO4), filtered and evaporated to give a residue that was purified using flash chromatography to afford a pale pink oil (112 mg, 85%), single spot at Rf 0.53 (1:1 hexane:ethyl acetate). HPLC 25 purity 99+% (tR 2.38 min in 10% water-acetonitrile) 1 H NMR (CDC 3 ): 6 7.64 (2H, dt, J=8.1, 1.8 Hz), 7.31 (2H, m), 7.18 (2H, d, J=8.4 Hz), 7.06 (1H, dd, J=8.6, 2.4 Hz), 2.59 (5H, m), 1.58 (2H, sextet, J=7.3 Hz), 0.89 (3H, t, J=7.3 Hz). LCMS: 329.15 (M-). FAB MS (MH+, C 1 7

H

18

N

2 0 3 S): calcd 331.1116, found 331.1118. 30 Synthesis of 2,5-dichloro-N-(2-methyl-benzooxazol-5-yl)-benzenesulfonamide, STX 846 (KRB01016): WO 2004/037251 PCT/GB2003/004590 128 Cl 26 N ClO H To a solution of 2,5-dichlorobenzenesulphonyl chloride (52 mg, 0.21 mmol) in dichloromethane (1.5 mL) was added pyridine (40 pL, 0.5 mmol) and the mixture was 5 stirred under N 2 for 5 min, after which time 5-amino-2-methylbenzoxazole (30 mg, 0.20 mmol) was added. The resulting mixture was stirred for 1 h at room temperature, then saturated NaHCO 3 solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na 2

SO

4 ), filtered and evaporated to give a residue that was purified using flash chromatography to afford a 10 pale pink solid (45 mg, 63%), single spot at Rf 0.53 (1:1 hexane:ethyl acetate). mp 193.5-193.90C, HPLC purity 98% (tR 2.27 min in 10% water-acetonitrile). 1 H NMR

(CDC

3 ): 6 7.86 (1H, d, J=2.2 Hz), 7.39 (4H, m), 7.12 (1H, dd, J=8.4, 1.8 Hz), 2.58 (3H, s). LCMS: 355.07 (M-). FAB-MS (MH+, C 14

H

1

OCI

2

N

2 0 3 S): calcd 356.9867, found 356.9878. 15 All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with 20 specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims. 25 WO 2004/037251 PCT/GB2003/004590 129 REFERENCES 1. Hammond, GH (1990) : Molecular properties of corticosteroid binding globulin and sex-steroid binding proteins. Endocr. Rev. 11, 65- 79. 5 2. Gomez-Sanchez EP,Gomex-Sanchez CE (1997): First there was one, then two ..why not more 11 1-Hydroxysteroid Dehydrogenases? Endocrinology vol. 138, 12. 3. Krozowski ZS, Funder JW (1983): Renal mineralocorticosterone receptors and hippocampal corticosterone binding species have identical intrinsic steroid specificity . Proc. Natl. Sci. USA 80: 6056-60 10 4. Ulick S, Levine LS, Gunczler P, Zanconato G, Rarnirez LC, Rauh W, Rosier A, Bradlow HL, Mew MI (1979): A syndrome of apparent mineralocorticoid excess associated with defects in the peripheral metabolism of cortisol. J. Clin. Endo. And Metab. 49: 757-64. 5. Edwards CRW, Stewart PM, Burt D, Brett L, Mcintyre MA, Sutanto WS, Kloet ER, 15 Monder C (1998): Localisation of 11 1-HSD-tissue specific protector of the mineralocorticoid receptor. Lancet 2: 986-989. 6. Moore CCD, Melloh SH, Mural I, Siiteri PK, Miller WL (1993): Structure and function of the hepatic form of 11 1-HSD in the squirrel monkey, an animal model of glucocorticoid resistance. Endocrinology 133: 368-375. 20 7. Kotelevtsev YV, larnieson PM, Best R, Stewart F, Edwards CRW, Seckl JR, Mullins II ( 1996): Inactivation of 11 -HSD type 1 by gene targeting in mice. Endocrinology Res. 22: 791-792. 8. Ricketts ML, Verhaeg JM, Bujalska I, Howie AJ, Rainey WE, Stewart PM (1998): Irnmunohistochemicallocalisation of type 1 11 P-HSD in human tissues. I. Clin. Endoc. 25 Metab. 83: 1325-35. 9. Stewart PM, Sheppard MC (1992): Novel aspects of hormone action: intracellular ligand supply and its control by a series of tissue specific enzymes. Molecular and Cellular Endocrinology 83: C13-C18. 10. Seckl JR, Chapman KE (1997): The 11 P3-HSD system, a determinant of 30 glucocorticoid and mineralocorticoid action. Medical and physiological aspects. European 1. Biochem. 249: 361-364. 11. Maser E (1998): 11 1-HSD responsible for carbonyl reduction of the tobacco specific nitrosoamine in mouse lung microsomes. Cancer Res. 58: 2996-3003. 12. Walker BR, Stewart PM, Shackleton C H L, Padfield PL, Edwards CRW (1993): 35 Deficient inactivation of cortisol by 11 -HSD in essential hypertension. Clin. Endocr. 38: 221-227.

WO 2004/037251 PCT/GB2003/004590 130 13. Daynes RA, Araneo BA (1998) : Contrasting effects of glucocorticoids on the capacity of T -cells to produce the growth factors interleukin-2 and interleukin-4. Eur. J. Immunol. 19: 2319-2324. 14. Bradford MM (1976): A rapid and sensitive method for the quantitation of microgram 5 quantities of protein utilizing the principle ofprotein-dye binding. Anal. Biochem. 72: 248-254. 15. Barf, T. et al., (2002), Arylsulfonamidothiazoles as a new class of potential antidiabetic drugs. Discovery of potent and selective inhibitors of the 11 P3-Hydroxysteroid Dehydrogenase Type 1. J. Med. Chem., 45, 3813-3815. 10 16. Stewart, P.M. and Mason, J.l., (1995), Cortisol to cortisone: Glucocorticoid to mineralocortcoid. Steriods, 60,143-146. 17. Escher, G. et al., (1995), Furosemide inhibits 11 1-Hydroxysteroid Dehydrogenase in vitro and in vivo. Endocrinology, 136, 1759-1765. 18. Hult, M. et. al., (1998), Selective inhibition of human type 1 11-hydroxysteroid 15 dehydrogenase by synthetic steroids and xenobiotics. FEBS Letters, 441, 25-28. 19. Diederich, S. et al., (2002), In the search for specific inhibitors of human 1113 hydroxysteroid-dehydrogenase (11 1-HSDs): chenodeoxycholic acid selectively inhibits 1113-HSD-1. Eur. J. Endocrinol., 142, 200-207. 20. Takahashi; Okada; Yakugaku Zasshi; 73; 1953; 802, 804; Chem.Abstr.; 1954; 20 9364. 21. Hunt, Richard el.al. J Chem Soc C, 1966, 344, 184-185. 22. DeGraw, Joseph and Goodman, Leon; J. Med. Chem.; 7; 1964; 213. 23. Fries, et.al.; Justus Liebigs Ann. Chem.; 454; 1927; 204.

Claims (51)

1. A compound having Formula I R1 N Formula I NN Z R3 --- X x R 2 wherein one of R 1 and R 2 is a group of the formula O 0 11 R 5 -S L N 5 R4 wherein R 4 is selected from H and hydrocarbyl, Rs is a hydrocarbyl group and L is an optional linker group, or R 1 and R 2 together form a ring substituted with the group O 0 L) I N4 R 4 10 wherein R 3 is H or a substituent and wherein X is selected from S, O, NR 6 and C(R 7 )(R 8 ), wherein R. is selected from H and hydrocarbyl groups, wherein each of R 7 and R 8 are independently selected from H and hydrocarbyl groups. 15

2. A compound according to claim 1 having Formula 11 RN Formula II -R 3 S R2

3. A compound according to claim 1 or 2 wherein L is not present.

4. A compound according to claim 1, 2 or 3 wherein R 1 and R 2 together form a ring WO 2004/037251 PCT/GB2003/004590 132 substituted with the group O 11 R5- S L 1N o R4

5. A compound according to any one of the preceding claims wherein R 1 and R 2 5 together form a carbocyclic ring.

6. A compound according to any one of the preceding claims wherein R 1 and R 2 together form a six membered ring. 10

7. A compound according to any one of the preceding claims wherein R, and R 2 together form an aryl ring.

8. A compound according to any one of the preceding claims having Formula III. 0 N Formula III R -S L R O-6S..N -R3 R4 15

9. A compound according to any one of the preceding claims having Formula IV. O N Formula IV R 5 - S L1 ° R R4 WO 2004/037251 PCT/GB2003/004590 133

10. A compound according to any one of the preceding claims having Formula V. O Formula V R5-S R4

11. A compound according to any one of the preceding claims having Formula VI o Formula VI 11 R5-S-N O R 4 R3 5

12. A compound according to any one of claims 1 to 10 having Formula VII N Formula VII O -R3 R5-S-N S I I I O R 4

13. A compound according to any one of the preceding claims wherein R 3 is selected from H, hydrocarbyl, -S-hydrocarbyl, -S-H, halogen and N(R)(R 10 ), wherein each of R 9 10 and Ro 1 0 are independently selected from H and hydrocarbyl groups.

14. A compound according to any one of the preceding claims wherein Ra is selected from H and C,-CI 0 alkyl groups, such as C-C6 alkyl group, and Cj-C3 alkyl group.

15 15. A compound according to any one of the preceding claims wherein R, is -CH 3 . WO 2004/037251 PCT/GB2003/004590 134

16. A compound according to any one of claims 1 to 7 having Formula VIII. O Formula VIll R 5 -S L R4

17. A compound according to according to any one of claims 1 to 7 having Formula 5 IX. O Formula IX Rs-S N IKS "- N R3 0S R4

18. A compound according to according to any one of claims 1 to 7 having Formula X O Formula X 1I R 5 -S- N II I O R 4 R 3 S

19. A compound according to any one of claims 1 to 7 having Formula Xl N Formula XI R 5 -S-N S 10

20. A compound according to any one of claims 16 to 19 wherein R 3 is selected from O, hydrocarbyl, and N(R 9 ) wherein R 9 is selected from H and hydrocarbyl groups.

21. A compound according to any one of the preceding claims wherein R 3 is selected WO 2004/037251 PCT/GB2003/004590 135 from O, C1-Co0 alkenyl groups, such as C 1 -C 6 alkenyl group, and C1-C3 alkenyl group, NH and N-CI-C 1 0 alkyl groups, such as N-CI-C. alkyl group, and N-C 1 -C 3 alkyl groups.

22. A compound according to any one of the preceding claims wherein R 4 is selected 5 from H and C,-Co alkyl groups, such as C,-C6 alkyl group, and C-C3 alkyl group.

23. A compound according to any one of the preceding claims wherein R 4 is H.

24. A compound according to any one of claims 1 to 21 wherein R 4 is a group of the 10 formula. 'T O=S O I R5

25. A compound according to any one of the preceding claims wherein Rs is a substituted ring. 15

26. A compound according to any one of the preceding claims wherein Rs is a carbocyclic ring.

27. A compound according to any one of the preceding claims wherein R 5 is a six 20 membered ring.

28 A compound according to any one of the preceding claims wherein R, is an aryl ring. 25

29 A compound according to any one of the preceding claims wherein RS is a group having the formula R 1 4 R13 R 15 R12 RI, WO 2004/037251 PCT/GB2003/004590 136 wherein each of R 11 , R 1 2 , R 13 , R 14 and R 1 5 are independently selected from H, halogen, and hydrocarbyl groups.

30 A compound according to claim 29 wherein each of R, 1 , R 1 2 , R 13 , R 14 and R 1 5 are 5 independently selected from H, halogen, alkyl, phenyl, O-alkyl, O-phenyl, nitrile, haloalkyl, carboxyalkyl, -CO 2 H, CO 2 alkyl, and NH-acetyl groups..

31. A pharmaceutical composition comprising a compound according to any one of claims 1 to 30 optionally admixed with a pharmaceutically acceptable carrier, diluent, 10 excipient or adjuvant.

32. A compound according to any one of claims 1 to 30 for use in medicine.

33. Use of a compound according to any one of claims 1 to 30 in the manufacture of 15 a medicament for use in the therapy of a condition or disease associated with 1 13-HSD.

34. Use according to claim 33 wherein the condition or disease is selected from the group consisting of metabolic disorders such as diabetes and obesity; cardiovascular disorders such as hypertension; glaucoma; inflammatory disorders such as arthritis or 20 asthma; immune disorders; bone disorders such as osteoporosis; cancer; intra-uterine growth retardation; apparent mineralocorticoid excess syndrome (AME); polycystic ovary syndrome (PCOS); hirsutism; acne; oligo- or amenorrhea; adrenal cortical adenoma and carcinoma; Cushing's syndrome; pituitary tumours; invasive carcinomas; breast cancer; and endometrial cancer. 25

35. Use of a compound according to any one of claims 1 to 30 in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 P-HSD levels. 30

36. Use of a compound according to any one of claims 1 to 30 in the manufacture of a pharmaceutical for modulating 11 3-HSD activity.

37. Use of a compound according to any one of claims 1 to 30 in the manufacture of a pharmaceutical for inhibiting 11 -HSD activity. 35

38. A method comprising (a) performing a 113-HSD assay with one or more WO 2004/037251 PCT/GB2003/004590 137 candidate compounds having the formula as defined in any one claims 1 to 30; (b) determining whether one or more of said candidate compounds is/are capable of modulating 113-HSD activity; and (c) selecting one or more of said candidate compounds that is/are capable of modulating 11 P-HSD activity. 5

39. A method comprising (a) performing a 113-HSD assay with one or more candidate compounds having the formula as defined in any one of claims 1 to 30; (b) determining whether one or more of said candidate compounds is/are capable of inhibiting 11 p-HSD activity; and (c) selecting one or more of said candidate compounds 10 that is/are capable of inhibiting 11-HSD activity.

40. A compound identified by the method according to claim 38 or claim 39.

41. A compound according to claim 40 for use in medicine. 15

42. A pharmaceutical composition comprising the compound according to claim 40 optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. 20

43. Use of a compound according to claim 40 in the manufacture of a medicament for use in the therapy of a condition or disease associated with 1 13-HSD.

44. Use according to claim 43 wherein the condition or disease is selected from the group consisting of metabolic disorders such as diabetes and obesity; cardiovascular 25 disorders such as hypertension; glaucoma; inflammatory disorders such as arthritis or asthma; immune disorders; bone disorders such as osteoporosis; cancer; intra-uterine growth retardation; apparent mineralocorticoid excess syndrome (AME); polycystic ovary syndrome (PCOS); hirsutism; acne; oligo- or amenorrhea; adrenal cortical adenoma and carcinoma; Cushing's syndrome; pituitary tumours; invasive carcinomas; breast cancer; 30 and endometrial cancer.

45. Use of a compound according to claim 40 in the manufacture of a medicament for use in the therapy of a condition or disease associated with adverse 11 P-HSD levels. 35

46. The invention of any one of claims 33 to 45 wherein 1 13-HSD is 1113-HSD Type 1. WO 2004/037251 PCT/GB2003/004590 138

47. The invention of any one of claims 33 to 45 wherein 11 j3-HSD is 11 P3-HSD Type 2. 5

48. A compound as substantially hereinbefore described with reference to any one of the Examples.

49. A composition as substantially hereinbefore described with reference to any one of the Examples. 10

50. A method as substantially hereinbefore described with reference to any one of the Examples.

51. A use as substantially hereinbefore described with reference to any one of the 15 Examples.