AU769753B2 - Steroid sulphatase inhibitors - Google Patents

Description

FV

7 P/00/011 28/5/91 Regulation 3.2 I,

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Actual Inventors: Address for service is: Sterix Limited Michael John REED Barry Victor Lloyd POTTER WRAY ASSOCIATES 239 Adelaide Terrace Perth, WA 6000 Attorney code: WR Invention Title: "Steroid Sulphatase Inhibitors" This application is a divisional application by virtue of Section 39 of Australian Patent Application 10130/00 filed on 6 January 2000.

The following statement is a full description of this invention, including the best method of performing it known to me:- IY.IIXTrll 1( Il)tllll--il 1ll1L1~1.1. IIIIICII..LtY13-I._i~-ir*- I II_ I I ~.Y~IYL _I i~i I) 1/2 STEROID SULPHATASE INHIBITORS This application is a divisional application of 10130/00 which in turn is a divisional application of 71952/98 which in turn is a divisional of 58373/96 which in turn is a divisional of 24905/92. The entire disclosure of these applications is incorporated herein by reference.

FIELD OF INVENTION This invention relates to novel compounds for use as steroid sulphatase inhibitors, and pharmaceutical compositions containing them.

BACKGROUND AND PRIOR ART Steroid precursors, or pro-hormones, having a sulphate group in the 3position of the steroid nucleus, referred to hereinafter simply as steroid sulphates, are known to play an important part as intermediates in steroid metabolism in the human body. Oestrone sulphate and dehydroepiandrosterone (DHA) sulphate, for example, are known to play an important role as intermediates in the production, in the body, of oestrogens :such as oestrone and oestradiol. Oestrone sulphate, in particular, is known, 20 for example, to represent one of the major circulating oestrogen precursors particularly in post-menopausal women and oestrone sulphatase activity in breast tumours is 100-1000 fold greater than that of other enzymes involved in oestrogen formation (James et al., Steroids, 50, 269-179 (1987)).

Not only that, but oestrogens such as oestrone and oestradiol, 25 particularly the over-production thereof, are strongly implicated in malignant conditions, such as breast cancer, see Breast Cancer, Treatment and Prognosis: Ed. R.A. Stoll, pp. 156-172, Blackwell Scientific Publications (1986), i and the control of oestrogen production is the specific target of many anticancer therapies, both chemotherapy and surgical, e.g. oophorectomy and adrenalectomy. So far as endocrine therapy is concerned, efforts so far tended to concentrate on aromatase inhibitors, i.e. compounds which inhibit aromatase activity, which activity is involved, as the accompanying oestrogen 1 i-i_ il .ilL.II. .li **si LII~I~iii.i-~l~_l lli~-i~ metabolic flow diagram (Figure 1) shows, in the conversion of androgens such as androstenedione and testosterone to oestrone and oestradiol respectively.

In recently published International Application W091/13083 a proposal has been made to target a different point in the oestrogen metabolic pathway, or rather two different points, that is to say the conversion of DHA sulphate and oestrone sulphate to DHA and oestrone, respectively, by steroid sulphatase activity, and using 3-monoalkylthiophosphonate steroid esters as a steroid sulphatase inhibitor, more especially oestrone-3-monomethylthiophosphonate.

OBJECTS OF THE INVENTION A first object of the present invention is to provide new compounds capable of inhibiting steroid sulphatase activity in vitro and in vivo.

A second object of the present invention is to provide new compounds having improved activity as steroid sulphatase inhibitors both in vitro and in vivo.

A third object of the invention is to provide pharmaceutical compositions effective in the treatment of oestrogen dependent tumours.

A fourth object of the invention is to provide pharmaceutical compositions effective in the treatment of breast cancer.

o 0 o 20 A fifth object of the invention is to provide a method for the treatment of oestrogen dependent tumours in mammals, especially humans.

A sixth object of the invention is to provide a method for the treatment of breast cancer in mammals and especially in women.

o.

SUMMARY OF INVENTION The invention is based on the discovery of novel compounds having :steroid sulphatase inhibitory activity, in some cases, with extremely high activity levels.

In its broadest aspect the present invention provides a ring system compound; 'wherei the ring system compound comprises a ring to which is attached a sulphamate group of the formula M11

=I-

3 R 0

N-S-O

R/ II R2 O wherein each of R, and R 2 is independently selected from H, alkyl, alkenyl, cycloalkyl and aryl, or together represent alkylene optionally containing one or more hetero atoms or groups in the alkylene chain; wherein R 1 or R 2 is H; wherein said compound is an inhibitor of an enzyme having steroid sulphatase activity and wherein if the sulphamate group of said compound is replaced with a sulphate group to form a sulphate compound it would be a substrate for. a steroid sulphatase enzyme I: n a further aspect the present invention provides a ring system 20 compound; wherein the ring system compound comprises a ring to which is attached a sulphamate group of the formula R 0 R 0 11

N-S-O

R/ OII wherein each of R, and R 2 is independently selected from H, alkyl, alkenyl, cycloalkyl and aryl, or together represent alkylene optionally containing one or more hetero atoms or groups in the alkylene chain; wherein R, or R 2 is H; ,n-ri wherein said compound is an inhibitor of an enzyme having steroid sulphatase activity and wherein if the sulphamate group of said compound is replaced with a sulphate group to form a sulphate compound and incubated with a steroid sulphatase enzyme at a pH 7.4 and 37 0 C it would provide a Km value of less than 50 pM.

In an even further aspect the present invention provides a ring system compound; wherein the ring system compound has the formula R O II -Poly cycle N-S-0 i R O 0 wherein each of R, and R 2 is independently selected from H, alkyl, alkenyl, cycloalkyl and aryl, or together represent alkylene optionally containing one or more hetero atoms or groups in the alkylene chain; wherein R, or R 2 is H; wherein the group Poly cycle is a polycyclic ring structure wherein said compound is an inhibitor of an enzyme having steroid sulphatase activity and wherein if the sulphamate group of said compound is replaced with a sulphate group to form a sulphate compound it would be a substrate for a steroid sulphatase enzyme "4A i S Fw- 4 W -111111W A P I In yet an even further aspect the present invention provides a ring system compound; wherein the ring system compound has the formula R 0 II /Poly cycle

N-S-O

R/ II R2 O wherein each of R, and R 2 is independently selected from H, alkyl, alkenyl, cycloalkyl and aryl, or together represent alkylene optionally containing one or more hetero atoms or groups in the alkylene chain; wherein R, or R 2 is H; wherein the group Poly cycle is a steroidal ring structure that wherein said compound is an inhibitor of an enzyme having steroid Ssulphatase activity and S: 20 wherein if the sulphamate group of said compound is replaced with a sulphate group to form a sulphate compound it would be a substrate for a Sr steroid sulphatase enzyme 9j In a preferred aspect the compounds are the sulphamic acid esters of 25 polycyclic alcohols, being polycyclic alcohols the sulphate of which is a substrate for enzymes having steroid sulphatase (EC 3.1.6.2) activity, the N-alkyl and N-aryl derivatives of those sulphamic acid esters, and their 9 pharmaceutically acceptable salts.

Broadly speaking, the preferred compounds of this invention are compounds of the Formula (I) r~ FORMULA (I) R 0 II /Poly cycle

N-S-O

R/ O

P

2 0 where:

R

1 and R 2 are each independently selected from H, alkyl, cycloalkyl, alkenyl and aryl, or together represent alkylene optionally containing one or more hetero atoms or groups in the alkylene chain; and the group polycycle represents the residue of a polycyclic alcohol, the sulphate of which is a substrate for enzymes having steroid sulphatase activity (EC 3.1.6.2).

As used herein the reference to polycyclic alcohols, the sulphate of which is a substrate for enzymes having steroid sulphatase activity refers to polycyclic alcohols, the sulphate of which, viz: the derivatives of the Formula: II Polycycle HO S O

II

when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37 C, provides a Km value of less than Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

LI ,lr ~LI i-iinll-*.,* BRIEF DESCRIPTION OF DRAWINGS The activity of the present compounds as steroid sulphatase inhibitors is illustrated in the accompanying drawings: Figure 1 is a schematic chart showing the metabolic pathways, enzymes and steroid intermediates associated with the production of oestradiol in vivo.

Figure 2 is a histogram showing the dose-dependent inhibitory effect of oestrone-3-sulphamate on steroid sulphatase activity in human MCF-7 cells in vitro.

Figure 3 is a histogram showing the dose-dependent inhibitory effect of oestrone-3-N,N-dimethylsulphamate on steroid sulphatase activity in human MCF-7 cells in vitro.

Figure 4 is a graph comparing the log dose-response curves for oestrone-3-sulphamate and oestrone-3-N,N-dimethylsulphamate on steroid sulphatase activity in human MCF-7 cells in vitro.

15 Figure 5 is a graph showing the dose-dependent inhibitory effect of S'oestrone-3-sulphamate, together with its IC 5 0 value (concentration required to produce 50% inhibition), on steroid sulphatase activity in human placental microsomes in vitro.

20 DETAILED DESCRIPTION In a preferred aspect the present invention provides, as novel compounds, the sulphamic acid esters of polycyclic alcohols, being polycyclic alcohols the sulphate of which is a substrate for enzymes having steroid sulphatase activity in accordance with the definition already provided, and their 25 N-alkyl, N-cycloalkyl, N-alkenyl and N-aryl derivatives. These compounds are of Formula I hereinbefore given.

Preferably the polycyclic group will contain, inclusive of all substituents, a maximum of about 40 carbon atoms, more usually no more than about Preferred polycycles are those containing a steroidal ring structure, that is to say a cyclopentanophenanthrene skeleton. Preferably, the sulphamyl or substituted sulphamyl group is attached to that skeleton in the 3-position, that is to say are compounds of the Formula II: 1~~7 ,r I I~~*I.HCI I .llrlULY~ ,cn~ 8 FORMULA (11) RZ 1 0 where R, and R 2 are as above defined and the ring system ABOD represents a substituted or unsubstituted, saturated or unsaturated steroid nucleus, preferably oestrone or dehydroepiandrosterone.

Other suitable steroid ring systems are: substituted aestrones, viz: 2-OH-oestrone 2-methoxy-oestrone 4-OH-oestrone 6 -OH-oestrone 7 -OH-oestrone 16 -OH-oestronel6 -OH-oestrone oestradiols and substituted oestradiols, viz: 2-OH-i 7-oestradiol 2-methoxy-1 7-oe-stradiol 4-OH-i 7-oestradiol 6 -OH-17 -oestradiol 7 -OH-17 -oestradiol 16 -OH-17 -oestradiol 20 16 -OH-17-oestradiol 16 -OH-17 -oestradiol 17 -oestradiol 1 7 -oestradiol 17 -ethinyl-i 7 -oestradiol oestriols and'substituted oestriols, viz: oestriol 2-OH-oestriol 2-methoxy-oestriol *.*:4-OH-oestriol 6 -OH-oestriol 7 -OH-oestriol 25 substituted dehydroepiandrosterones, viz: 6 -OH-dlehydroepiandrosterone 7 -O H-dehydroepiandrosterone 16 -OH-dehydroepiandrosterone 16 -OH-dehydroepiandrosterone In general terms the steroid ring system ABCD may contain a variety of non- interfering, su bstituents. In particular, the ring syste m ABOD may contain one or more hydroxy, alkyl especially lower (C 1

-C

6 alkyl, e.g. methyl, ethyl, npropyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl 9 isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C 1

-C

6 alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g.

fluoro substituents.

Suitable non-steroidal ring systems include: diethylstilboestrol, stilboestrol and other ring systems providing sulfates having Km values of less than 50moles with steroid sulphatase EC3.1.6.2.

When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl,: N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms.

When R 1 and/or R 2 is alkyl, the preferred values are those where R 1 and R 2 are each independently selected from lower alkyl groups containing from 1 to carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R 1 and R 2 are both methyl. When R 1 and/or R 2 is aryl, typical values are phenyl and tolyl (-PhCH3; m- or Where R 1 and R 2 represent cycloalkyl, typical values 15 are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R 1 and R 2 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. or -NH- to provide a 6- or 7- membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.

20 Within the values alkyl, cycloalkyl, alkenyl and aryl we include substituted groups containing as substituents therein one or more groups which do not interfere with the sulphatase inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.

Most preferred are compounds of the Formula III and IV: FORMULA (1l1) 0 R,0 0 FORMULA (IV) 100 *where R, and R 2 are H or 01-0.5 alkyl, i.e. oestrone-3-sulpharnate and oo dehydroepiandrosterone-3-suiphamate and their N-(C 1

-C

5 alkyl derivatives, especially the dimethyl derivatives, R 1 R2= CH 3 :The sulphamic acid esters of this invention are typically prepared by reacting a ring system alcohol, e.g. oestrone or dehydroepiandrosterone, with a .0*0 sulfamoyl chloride R 1

R

2

NSO

2 CI, i.e. the reaction scheme I 11 REACTION SCHEME I o RRNSO,CI R, II NaH RK I HO O Oestrone Conditions for carrying out reaction scheme I are as follows: Sodium hydride and a sulphamoyl chloride are added to a stirred solution of oestrone in anhydrous dimethyl formamide at 0 C. Subsequently, the reaction is allowed to warm to room temperature whereupon stirring is continued for a further 24 hours. The reaction mixture is poured onto a cold 15 saturated solution of sodium bicarbonate and the resulting aqueous phase is extracted with dichloromethane. The combined organic extracts are dried over anhydrous MgSO 4 Filtration followed by solvent evaporation in vacuo and coevaporation with toluene affords a crude residue which is further purified by flash chromatography.

Where necessary, functional groups in the polycyclic alcohol (sterol) may be protected in known manner and the protecting group or groups removed at the end of the reaction.

For pharmaceutical administration, the steroid sulphatase inhibitors of this invention can be formulated in any suitable manner utilising conventional 25 pharmaceutical formulating techniques and pharmaceutical carriers, exipients, diluents etc. and usually for parenteral administration. Approximate effective dose rates are in the range 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average 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 12 several days. For oral administration they may be formulated in tablets, capsules, solution or suspension 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 bodyweight of the patient, such variations being within the skill and judgement of the physician.

For particular applications, it is envisaged that the steroid sulphatase inhibitors of this invention may be used in combination therapies, either with another sulphatase inhibitor, or, for example, in combination with an aromatase inhibitor, such as for example, 4-hydroxyandrostenedione (4-OHA).

The invention is illustrated by the following preparative Examples and test data: Example 1 Preparation of oestrone-3-sulphamate Sodium hydride (60% dispersion; 2 eq) and sulphamoyl chloride (2 eq) were added to a stirred solution of oestrone (1 eq) in anhydrous dimethyl formamide at 0 C. Subsequently, the reaction was allowed to warm to room temperature whereupon stirring was continued for a further 24 hours.

oThe reaction mixture was poured onto a cold saturated solution of sodium bicarbonate and the resulting aqueous phase was extracted with 25 dichloromethane. The combined organic extracts were dried over anhydrous o" MgSO 4 Filtration followed solvent evaporation in vacuo and co-evaporation with toluene afforded a crude residue which is further purified by flash chromatography.

Analysis showed the following data: r-I 1 H (270MHz; CD 3 OD): 0.91 3H, C 1 8 1.40-2.55 (series of m, 13H), 2.90-2.92 (in, 2H), 7.04 (br d, 2H, J=1 0.44Hz), 7.33 (br d, 1 H, J=8.42Hz).

13 C (67.8MHz; CD 3 OD): 14.53 C 18 22.80 27.24 27.73 30.68 33.05 37.01 39.76 45.73 C 18 51.86 120.76 123.54 127.89 139.83 150.27 223.87 C=O).

mlz 349 270 (100), 213 185 172 159 146 91 69 57 43 29 (24).

Microanalysis: C H N Expected: 61.87% 6.63% 4.01% Found: 61 .90% 6.58% 3.95% is Example 2 Preparation of oestrone-3-N-methvlsulphamate *The procedure of Example 1 was repeated save that suiphamoyl chloride was replaced by the same quantity of N-methylsulphamoyl chloride.

Analysis showed the following data: 1 H (270MHz; CDCI 3 0.91 3H, C 18 1.28-1.68 (in, 6H), 1.93-2.60 (series of m, 7H), 2.90-2.95 2H), 2.94 3H, J=5.13 Hz, MeN-), 4.68-4.71 (br in, exchangeable, 1 H, 7.02-7.07 (mn, 2H), 7.26-7.32 (in, 1 H).

mlz 364 [M+HI1+ Example 3 Preparation of oestrone-3-N.N-dimethvlsulphamate The procedure of Example 1 was repeated save that sulphamoyl chloride was replaced by the same quantity of N,N-dimethylsulphamoyl chloride.

Analysis showed the following data: 1H (270MHz; CDCI 3 0.92 3H, C 1 8 1.39-1.75 5H), 1.95-2.60 (series of m, 6H), 2.82 3H, MeN-), 2.96-3.00 4H), 2.98 3H, MeN-), 7.04 (br d, 2H, J=7.69Hz), 7.29 (br d, 1H, J=7.88Hz).

m/z 377 [M] Microanalysis: C H N Expected: 63.63% 7.21% 3.71% Found: 63.50% 7.23% 3.60% Example 4 Inhibition of Steroid Sulphatase Activity in MCF-7 cells by oestrone-3sulphamate Steroid sulphatase is defined as: Steryl Sulphatase EC 3.1.6.2.

Steroid sulphatase activity was measured in vitro using intact MCF-7 25 human breast cancer cells. This hormone dependent cell line is widely used to study the control of human breast cancer cell growth. It possesses significant steroid sulphatase activity (Maclndoe et al. Endocrinology, 123, 1281-1287 (1988); Purohit Reed, Int. J. Cancer, 50, 901-905 (1992)) and is available in the U.S.A. from the American Type Culture Collection (ATCC) and in the U.K.

from The Imperial Cancer Research Fund). Cells were maintained in Minimal Essential Medium (MEM) (Flow Laboratories, Irvine, Scotland) containing 20 mM HEPES, 5% foetal bovine serum, 2 mM glutamine, nonessential amino acids and 0.075% sodium bicarbonate. Up to 30 replicate cm 2 tissue culture flasks were seeded with approximately I x 105 cells/flask using the above medium. Cells were grown to 80% confluency and medium was changed every third day.

Intact monolayers of MCF-7 cells in triplicate 25 cm 2 tissue culture flasks were washed with Earle's Balanced Salt Solution (EBSS from ICN Flow, High Wycombe, and incubated for 3-4 hours at 37 C with 5 pmol (7 x 105 dpm) [6,7- 3 H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass., in serum-free MEM (2.5 ml) together with oestrone-3-sulphamate (11 concentrations: 0; 1fM; 0.01pM; 0.1pM; 1pM; 0.01nM; 0.1nM; 1nM; 0.01pM; 0.1pM; 1pM). After incubation each flask was cooled and the medium (1 ml) was pipetted into separate tubes containing 1 4 C]oestrone (7 x 103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, The mixture was 15 shaken thoroughly for 30 seconds with toluene (5 ml). Experiments showed that >90% 14 C]oestrone and 3 H]oestrone-3-sulphate was removed from the aqueous phase by this treatment. A portion (2 ml) of the organic phase was removed, evaporated and the 3 H and 1 4 C content of the residue determined by scintillation spectrometry. The mass of oestrone-3-sulphate p p 20 hydrolysed was calculated from the 3 H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of l.

[(1 4 C]oestrone added) and the specific activity of the substrate. Each batch of experiments included incubations of microsomes prepared from a sulphatasepositive human placenta (positive control) and flasks without cells (to assess 25 apparent non-enzymatic hydrolysis of the substrate). The number of cell nuclei per flask was determined using a Coulter Counter after treating the cell monolayers with Zaponin. One flask in each batch was used to assess cell membrane status and viability using the Trypan Blue exclusion method (Phillips, H.J. (1973) In: Tissue culture and applications, [eds: Kruse, D.F. Patteison, pp. 406-408; Academic Press, New York).

16 Data for oestrone-3-sulphamate are shown in Table I and Figures 2 and 4. Results for steroid sulphatase activity are expressed as the mean 1 S.D. of the total product (oestrone oestradiol) formed during the incubation period hours) calculated for 106 cells and, for values showing statistical significance, as a percentage reduction (inhibition) over incubations containing no oestrone- 3-sulphamate. Unpaired Student's t-test was used to test the statistical significance of results.

TABLE I Steroid Suiphatase Activity in MCF-7 cells in the presence of Oestrone-3-sulphamate Oestrone-3sumphamate concentration Steroid Sulphatase Activity (fmol/20 hr/106 cells) reduction over control inhibition)

S

5

S.

0 (control) lfM 0.01pM 0.1pM 1pM 0.01nM 0.1nM inM 0.01 pM 0.1pM 1pM 319.7 18.5 353.3 39.0 362.3 21.2 330.7 17.8 321.8 6.2 265.1 11.0* 124.8 12.4*** 16.49 3.92 0.4** 2.53 1.68 S. S S S

S.

S

S. S S S

S.

S. S S S

S.

17.2% 60.9% 95.0% 98.8% 99.2% 99.5% I mean 1 S.D. n=3 p 0.05 p 0.001 Example Inhibition of Steroid Sulphatase Activity in MCF-7 cells by oestrone-3-NN dimethylsulphamate 0 0*O0SS

S@

S S

S@

S

060055

S

S. Se 0

S

0* S S S

S

S

S

SO..

S. S C S *5 .5.5 5 0055 5* S S .5 @0 S 0@

S.

An identical experimental protocol to that described in Example 4 was used to generate results for oestrone-3-N,N-dimethylsulphamate except that incubations contained oestrone-3-NN-dimethylsulphamate (5 concentrations: 0;1 0.001 pM; 0.01 pM; 0.1 pM; 1 pM) in place of oestrone-3-sulphamate.

Results for oestrone-3-NN-dimnethylsulphamate are shown in Table 11 and Figure 3 and are expressed in an identical manner to Table I and Figure 2 respectively. Additionally the log dose-response curve is compared with oestrone-3-sulphamate in Figure 4.

TABLE 11 Steroid Sulphatase Activity in MCF-7 cells in the presence of oestrone-3-1 N,N-dimethylsulphamate Oestrone-3-N,N- .Steroid Sulphatase reduction over dimethylsulphamate Activity (fmol/20 hr/i 06 control inhibition) concentration cells) 0 (control) 82.63 t 3.6 0.001 pM 68.33 17.3% 0.Ol pM 46.0 4.9* 44.3%' 0.1 pM 17.43 4.3" 78.9% 1pM 11.89 85.6% mean ±1 S.D. n=3 p 0.01 p 0.001 Example 6 Inhibition of Steroid Sulphatase Activity in MCF-7 cells by bre-treatment with oestrone-3-N .N-dimethvlsulphamate and oestrone-3-NN-dimethylsulphamate A similar experimental protocol to that described in Example 4 was used to determine the effect of pre-treating MCF-7 cells with oestrone-3-sulphamate and oestrone-3-N ,N-dimethylsulphamnate respectively.

Intact monolayers were initially incubated for 2 hours at 37 C with 0.1 PM oestrone-3-sulphamate, oestrone-3-N,N-dimethylsulphamate or medium alone *C (control). The medium bathing the cells was then removed by aspiration and cells were washed 3 times successively with 5 ml of medium on each occasion.

The resultant 'washed' cells were then re-suspended and incubated for 3-4 hours at 37 C in medium containing 5 pmol (7 x 105 dpm) [6,7- 3 H]oestrone-3sulphate. All other aspects were identical to those described in Examples 3 and 4.

Results for oestrone-3-sulphamate and oestrone-3-N,N-dimethylsulphamate are shown in Table III and are expressed in a similar manner to Table I.

TABLE III Steroid Sulphatase Activityin MCF-7 cells pre-incubated with oestrone-3sulphamates Pre-treatment Steroid Sulphatase reduction over Activity (fmol/20 control hr/106 cells) inhibition) Control 65.4 6.4 Oestrone-3-sulphamate 1.7 9714% Oestrone-3-N,N- 53.1 3.4* 18.8% dimethylsulphamate mean 1 S.D. n=3 *p 0.05 p 0.001 Example 7 Inhibition of Steroid Sulphatase Activity in Placental Microsomes by Oestrone- 3-sulphamate Sulphatase-positive human placenta from normal term pregnancies (Obstetric Ward, St. Mary's Hospital, London) were thoroughly minced with scissors and washed once with cold phosphate buffer (pH 7.4, 50 mM) then resuspended in cold phosphate buffer (5 ml/g tissue). Homogenisation was accomplished with an Ultra-Turrax homogeniser, using three 10 second bursts 19 separated by 2 minute cooling periods in ice. Nuclei and cell debris were removed by centrifuging (4 C) at 2000g for 30 minutes and portions (2 ml) of the supernatant were stored at -20 C. The protein concentration of the supernatants was determined by the method of Bradford (Anal. Biochem., 72, 248-254 (1976)).

Incubations (1 ml) were carried out using a protein concentration of 100 pg/ml, substrate concentration of 20 pM [6,7- 3 H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass., and an incubation time of 20 minutes at 37 C. Eight concentrations of oestrone-3sulphamate were employed: 0 control); 0.05pM; 0.1pM; 0.2pM; 0.4pM; 0.6pM; 0.8pM; 1.OpM. After incubation each sample was cooled and the medium (1 ml) was pipetted into separate tubes containing 1 4 C]oestrone (7 x 3 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, The mixture was shaken thoroughly 15 for 30 seconds with toluene (5 ml). Experiments showed that 1 4 C]oestrone and 3 H]oestrone-3-sulphate was removed from the aqueous phase by this treatment. A portion (2 ml) of the organic phase was removed, evaporated and the 3 H and 1 4 C content of the residue determined by scintillation spectrometry. The mass of oestrone-3-sulphate hydrolysed was 20 calculated from the 3 H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [1 4 C]oestrone added) and the specific activity of the substrate.

Results for oestrone-3-sulphamate are shown in Table IV and Figure Results for steroid sulphatase activity are expressed in Table IV as total product 25 (oestrone oestradiol) formed during the incubation period (time) and as a percentage reduction (inhibition) over incubations containing no oestrone-3sulphamate which acted as control. Results for steroid sulphatase activity are expressed in Figure 4 as percentage reduction (inhibition) over control against concentration of oestrone-3-sulphamate and include the calculated IC 5 0 value the concentration of oestrone-3-sulphamate which produces 50% inhibition in relation to control) of 0.07pM.

i.ii~ll 1*s*1111-* ^l ^L LI.I- III*i

V

TABLE IV Steroid Sulphatase Activity in placental microsornes in the presence of Oestrone-3-sulphamate Oestrone-3- Steroid Sulphatase Activity reduction over sulphamate (pmol/hr/0.1 mg protein) control inhibition) concentration 0 (control) 768.6 0.05pM 430.4 44.0% 0.1pM 305.9 60.2% 0.2pM 140.0 81.8% 0.4pM 83.3 89.2% 0.6pM 61.8 92.0% 0.8pM 49.2 93.6% 51.6 93.3% mean of 2 estimates Example 8 Inhibition of Steroid Sulphatase Activity in Liver Microsome Preparations from Rats treated with subcutaneous Oestrone-3-sulphamate Four groups of 3 female Wistar rats (weight range 80-110g) were given 100 pl subcutaneous injections (once daily for 7 days, vehicle: propylene glycol) of either: Propylene glycol (vehicle control) Oestrone-3-sulphamate (10 mg/kg/day) Oestrone-3-sulphate (10 mg/kg/day) (substrate control) Oestrone-3-sulphate (10 mg/kg/day) Oestrone-3-sulphamate mg/kg/day) On the eighth day all rats were sacrificed and livers were removed by dissection. Liver microsomal preparations were prepared by an identical protocol to that described in Example 6 except that the tissue source was rat 21/1 liver .and that duplicate experiments to determ Ine' steroid sulphatas .e activity were performed using [6,7 3 Hoestrone-3-sulphate and 3 H]dehydroepiandrosterone-3-sulphate as separate substrates.

Results tor steroid sulphatase activity are shown in Table V and are expressed as total product formed during the incubation period in the form of mean ±1S.D. Results for incubations of tissue obtained from groups of rats treated with oestrone-3-sulphamate are also expressed as a percentage reduction (inhibition) in steroid sulphatase activity compared to their respective controls.

TABLE V Steroid Sulphatase Activity in Liver Micro-some Preparations from Rats treated with subcutaneous Oestrone-3-sulphamaie Treatment Group Assay Steroid Sulphatase reduction Substrate Activity (nmol/30 over control mi200 p g protein) *control (vehicle) E, -S 20.95 0.2 E-S0 3

NH

2 El -S 0.34 ±-984 Scontrol (El El-S 20.6 ±0.4- E-S+ E, -S0 3

NH

2 1l- 0.21 0.03* 99.00/ control (vehicle) DHA1 .73 +0.4 El -S0 3

NH

2 DHA-S 0.1 0.01 94.2% control (El-S) DHA-S 1.71 0.1 E-+EDHA-S 0.09.± 0.01 94.7%

E

1 S E-S0 3

NH

2 mean 1 S.D. n=3 0.001 1s E, 1 -S =oestrorne-3-sulphamate DHA-S dehydroepiandrosterone3sulphate Ej -S0 3

NH

2 oestrone-3-N,N-dimethylsulphamate 21/2 Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claims (3)

1. A purified compound selected from compounds of the formulae OH~ OH H H HO0 H H HO H0 N-S-O -j 17a ethinyl 17j3 oestradiol-3-sulphamate 1713 oestradiol-3-sulphamate OH 0 H H HO H H HO0 H H 0 HO0 1 7a oestradiol-3-sulphamate oestrone-3-sulphamate (EMATE) OH OH .H \0 H H HO0 ****oestriol-3-sulphamate t~t*~X#-"fc

2. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a compound selected from compounds of the formulae HO 1 H H HO0 17at ethinyl 17f3 oestradiol-3-sulphamate OH H N-S-0 11 H 0 1 7(x oestradiol-3-sulphamate OH OH H H 0 oestriol-3-sulphamate H H N-S-O 11 H o 1 7P oestradiol-3-sulphamate 0 H HO0 II H 0 oestrone-3-sulphamate (EMATE) a a a a

3. Use of a compound in the manufacture of a medicament to inhibit steroid suiphatase activity, wherein the compound is selected from compounds of the formulae 17at ethinyl 17f3 oestradiol-3-sulphamate OH OH H HO0 17P oestradiol-3-sulphamate 0 H H 0 oestrone-3-sulphamate (EMATE) S S 1 7a oestradiol-3-sulphamate HO H N-S-O 11 HO, oestriol-3-sulphamate Dated this TWENTY SECOND day of FEBRUARY 2001. STERIX LIMITED Wray Associates Perth, Western Australia Patent Attorneys for the Applicant