GB2371299A - Sulfamate oestrone sulfatase inhibitors - Google Patents

Abstract

A family of non-steroidal sulfamate compounds is described which are suitable for use as oestrone sulfatase inhibitors. The compounds have the general formula (C),: <EMI ID=1.1 HE=38 WI=61 LX=745 LY=630 TI=CF> <PC>wherein:<BR> R<SB>1</SB>-R<SB>5</SB> are independently selected from H, halo, alkyl, nitro, optionally substituted alkoxy, optionally substituted optionally substituted aryl, optionally substituted aryloxy, optionally substituted alkylamino, optionally substituted arylamino, a further group COOR<SB>6</SB>, or a sulfamate group; or R<SB>1</SB> and R<SB>2</SB> taken together with the adjacent carbon atoms, or R Þ and R ý taken together with the adjacent carbon atoms, represent a second optionally substituted aromatic ring;<BR> but wherein at least one of R Ü -R Å is a sulfamate group or aryl, aryloxy, or arylamino substituted with a sulfamate group, or R Ü and R Þ taken together with the adjacent carbon atoms, or R Þ and R ý taken together with the adjacent carbon atoms, represent an aromatic ring substituted with a sulfamate group; and<BR> R Û is independently selected from H, aryl, or alkyl and substituted variants thereof;<BR> or a pharmaceutically acceptable salt thereof.

Description

PHENOLIC COMPOUNDS The present invention relates to members of a family of non-steroidal compounds which have been found to possess inhibitory activity against the enzyme oestrone sulfatase.

Although plasma oestrogen concentrations are found to be similar in women with or without breast cancer, breast tumour levels of oestrone and oestradiol are significantly higher than in normal breast tissue or blood. Synthesis of oestrogens within tumours is thought to make an important contribution to these high levels of the female hormones. Oestrogens are suggested to be the major mitogens involved in promoting the growth of tumours in endocrine-dependent tissues, such as the breast and therefore specific inhibitors of oestrogen biosynthesis are of potential value for the treatment of endocrine-dependent tumours.

Recently, there has been considerable interest in the development of inhibitors of the cytochrome P-450 enzyme aromatase (AR) -a pathway which is responsible for the conversion of the androgen precursor androstenedione to oestrone.

There is now evidence, however, that the oestrone sulfatase (EI-STS) pathway [the hydrolysis of oestrone sulfate (EIS, 2) to oestrone to (El, 1)] :

is the major source of oestrogen in breast tumours 2 as opposed to the AR pathway. This is supported by a modest reduction of plasma oestrogen concentration in postmenopausal women with breast cancer treated by AR inhibitors, such as

3, 4, 5 aminoglutethimide (AG) and 4-hydroxyandrostenedione The oestrone sulfatase inhibitors are sulfamate esters, such as oestrone-3-sulfamate (3) (otherwise known as"EMATE") :

EMATE is a potent E l-STS inhibitor and displays more than 99% inhibition of EI-STS activity in intact MCF-7 cells at 0. 1 uM concentration. EMATE also inhibits dehydroepiandrosterone sulfatase (DHA-STS), an enzyme that is believed to have a crucial role in regulating the biosynthesis of the oestrogenic steroid androstenedioll. Furthermore, there is now evidence to suggest that androstenediol

6 may be of even greater importance as a promoter of breast tumour growth.

Although potency for the inhibition of EI-STS may have been attained in EMATE, it has been suggested that oestrone may be released during sulfatase inhibition7 and that EMATE and its analogues may possess oestrogenic activity 8.

This present invention therefore seeks to provide compounds suitable for the inhibition of EI-STS but which possess no, or a minimal, oestrogenic effect.

Co-pending application PCT/GBOO/02592 discloses compounds of formula (A):

where X is preferably 0 and R6 is preferably an alkyl, aryl or substituted aryl group.

The present invention is concerned with further compounds belonging to this general class.

According to a first aspect of the present invention there is provided a non-steroidal sulfamate compound suitable for use as an inhibitor of oestrone sulfatase, wherein the compound has a ring structure wherein the ring mimics the A ring of oestrone. The compound has the general formula (C):

wherein : RI-Rs are independently selected from H, halo, alkyl, nitro, optionally substituted alkoxy, optionally substituted aryl, optionally substituted aryloxy, optionally substituted alkylamino, optionally substituted arylamino, a further group COR6, or a sulfamate group; but wherein at least one of Ri-rus is a sulfamate group or aryl, aryloxy, or arylamino substituted with a sulfamate group; and ri is independently selected from H, aryl, or alkyl and substituted variants thereof ; or a pharmaceutically acceptable salt thereof.

Thus the invention includes derivatives of the following compounds derived from benzoic acid and biphenyl-4-carboxylic acid:

where X is COR6, as well as structural isomers thereof and other variants, as specified above.

By"optionally substituted"is meant a group substituted with halo, alkyl, nitro, alkoxy, aryloxy, alkylamino, arylamino or a sulfamate group.

The term"alkyl", as used herein, includes straight-chain and branched groups, as well as saturated and unsaturated groups, although saturated groups are preferred.

It includes substituted or non-substituted groups. They are preferably Cl-C, 3, more preferably Cl-Cg, especially CI-C6.

The alkyl group may also be a cyclic alkyl group.

Aryl includes heteroaryl. Aryl groups are preferably up to C15.

The term"sulfamate"as used herein includes an ester of sulfamic acid, or an ester of an N-substituted derivative of sulfamic acid, or salt thereof. Thus the term includes functional groups of the formula-O-SO2-NR7R8, where R7 and Rg are

independently selected from H, alkyl, aryl or any other suitable group.

Further compounds derived from naphthoic acid :

are also contemplated as forming part of the invention, and the invention may be extended to include a compounds in which R) and R2 taken together with the adjacent carbon atoms, or R2 and R3 taken together with the adjacent carbon atoms, represent a

second optionally substituted aromatic ring. In such case at least one of Rl-R5 may represent a sulfamate group or aryl, aryloxy, or arylamino substituted with a sulfamate group, or R, and R2 taken together with the adjacent carbon atoms, or R2 and R3 taken together with the adjacent carbon atoms, may represent an aromatic ring substituted with a sulfamate group. This includes compounds of the general formula (E) or (F):

wherein Rj-Rs and R9-R12 are independently selected from H, halo, alkyl, nitro, optionally substituted alkoxy, optionally substituted alkylamino, a further group COOR6, or a sulfamate group; but wherein at least one of R)-Rs and Rg-R, 2 is a sulfamate group.

Note that esters of formula (D):

wherein R is methyl or ethyl and R'is H, methyl, Br or Cl, have been disclosed in J. Org. Chem. 1988, 53, 41124114 (Kamal et al.), though they have never been

previously been used pharmaceutically. Thus, according to a second aspect of the present invention there is provided a non-steroidal compound for use as a pharmaceutical product.

According to a third aspect of the present invention there is provided a non-steroidal compound for inhibiting oestrone sulfatase According to a fourth aspect of the present invention there is provided a pharmaceutical composition comprising a non-steroidal compound; and a pharmaceutically acceptable carrier, excipient or diluent. (Such materials are well-known to those skilled in the art, and are too diverse to be stated here.) According to a fifth aspect of the present invention there is provided the use of a non-steroidal compound in the manufacture of a pharmaceutical product for use in the treatment or prophylaxis of a condition associated with oestrone sulfatase, such as an endocrine-dependent cancer.

Preferably RI-R5 are independently selected from H, alkyl, haloalkyl, a further group COR6, a sulfamate group, and alkylphenyl substituted with a sulfamate group.

Preferably R3 is-OSO2NH2 or-OSO2NH2.

Preferably the compound is any one ofpentyl 4- [ (aminosulfonyl) oxy] benzoate (14), hexyl 4-[ (aminosulfonyl) oxy]benzoate (16), heptyl 4-[ (aminosulfonyl) oxy]benzoate (18), octyl 4-[ (aminosulfonyl) oxy]benzoate (20), ethyl 4'-[ (aminosulfonyl)oxyl-1, 1'-biphenyl-4-carboxylate (30), and butyl 4'-[ (aminosulfonyl) oxy] -1, 1'-biphenyl-4-carboxylate (32) :

Preferably the compound is hexyl 4-[ (aminosulfonyl) oxy]benzoate (16) or heptyl 4-[ (aminosulfonyl) oxy]benzoate (18). The derivatisation of the phenyl ring with electron withdrawing groups (such as NO2) is expected to increase potency of these compounds against oestrone sulfatase.

The term"sulfamate"as used herein includes an ester of sulfamic acid, or an ester of an N-substituted derivative of sulfamic acid, or a salt thereof. Thus, the term includes functional groups of the formula :-0-S (O) (O)-N (R7) (rus) where R7 and Rg are independently selected from H, halo, linear or branched alkyl which may be saturated or unsaturated and/or substituted or non-substituted, aryl, or any other suitable group.

Preferably, at least one of R7 and Rg is H. In a preferred embodiment, each of R7 and R8 is H.

The term"mimic"as used herein is used in its normal sense-namely having a different structure but having a similar functional effect.

A key advantage of the non-steroidal compound of the present invention is that it has less oestrogenic activity and can therefore be deemed to be a "non-oestrogenic compound".

The term"non-oestrogenic compound"as used herein means a compound exhibiting no or substantially reduced oestrogenic activity.

The present invention therefore provides non-steroidal compounds which have a reduced oestrogenic activity. In this regard, the non-steroidal compounds of the present invention act as E I-STS inhibitors.

Another advantage is that the compounds may not be capable of being metabolised to compounds which display or induce hormonal activity.

The compounds of the present invention are further advantageous in that the sulfamate compounds may have an irreversible effect.

The compounds of the present invention are further advantageous in that they may also inhibit DHA-STS.

Thus, in a preferred embodiment, the non-steroidal compounds are useful for the treatment of breast cancer. In addition, the non-steroidal compounds are useful for the treatment of non-malignant conditions, such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age.

A particularly preferred non-steroidal compound according to the present invention is hexyl 4-[(aminosulfonyl) oxy] benzoate (16) or heptyl 4-[ (aminosulfonyl)- oxy] benzoate (18).

A highly preferred embodiment of the present invention therefore relates to pharmaceutical composition comprising hexyl 4- [ (aminosulfonyl) oxy] benzoate (16) or heptyl 4- [ (aminosulfonyl) oxy] benzoate (18) and a pharmaceutically acceptable carrier, excipient or diluent.

The present invention therefore relates to non-steroidal compounds which are suitable for use as sulfatase inhibitors.

Of the preferred compounds, hexyl 4-[ (aminosulfonyl) oxy]benzoate (16) together with heptyl 4-[ (aminosulfonyl) oxy]benzoate (18) appear active in vitro. In

this regard, hexyl 4-[ (aminosulfonyl) oxy]benzoate (16) inhibited placental microsomal by 72.6% at 10 M with an IC50, of 3.8 uM. Heptyl 4- [ (aminosulfonyl) oxy] benzoate (18) also inhibited placental microsomal oestrone

sulfatase by 69. 5% at 10 uM with an IC5o, of 3. 4 uM. This inactivation was shown to be in a similar way to EMATE which inhibited placental microsomal oestrone sulfatase with an ICso, of 0. 5 uM.

The non-steroidal compounds of the present invention, in particular the preferred nitrated and non-nitrated sulfamates, represent important compounds for the

optimisation of non-steroidal sulfatase inhibition. The compounds are also believed to have therapeutic uses other than for the treatment of endocrine-dependent cancers, such as the treatment of autoimmune diseases. The compounds may also have uses in the increasing of short (and long) term memory.

Aspects of the present invention will now be described further by way of example.

EXAMPLES A general method for the synthesis of the 4-sulfamate derivative of substituted benzoic acid derivatives of the present invention is shown in the following scheme, where: a = ROH/A/toluene ; b = NaH/H2NSO2Cl/toluene :

Aminosulfonyl chloride (4): Methanoic acid (1.00 ml, 26. 50 mmol) was added dropwise to chlorosulfonyl isocyanate (2.31 ml, 26.50 mmol, dried over B203), under nitrogen at 0-4'C. After evolution of gas, anhydrous toluene (20 ml) was added to dissolve the product, and the solution stirred for 1 hour. Insoluble by products were removed by filtration.

Removal of toluene under vacuum ( < 30 C) gave (4) as a yellow/orange solid, m. p.

33-38 Oc (expected 40 oC Appel & Berger) ; Rf = 0. 93 compared to chlorsulfonyl isocyanate Rf= 0. 15 [DCM].

Methyl 4-hydroxybenzoate (5): Concentrated sulfuric acid (H2SO4) (3 ml, 10 M) was carefully added to a suspension of 4-hydroxy benzoic acid (3 g, 21.74 mmol) in methanol (20 ml) and the solution refluxed for 1 h. After cooling to room temperature, NaOH (-15 ml) was added to neutralise the solution. The resulting mixture was allowed to stand for 15 min, before being poured into a cool beaker, and made up to 500 ml with water. The white precipitate was filtered, and dried (80 OC), to

give (5) (3. 3 g, 99. 9%) as a white crystalline solid [m. p. 112-115 C ; Rf 0. 47 diethyl ether/petroleum ether 40-60 C (50/50)].

V (may) (film) cm-l : 3263. 0 (OH) 1688. 2 (C = 0). on (CDCI3) : 7. 95 (2H, d, J = 8 Hz, ArH), 6. 89 (2H, d, J = 8 Hz, ArH), 6. 06 (1H, s, OH), 3. 90 (3H, s, CH3). 5c (CD13) : 167.2 (C = 0), 160.0, 131.8, 122.3, 115.1 (CAr), 52.0 (CH3). GCMS tR 9. 176 m/z 152 (M+).

Methyl 4-[(aminosulfonyl)oxy]benzoate (6): Sodium hydride (NaH) (60% dispersion in mineral oil, 0.16 g, 4 mmol) was added to a stirred solution of (5) (0.5 g, 3.29 mmol) in dimethyl formamide (DMF) (10 ml) under an atmosphere of nitrogen gas at 0 C. After evolution of hydrogen had ceased (30 min), aminosulfonyl chloride in toluene (10 ml,-10 mmol) was added in one portion and the reaction allowed to stir for 10 h. The reaction was then quenched with saturated sodium bicarbonate (NaHCO3) solution (50 ml), extracted into dichloromethane (DCM) (2 x 50 ml), washed with water (3 x 30 ml) and dried over anhydrous magnesium sulfate (MgS04). The mixture was filtered and the solvent removed under vacuum to give a yellow oil which solidified on addition of water. The crude product was purified using flash chromatography to give (6) (0.24 g, 31.6%) as a pure white solid [m. p. 118-121 OC ; Rf 0.24 diethyl ether / petroleum ether 40-60 C

(50/50)]. v (may) (film) cm-l : 3376. 1, 3274. 0 (NH2), 1704. 3 (C = 0), 1376. 7, 1156. 9 (S = 0). aH (CDC13) : 8. 08 (2H, d, J = 9 Hz, ArH), 7. 41 (2H, d, J 9 Hz, ArH), 5. 10 (2H, s, NH2), 3. 93 (3H, s, H3C-). c (CD13) : 165. 2 (C = 0), 154. 0, 149. 8, 131. 6, and 121. 9 (CAr), 52. 4 (CH3). MS m/z found : W 231. 0198, (CgH9NOsSt requires 231. 0201.

Ethyl 4-hydroxybenzoate (7) : Compound (7) was synthesised following the same procedures as for compound (5) except conc. H2SO4 (3 ml) was added to 4-hydroxybenzoic acid (3 g, 21.74 mmol) in ethanol (20 ml) to give (7) (2.91 g, 81%) as a white solid [m. p. 122-124 OC ; Rf 0.66 diethyl ether/petroleum ether 40 60 C (70/30)]. v (max) (film) cm-I : 3219.5 (OH) 1672.6 (C = 0). 90 MHz #H (CDCl3) : 6.9 (2H, d, J = 8 Hz, ArH), 8.0 (2H, d, 8 Hz, ArH), 6.5 (IH, s, OH), 4.3 (2H, q, J = 7 Hz,

CHzCH3), 1. 3 (3H, t, J = 7 Hz, CH2CH3). GCMS tR 10. 308 m/z 166 (M+).

Ethyl 4-[(aminosulfonyl)oxy]benzoate (8): Compound (8) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.2 g, 5 mmol) was added to a stirred solution of (7) (0.5 g, 3.01 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml, #10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (8) (0.29 g,

39. 3%) as a pure white solid [m. p. 83-86 oC ; Rf 0. 55 diethyl ether/petroleum ether 40-60 Oc (70/30)].

V () (film) cm-' : 3341. 4, 3217. 7 (NH2), 1693. 7 (C = 0), 1383. 1, 1174. 7 (S = 0). 5H (CDC13) : 8. 08 (2H, d, J = 9 Hz, ArH), 7. 40 (2H, d, J = 9 Hz, ArH), 5. 21 (2H, s, NH2), 4. 33 (2H, q, J = 7 Hz, CH2), 1.40 (3H, t, J = 7 Hz, CH3). lie (CDCl3) : 165.8 (C - 0), 154.7, 131.5, 129.3, 121.8 (CAr), 61.4 (CH2), 13.3 (CH3). MS m/z 246 (MM.

Propyl 4-hydroxybenzoate (9): Compound (9) was synthesised following the same procedures as for compound (5) except conc. H2SO4 (3 ml) was added to 4-hydroxybenzoic acid (3 g, 21.74 mmol) in 1-propanol (20 ml) to give (9) (3.11 g, 79.5%) as a white crystalline solid [m. p. 88-90 oC ; Rf 0.48 diethyl ether/petroleum ether 40-60 C (50/50)].

v (max) (film) cm : 3285. 8 (OH) 2967. 0, 2866. 3 (CH), 1679. 1 (C = 0). on (CDCl3) : 7. 95 (2H, d, J = 8 Hz, ArH), 6. 91 (2H, d, J = 8 Hz, ArH), 6. 71 (IH, s, OH), 4. 26 (2H, t, J = 6 Hz, OCH2), 1. 78 (2H, m, OCH2CH2), 1. 03 (3H, t, J-6 Hz, CHs). 8c 2), 1. 03 (3H, t, J = 6 Hz, CH3) (CDCl3) : 167. 1 (C = 0), 160. 3, 131. 9, 122. 5, 115. 3 (CAr), 66. 6 (OCH2), 22. 1 (OCH2CH2), 10. 5 (CH3). GCMS tR 12. 786 m/z 180 (M+).

Propyl 4-[ (aminosulfonyl) oxvlbenzoate (10) : Compound (10) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.13 g, 4 mmol) was added to a stirred solution of (9) (0.5 g, 2.78 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml, #10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (10) (0.34 g, 47.2%) as a pure white solid [m. p. 58-60 oC ; Rf 0.35 diethyl ether/petroleum ether 40-60 OC (50/50)]. v (max) (film) cm-' : 3352. 5,3265. 7 (NH2), 1702.4 (C = 0), 1389.6, 1156.0 (S = 0). #H(CDCl3) : 8. 04 (2H, d, J = 9 Hz, ArH), 7.37 (2H, d, J = 9 Hz, ArH), 5.43 (2H, s,

NH2), 4. 26 (2H, t, J = 7 Hz, OCH2), 1. 78 (2H, m, (XCHb), 1. 01 (3H, t, J = 7 Hz, CH3). 300 MHz #c (CDCl3) : 166.6 (C = 0), 153.4, 131.5, 129.2, 122.0 (CAr), 67.0 (OCH2), 22.0 (OCH2CH2), 10.5 (CH3). MS mlz 259 (M+).

Butyl 4-hydroxybenzoate (11) : Cone. H2SO4 was added to a stirred solution of 4-hydroxybenzoic acid (4 g, 29. 9 mmol) and 1-butanol (2. 7 ml, 30 mmol) in toluene (50 mil), and the solution refluxed using Dean and Stark apparatus until it became clear (2-4 h). The toluene was removed under vacuum and the remaining

residue quenched with saturated NaH03. The organic product was extracted into DCM (2 X 30 ml), washed with water (2 X 30 ml), dried (MgSO4), and the solvent removed under vacuum to give (11) (2.76 g, 47.4%) as a white solid [m. p. 64-66 oC ; Rf 0.52 diethyl ether/petroleum ether 40-60 C (50/50)]. v (max) (fim) cm-' : 3385.0 (OH) 2955.1, 2873.6 (CH), 1679.5 (C = 0). bH (CDCl3) : 7.95 (2H, d, J = 8 Hz, ArH), 6.91 (2H, d, J = 8 Hz, ArH), 6.92 (1H, s, OH), 4.31 (2H, t, J = 6 Hz, OCH2), 1.74 (2H, m, OCH2CH2), 1.47 (2H, m,-CH2CH3), 0.97

(3H, t, J = 7 Hz, CH3). be (CDCI3) : 167. 3 (C = 0), 160. 4, 131. 9, 122. 4, 115. 3 (CAr), 64. 9 (OCH2), 30. 5 (OCH2CH2), 19. 3 (O (CH2) 2CH2) 13. 8 (CH3). GCMS tR 12. 786 m/z 194 (M).

Butyl 4-[(aminosulfonyl)oxy]benzoate (12): Compound (12) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.05 g, 1.25 mmol) was added to a stirred solution

of (11) (0. 17g, 0. 88 mmol) in DMF (10ml). Aminosulfonyl chloride in toluene (10 ml,-10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (12) (0.06 g, 25%) as a pure white solid [m. p. 71-74 C ; Ra 0. 31 diethyl ether/petroleum ether 40 60 C (50/50)].

V (max) (film) cm-l : 3362.3, 3264.6 (NH2), 1703.4 (C = 0), 1387.7, 1156.1 (S = 0). aH (CDC13) : 8.04 (2H, d, J = 9 Hz, ArH), 7.39 (2H, d, J = 9 Hz, ArH), 5.37 (2H, s, NH2), 4.31 (2H, t, J = 6 Hz, OCH2), 1.74 (2H, m, OCH2CH2), 1.47 (2H, m, CFCH3), 0.98 (3H, t, J = 7 Hz, CH3). lie (CDCl3) : 165.7 (C = 0), 153.2, 131. 4,129. 2,121. 9 (CAr), 65.3 (OCH2), 30.6 OCH2CH2), 19.1 (O (CH2) 2CH2), 13. 7 (CH3). MS m/z 274 (MH+).

Pentyl 4-hydroxybenzoate (13): Compound (13) was synthesised following the same procedures as for compound (11) except that conc. H2SO4 (1 ml) was added to 4-hydroxybenzoic acid (4.5 g, 33 mmol) and l-pentanol (3.4 ml, 33 mmol) in toluene (50 ml). Compound (13) was obtained as a clear oil (2.78 g, 41.1%) [Rf 0.57 diethyl ether/petroleum ether 40-60 C (50/50)]. v (max) (film) cm-l : 3276.4 (OH) 2954.3, 2872.0 (CH), 1682.9 (C = 0). SH

(CDCl3) : 7. 95 (2H, d, J = 8 Hz, ArH), 6. 91 (2H, d, J = 8 Hz, ArH), 7. 78 (1H, s, OH), 4. 31 (2H, t, J = 6 Hz, OCH2), 1. 77 (2H, m, OCH2CH2), 1. 40 (4H, m, (CP2CH3), 0. 92 (3H, t, J = 7 Hz, CH3). Ôc (CDCl3) : 167. 9 (C = 0), 161. 1, 132. 0, 121. 8, 115. 8 (CAr), 65. 5 (OCH2), 28. 4 (OCH2CH2), 28. 2 (O (CH2) 2CH2) 22. 365 (O (CH2) 3CH2), 13. 993 (CH3). GCMS tR 14. 009 m/z 208 (M+).

Pentyl 4-) (aminosulfonvl) oxvlbenzoate (14) : Compound (14) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.12 g, 3 mmol) was added to a stirred solution of (13) (0.5 g, 2.40 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml, - 10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (14) (0.34 g, 49.4%) as a pure white solid [m. p 48-49 C ; Rf 0.31 diethyl ether/petroleum ether 40-60'C (50/50)].

V (may) (film) cm-I : 3362. 3, 3264. 1 (NH2), 1704. 2 (C = 0), 1386. 6, 1156. 5 (S = 0). OH (CDC13) : 8. 00 (2H, d, J = 9 Hz, ArH), 7. 37 (2H, d, J = 9 Hz, ArH), 5. 59 (2H, s, NH2), 4.28 (2H, t, J = 6Hz, OCH2), 1.74 (2H, m, OCH2CH2), 1.38 (4H, m, (CH2)2CH3), 0.92 (3H, t, J = 7 Hz, CH3). #c (CDCl3) : 165.9 (C = 0), 153.4, 131.5, 129.2, 122.0 (CAr), 65.7 (OCH2), 28.3 (OCH2CH2), 28. 1 (0 (CH2) 2CH2), 22. 3

(O (CH2) 3CH2), 14. 0 (CH3). MS m/z 288 (MIt) Hexyl 4 : h droxybenzoate (15) : Compound (15) was synthesised following the same procedures as for compound (11) except that conc. H2SO4 (I ml) was added to 4-hydroxybenzoic acid (4.5 g, 33 mmol) and 1-hexanol (4.05 ml, 33 mmol) in toluene

(50 ml). Compound (15) was obtained as a clear oil (3. 9 g, 53. 2%) [Rf 0. 57 diethyl ether /petroleum ether 40-60"C (50/50)]. v (max) (film) cm-1 : 3281.4 (OH) 2959.0, 2958.1 (CH), 1682.8 (C = O). #H (CDC13) : 7.94 (2H, d, J = 8 Hz, ArH), 6.92 (2H, d, J = 8 Hz, ArH), 7.78 (1H, broad s, OH), 4.30 (2H, t, J = 6 Hz, OCH2), 1.75 (2H, m, OCH2CH2), 1.38 (6H, m,- (CH2) 3CH3),

0. 89 (3H, t, J = 7 dz, CH3). c (CDC) : 167. 5 (C = 0), 160. 7, 131. 9, 122. 2, 115. 4 (CAr), 65. 3 (OCH2), 31. 5 (OCH2CH2), 28. 7 (O (CH2) 2CH2) 25. 7 (O (CH2) 3CH2), 22. 6 (O (CH2) 4CH2), 14. 1 (CH3). GCMS tir 5. 114 m/z 222 (M+).

Hexyl 4- [ (aminosulfonyl) oxv] benzoate (16) : Compound (16) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.12 g, 3 mmol) was added to a stirred solution of (15) (0.5 g, 2.25 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml, #10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (16) (0.24 g, 35.4%) as a pure white solid [m. p. 37-39 C ; Rf 0.37 diethyl ether/petroleum ether 40-60'C (50/50)].

v (max) (film) cm-l : 3365. 4, 3265. 1 (NH2), 1703. 0 (C = 0), 1389. 2, 1181. 7 (S = 0). < H (CDC13) : 8. 01 (2H, d, J = 9 Hz, ArH), 7. 37 (2H, d, J = 9 Hz, ArH), 5. 58 (2H, s, NH2), 4. 28 (2H, t, J = 6 Hz, OCH2), 1. 74 (2H, m, OCH2CH2), 1. 35 (6H, m, (Cfu) 3CH3), 0. 90 (3H, t, J = 7 Hz, CH3). Oc (CDCl3) : 165. 8 (C = 0), 153. 3, 131. 4, 129. 1, 121. 9 (CAr), 65. 6 (OCH2), 31. 4 (OCH2CH2), 28. 5 (O (CH2) 2CH2), 25. 6 (O (CH2) 3CH2), 22. 5 (O (CH2) 4CH2), 13. 9 (CH3). MS m/z 302 (MH).

Heptyl 4-hvdroxybenzoate (17) : Compound (17) was synthesised following the same procedures as for compound (11) except that H2SO4 (0. 5ml) was added to 4-hydroxybenzoic acid (2 g, 14.5 mmol) and 1-heptanol (2.1 ml, 14.6 mmol) in toluene (50 ml). Compound (17) was obtained as a clear oil (1.45 g, 42.4%) [Rf 0.57 diethyl ether/petroleum ether 40-60 C (50/50)].

#(max) (film) cm-1 : 3348.6 (OH) 2956.6, 2929.9 (CH), 1682.5 (C = 0). OH (CDCI3) : 7.94 (2H, d, J = 8 Hz, ArH), 6.92 (2H, d, J = 8 Hz, ArH), 7.90 (1H, broad s, OH), 4.30 (2H, t, J = 6 Hz, OCH2), 1.75 (2H, m, OCH2CH2), 1.37 (8H, m,- (Cfu) 4CH3),

0. 88 (3H, t, J = 7Hz, CH3). Sc (CDC/j ;) : 167. 7 (C = 0), 161. 0, 132. 0, 121. 9, 115. 4 (CAr), 65. 4 (OCH2), 31. 7 (OCH2CH2), 29. 0 (O (CH2) 2CH2) 28. 7 (O (CH2) 3CH2), 26. 0 (O (CH2) 4CH2), 22. 6 (O (CH2) 5CH2), 14. 1 (CH3). GCMS tR 16. 236 m/z 236 (M+).

Heptyl 4-r (aminosulfonvl) oxvlbenzoate (18) : Compound (18) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.05 g, 1.25 mmol) was added to a stirred solution of (17) (0. 18 g, 0.74 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml, #10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (18) (0. 06 g, 25.7%) as a pure white solid [m. p. 54-56 OC ; Rf 0.37 diethyl ether/ petroleum ether 40-60 C (50/50)]. v (max) (film) cm-1 : 3362.3, 3272.0 (NH2), 1704.4 (C = 0), 1388.1, 1156.6 (S =

0). aH (CDC13) : 8. 02 (2H, d, J = 9 Hz, ArH), 7. 39 (2H, d, J = 9 Hz, ArH), 5. 41 (2H, s, NH2), 4. 28 (2H, t, J = 6 Hz, OCH2), 1. 75 (2H, m, OCH2CH2), 1. 36 (8H, m, (CH2) 4CH3), 0. 89 (3H, t, J = 7Hz, CH3). (CDC : 165. 8 (C = 0), 153. 3, 131. 5, 129. 3, 122. 0 (CAr), 65. 7 (OCH2), 31. 7 (OCH2CH2), 28. 9 (O (CH2) 2CH2), 28. 6 (O (CH2) 3CH2), 26. 0 (O (CH2) 4CH2), 22. 6 (O (CH2) 5CH2), 14. 1 (CH3). MS m/z 333 (MNH4+).

Octyl 4-hydroxybenzoate (19) : Compound (19) was synthesised following the same procedures as for compound (11) except that conc. H2SO4 (5 drops) was added to 4-hydroxybenzoic acid (1 g, 7. 24 mmol) and 1-octanol (1. 2 ml, 7. 6 mmol) in toluene (50 ml). The crude oil was purified using flash chromatography to give (19) as a clear oil (1.05 g, 58.0%) [Rf 0. 61 diethyl ether/petroleum ether 40-60 C (50/50)].

V (max) (film) cm-l : 3349. 7 (OH) 2924. 7, 2854. 5 (CH), 1684. 2 (C = 0). OH (CDC ! j) : 7. 94 (2H, d, J = 8 Hz, ArH), 6. 89 (2H, d, J = 8 Hz, ArH), 6. 09 (1H, broad s, OH), 4. 29 (2H, t, J = 6Hz, OCH2), 1. 75 (2H, m, OCH2CH2), 1. 36 (IOH, m, (CH2) 5CH3), 0. 85 (3H, t, J = 7 Hz, CH3). c (CDC13) : 166. 9 (C = 0), 160. 1, 132. 0, 123. 0, 115. 3 (CAr), 65. 2 (OCH2), 31. 9 (OCH2CH2), 29. 8 (O (CH2) 2CH2) 29. 3 (O (CH2) 3CH2), 28. 9 (O (CH2) 4CH2), 26. 2 (O (CH2) sCH2), 27. 8 (O (CH2) 6CH2), 14. 2 (CH3). GCMS tR 17. 206 m/z 250 (M).

Octyl 4-[(aminosulfonyl)oxy]benzoate (20): Compound (20) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.5 g, 12.5 mmol) was added to a stirred solution of (19) (2. 38 g, 9. 52 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (20 ml,-20 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (20) (1. 01 g, 32.2%) as a pure white solid [m. p. 62-64 oC ; Rf 0.45 diethyl ether/ petroleum ether 40-60 C (50/50)]. v (max) (film) cm-1 : 3364.3, 3267.7 (NH2), 1704.2 (C = 0), 1391.2, 1157.0 (S = 0). #H (CDCl3) : 7.99 (2H, d, J = 9 Hz, ArH), 7.35 (2H, d, J = 9 Hz, ArH), 5.16 (2H, s, NH2), 4.26 (2H, t, J = 7 Hz, OCH2), 1.73 (2H, m, OCH2CH2), 1.35 (lOH, m,

(CH CDC13 (cru) 5CH3), 0. 87 (3H, t, J = 7 Hz, CH3). CDC : 165. 9 (C = 0), 153. 4, 131. 5, 129. 1, 122. 0 (CAr), 65. 7 (OCH2), 31. 8 (OCH2CH2), 29. 0 (O (CH2) 2CH2), 28. 6 (O (CH2) 3CH2), 26. 0 (O (CH2) 4CH2), 25. 4 (O (CH2) sCH2), 22. 6 (O (CH2) 6CH2), 14. 1 (CH3). MS m/z 347 (MNH4+).

Nonyl 4-hydroxybenzoate (21) : Compound (21) was synthesised following the same procedures as for compound (11) except that conc. H2SO4 (0. 5ml) was added to 4-hydroxybenzoic acid (2 g, 14.5 mmol) and 1-nonanol (2.5 ml, 14.4 mmol) in toluene (50 ml). The crude oil was purified using flash chromatography to give (21) as a clear oil (0.95 g, 24.8%) [Rf 0.57 diethyl ether/petroleum ether 40-60 Oc (50/50)]. v (max) (film) cm-l : 3352.2 (OH) 2926.6. 2855.8 (CH), 1682.3 (C = 0). OH (CDCl3) : 7.93 (2H, d, J = 8 Hz, ArH), 6.94 (2H, d, J = 8 Hz, ArH), 7.11 (1H, broad s, OH), 4.30 (2H, t, J = 6Hz, OCH2), 1.76 (2H. m, OCH2CH2), 1.36 (12H, m, -

(C ! h) 6CH3), 0. 88 (3H, t, J = 7 Hz, CH3). c (CDC13) : 167. 7 (C = 0), 161. 0, 132. 0, 121. 9, 115. 4 (CAr), 65. 4 (OCH2), 32. 4 (OCH2CH2), 29. 5 (O (CH2) 2CH2) 29. 3 (O (CH2) 3CH2), 28. 7 (O (CH2) 4CH2), 26. 0 (O (CH2) CH2), 25. 7 (O (CH2) 6CH2), 22. 7 (O (CH2) ? CH2), 14. 1 (CH3). GCMS tR 18. 406 m/z 264 (M+).

Nonyl 4-[(aminosulfonyl)oxy]benzoate (22): Compound (22) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.2 g, 5 mmol) was added to a stirred solution of (21) (0.5 g, 1.90 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml, #10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (22) (0.07 g, 10.7%) as a pure white solid [m. p. 58-61 oC ; Rf 0.24 diethyl ether/petroleum ether 40-60 C (30/70)].

V (maux) (film) cm-l : 3362. 3, 3264. 2 (NH2), 1704. 3 (C= 0), 1391. 8, 1181. 8 (S = 0). aH (CDC13) : 8. 02 (2H, d, J = 9 Hz, ArH), 7. 38 (2H, d, J = 9 Hz, ArH), 5. 46 (2H, s, NH2), 4. 28 (2H, t, J = 6Hz, OCH2), 1. 74 (2H, m, OCH2CH2), 1. 31 (12H, m, (C ! h) 6CH3), 0. 88 (3H, t, J = 7 Hz, CH3). lie (CDC13) : 165. 7 (C = 0), 153. 3, 131. 5, 129. 2, 122. 0 (CAr), 65. 7 (OCH2), 31. 8 (OCH2CH2), 29. 5 (O (CH2) 2cl2), 29. 3 (O (CH2) 3CH2), 29. 2 (O (CH2) 4CH2), 28. 6 (O (CH2) 5CH2), 26. 0 (O (CH2) 6CH2), 22. 7 (O (CH2) 7CH2), 14. 1 (CH3). MS m/z found : MNH4+ 361. 1805, (CtNO) NH4+ requires 361. 1797.

Decyl 4-hydroxybenzoate (23): Compound (23) was synthesised following the same procedures as for compound (l l) except that conc. H2SO4 (0.5 ml) was added to 4-hydroxybenzoic acid (2 g, 14.5 mmol) and 1-decanol (2.8 ml, 14.7 mmol) in toluene (50 ml). The crude oil was purified using flash chromatography to give (23) as a white solid (1.06 g, 26.3%) [m. p. 30-34 oC ; Rf 0.57 diethyl ether/petroleum ether 40-60 Oc (50/50)].

v (max) (film) crn-l : 3354. 7 (OH) 2924. 2, 2854. 1 (CH), 1683. 7 (C = 0). OH (CDCl3) : 7.94 (2H, J = 8 Hz, ArH), 6.93 (2H, d, J = 8 Hz, ArH), 6. 37 (1H, broad s, OH), 4.30 (2H, t, J = 6Hz, OCH2), 1.76 (2H, m, OCH2CH2), 1.36 (14H, m, (CH2) 7CH3), 0.88 (3H, t, J = 7 Hz, CH3). #c (CDC13) : 167.5 (C = 0), 160.7, 131.9, 122.2,

115. 3 (CAr), 65. 3, 31. 9, 29. 5, 29. 3, 28. 7, 26. 0, 22. 7, 14. 1 (only 8 aliphatic carbons visible). GCMS tR 19. 195 m/z 278 (M+).

Decyl 4-[(aminosulfonyl)oxy]benzoate (24): Compound (24) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.1 g, 2.5 mmol) was added to a stirred solution of (23) (0.5 g, 1.8 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml, #10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (24) (0.21 g, 32.7%) as a pure white solid [m. p. 48-51 OC ; Rf 0.41 diethyl ether/petroleum ether 40-60"C (50/50)].

V (max) (film) cm-l : 3394. 0, 3276. 2 (NH2), 1715. 9 (C-0), 1377. 2, 1181. 7 (S = 0). #H (CDCI3) : 8.04 (2H, d, J = 9 Hz, ArH), 7.39 (2H, d, J = 9 Hz, ArH), 5.39 (2H, s, NH2), 4.29 (2H, t, J = 6 Hz, OCH2), 1.75 (2H, m, OCH2CH2), 1.34 (14H, m, (CH2) 7CH3), 0.88 (3H, t, J = 7 Hz, CH3). (CDCI3) : 165.7 (C = 0), 153.3, 131.5, 129.3, 121.9 (CAr), 65.7, 31.9, 29.5, 29.3, 28.6, 26.0, 22.7, 14.1 (only 8 aliphatic peaks visible). MS m/z found: MNH4+ 375. 1961, (C17H27NO5S) NI44-' requires 375.1954.

Methyl (4'-hydroxy)-1,1'-biphenyl-4-carboxylate (25): Compound (25) was synthesised following the same procedures as for (5), except that conc. H2SO4 (1 ml) was added to a suspension of 4-hydroxybiphenyl carboxylic acid (0.6 g, 2.8 mmol) in methanol (20 ml). The white precipitate was filtered and dried (80 OC), to give (25) (0.58 g, 90.2%) as a white solid [m. p. 220-222 C (literature 224-225 oc, Yang et al, 1972); Rf 0. 55 diethyl ether/petroleum ether 40-60 C (70/30)].

#(max) (film) cm'' : 3408.4 (OH), 2926.5 (CH aliphatic), 1696.0 (C = 0).

90 MHz 8H (d6-Acetone) : 7.5 (8H, m, ArH), 3.9 (3H, s, CH3), 2.8 (1H, s, OH). GCMS tR 18.208 m/z 228 (M+).

Ethyl (4'-hydroxy)-1,1'-biphenyl-4-carboxylate (26): Compound (26) was synthesised following the same procedures as for compound (5), except that conc.

H2SO4 (1 ml) was added to a suspension of 4-hydroxybiphenyl carboxylic acid (0.5 g, 2.34 mmol) in ethanol (20 mol). The white precipitate was filtered and dried (80 C), to

give (26) (0. 46 g, 81. 23%) as a white solid [m. p. 141-143 oC ; Rf 0. 63 diethyl ether/ petroleum ether 40-60 C (70/30)]. v (max) (film) cm-l : 3335. 9 (OH), 1681. 4 (C = 0). 6H (CDCIa) : 8. 00 (2H, d, J = 8 Hz, ArH), 7. 60 (2H, d, J = 8 Hz, ArH), 7. 53 (2H, d, J = 8 Hz, ArH), 6. 95 (2H, d, J = 8 Hz, ArH), 5. 81 (1H, s, OH), 4. 41 (2H, q, J = 7 Hz, CH2CH3), 1. 42 (3H, t, J = 7 Hz, CH2CH3). oc (CDCl3) : 167. 2 (C = 0), 157. 3, 145. 6, 132. 5, 130. 1, 128. 6, 128. 4, 126. 4, 115.9 (CAr), 61.1 (OOCH3), 14.3 (OCCHs). GCMS tR 19.223 m/z 242 (M+).

Propyl (4'-hydroxy)-1, I'-biphenyl-4-carboxylate (27): Compound (27) was synthesised following the same procedures as for compound (5), except that conc.

H2SO4 (1 ml) was added to a suspension of 4-hydroxybiphenyl carboxylic acid (0.6 g, 2.8 mmol) in 1-propanol (20 ml). The white precipitate was filtered and dried (80 C), to give (27) (0.65 g, 88.9%) as a white solid [m. p. 117-121 OC ; Rf 0.67 diethyl ether/ petroleum ether 40-60 C (70/30)]. vax.) (film) cm-l : 3317. 2 (OH), 2964.1 (CH aliphatic), 1714.1 (C = 0)-6H (CDC13) : 8.10 (2H, d, J = 9 Hz, ArH), 7.61 (2H, d J = 9 Hz, ArH), 7.46 (2H, d, J =

9 Hz, ArH), 6. 96 (2H, d, J = 9 Hz, ArH), 5. 70 (1H, s, OH), 4. 32 (2H, t, J = 7 Hz, OCH2-), 1. 81 (2H, m, OCH2Cfu-), 1. 06 (3H, t, J = 7 Hz,-CH3). Sc (CDCI3) : 167. 0 (C = 0), 156. 1, 145. 3, 132. 5, 130. 1, 128. 6, 128. 5, 126. 5, 115. 9 (C-Ar), 66. 7 (OCH2), 22. 1 (CH2), 10. 5 (CH3). GCMS tu 20. 170 m/z 256 (M").

Butyl (4'-hydroxy)-1, 1'-biphenyl-4-carboxylate (28) : Compound (28) was synthesised following the same procedures as for compound (5), except that conc.

H2SO4 (1 ml) was added to a suspension of 4-hydroxybiphenyl carboxylic acid (0.6 g, 2.8 mmol) in l-butanol (20 ml). After quenching in ice water, the product was extracted into ether (3 x 30 ml). Removal of the solvent under vacuum, followed by distillation to remove excess 1-butanol, gave crude (28) (0.35 g, 46.1%) as a pale brown solid [Rf 0.71 diethyl ether/petroleum ether 40-60 C (70 : 30)]. GCMS tR 21. 585 m/z 270 (M+). The product from this reaction was used directly in the synthesis of (32).

Methyl 4'-[(aminosulfonyl)oxy]-1,1'-biphenyl-4-carboxylate (29) : Compound (29) was synthesised following the same procedure as for compound (6)

except that NaH (60% dispersion in mineral oil, 0. 1 g, 2. 5 mmol) was added to a stirred solution of (25) (0. 4 g, 1. 75 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (20 ml,-20 mmol) was added after 30 min. Removal of the solvent under vacuum produced a yellow oil, which was purified using flash chromatography to give (29) (0.2 g, 37.6%) as a pure white solid [m. p. 162-165 oC ; Rf 0.52 diethyl ether/petroleum ether 40-60 C (70/30)].

#(max) (Film) cm-1 : 3363.6, 3234.7 (NH2), 1708.0 (C = 0), 1388.6, 1161.1 (S = O). #H (d6-Acetone) : 8. 11 (2H, d, J = 9 Hz, ArH), 7.82 (2H, d, J = 9 Hz, ArH), 7.81 (2H, d, J = 9 Hz, ArH), 7.45 (2H, d, J = 9 Hz, ArH), 7.23 (2H, s, NH2), 3.91 (3H, s, OCH3). #C (d6-Acetone) : 151.5, 144.8, 138.8, 130. 6,130. 0,129. 1,127. 7,123. 6 (CAr), 52.2 (OCH3). MS m/z found: MNH 325. 0854, (Cl4Hl3NO5S) NW requires 325.0858.

Ethyl 4'-f (aminosulfonyl) oxy]-1,1'-biphenyl-4-carboxylate (30): Compound (30) was synthesised following the same procedure as for compound (6) except that NaH (60% dispersion in mineral oil, 0.05 g, 1.25 mmol) was added to a stirred solution of (26) (0.2 g, 0.83 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml,-10 mmol) was added after 30 min. Removal of the solvent under vacuum produced a solid, which was purified using flash chromatography to give (30) (0.08 g, 30.0%) as a pure white solid [m. p. 171.2-173. 5 C ; Rf 0. 30 petroleum ether 40-60 C : ethyl acetate (65/35)]. x) (Film) cm-l : 3343.7, 3222.2 (NH), 1693.9 (C = 0) 1401.2, 1162.5 (S = 0). OH (CDC13) : 8.12 (2H, d, J = 9 Hz, ArH), 7.80 (2H, d J = 9 Hz, ArH), 7.79 (2H, d, J = 9 Hz, ArH), 7.44 (2H, d, J = 9 Hz, ArH), 5.03 (2H, s, NH2), 4.41 (2H, q, J = 7 Hz,

CH2-), 1. 42 (3H, t, J = 7 Hz, CH2CH -Acetone) : 199. 7 (C = 0), 154. 2, 135. 0, 131.4, 130.3, 122.7, 115.2 (C-Ar), 32.0 (OCH2), 8.2 (CH3). MS m/z 321 (M+).

Propyl 4'-[(aminosulfonyl)oxy]-1,1'-biphenyl-4-carboxylate (31) : Compound (27) was synthesised following the same procedure as for compound (6) except that NaH (60% dispersion in mineral oil, 0. 1 g, 2.5 mmol) was added to a stirred

solution of (27) (0. 48 g, 1. 86 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (20 ml,-20 mmol) was added after 30 min. Removal of the solvent under vacuum produced a yellow oil, which was purified using flash chromatography to

give (31) (0. 19 g, 30. 5%) as a pure white solid [m. p. 136-139 oC ; Rf 0. 55 diethyl ether/petroleum ether 40-60 C (70/30)].

#(max) (Film) cm'-' : 3324.6, 3197.1 (NH), 1687.8 (C = 0) 1369.7, 1193.3 (S = 0). OH (CDC13) : 8.13 (2H, d, J = 9 Hz, ArH), 7.65 (2H, d, J = 9 Hz, ArH), 7.63 (2H, d, J = 9 Hz, ArH), 7.44 (2H, d, J = 9 Hz, ArH), 5. 01 (2H, s, NH2), 4.32 (2H, t, J = 7 Hz, OCH2-), 1.83 (2H, m, OCH2CH2-), 1.06 (3H, t, J = 7 Hz,-CH3). < %/ (CDCI3) : 166.2 (C = 0), 151.3, 144.6, 138.7, 130.5, 130.1, 129.0, 127.5, 123.5 (C-Ar), 66.7

(OCH2), 22. 4 (CH2), 10. 4 (CH3). MS m/z found : MH 336. 0900, ( (HNCS) requires 336.0905.

Butyl 4'-[(aminosulfonyl)oxy]-1,1'-biphenyl-4-carboxylate (32): Compound (32) was synthesised following the same procedure as for compound (6) except that NaH (60% dispersion in mineral oil, 0. 1 g, 2. 5 mmol) was added to a stirred

solution of (28) (0. 3 g, 1. 11 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (20 ml,-20 mmol) was added after 30 min. Removal of the solvent under vacuum produced a yellow oil, which was purified using flash chromatography to give (32) (0.11 g, 28.4%) as a pure white solid [m. p. 100-104 C ; Rf 0.57 diethyl ether/petroleum ether 40-60"C (70/30)]. ax) (Film) cm-1 : 3415.0, 3300.3 (NH), 1712.5 (C = 0) 1378.9, 1179.0 (S = O). #H (CDCl3) : 8.11 (2H, d, J = 9 Hz, ArH), 7.61 (2H, d, J = 9 Hz, ArH), 7.59 (2H,

d, J = 9 Hz, ArH), 7. 43 (2H, d, J = 9 Hz, ArH), 5. 01 (2H, s, NH2), 4. 35 (2H, t, J = 7 Hz, OCH2-), 1. 77 (2H, m, OCH2CH2-), 1. 50 (2H, m, OCH2CH2CH2-) 1. 00 (3H, t, J = 7 Hz,-CH3). (CDC) : 150. 0 (C = 0), 130. 1, 128. 8, 127. 0, 122. 6 (CAr), 65. 0 (OCH2), 30. 8 (OCH2CH2), 19. 3 (OCH2CH2CH2), 13. 8 (CH3). MS m/z 349 (M+).

5-Hydroxyisophthalic acid dimethyl ester (33) : Compound (33) was synthesised following the same procedures as for compound (5) except conc.

H2SO4 (3 ml) was added to 5-hydroxyisophthalic acid (1 g, 5.5 mmol) in methanol (20 ml) to give (33) (1.12 g, 97%) as a white solid [Rf 0.52 diethyl ether/petroleum ether 40-60 C (70/30)].

#(max.) (film) cm-1 : 3218.3 (OH) 1674.9 (C = 0). 300 MHz #H (CDCI3) : 8.37 (1H, s, ArH), 7.98 (2H, s, ArH), 6.5 (1H, s, OH), 3. 92 (6H, s, CH3).

5-Sulfamoyloxyisophthalic acid dimethyl ester (34) : Compound (34) was synthesised following the same procedures as for compound (6) except that NaH (60% dispersion in mineral oil, 0.2 g, 5 mmol) was added to a stirred solution of (29) (0.5 g, 2.38 mmol) in DMF (10 ml). Aminosulfonyl chloride in toluene (10 ml, #10 mmol) was added after 30 min. Removal of the solvent under vacuum yielded a yellow oil, which was purified using flash chromatography to give (34) (0.18 g, 26.2%) as a pure white solid [Rf 0.29 diethyl ether/petroleum ether 40-60 oc

(50/50)]. v (max) (film) cm-l : 3338. 7, 3217. 7 (NH2), 1698. 7 (C = 0), 1379. 3, 1174. 6 (S = 0).. 5H 4c : 8. 37 (1H, s, ArH), 8. 00 (2H, s, ArH), 7. 25 (2H, s, NH2), 3. 92 (6H, s, CHs). (d6-Acetone) : 165. 3 (C = 0), 151. 4, 133. 1, 128. 6, 128. 1 (CAr), 52. 9 (CH3).

In vitro biological testing The total assay volume was 1 ml. 3H-oestrone sulfate (25 ul, 20 uM/tube ; 300,000 dpm/tube) and the inhibitors (25 Ill in various concentrations) dissolved in ethanol were added to a 10 ml assay tube, and the ethanol removed with a stream of

nitrogen. Tris-cl buffer (0. 05 M, pH 7. 2, 0. 2 ml) was added to each tube. Placental microsomes were then diluted with Tris-HCl buffer (115 ug/ml). The microsomes and assay tubes were preincubated for 5 min at 37 Oc in a shaking water bath prior to the addition of the microsomes (0. 8 ml) to the tubes. After 20 min incubation (at 37 OC), toluene (4 ml) was added to quench the assay, and the tubes placed in ice. The quenched samples were vortexed for 45 s and centrifuged (3000 rpm, 10 min). I ml of toluene was added to 5 ml scintillation cocktail (TRITON-X). The aliquots were counted for 3 min. All samples were run in triplicate. Control samples with no inhibitor were incubated simultaneously. Blank samples were obtained by incubating with boiled microsomes.

Irreversible ES assay : The irreversible inhibition was determined using the procedure described by Purohit et al (1995) 9 using EMATE (10 uM), COUMATE (100 uM) and sulfamated esters (700 M). Placental microsomes (18 mg/ml, 55 l)

were incubated with each of the inhibitors (25 ul in ethanol, removed with a stream of nitrogen) in Tris-cl buffer (50 mM, pH 7. 2, 945 u. l) at 37 Oc for ten minutes. A

control tube with no inhibitor was incubated simultaneously (100% tubes). An aliquot (100 u. l) in triplicate, was taken from each sample and tested for ES activity using the procedure above, except that 900 l of Tris-HCl buffer was added to the assay tubes. A second aliquot (100 u. l) in triplicate, was subjected to dialysis at 4 C for 16 h, with regular changes of Tris-HCl buffer. The microsomes were then removed from the dialysis tubing and tested for ES activity as described above.

Table I shows the inhibitory data for the two standard compounds EMATE

and COUMATE : Table 1

Compound Percentage inhibition IC50 50 M 10 M 0. 5 M EMATE-68. 6% 50% 0.5 M ~ 0.001 COUMATE 83. 1% 47. 6% - 12 M ~ 0. 16 Table 2 shows the inhibitory data for the synthesised compounds derived from benzoic acid: Table 2

Compound R Percentage inhibition % IC50 M 50 gm 10 gm (6) CH3 74.7 25.4 31.6 ~ 1.23 (8) C2H5 63.4 25.6 31.6 ~ 1.95 (10) C37 76.9 42.6 13.2 ~ 0.4 (12) C4H9 - 48.3 10.5 ~ 0. 28 (14) C5H11 86.2 64.4 5. 9 ~ 0. 44 (16) C6H13-72. 63. 8 ~ 0. 16 (18) C2H15 83.7 69.5 3. 4 0. 25

(20) C8H7 80.4 63.0 5 ~ 0.26 (22) C9H19 76.2 - 4.8 ~ 0.17 (24) C10H21 - 36.5 22.4 ~ 0.48 Table 3 shows the inhibitory data for the synthesised compounds derived from biphenyl-4-carboxylic acid: Table 3

Compound X Percentage inhibition % ICsopM 250 100pu (29) CO2CH3 50.0 - 250 ~ 9. 9 (30) CO2C2H5 - 67.2 50 ~ 50 ~ 2. 3 (31) CO2C3H7 54.8 37.3 204 ~ 11. 6 (32) CO2C4H9 44.1 39.7

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Claims (7)

1. A compound of formula (C) :

wherein: R1-R5 are independently selected from H, halo, alkyl, nitro, optionally substituted alkoxy, optionally substituted aryl, optionally substituted aryloxy, optionally substituted alkylamino, optionally substituted arylamino, a further group COOR6, or a sulfamate group; but wherein at least one of Ri-rus is a sulfamate group or aryl, aryloxy, or arylamino substituted with a sulfamate group; and R6 is independently selected from H, aryl, or alkyl and substituted variants thereof ; or a pharmaceutically acceptable salt thereof.

2. A compound as claimed in claim 1 wherein R1-R5 are independently selected from H, alkyl, haloalkyl, a further group COOP,. a sulfamate group, and alkylphenyl substituted with a sulfamate group.

3. A compound as claimed in claim 1 or claim 2 wherein R3 is -OSO2NH2 or C6H4OSO2NH2.

4. A compound of formula (C):

wherein: Ri-rus are independently selected from H, halo, alkyl, nitro, optionally substituted alkoxy, optionally substituted optionally substituted aryl, optionally substituted

aryloxy, optionally substituted alkylamino, optionally substituted arylamino, a further group COR6, or a sulfamate group ; or R, and R2 taken together with the adjacent carbon atoms, or R2 and R3 taken together with the adjacent carbon atoms, represent a second optionally substituted aromatic ring;

but wherein at least one of RI-Rs is a sulfamate group or aryl, aryloxy, or arylamino substituted with a sulfamate group, or Rl and R2 taken together with the adjacent carbon atoms, or R2 and R3 taken together with the adjacent carbon atoms, represent an aromatic ring substituted with a sulfamate group; and Ru is independently selected from H, aryl, or alkyl and substituted variants thereof; or a pharmaceutically acceptable salt thereof.

5. A compound as claimed in any preceding claim for use as a pharmaceutical product.

6. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 4 and a pharmaceutically acceptable carrier, excipient or diluent.

7. Use of a compound as claimed in any one of claims 1 to 4 in the manufacture of a pharmaceutical product for use in the treatment or prophylaxis of a condition associated with oestrone sulfatase.