COMPOUNDS WITH A SULFAMATE GROUP

Abstract: A sulphamate compound is described. The compound is suitable for use as an inhibitor of both oestrone sulphatase activity and aromatase activity. A preferred compound has general formula (II) wherein F represents a phenolic ring structure (the first ring structure), J represents the third ring structure, I represents a phenolic ring structure (the second ring structure), G is an optional double bond, H is a link joining the second ring structure to the third ring structure, and Y represents a suitable second group; wherein any one of ring structures F, J and I has bound thereto a sulphamate group.

Description: COMPOUNDS WITH A SULFAMATE GROUP The present invention relates to a compound In particular the present invention relates to a pharmaceutical composition comprising the compound Breast and endometπal cancers are major causes of death in Western women In particular, rumours in endocrine-dependent tissues, such as the breast and endometπum, occur most frequently in postmenopausal women at a time when the ovaries have ceased their production of oestrogens Evidence suggests that oestrogens are the major mitogens involved in stimulating and promoting the growth ot tumours in endocrine-dependent tissues, such as the breast and endometπum 21 Although plasma oestrogen concentrations are similar in women with or without breast cancer, breast tumour oestrone and oestradiol levels are significantly higher than in normal breast tissue or blood In addition, in postmenopausal women oestrogens continue to be produced by extraglanduiar production in adipose tissue but also in normal and malignant breast tissues" Figures 1 and 2 are schematic diagrams showing some of the enzymes involved in the tn situ synthesis of oestrone from oestrone sulphate, oestradiol and androstenedione In Figure 2, which schematically shows the origin of oestrogenic steroids in postmenopausal women, "ER" denotes Oestrogen Receptor, "DHA/-S ' denotes Dehydroepιandrosterone/-Sulphate, "Adiol" denotes Androstenediol "El-STS" denotes Oestrone Sulphatase, "DHA-STS" denotes DHA-sulphatase, Adioi-STS" denotes Adiol Sulphatase, and " 17B-HSD" denotes Oestradiol 17B-hydroxγsteroιd dehydrogenase As can be seen, the ma two enzymes that are involved in the peripheral synthesis of oestrogens are the aromatase enzyme and the enzyme oestrone sulphatase In short, the aromatase enzyme converts androstenedione, which is secreted in large amounts by the adrenal cortex, to oestrone Recent reports have suggested that some flavones could inhibit aromatase activity- 15 J6 Much of the oestrone so formed, however, is convened to oestrone sulphate (EIS) and there is now a considerable body of evidence showing that EIS in plasma and tissue acts as a reservoir tor the formation of oestrone by the action of oestrone sulphatase 23 In this regard, it is now believed that the oestrone sulphatase (El-STS) pathway - I e the hydrolysis ot oestrone sulphate to oestrone (EIS to El) is the major source of oestrogen in breast tumours' 2 This theory is supported by a modest reduction of plasma oestrogen concentration in postmenopausal women with breast cancer treated by aromatase inhibitors, such as ammoglutethimide and 4-hydroxyandrostenedιone- 1 4 and also by the fact that plasma EIS concentration in these aromatase inhibitor-treated patients remains relatively high The long half-life of EIS in blood (10- 12 h) compared with the unconjugated oestrogens (20 min) 5 and high levels of steroid sulphatase activity in liver and, normal and malignant breast tissues, also lend suppoπ to this theory 6 Thus, oestrogen formation in malignant breast and endometnal tissues via the sulphatase pathway makes a major contribution to the high concentration of oestrogens which are present in these tumours 24 25 PCT/GB92/01587 teaches novel steroid sulphatase inhibitors and pharmaceutical compositions containing them for use in the treatment of oestrone dependent rumours, especially breast cancer These steroid sulphatase inhibitors are sulphamate esters such as N.N-dimethyl oestrone-3-sulphamate and, preferably, oestrone-3-sulphamate (otherwise known as "EM ATE") EMATE is a potent El -STS inhibitor as it displays more than 99% inhibition of El - STS activity in intact MCF-7 ceils at 0 1 μM EMATE also inhibits the El-STS enzyme in a time-dependent and concentration-dependent manner, thereby indicating that it acts as an active site-directed inactivator 7 3 Although EMATE was originally designed for the inhibition of El-STS, it also inhibits dehydroepiandrosterone sulphatase (DHA-STS), which is an enzyme that is believed to have a pivotal role in regulating the biosynthesis of the oestrogenic steroid androstenediol 8 9 This is of significance as there is now evidence to suggest that androstenediol may be of even greater importance as a promoter of breast tumour growth 10 EMATE is also active in vivo as almost complete inhibition of rat ver El-STS (99%) and DHA-STS (99%) activities resulted when it is administered either orally or subcutaneously" In addition, EMATE has been shown to have a memory enhancing effect in rats' 4 Studies in mice have suggested an association between DHA-STS activity and the regulation of part of the immune response. It is thought that this may also occur in humans' 5 16 . The bridging O-atom of the sulphamate moiety in EMATE is believed to be important for inhibitory activity Thus, when the 3-( -atom is replaced by other heteroatoms - as in oestrone-3- V-sulphamate and oestrone-3-5- sulphamate - these analogues are weaker non-time-dependent inactivators' 2 Thus, EMATE is a potent steroid sulphatase inhibitor which blocks the hydrolysis of both EIS and DHA-S 29'31 This inhibitor, therefore, not only blocks the synthesis of oestrone from EIS but also the formation of androstenediol from DHA-S In addition to oestrone, the other major steroid with oesirogemc properties which is produced by postmenopausal women is androstenediol (see Figure 2) Androstenediol, although an androgen, can bind to the oestrogen receptor (ER) and can stimulate the growth ot ER positive breast cancer cells and the growth of carcinogen-induced mammary tumours in the rat 26 27 Importantlv in postmenopausal women 90% of the androstenediol produced originates from the androgen dehydroepiandrosterone sulphate (DHA-S) which is secreted in large amounts by the adrenal cortex DHA-S is convened to DHA by DHA sulphatase, which may be the same as, or different from, the enzyme, oestrone sulphatase, which is responsible for the hydrolysis of EIS 28 During the last 10-15 years considerable research has also been earned out to develop potent aromatase inhibitors, some of which are currently undergoing clinical evaluation However, in three recent reports of postmenopausal women with breast cancer who received aromatase inhibitor therapy, plasma EIS concentrations remained between 400-1000 pg/ml" "34 In summation therefore in situ synthesis of oestrogen is thought to make an important contribution to the high levels of oestrogens in rumours and therefore specific inhibitors of oestrogen biosynthesis are of potential value tor the treatment of endocrine-dependent rumours Moreover, even though oestrogen formation in malignant breast and endometnal tissues via the sulphatase pathway makes a major contribution to the high concentration of oestrogens, there are still other enzymatic pathways that contnbute to in vivo synthesis of oestrogen Thus, there is an urgent need to develop new therapies for the treatment of these cancers The present invention therefore seeks to overcome one or more of the problems associated with the prior art methods of treating breast and endometnal cancers _) According to a first aspect of the present invention there is provided a sulphamate compound suitable for use as an inhibitor of both oestrone sulphatase activity and aromatase activity In a highly preferred embodiment, the compound of the present invention is a non- steroidal compound. According to a second aspect of the present invention there is provided a compound having the general formula II wherein F represents a phenolic ring structure (a first ring structure), J represents a third ring structure, I represents a phenolic ring structure (a second ring structure), G is an optional double bond, H is a link joining the second ring structure to the third ring structure, and Y represents a suitable second group, wherein any one of ring structures F, J and I has bound thereto a sulphamate group. According to a third aspect of the present invention there is provided a compound according to the present invention for use as a pharmaceutical. According to a fourth aspect of the present invention there is provided a compound according to the present invention for inhibiting oestrone sulphatase activity and aromatase activity. According to a fifth aspect of the present invention there is provided a pharmaceutical composition comprising a compound according to the present invention, and a pharmaceutically acceptable carrier, excipient or diluent. According to a sixth aspect of the present invention there is provided the use of a compound according to the present invention in the manufacture of a pharmaceutical for inhibiting oestrone sulphatase activity and aromatase activity According to a seventh aspect of the present invention there is provided a Drocess tor preparing a compound according to the present invention, the process comprising sulphating a flavone, isofiavone or a flavanone According to an eighth aspect of the present invention there is provided a process for preparing a compound according to the present invention, the process comprising sulphamoylating a flavone, isofiavone or a flavanone In one aspect, therefore, the present invention provides a compound, or a pharmaceutical composition comprising the same, that can affect, such as substantially inhibit, not only the oestrone sulphatase pathway - which pathway converts oestrone to and from oestradiol - but also the aromatase pathway - which pathway converts the androgen precursor androstenedione to oestrone This aspect of the present invention is advantageous because by the administration of one type of compound it is possible to block the synthesis of oestrone from both androstenedione and EIS In addition, the present invention is further advantageous in that it may also be possible to block the formation of androstenediol from DHA-S Hence, the present invention provides compounds that have considerable therapeutic advantages, particularly for treating breast and endometnal cancers The compounds of the present invention are different from those disclosed in the prior art because they can act as therapeutic agents that possess both aromatase and steroid sulphatase inhibitory properties In a preferred embodiment the compound of the present invention compnses a first ring structure and a sulphamoyl group, which first ring structure may be substituted and/or unsaturated. Preferably the first ring structure is a phenolic ring structure, which phenolic ring mav be substituted. Preferably, the compound of the present invention further comprises a second ring structure, which second ring structure may be substituted and/or unsaturated. Preferably the second ring structure is a phenolic ring structure, which phenolic ring may be substituted. Preferably, the compound of the present invention further comprises a third ring structure which is intermediate the first ring structure and the second ring structure, which third ring structure may be substituted and/or unsaturated. In a prefered embodiment, the compound of the present invention is a sulphamate of a flavone, an isofiavone or a flavanone. Alternatively, the compound of the present invention is a sulphamate of a benzoflavone - such as the benzoflavone of Figure 10 wherein R is H or OH (ref. 38). The present invention also covers substituted variants of the sulphamate of the benzoflavone of Figure 10. The present invention will now be described by reference to the Formulae presented in Figures 3-9. In this regard, its is generally preferred that the compound of the present invention has the general formula I wherein A represents the first ring structure, B represents the third ring structure, D represents the second ring structure, C is an optional double bond, E is a link joining the second ring structure to the third ring strucmre, X represents a suitable first group, and Y represents a suitable second group; wherein any one of ring structures A, B and D is a phenolic ring; and wherein any one of ring structures A, B and D has bound thereto a sulphamate group Each of the ring structures can independently comprise from 3 to 20 atoms in the ring, preferably from 4 to 8 atoms in the ring. Preferably, ring A and ring D comprise 6 atoms in the ring. A further cyclic group may be linked to ring A or D. This cyclic group may be linked to two spaced-apart atoms in ring A or ring D, such as the structure shown in figure 10. Preferably , the first ring strucmre and the second ring strucmre are substituted. Preferably, any one of ring structures A and D has bound thereto a sulphamate group. Preferably, each of the first ring and the second ring is a homogeneous ring strucmre - i.e. the ring is made up of the same atoms. Preferably, each of the first ring and the second ring comprises only carbon atoms in the ring. Preferably, X is C =O. Preferably, the compound of the present invention has the general formula II wherein F represents a phenolic ring structure (the first ring structure), J represents the third ring structure. I represents a phenolic ring strucmre (the second ring structure), G is an optional double bond, H is a link joining the second ring structure to the third ring structure, and Y represents a suitable second group; wherein any one of ring structures F, J and I has bound thereto a sulphamate group Preferably, the third ring structure is a heterogeneous ring structure - I e different atoms are in the ring. Preferably, Y is O Preferably any one of the ring structures F and I has bound thereto a sulphamate group Preferably, link E or link H is a bond Preferably, the compound of the present invention is a sulphamate ot any one of a flavone, an isofiavone or a flavanone Preferably, the compound of the present invention is any one of a compound of the general formula IV, a compound of the general formula V, or a compound of the general formula VI; wherem R t -Rι 2 are independently selected from H, OH, a halogen, an amme, an amide, a sulphonamme, a sulphonamide, any other sulphur containing group, a samrated or unsaturated C, t0 alkyl, an aryl group, a saturated or unsaturated C,. 10 ether, a saturated or unsamrated C,. l0 ester, a phosphorus containing group: and wherein at least one of R,- i 2 1S a sulphamate group. Preferably, the sulphamate group has the general formula OSO NR π R 14 wherein R 13 and R| 4 are independently selected from H, OH, a halogen, a samrated or unsamrated C|., o alkyl. an aryl group, a samrated or unsamrated C, .10 ether, a saturated or unsamrated C,., 0 ester. Each of R !3 and R may be other suitable groups. Preferably, the compound of the present invention is any one of a compound of the general formula IV, a compound of the general formula V, or a compound of the general formula VI; wherein R]-R I2 are independently selected from H, OH, OSO 2 NR 13 R| 4l 0-CH 3 ; wherein at least one of R . -R, 2 is OSO 2 NR l3 R, 4 , and wherein R 13 and R are defined as above. Preferably, at least one of R I3 and R is H. Preferably, each of R 13 and R 14 is H. Preferably, the compound of the present invention is a sulphamate of any one of the flavone of formula VII, the isofiavone of formula VIII, or the flavanone of formula IX. Preferably, the compound of the present invention is the sulphamate of any one of formula VII, formula VIII or formula IX. Preferably, the compound of the present invention is a sulphamate of any one of a flavone, an isofiavone or a flavanone; and wherein the sulphamoyl group is on the C4' atom of the flavone, isofiavone or flavanone. The C4' position has been shown in general Formula III according to the present invention. Preferably, the compound of die present invention is a flavonoid or flavanoid sulphamate. In summation the present invention provides compounds that avoid the need for polytherapy. In this regard, the compounds of the present invention can act as therapeutic agents that possess both aromatase and steroid sulphatase inhibitory properties. Preferably, if the sulphamate group of the compound of the present invention were to be replaced with a sulphate group so as to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity. The compound of the present invention may have one or more sulphamate groups. For example, the compound may be a mono-sulphamate or a bis-sulphamate. For example, in Figures 7, 8 and 9 R 3 and R, may be each a sulphamate. Figures 1 and 2 present schematic pathways; Figure 3-10 present chemical formulae; and Figure 11 presents a graph. The present invention will now be described only by way of example. COMPOUNDS SYNTHESISED The following sulphamate derivatives were synthesised from the following parent compounds: wherein 1 = 6-hydroxy flavone 2 — flavone-6-sulphamate 3 = 7-hydroxy flavone 4 = flavone-7-sulphamate 5 = 5 ,7-dιhydroxy flavone 6 = 5-hydrσxy-flavone-7-suiphamate 7 = 5,7-dιhydroxy-4'-hydroxy-flavone 8 = 5,7-dιhydroxy flavanone-4' -flavanone sulphamate 9 = 5,7-dιhydroxy-4'-methoxy-ιsoflavone 10 = 5-hydroxy-4 ' -methoxy-ιsoflavone-ιsoflavone-7-sulphamate The formulae are presented in Figures 7-9 SYNTHESIS The sulphamate derivatives were prepared essentially as described previously 29 In this regard, a solution of the appropriate flavone, isofiavone or flavanone in anhydrous DMF was treated with sodium hydride (60% dispersion; 1 equiv for 2 and 4; 2 equiv for 6, 8 and 10) at 0°C under an atmosphere of N 2 After evolution of hydrogen had ceased, sulfamoyl chloride (2 equiv except for 8, 5 equiv) was added and the reaction mixture was poured into brine after warming to room temperature overnight and diluting with ethyl acetate. The organic fraction was washed exhaustively with brine, dried (MgS0 4 ), filtered and evaporated The crude product obtained was purified by flash chromatography and recrystalhsation to give the corresponding sulfamate. Flavone 6-O-sulphamate (2) 6-Hydroxyflavone (1.0 g, 4.113 mmol) gave crude product (1 21 g) which was fractionated on silica (200 g) with ethyl acetate. Upon evaporation, the first fraction gave a creamy residue (760 mg, 58.2%) which was recrystaliised in warm acetone/hexane (3:2) to give 2 as creamy rod-shaped crystals (557 mg), m.p. 190- 191°C; R,s = 0.71 (ethyl acetate), 0.51 (ethyl acetate/hexane, 2.1), vmax (KBr) 3260, 3040, 1620, 1600, 1580, 1370, 1 180 cm 1 , <5 H (acetone-d 6 ) 6 917 (IH, s, Co¬ li), 7 355 (2H, br s, exchanged with D 2 0, -OSO : H : ), 7 64 (3H, , C-3'-H, C-4'-H and C-5' -H), 7 75 (IH, dd, J C 8 . H C 7 H = 9 Hz and J C 5 H C 7 H = 3 Hz, C-7-H). 7 87 ( IH, d, J C 7 H C 8 - H = 9 Hz, C-8-H), 8 02 (IH, d, J C , H C S H = 3 Hz, C-5-H) and 8 13 (2H. m, C-2'-H and C-6'-H) MS m/z (E.I , rel intensity) 317 0(11). 304 2(6), 238 0(96), 210 0(16), 187 1(14), 152 0(8) 136 0(100) Ace MS (E I ) m/z 317 0296, C,jH„NO 5 S requires 317 0358 Found C, 56 7, H, 3 44, N. 4 31 C .J H„NO J S requires C, 56 78, H, 3 49, N, 4 41 % Flavone 7-0- sulphamate (4) 7-Hydroxy flavone (700 mg, 2.938 mmol) gave crude product (770 mg) which was fractionated on silica (200 g) with ethyl acetate. Upon evaporation, the first fraction gave a light brown residue (132 mg) which was recrystallised in hot isopropyl alcohol to give 4 as white needle-shaped crystals (60 mg), m.p. 172-174°C (dec ), RrS = 0 78 (ethyl acetate), 0.56 (ethyl acetate/hexane, 4 1); vmax (KBr) 3260, 3100, 1630, 1600, 1400, 1580, 1200, 1150 cm ', <5 H (DMSO-d 6 /CDCl 3 , ca. 1.20) 6 824 (IH, s, C-3-H), 7 396 (IH, dd, J C S . H . C ^ H = 8 8 HZ and J C . J . H . C * H = 2-2 Hz, C-6-H), 7 47 (2H, br s, exchanged with D-O, -OSO 2 NH 2 ), 7 55 (3H, m, C-3'-H, C-4'-H and C-5'- Hi, 7 639 (IH. d, J C ^ H . C - S - H = 2.2 Hz, C-8-H), 7 92 (2H, m, C-2'-H and C-6 ' -H) and 8 220 (IH, d, J C ^ H . C - 5 - H = 8.8 Hz, C-5-H) Found C, 56 5. H, 3 36. N, 4 19 C IS H„NO,S requires C, 56.78; H, 3 49, N, 4 41 % 5 -Hydroxy flavone 7-O-Sulphamate (6) 5,7-Dihydroxyflavone (1,0 g. 3.933 mmol) gave crude product (1 13 g) which was fractionated on silica (200 g) with chloroform/acetone (8 1) Upon evaporation, the second fraction gave a yellow residue (324 mg. 24 7%) which was recrystallised in emyl acetate/hexane (1.1) to give 6 as yellow crystals (213 mg), m p 195-200°C (dec ), R f S = 0.21 , 0.25 and 0.44 for chloroform/acetone 12. 1, 8 1 and 4 1 respectively vmax (KBr) 3360, 3250, 2925-2850, 1650, 1610 1380 cm 1 , <5 H (acetone-d 6 75, 6 98, 7 17 (3H, three s, C-3-H, C-6-H, C-8-H), 7 63 (2H, br s exchanged with D 0 -OSO NH 2 ), 7 65 (3H, m, C-3'-H, C-4 -H and C-5 -H), 8 15 (2H, d, J = 7 7 Hz, C-2'-H and C-ό ' -H) and 13 0 (IH, br s exchanged with D 2 0, C-5-OH) MS m/z ( + ve ion FAB m m-NBA, rel intensity) 440 1( 10) 389 3( 10), 334 1 [100, (M + H) * ] , 288 1( 17), 255 0[25, (M + H-79) + ] , 169 1.30) MS m/z (-ve ion FAB in m-NBA, rel intensity) 499 0(30), 484 1(14, (M-2H + 153) ] , 475 1(20), 443 1(24), 332 1[100, (M-H) ] , 308 1(28), 274 1(20), 253 1 [50, (M-H-79) ], 195 1(24) Ace MS ( + ve ion FAB in m-NBA) m/z 334 0392. C 15 H 12 N0 5 S requires 334 0385 Found C, 54 0, H, 3 39, N, 4 21 C l5 H π N0 6 S requires C, 54 03, H, 3 33 , N, 4 20% 5 7 -Dihydrox flavanone 4 ' -O -sulphamate (8) 4' ,5,7-Tπhydroxyflavanone ( I 0 g, 3 675 mmol) gave crude product (965 mg) which was fractionated on silica (200 g) with ethyl acetate/hexane (4 1) to give a mixmre of the starting flavanone and product This mixmre was further fractionated on silica (200 g) with chloroform/acetone (4 1) and upon evaporation, the second fraction gave a pale yellow oil (345 mg, 34%) which solidified on standing Subsequent recrystallisation of this solid in ethyl acetate/hexane (1 1) gave 8 as white crystals (259 mg), m p 211-213°C, R, = 0 21 (chloroform/ acetone, 4 1), vmax (KBr) 3420, 3340, 3260, 3140, 1640, 1510, 1380, 1160 cm ' , δ H (acetone-d 6 ) 2 84 (IH, dd. J ΛB = 17 4 Hz and J„ „, = 3 1 Hz, C-3-H B ), 3 19 (IH, dd, J BA = 16 9 Hz and J » „ = 12 8 Hz, C-3-H Λ ), 5.62 (IH, dd, J„ „ = 3 1 Hz and J„ „ = 12 8 Hz, C-2-H), 5 98 (IH, d, J= 2 0 Hz, C-6-H or C-8-H). 6 01 (IH, d, J = 2 0 Hz, C-6-H or C-8-H), 7 20 (2H, br s, exchanged with D,O. -OSO 2 NH 2 ), 7 40 (2H, d, J = 8 7 Hz, C-2'-H and C-6'-H), 7 66 (2H, d, J = 8 7 Hz, C-3'-H and C-5'-H), 9 65 (IH. br s, C-7- OH) and 12 15 (IH, s, C-5-OH) MS m/z ( +ve ion FAB m-NBA, rel intensity) 352 0[100, (M +H)*] , 288 1(10), 272 1[14, (M-79) * ], 255 2(9), 169 0(13) MS m/z (-ve ion FAB in m-NBA, rel intensity) 701.2(12), 606 2(10). 517 1(42). 504 1[20, (M + 153) ], 473 2(10), 350 1[100. (M-H) ] , 271 1[45, (M-H-79) ] , 182 0(8) Ace MS ( + ve ion FAB in m-NBA) m/z 352 0496, C . jH w NO 7 S requires 352 0491 Found C 51 1 , H, 3 68, N, 3 98 C 15 H 13 N0 7 S requires C, 5 1 28. H. 3 73 , N 3 99% 5-Hydroxy-4'-methoxyιsoflavone 7 -O-sulphamate (10) 5 ,7-Dιhydroxy-4'-methoxyιsoflavone (800 mg, 2 817 mmol) gave crude product (650 mg) which was fractionated on silica (200 g) with chloroform/ acetone (8 1) Upon evaporation, the second fraction gave a yellow residue (266 mg, 26%) which was recrystallised in ethyl acetate/hexane (1 1) to give 10 as yellow crystals (21 1 mg), m p 184- 188°C, Rfi = 0 22 and 0 59 for chloroform/acetone 8 1 and 4 1 respectively, vmax (KBr) 3300-3020, 1660, 1610, 1400 cm 1 , δ H (acetone-d 6 ) 3 86 (3H, s, -OCHj), 6 75 (IH, d, J = 2 2 Hz, C-6-H or C-8-H), 7 04 (3H. m, C-6-H or C-8-H and C-3'-H and C-5'-H), 7 49 (2H, br s, exchanged with D 2 0, -OS0 2 NH 2 ), 7 58 (2H, d. J= 7 Hz, C-2'-H and C-6'-H), 8 A 1 (IH, s, C-2-H), 13 05 (IH, br s, exchanged with D 2 O, C-5-OH) MS m/z ( + ve ion FAB in m-NBA, rel intensity) 393 3(12), 364 0[100, (M + H) + ], 284 1[12, (M-79) + ], 169 1(24), 134 0(22) MS m/z (-ve ion FAB in m-NBA, rel intensity) 529 1(25), 515 1[12, (M-H + 153) ], 442.1(20), 362 1[100, (M-H) ], 308 1(34), 283 1[70, (M-H-79) ] , 170 1(26) Ace MS ( + ve ion FAB in m-NBA) m/z 364 0494, C 16 H 14 NO 7 S requires 364 0491 Found C, 52 8, H, 3 65, N, 3 81 C 16 H l3 NO 7 S requires C, 52 89, H, 3 61 , N, 3 85% 5-Hydroxy Isoflavone-4',7-c9, 0-Disulphamate (11) and 5,7-Dihydroxy Isoflavone- 4'-0-Sulphamate (12) 4 " .5.7-Trihydroxy isofiavone (0.5 g, 1.85 mmol) upon sulphamoylation gave a crude product (0.65 g) which was fractionated on silica (200 g) with chloroform/acetone (4: 1), and upon evaporation the third fraction gave a light yellow residue (0.329 g, 51 %) which was recrystallized in ethylacetate/hexane (1 :2) to give compound (11) as beige crystals (0.197 g); m.p = > 198°C (dec); R,s = 0.14 and 0.24 for chloroform/acetone 4: 1 and 2: 1 respectively; v™* (KBr) 3460 (-NH 2 ), 165 ° (C =O), 1400 (-SO 2 N-) cm 1 ; δ H (acetone-d 6 ) 6.78 (IH, d, J = 2.2 Hz, C-6-H or C-8-H, 7.03 (IK, d, J = 2.2 Hz, C-8-H or C-6-H), 7.4 (4H, br s, exchanged with D 2 O, C-4'-OSO 2 NH 2 and C-7-OSO 2 NH 2 ), 7.43 (2H, d, J = 8.4 Hz, C-3'-H and C-5'-H or C-2'-H and C-6'-H and C-6'-H), 7.72 (2H, d, J = 8.4 Hz, C-2'-H and C-6'-H or C-3 ' -H and C-5'-H), 8.51 (IH, s, C-2-H) and 12.93 (IH, s, C-5-OH). MS: m/z (+ve ion FAB in - NBA, rel. intensity) 428.9 [100, (M + H) * ], 350.0 [20, (M + H-SO 2 NH 2 ) + ], 272.1 [30, (M-H-SO 2 NH 2 ) + ]. MS: m/z (-ve ion FAB in m- NBA. rel. intensity) 426.9 [100, (M-H) ], 347.9 [95, (M-H-SO 2 NH 2 ) ] , 269.0 [30, (M-H-SO 2 NH 2 ) ]. Ace. MS: m/z (FAB) + 429.0083 C 15 H 13 N 2 O 9 S 2 requires 429.0063. Found C, 42.0; H, 2.91; N, 6.45; C 15 H 12 N 2 O 9 S 2 requires C, 42.06; H, 2.82; N, 6.54%. The second fraction was collected and upon evaporation gave light yellow residue (0.112 g, 17%) which was recrystallized in ethylacetate/hexane (1 :3) to give compound (12) as pale white crystals (0.068 g); m.p. = 189-192°C R,s = 0.23 and 0.33 for chloroform/acetone 4: 1 and 2: 1 respectively; \™ x (KBr) 3500-3300 (-NH 2 ), 3200 (H-bonded-OH), 1680 (C =O), 1610, 1400 (-SO 2 N-)cm '; δ„ (acetone-d 6 ) 6.32 (IH, d, J = 2.2 Hz, C-6-H or C-8-H), 6.46 (IH, d, J = 2.2 Hz, C-8-H or C-6-H), 7.32 (2H, br s, exchanged with D 2 O,-SO 2 NH 2 ), 7.42 (2H, t, J = 8.4 Hz. C-3'-H and C-5'-H or C-2'-H and C-6'-H, 7.69 (2H, d, J = 8.4 Hz, C-2'-H and C-6'-H or C-3'-E and C-5'-H), 8.31 (IH, s, C-2-H), 9.53 (IH, s, C-7-OH) and 12.9 (IH, s, C-5-OH). MS: m/z (+ve ion FAB in m- NBA, rel. intensity) 350.0 [100, (M+H) + ], 271.1 [15, (M+H-SO 2 NH 2 ) + ]. MS: m/z (-ve ion FAB in m- NBA, rel. intensity) 347.9 [100, (M-H) " ], 269.0 [20, (M-H-SO 2 NH 2 ) ] . Ace. MS: m/z (FAB) + 350.0347 C 15 H !2 NO 7 S requires 350.0335. Found C, 51.0; H, 3.16; N, 3.90; C, 5 H u NO 7 S requires C, 51.58; H, 3.17; N, 4.01 % . Isoflavone-4\7-C,0-Disulph_uιιate (13) 4' .7-Dihydroxy isofiavone (0.45 g, 1.77 mmol) upon sulphamoylation gave a crude product (0.769 g) which was fractionated on silica (200 g) with chloroform/acetone (4: 1), and upon evaporation the second fraction gave a pale white residue (0.553 g, 72%) which was recrystallized in acetone/hexane (1:2) to give the compound (13) as white crystals (0.327 g); m.p. > 195°C (dec); R,s = 0.21 and 0.40 for chloroform/acetone 4: 1 and 2: 1 respectively; v ma * (KBr) 3400 (-NH 2 ), 1640 (C=O), 1360 (-SO 2 N-) cm 1 . δ H (DMSO-d 6 ), 7.37 (2H, d, J = 8.8 Hz, C-3'-H and C-5'-H or C-2'-H and C-6'-H, 7.42 (IH, dd, H . C - S -H = 2.2 Hz, J C . 6 - H . c - J - H = 8 - 8 Hz > C-6-H), 7.7 (2H, d, J = 8.8 Hz, C-2'-H and C-6 * -H or C-3'-H and C-5'-H), 8.09 (2H, br s, exchanged with D 2 O, 8.24 (IH, d, J = 8.8 Hz, C-5-H, 8.36 (2H, br s, exchanged with D 2 O, -OSOjNty, 8.63 (IH, s, C-2-H). MS: m/z ( + ve ion FAB in m- NBA, rel. intensity) 412.9 [100, (M+H) + ], 334.0 [25, (M+H-SO 2 NH 2 ) + ], 255.1 [20, (M+H-SO 2 NH 2 ) + ]. MS: m/z (-ve ion FAB in m- NBA, rel. intensity) 410.9 [100, (M-H) ], 332.0 (70, (M-H-SO 2 NH 2 )"] , 253.0 [30, (M-H-SO 2 NH 2 ) ]. Ace. MS: m/z (FAB) + 413.0119 C 15 H I3 N 2 O g S 2 requires 413.0113. Found C, 44.0; H, 2.94; N, 6.62; C 15 H 12 N 2 O 8 S 2 requires C, 43.69; H, 2.93; N, 6.79%. ASSAY OF INHIBITION OF SULPHATASE AND AROMATASE ACTIVITIES Sulphatase inhibition was assessed using placental microsome (100,000 g) preparations or intact MCF-7 breast cancer cells as described previously 29,3 °. Placental microsomes were incubated with 3 H EIS, adjusted to 20 μM with unlabelled substrate, in the absence or presence of inhibitor. Placental microsomes were also used to assess the aromatase inhibitory properties of the flavanoid sulphamates using a tritiated water release assay 37 . Further placental microsomes (200 μl) were incubated with [ 10- _T] androstenedione, 60 nM and 1 mM NADPH in the absence or presence of inhibitor. INHIBITION OF SULPHATASE AND AROMATASE ACTIVITIES Inhibition of oestrone sulphatase and aromatase activities in placental microsomes by the flavanoid sulphamate derivatives is shown in the Table below. From the results, it can be seen that potent inhibition of sulphatase and aromatase activities was detected. For sulphatase inhibition this ranged from 21 % at 10 μM by 5-hydroxy flavone-7-sulphamate, to 98% by 5,7-dihydroxy flavanone-4' -sulphamate at 10 μM. Potent aromatase inhibition was also achieved ranging from 6.5 % by flavone-6-sulphamate at 10 μM to 86% by flavone-7-sulphamate at 10 μM. FURTHER IN VITRO TESTING The following Table presents in vitro data for three isoflavones that were tested. IN VITRO ACTIVITY Compound Isofiavone 5-hydroxy- 4' ,7-bissulphamate Isofiavone 5,7-dihydroxy- 4 '-sulphamate Isoflavone-4',7- bissulphamate IN VIVO TESTING Figure 11 presents in vivo inhibition of oestrone sulphatase activity in rat liver for two isoflavones according to die present invention. In this regard, BH22F1 = 5-hydroxy isoflavone-4',7-bissulphamate; BH22BF1 = 5,7-dihydroxy isoflavone-4' -sulphamate. Compounds were administered as a single 10 mg/Kg dose. Oestrone sulphatase activity was assayed in tissue samples obtained 24 h after drug administration. Other modifications of the present invention will be apparent to those skilled in the art. REFERENCES ( 1) Santner, S J , Feil, P D Santen, R J In situ oestrogen production via the oestrone sulphatase pathway in breast tumors relative importance vs the aromatase pathway J Clin Endocnnol Metab 1984, 59, 29-33 (2) Yamamoto T , Kitawaki, J , Urabe, M , Honjo, H , Tamura, T , Noguchi, T , Okada, H , Sasaki, H , Tada, A , Terashima, Y , Nakamura, J , Yoshihama, M Oestrogen productivity of endometπum and endometnal cancer tissue - influence of aromatase on proliferation of endometnal cancer cells J Steroid Biochem Mol (3) Santen, R J , Santner, S J , Davis, B , Veldhuis, J , Samojilik, E , Ruby, E Ammoglutheϋnmide inhibits extraglandular oestrogen production in post-menopausal women with breast carcinoma / Clin Endocnnol Metab 1978, 47, 1257-1265 .4) Reed, M J , Lai, L C , Owen, A M , Singh, A , Coldham, N G , Purohit, A , Ghilchik, M W , Shaikh, N A , James, V H T Effect of treatment with 4- hydroxy-androstenedione on the peripheral conversion ot androstenedione to oestrone and in vitro tumour aromatase activity in postmenopausal women with breast cancer Cancer Res 1990, 50, 193-196 (5) Ruder, H J , Lonaux, D L , Lipsett, M B Oestrone sulphate production rate and metabolism in man J Clin Invest 1972, 57 , 1020-1023 (6) James, V H T , McNeill, J M , Lai, L C , Newton. C J , Ghilchik, M W , Reed, M J Aromatase activity in normal breast and breast mmor tissues in vivo and in vitro studies Steroids 1987, 50, 269-279 (7) Howarth, N. M. ; Purohit, A. ; Reed, M. J. ; Potter, B. V. L. Oestrone sulphamates: potent inhibitors of oestrone sulphatase with therapeutic potential. J. Med. Chem. 1994, 37, 219-221. (8) Purohit, A. ; Williams, G. J. , Howarth, N. M. ; Potter, B. V. L. ; Rεed, M. J. Inactivation of steroid sulphatase by an active site -directed inhibitor, oestrone-3-O- sulphamate. Biochemistry 1995, 34, 11508-11514. (9) Purohit, A. ; Dauvois. S. ; Parker, M. G. ; Potter, B. V. L. ; Williams, G. J. ; Reed, M. J. The hydrolysis of oestrone sulphate and dehydroepiandrosterone sulphate by human steroid sulphatase expressed in transfected COS- 1 cells. J. Steroid Biochem. Mol. Biol. 1994, 50, 101- 104. (10) Dauvois, S. ; Labrie, F. Androstenedione and androst-5-ene-35\17|3-diol stimulate DMBA-induced rat mammary mmours - role of aromatase. Breast Cancer Res. Treat. 1989, 13, 61-69. (11) Purohit, A. ; Williams, G. J. ; Roberts, C. J. ; Potter, B. V. L.; Reed. M. J. In vivo inhibition of oestrone sulphatase and dehydroepiandrosterone sulphatase by oestrone-3- >-sulphamate. Int. J. Cancer 1995 , 63 , 106-111. (12) Woo, L. W. L.; Lightowler, M. ; Purohit, A. ; Reed, M. J. ; Potter, B. V. L. Heteroatom-substituted analogues of the active-site directed inhibitor oestra- 1.3,5(10)- trien-17-one -3 -sulphamate inhibit oestrone sulphatase by a different mechanism. /. Steroid Biochem. Mol. Biol. 1996 (in press). (13) Elger, W. ; Schwarz, S.; Hedden, A. ; Reddersen, G.; Schneider, B. Sulphamates of various oestrogens - prodrugs with increased systemic and reduced hepatic oestrogenicity at oral application. J. Steroid Biochem. Mol. Biol. 1995, 55, 395-403 ( 14) Li, P K; Rhodes, M. E , Jagannathan, S, Johnson, D A Memory enhancement mediated by the steroid sulphatase inhibitor oestrone 3-0-suIphamate. J. Endocnnol. 1995, 144, Abstr. P155. ( 15) Daynes, R. A. ; Araneo, B. A. ; Dowell, T. A. , Huang, K. , Dudley, D. Regulation of murine lymphokine production in vivo. 3 The lymphoid tissue micro- environment exerts regulatory influences over T-helper cell function. J. Exp. Med. 1990, 171, 979-996. (16) Rook, G. A. W. ; Hernandez-Pando, R. ; Lightman, S. Hormones, peripherally activated prohormones and regulation of the TH1/TH2 balance Immunol. Today 1994, 15, 301-303. (21) James & Reed (1980) Prog Cancer Res Ther 14: 471-487. (22) James et al (1987) Steroids 50: 269-279. (23) Reed et ai (1994) Drugs for the Future 19: 673-680. (24) Santner et al (1984) J Clin Endocr Metab 59: 29-33. (25) Yamamoto et al (1993) J Steroid Biochem Molec Biol 44: 463-468. (26) Poulin & Labrie (1986) Cancer Res 46: 4933-4937. (27) Dauvois &. Labrie (1989) Breast Cancer Res Treat 13: 61-69. (28) Purohit et al (1994) J Steroid Biochem Mol Biol 50: 101-104 (29) Howarth et al (1994) J Med Chem 37: 219-221 . (30) Purohit et al ( 1995) Biochemistry 34: 11508- 1 1514. (31) Purohit et al (1995) Int J Cancer 62: 106-1 11. (32) Svenstrup et al ( 1994) Eur J Cancer 30A: 1254-1258. (33) Johnston et al (1994) Cancer Res 54: 5875-5881. (34) Dowsett et al (1994) Eur J Cancer 30A: 1453-1458. (35) Kellis et ai (1986) Biochem Pharmacol 35: 2887-2891. (36) Campbell & Kurzer (1993) J Steroid Biochem Molec Biol 46- 381-388. (37) Newton et al (1986) J Steroid Biochem 24: 1033-1039. (38) Kellis et al (1986) Biochem Pharmacol 35: 2887

Claims: CLAIMS 1 A sulphamate compound suitable for use as an inhibitor of both oestrone sulphatase activity and aromatase activity 2 A compound according to claim 1 wherein the compound comprises a first ring strucmre and a sulphamoyl group, which first ring strucmre may be substituted and/or unsamrated 3 A compound according to claim 2 where the first ring strucmre is a phenolic ring strucmre, which phenolic ring may be substituted 4 A compound according to any one of claims 1 to 3 wherein the compound further comprises a second ring strucmre, which second ring strucmre may be substituted and/or unsamrated 5 A compound according to claim 4 wherein the second ring strucmre is a phenolic ring strucmre, which phenolic ring may be substituted 6 A compound according to any one of claims 1 to 5 wherein the compound further comprises a third ring strucmre which is intermediate the first ring strucmre and the second ring strucmre, which third πng strucmre may be substituted and/or unsamrated 7 A compound according to claim 6 wherein the compound has the general formula I. wherem A represents the first ring strucmre, B represents the third ring strucmre, D represents the second ring strucmre, C is an optional αouble bond. E is a link joining the second ring strucmre to me third ring strucmre, X represents a suitable first group, and Y represents a suitable second group, wherein any one of ring strucmres A, B and D is a phenolic πng, and wherein any one of πng strucmres A, B and D has bound thereto a sulphamate group 8 A compound according to claim 7 wherein the compound has the general formula II wherein F represents a phenolic πng strucmre (the first πng strucmre), J represents the third πng strucmre, I represents a phenolic πng strucmre (the second πng strucmre), G is an optional double bond, H is a link joimng the second πng strucmre to the third πng strucmre, and Y represents a suitable second group, wherein any one of ring strucmres F, J and I has bound thereto a sulphamate group 9 A compound having the general formula II wherein F represents a phenolic πng strucmre (a first ring strucmre), J represents a third πng strucmre, I represents a phenolic ring strucmre (a second ring strucmre), G is an optional double bond, H is a link joining the second ring strucmre to the third ring strucmre, and Y represents a suitable second group; wherein any one of ring strucmres F, J and I has bound thereto a sulphamate group 10 A compound according to any one of claims 7 to 9 wherein the first ring strucmre and the second πng strucmre are substituted 11. A compound according to any one of claims 7 to 10 wherem any one of ring strucmres A or F and D or I has bound thereto a sulphamate group 12 A compound according to any one of claims 7 to 11 wherein the third πng strucmre is a heterogeneous ring strucmre 13 A compound according to any one of claims 7 to 12 wherein X is C = 0 14 A compound according to any one of claims 7 to 13 wherem Y is O 15 A compound according to any one of the preceding claims wherein the compound of the present invention is a sulphamate of any one of a flavone, an isofiavone or a flavanone 16 A compound according to claim 15 wherein the compound is any one of a compound of the general formula IV, a compound of the general formula V or a compound of the general formula VI, where R,-R 12 are independently selected from H. OH, a halogen, an amine, an amide, a sulphonamme, a sulphonamide, any other sulphur containing group, a samrated or unsamrated C, 10 alkyl, an aryl group, a samrated or unsamrated C L 10 ether, a samrated or unsamrated C, 10 ester, a phosphorus containing group, and wherem at least one of R,-R 12 is a sulphamate 17 A compound according to any one of the preceding claims wherein the sulphamate group has the general formula OS0 2 NR 13 R 14 wherein R 13 and R 14 are independently selected from H, OH, a halogen, a samrated or unsamrated C M0 alkyl, an aryl group, a samrated or unsamrated C,. 10 ether, a samrated or unsamrated C, I0 ester 18 A compound according to any one of the preceding claims wherein the compound is any one of a compound of the general formula IV, a compound of the general formula V or a compound of the general formula VI, wherein R,-Rι. 2 are independently selected from H, OH, OSO 2 NR l3 R 14> 0-CH 3 , wherein at least one of R,-R l2 is OSO 2 NR 13 R 14 , and wherein R l3 and R 14 are defined in claim 17 19 A compound according to claim 16 or claim 17 wherein R 13 and R 14 are H 20 A compound according to any one of the preceding claims wherein the compound is a sulphamate of any one of the flavone of formula VII, the isofiavone of formula VIII or the flavanone ot formula IX 21. A compound according to any one of the preceding claims wherein the compound is the sulphamate of any one of formula VII, formula VIII or formula IX. 22. A compound according to any one of the preceding claims wherein the compound is a sulphamate of any one of a flavone, an isofiavone or a flavanone; and wherein the suiphamoyl group is on the C4' atom of the flavone, isofiavone or flavanone. 23. A compound according to any one of the preceding claims wherein the compound is a flavanoid sulphamate. 24. A compound according to any one of the preceding claims for use as a pharmaceutical. 25. A compound according to any one of claims 1 to 23 for inhibiting oestrone sulphatase activity and aromatase activity. 26. A pharmaceutical composition comprising a compound according to any one of claims 1 to 23; and a pharmaceutically acceptable carrier, excipient or diluent. 27. Use of a compound according to any one of claims 1 to 23 in the manufacture of a pharmaceutical for inhibiting oestrone sulphatase activity and aromatase activity. 28. A process for preparing a compound according to any one of claims 1 to 23, the process comprising sulphating a flavone, isoflavone or a flavanone. 29. A process for preparing a compound according to any one of claims 1 to 23, the process comprising sulphamoylating a flavone, isoflavone or a flavanone. 30. A compound substantially as described herein. 31. A process of preparing a compound substantially as described herein.

Inventors: POTTER BARRY VICTOR LLOYD, REED MICHAEL JOHN, POTTER, BARRY, VICTOR, LLOYD, REED, MICHAEL, JOHN
Applicants: IMPERIAL COLLEGE, POTTER BARRY VICTOR LLOYD, REED MICHAEL JOHN, UNIV BATH, IMPERIAL COLLEGE OF SCIENCE TECHNOLOGY AND MEDICIN, POTTER, BARRY, VICTOR, LLOYD, REED, MICHAEL, JOHN, UNIVERSITY OF BATH
Application year: 1997
Main classification: C07D311
IPC codes: A61K31, A61P5, A61P43, C07D311