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.

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