Patent Grant
7098199
This invention relates to novel compounds for use as steroid sulphatase inhibitors, and pharmaceutical compositions containing them.
Steroid precursors, or pro-hormones, having a sulphamate group in the 3-position of the steroid nucleus, referred to hereinafter simply as steroid sulphates, are known to play an important part as intermediates in steroid metabolism in the human body. Oestrone sulphate and dehydroepiandrosterone (DHA) sulphate, for example, are known to play an important role as intermediates in the production, in the body, of oestrogens such as oestrone and oestradiol. Oestrone sulphate, in particular, is known, for example, to represent one of the major circulating oestrogen precursors particularly in post-menopausal women and oestrone sulphatase activity in breast tumorous is 100–1000 fold greater than that of other enzymes involved, in oestrogen formation (James et al., Steroids, 50, 269–279 (1987)).
Not only that, but oestrogens such as oestrone and oestradiol, particularly the over-production thereof, are strongly implicated in malignant conditions, such as breast cancer, see Breast Cancer, Treatment and Prognosis: Ed. R. A. Stoll, pp. 156–172, Blackwell Scientific Publications (1986), and the control of oestrogen production is the specific target of many anti-cancer therapies, both chemotherapy and surgical, e.g. oöphorectomy and adrenalectomy. So far as endocrine therapy is concerned, efforts have so far tended to concentrate on aromatase inhibitors, i.e. compounds which inhibit aromatase activity, which activity is involved, as the accompanying oestrogen metabolic flow diagram (
In recently published International Application WO91/13083 a proposal has been made to target a different point in the oestrogen metabolic pathway, or rather two different points, that is to say the conversion of DHA sulphate and oestrone sulphate to DHA and oestrone, respectively, by steroid sulphatase activity, and using 3-monoallylthiophosphonate steroid esters as a steroid sulphatase inhibitor, more especially oestrone-3-monomethyl-thiophosphate.
A first object of the present invention is to provide new compounds capable of inhibiting steroid sulphatase activity in vitro and in vivo.
A second object of the present invention is to provide new compounds having improved activity as steroid sulphatase inhibitors both in vitro and in vivo.
A third object of the invention is to provide pharmaceutical compositions effective in the treatment of oestrogen dependent tumours.
A fourth object of the invention is to provide pharmaceutical compositions effective in the treatment of breast cancer.
A fifth object of the invention is to provide a method for the treatment of oestrogen dependent tumours in mammals, especially humans.
A sixth object of the invention is to provide a method for the treatment of breast cancer in mammals and especially in women.
The invention is based on the discovery of novel compounds having steroid sulphatase inhibitory activity, in some cases, with extremely high activity levels.
In one aspect, the present invention provides a method of inhibiting steroid sulphatase activity in a subject in need of same.
In another aspect, the present invention provides compounds and compositions useful in that method of inhibiting steroid sulphatase activity.
The method of the present invention comprises administering to said subject a steroid sulphatase inhibiting amount of a ring system compound; which ring system compound comprises a ring to which is attached a sulphamate group of the formula
wherein each of R1 and R2 is independently selected from H, alkyl alkenyl, cycloalkyl and aryl, or together represent alkylene optionally containing one or more hetero atoms or groups in the alkylene chain; and wherein said compound is an inhibitor of an enzyme having steroid sulphatase activity (E.C.3.1.6.2); and if the sulphamate group of said compound is replaced with a sulphate group to form a sulphate compound and incubated with a steroid sulphatase enzyme (E.C.3.1.6.2) at a pH 7.4 and 37° C. it would provide a Km value of less than 50 μM.
The compounds that are useful in the method of the present invention are sulphamic acid ester ring system compounds, the sulphate of which is a substrate for enzymes having steroid sulphatase (EC 3.1.6.2) activity, the N-alkyl and N-aryl derivatives of those sulphamic acid esters, and their pharmaceutically acceptable salts.
In one aspect of the present invention, compounds for use in the method of the present invention are the sulphamic acid esters of polycyclic alcohols, being polycyclic alcohols the sulphate of which is a substrate for enzymes having steroid sulphatase (EC 3.1.6.2) activity, the N-alkyl and N-aryl derivatives of those sulphamic acid esters, and their pharmaceutically acceptable salts.
For one aspect of the present invention, broadly speaking, the novel compounds of this invention are compounds of the Formula (I)
where:
As used herein the reference to polycyclic alcohols, the sulphate of which is a substrate for enzymes having steroid sulphatase activity refers to polycyclic alcohols, the sulphate of which, viz: the derivatives of the Formula:
when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37° C. provides a Km value of less than 50 μmoles.
The activity of the present compounds as steroid sulphatase inhibitors is illustrated in the accompanying drawings, in which:
a, 16b, 16c, and
Thus, in one aspect, the present invention provides a method of inhibiting steroid sulphatase activity in a subject in need of same, the method comprising administering to said subject a steroid sulphatase inhibiting amount of a ring system compound; which ring system compound comprises a ring to which is attached a sulphamate group of the formula
wherein each of R1 and R2 is independently selected from H, alkyl, alkenyl, cycloalkyl and aryl, or together represent alkylene optionally containing one or more hetero atoms or groups in the arylene chain; and wherein said compound is an inhibitor of an enzyme having steroid sulphatase activity (E.C.3.1.6.2); and if the sulphamate group of said compound is replaced with a sulphate group to form a sulphate compound and incubated with a steroid sulphatase enzyme (E.C.3.1.6.2) at a pH 7.4 and 37° C. it would provide a Km value of less than 50 μM.
In one aspect the present invention provides, as novel compounds, the sulphamic acid esters of polycyclic alcohols, being polycyclic alcohols the sulphate of which is a substrate for enzymes having steroid sulphatase activity in accordance with the definition already provided, and their N-alkyl, N-cycloalkyl, N-alkenyl and N-aryl derivatives. These compounds are of Formula I hereinbefore given.
Preferably the polycyclic group will contain, inclusive of all substituents, a maximum of about 40 carbon atoms, more usually no more than about 30.
Preferred polycycles are those containing a steroidal ring structure, that is to say a cyclopentanophenanthrene skeleton. Preferably, the sulphamyl or substituted sulphamyl group is attached to that skeleton in the 3-position, that is to say are compounds of the Formula II.
where R1 and R2 are as above defined and the ring system ABCD represents a substituted or unsubstituted, saturated or unsaturated steroid nucleus, preferably oestrone or dehydroepiandrosterone. Other suitable steroid ring systems are:
In general terms the steroid ring system ABCD may contain a variety of non-interfering substituents. In particular, the ring system ABCD may contain one or more hydroxy, alkyl especially lower (C1–C6) allyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C1–C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
Suitable non-steroidal ring systems (i.e. ring system compounds) include:
Examples of some non-steroidal ring systems are presented in the Examples section.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R1 and/or R2 is allyl, the preferred values are those where R1 and R2 are each independently selected from lower alkyl groups containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R1 and R2 are both methyl. When R1 and/or R2 is aryl, typical values are phenyl and tolyl (—PhCH3; o—, m— or p—). Where R1 and R2 represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R1 and R2 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. —O— or —NH— to provide a 5-, 6- or 7) -membered heterocycle, e.g. morpholino pyrrolidino or piperidino.
Within the values ally, cycloalkyl, alkenyl and aryl we include substituted groups containing as substituents therein one or more groups which do not interfere with the sulphatase inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, allyl and aryl.
Most preferred are compounds of the Formula III and IV:
where R1 and R2 are H or C1–C5 alkyl, i.e. oestrone-3-sulphamate and dehydroepiandrosterone-3-sulphamate and their N—(C1–C5) allyl derivatives, especially the dimethyl derivatives, R1═R2═CH3.
The sulpnamic acid esters of this invention are prepared by reacting the polycyclic alcohol, e.g. oestrone or dehydroepiandrosterone, with a sulfamoyl chloride R1R2NSO2Cl, i.e. the Reaction Scheme I
Conditions for carrying out Reaction Scheme I are as follows:
Sodium hydride and a sulphamoyl chloride are added to a stirred solution of oestrone in anhydrous dimethyl formamide at 0° C. Subsequently, the reaction is allowed to warm to room temperature whereupon stirring is continued for a further 24 hours. The reaction mixture is poured onto a cold saturated solution of sodium bicarbonate and the resulting aqueous phase is extracted with dichloromethane. The combined organic extracts are dried over anhydrous MgSO4. Filtration followed by solvent evaporation in vacuo and co-evaporation with toluene affords a crude residue which is further purified by flash chromatography.
Where necessary, functional groups in the polycyclic alcohol (sterol) may be protected in known manner and the protecting group or groups removed at the end of the reaction.
For pharmaceutical administration, the steroid sulphatase inhibitors of this invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, exipients, diluents etc. and usually for parenteral administration. Approximate effective dose rates are in the range 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70 kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day. They may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days. For oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of compound per unit dose. Alternatively and preferably the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
For particular applications, it is envisaged that the steroid sulphatase inhibitors of this invention may be used in combination therapies, either with another sulphatase inhibitor, or, for example, in combination with an aromatase inhibitor, such as for example, 4-hydroxyandrostenedione (4-OHA).
The invention is illustrated by the following preparative Examples and test data:
Preparation of Oestrone-3-sulphamate
Sodium hydride (60% dispersion; 2 eq) and sulphamoyl chloride (2 eq) were added to a stirred solution of oestrone (1 eq) in anhydrous dimethyl formamide at 0° C. Subsequently, the reaction was allowed to warm to room temperature whereupon stirring was continued for a further 24 hours.
The reaction mixture was poured onto a cold saturated solution of sodium bicarbonate and the resulting aqueous phase was extracted with dichloromethane. The combined organic extracts were dried over anhydrous MgSO4. Filtration followed solvent evaporation in vacuo and co-evaporation with toluene afforded a crude residue which is further purified by flash chromatography.
Analysis showed the following data:
δ1H (270 MHz; CD3OD): 0.91 (s, 3H, C18-Me), 1.40–2.55 (series of m, 13H), 2.90–2.92 (m, 2H), 7.04 (br d, 2H, J=10.44 Hz), 7.33 (br d, 1H, J=8.42 Hz).
δ13C (67.8 MHz; CD3OD): 14.53 (q, C18-Me), 22.80 (t), 27.24 (t), 27.73 (t), 30.68 (t), 33.05 (t), 37.01 (t), 39.76 (d), 45.73 (s, C18), 51.86 (d), 120.76 (d), 123.54 (d), 127.89 (d), 139.83 (s), 150.27 (s), 223.87 (s, C═O).
m/z (%): 349 (9) (m+), 270 (100), 213 (26), 185 (43), 172 (31), 159 (21), 146 (36), 91 (33), 69 (37), 57 (73), 43 (56), 29 (24).
Preparation of Oestrone-3-N-methylsulphamate
The procedure of Example 1 was repeated save that sulphamoyl chloride was replaced by the same quantity of N-methylsulphamoyl chloride.
Analysis showed the following data:
δ1H (270 MHz; CDCl3): 0.91 (s, 3H, C18-Me), 1.28–1.68 (m, 6H), 1.93–2.60 (series of m, 7H), 2.90–2.95 (m, 2H), 2.94 (d, 3H, J=5.13 Hz, MeN—), 4.68–4.71 (br m, exchangeable, 1H,—NH), 7.02–7.07 (m, 2H), 7.26–7.32 (m, 1H).
m/z (%): 364[M+H]+
Preparation of oestrone-3-N.N-dimethylsulphamate
The procedure of Example 1 was repeated save that sulphamoyl chloride was replaced by the same quantity of N,N-dimethylsulphamoyl chloride.
Analysis showed the following data:
δ1H (270 MHz; CDCl3): 0.92 (s, 3H, C18-Me), 1.39–1.75 (m, 5H), 1.95–2.60 (series of m, 6H), 2.82 (s, 3H, MeN—), 2.96–3.00 (m, 4H), 2.98 (s, 3H, MeN—), 7.04 (br d, 2H, J=7.69 Hz), 7.29 (br d, 1H, J=7.88 Hz).
m/z (%): 377 [M]+
Inhibition of Steroid Sulphatase Activity in MCF-7 cells by oestrone-3-Sulphamate
Steroid sulphatase is defined as: Steryl Sulphatase EC3.1.6.2.
Steroid sulphatase activity was measured in vitro using intact MCF-7 human breast cancer cells. This hormone dependent cell line is widely used to study the control of human breast cancer cell growth. It possesses significant steroid sulphatase activity (MacIndoe et al. Endocrinology, 123, 1281–1287 (1988); Purohit & Reed, Int. J. Cancer, 50, 901–905 (1992)) and is available in the U.S.A. from the American Type Culture Collection (ATCC) and in the U.K. (e.g. from The Imperial Cancer Research Fund). Cells were maintained in Minimal Essential Medium (MEM) (Flow Laboratories, Irvine, Scotland) containing 20 mM HEPES, 5% foetal bovine serum, 2 mM glutamine, nonessential amino acids and 0.075% sodium bicarbonate. Up to 30 replicate 25 cm2 tissue culture flasks were seeded with approximately 1×105 cells/flask using the above medium. Cells were grown to 80% confluency and medium was changed every third day.
Intact monolayers of MCF-7 cells in triplicate 25 cm2 tissue culture flasks were washed with Earle's Balanced Salt Solution (EBSS from ICN Flow, High Wycombe, U.K.) and incubated for 3–4 hours at 37° C. with 5 pmol (7×105 dpm) [6,7-3H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass., U.S.A.) in serum-free MEM (2.5 ml) together with oestrone-3-sulphamate (11 concentrations: 0; 1 fM; 0.01 pM; 0.1pM; 1pM; 0.01 nM; 0.1 nM; 1 nM; 0.01 μM; 0.1 μM; 1 μM. After incubation each flask was cooled and the medium (1 ml) was pipetted into separate tubes containing [14C]oestrone (7×103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, U.K.). The mixture was shaken thoroughly for 30 seconds with toluene (5 ml). Experiments showed that >90% [14C]oestrone and <0.1% [3H]oestrone-3-sulphate was removed from the aqueous phase by this treatment. A portion (2 ml) of the organic phase was removed, evaporated and the 3H and 14C content of the residue determined by scintillation spectrometry. The mass of oestrone-3-sulphate hydrolysed was calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C]oestrone added) and the specific activity of the substrate. Each batch of experiments included incubations of microsomes prepared from a sulphatase-positive human placenta (positive control) and flasks without cells (to assess apparent non-enzymatic hydrolysis of the substrate). The number of cell nuclei per flask was determined using a Coulter Counter after treating the cell monolayers with Zaponin. One flask in each batch was used to assess cell membrane status and viability using the Trypan Blue exclusion method (Phillips, H. J. (1973) In: Tissue culture and applications,[eds: Kruse, D. F. & Patterson, M. K.]; pp. 406–408; Academic Press, New York).
Data for oestrone-3-sulphamate are shown in Table I and
Inhibition of Steroid Sulphatase Activity in MCF-7 cells bv oestrone-3-N.N-dimethylsulphamate
An identical experimental protocol to that described in Example 4 was used to generate results for oestrone-3-N,N-diimethylsulphamate except that incubations contained oestrone-3-N,N-dimethylsulphaiate (5 concentrations: 0; 0.001 μM; 0.01 μM; 0.1 μM; 1 μM) in place of oestrone-3-sulphamate.
Results for oestrone-3-N,N imethylsulphamate are shown in Table II and
Inhibition of Steroid Sulphatase Activity in MCF-7 cells by pre-treatment with oestrone-3-N.N-dimethylsulphamate and oestrone-3-N.N-dimethylsulphamate
A similar experimental protocol to that described in Example 4 was used to determine the effect of pre-treating MCF-7 cells with oestrone-3-sulphainate and oestrone-3-N,N-dimethylsulphamate respectively.
Intact monolayers were initially incubated for 2 hours at 37° C. with 0.1 μM oestrone-3-sulphamate, oestrone-3-N,Nimethylsulphamate or medium alone (control). The medium bathing the cells was then removed by aspiration and cells were washed 3 times successively with 5 ml of medium on each occasion. The resultant ‘washed’ cells were then re-suspended and incubated for 3–4 hours at 37° C. in medium containing 5 pmol (7×105 dpm) [6,7-3H]oestrone-3-sulphate. All other aspects were identical to those described in Examples 3 and 4.
Results for oestrone-3-sulphamate and oestrone-3-N,N-dimethylsulphamate are shown in Table III and are expressed in a similar manner to Table I.
Inhibition of Steroid Sulphatase Activity in Placental Microsomes by Oestrone-3-sulphamate
Sulphatase-positive human placenta from normal term pregnancies (Obstetric Ward, St. Mary's Hospital, London) were thoroughly minced with scissors and washed once with cold phosphate buffer (pH 7.4, 50 mM) then re-suspended in cold phosphate buffer (5 ml/g tissue). Homogenisation was accomplished with an Ultra-Turrax homogeniser, using three 10 second bursts separated by 2 minute cooling periods in ice. Nuclei and cell debris were removed by centrifuging (4° C.) at 2000 g for 30 minutes and portions (2 ml) of the supernatant were stored at −20° C. The protein concentration of the supernatants was determined by the method of Bradford (Anal. Biochem., 72, 248–254 (1976)).
Incubations (1 ml) were carried out using a protein concentration of 100 μg/ml, substrate concentration of 20 μM [6,7-3H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass., U.S.A.) and an incubation time of 20 minutes at 37° C. Eight concentrations of oestrone-3-sulphamate were employed: 0 (i.e. control); 0.05 μM; 0.1 μM; 0.2 μM; 0.4 μM; 0.6 μM; 0.8 μM; 1.0 μM. After incubation each sample was cooled and the medium (1 ml) was pipetted into separate tubes containing [14C]oestrone (7×103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, U.K.). The mixture was shaken thoroughly for 30 seconds with toluene (5 ml). Experiments showed that >90% [14C]oestrone and <0.1% [3H]oestrone-3-sulphate was removed from the aqueous phase by this treatment. A portion (2 ml) of the organic phase was removed, evaporated and the 3H and 14C content of the residue determined by scintillation spectrometry. The mass of oestrone-3-sulphate hydrolysed was calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C]oestrone added) and the specific activity of the substrate.
Results for oestrone-3-sulphamate are shown in Table IV and
Inhibition of Steroid Sulphatase Activity in Liver Microsome Preparations from Rats Treated With Subcutaneous Oestrone-3-sulphamate
Four groups of 3 female Wistar rats (weight range 80–110g) were given 100 ml subcutaneous injections (once daily for 7 days, vehicle: propylene glycol) of either:
On the eighth day all rats were sacrificed and livers were removed by dissection. Liver microsomal preparations were prepared by an identical protocol to that described in Example 6 except that the tissue source was rat liver and that duplicate experiments to determine steroid sulphatase activity were performed using [6,7-3H]oestrone-3-sulphate and [7-3H]dehydroepiandrosterone-3-sulphate as separate substrates.
Results for steroid sulphatase activity are shown in Table V and are expressed as total product formed during the incubation period in the form of mean ±1 S.D. Results for incubations of tissue obtained from groups of rats treated with oestrone-3-sulphamate are also expressed as a percentage reduction (inhibition) in steroid sulphatase activity compared to their respective controls.
Starting with the appropriate parent compound, the ring system sulphamates according to the tile present invention were prepared essentially as follows. In this regard, a solution of the appropriate parent compound in anhydrous DMF was treated with sodium hydride [60% dispersion; 1.2 equiv.] at 0° C. under an atmosphere of N2. After evolution of hydrogen had ceased, sulfamoyl chloride in toluene [excess, ca. 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 (MgSO4), filtered and evaporated. The crude product obtained was purified by flash chromatography and recrystallisation to give the corresponding sulfamate. All the compounds were fully characterized by spectroscopic and combustion analysis.
Example compounds are as follows:
4-n-Heptyloxyphenol (1.0 g, 4.80 mmol) gave a crude product (1.41 g) which was fractionated on silica (200 g) with chloroform/acetone (8:1), and upon evaporation the second fraction gave a pale white residue (757 mg) which was recrystallized from acetone/hexane (1:5) to give 9a as white crystals (0.557 g).
Analytical results were as follows:
m.p.>42° C.(dec.) Rfs=0.56, 0.69 and 0.8 for chloroform/acetone 8:1, 4:1 and 2:1 respectively νmax (KBr) 3440, 3320 (—NH2), 1370 (—SO2N—) cm−1 δH (acetone-d6) (270 MHz) 0.89 (3H, t, C—4-C H3), 1.34 (8H, m, —(CH2)4CH3), 1.79 (2H, pentet, —CH2(CH2)4CH3), 4.0 (2H, t, J=6.4 Hz, —OCH2—), 6.95 (2H, dd, JC-3-H and C-5-H=2.2 Hz and JC-3-H and C-2-H =6.97 Hz, C-3-H and C-5-H), 7.01 (2H, br s, exchanged with D2O, —OSO2NH2), 7.23 (2H, dd, JC-2-H and C-6-H =2.4 Hz and JC-2-H and C-6-H=6.97 Hz, C-2-H and C-6-H) MS: m/z (+ve ion FAB in m-NBA, rel. intensity) 287.1 [100, (M)+], 208.2 [30, (M—SO2NH2)+] MS: m/z (−ve ion FAB in m-NBA, rel. intensity) 286.0 [100, (M−H)],96.0 (10) and 77.9 (20). Acc. MS: m/z (FAB)+288.1246 C13H22NO4S requires 288.1269. Found C, 54.2; H, 7.35; N, 4.7; C13H21NO4S requires C, 54.33; H, 7.37; N, 4.87%.
Biological Data:-
E-Capsaicin ((E)—N-(4-Hydroxy-3-methoxyphenyl)-methyl-γ-methyl-6-nonenamide) (100 mg, 0.3274 mmol) gave a beige crude product (130 mg) which was fractionated on silica (100 g) with chloroform/acetone (2:1), and upon evaporation the second fraction gave a pale white residue (85 mg) which was recrystallized from acetone/hexane (1:2) to give 9b as pale white crystals (63 mg, 50%).
Analytical results were as follows:
m.p=114–116° C.
RfS=0.4 and 0.15 for chloroform/acetone 2:1 and 4:1 respectively
νmax (KBr) 3490,3300 (—NH2), 1650 (CO), 1380 (—SO2N—) cm−1
δH (CDCl3) (270 MHz) 0.94 (6H, d, J=6.6 Hz, 2×-CH3), 1.4 (2H, pentet, —COCH2CH2- or —CH2CH2CH═CH—), 1.62 (2H, pentet, —CH2CH2CH═CH—or —COCH2CH2—), 2.0 (2H, q, —CH2CH═CH—), 2.2 (3H, t, —CH2CONH— and —CH(CH3)2), 3.87(3H, s, C-3—OCH3), 4.39 (2H, d, J=5.86 Hz, ArCH2NHCO), 5.14 (2H, br s, exchanged with D2O, —OSO2NH2), 5.34 (2H, m, —CH═CH—), 5.87 (1H, t, —NHCO—), 6.84 (1H, dd, J=C-6-H and C-2-H 1.92 Hz and J=C-6-H and C-5-H 8.15 Hz C-6-H), 6.86 (1H, d, J=C-1-H and C-6-H 1.83 Hz, C-1-H) and 7.26 (1H, d, J=C-5-H and C-6-H 8.08 Hz, C-5-H).
MS: m/z (+ve ion FAB in m-NBA, rel. intensity) 385.2 [100, (M+H)+], 304.2 (20), 287.1 (10) MS: m/z (−ve ion FAB in m-NBA, rel. intensity) 383.1 [100, (M−H)]302.2 (10), 95.9 (10) and 77.9 (35)
Found C, 56.2; H, 7.38; N, 7.29; C18H28N2O5S requires C, 56.23; H, 7.34; N, 7.29%.
Biological Data:
A stirred solution of 2-nitrophenol (1.391 g, 10.0 mmol) in anhydrous DMF (20 mL) was treated with sodium hydride (60% dispersion, 400 mg, 10.0 mmol) at 0° C. under an atmosphere of N2. After evolution of hydrogen had ceased, sulfamoyl chloride (2 eq.) was added. The reaction mixture was stirred at room temperature overnight and then poured into water (150 mL). The resulting mixture was extracted with ethyl acetate (150 mL) and the organic portion separated was washed with brine (5×100 mL), dried (MgSO4), filtered and evaporated in vacuo at 40° C. Purification by flash chromatography (ethyl acetate/hexane, 1:1) gave the crude 2-nitrophenol-O-sulfamate which was further purified by recrystallization from hot chloroform to afford the title compound as white crystals (333 mg, 745.8 μmol). The residue recovered, from the evaporation of the mother liquor, was recrystallized from chloroform/hexane to give further crops of the title compound (a total of 252 mg, 564.4 μmol, 26% overall).
Analytical results were as follows:
mp 102–103° C. 1H NMR (270 MHz, CDCl3) δ5.29 (2H, br s, exchanged with D2O, OSO2NH2), 7.48 (1H, m, C4-H or C5-H), 7.66 (1H, dd, J=1.5 and 8.3 Hz, C3-H or C6-H), 7.71 (1H, m, C4-H or C5-H) and 8.05 (1H, dd, J=1.5 and 8.3 Hz, C3-H or C6-H)
MS (EI, 70 eV) m/z (rel. intensity) 218(2, M+), 139[100, (M—SO2NH)+]; MS (CI, isobutane) m/z (rel. intensity) 219[27, (M+H)+], 202[10, (M+H—NH3)+], 140[100, (M+H—SO2NH)+], 122[15, (M—OSO2NH2)+]. Anal. (C6H6N2O5S) C, H, N.
Biological Data:
Starting with the appropriate phenolic parent compound (if there are two phenol groups, it may be necessary to protect one of them using standard protection techniques for at least a part of the reaction), the ring system sulphamates according to the present invention are prepared essentially as follows. Likewise, a solution of the appropriate parent compound in anhydrous DMF is treated with sodium hydride [60% dispersion; 1.2 equiv.] at 0° C. under an atmosphere of N2. After evolution of hydrogen has ceased, sulfamoyl chloride in toluene [excess, ca. 5 equiv.] is added and the reaction mixture is poured into brine after warming to room temperature overnight and diluting with ethyl acetate. The organic fraction is washed exhaustively with brine, dried (MgSO4), filtered and evaporated. The crude product obtained is purified by flash chromatography and recrystallisation to give the corresponding sulfamate. All the compounds are fully characterized by spectroscopic and combustion analysis.
The following compounds of the present invention are made and are found to be steroid sulphatase inhibitors in accordance with the present invention.
Starting with the appropriate phenolic parent compound (if there are two phenol groups, A it may be necessary to protect one of them using standard protection techniques for at least a part of the reaction), the ring system sulphamates according to the present invention are prepared essentially as follows. Here, a solution of the appropriate parent compound in anhydrous DMF is treated with sodium hydride [60% dispersion; 1.2 equiv.]0° C. under an atmosphere of N2. After evolution of hydrogen has ceased, N-methyl sulfamoyl chloride in toluene [excess, ca. 5 equiv.] is added and the reaction mixture is poured into brine after warming to room temperature overnight and diluting with ethyl acetate. The organic fraction is washed exhaustively with brine, dried (MgSO4), filtered and evaporated. The crude product obtained is purified by flash chromatography and recrystallisation to give the corresponding sulfamate. All the compounds are fully characterized by spectroscopic and combustion analysis.
The following compounds of the present invention are made and are found to be steroid sulphatase inhibitors in accordance with the present invention (In the following formulae, the methyl and H groups on the sulphamate group can be interchanged.)
Starting with the appropriate phenolic parent compound (if there are two phenol groups, it may be necessary to protect one of them using standard protection techniques for at least a part of the reaction), the ring system sulphamates according to the present invention are prepared essentially as follows. Here, a solution of the appropriate parent compound in anhydrous DMF is treated with sodium hydride [60% dispersion; 1.2 equiv.] at 0° C. under an atmosphere of N2. After evolution of hydrogen has ceased, N, N-dimethyl sulfamoyl chloride in toluene [excess, ca. 5 equiv.] is added and the reaction mixture is poured into brine after warming to room temperature overnight and diluting with ethyl acetate. The organic fraction is washed exhaustively with brine, dried (MgSO4), filtered and evaporated. The crude product obtained is purified by flash chromatography and recrystallisation to give the corresponding sulfamate. All the compounds are fully characterized by spectroscopic and combustion analysis.
The following compounds of the present invention are made and are found to be steroid sulphatase inhibitors in accordance with the present invention.
The compounds of the present invention may be prepared by a process that comprises a Packman synthesis step. Packman synthesis is known in the art.
The general procedure for the sulphamoylation of coumarins was as follows. A solution of an appropriate coumarin in anhydrous DMF (ca. 40 ml per g of coumarin) was treated with sodium hydride [60% dispersion; 1 equiv] at 0° C. under an atmosphere of N2. After evolution of hydrogen had ceased, sulphamoyl chloride in toluene [ca. 0.68 M, 1.5 equiv] was added and the reaction mixture was poured into water after warming to room temperature overnight and then the crude product was then quenched. The organic fraction in ethyl acetate (150 ml) was washed exhaustively with brine, dried (MgSO4), filtered and evaporated. The crude product obtained was purified by flash chromatography followed by recrystallisation to give the corresponding sulphamate. All new compounds were fully characterised by spectroscopic and combustion analysis. The synthesis of 4 methylcoumarin-7-O-sulphamate (14) is shown in
Following this general procedure, compounds 13–16 (as shown in FIG. 7)—i.e. coumarin-7-O-sulphamate (13), 4-methylcoumarin-7-O-sulphamate (14), 3,4,8-trimethylcoumarin-7-O-sulphamate (15) and 4-(trifluoromethylcoumarin)-7-O-sulphamate (16)—were prepared. More details on the synthesis of these compounds now follow.
The synthesis of compound 12 (as shown in
Following the above-mentioned general procedure, 7-Hydroxycoumarin (500 mg, 3.082 mmol) gave a crude product (605 mg) which was fractionated on silica (200 g) by gradient elution with chloroform/acetone (8:1, 500 ml; 4:1, 1000 ml and then 2:1, 500 ml). Upon evaporation, the second fraction gave a creamy yellow residue (389 mg, 52.3%) which was recrystallised in ethyl acetate/hexane (1:1) to give (13) as dull white crystals (239 mg).
Analytical data were as follows:
M.p. 170.0–171.5° C.; Rfs=0.48 (ether), 0.67 (ethyl acetate), 0.19 (chloroform/acetone, 4:1); νmax (KBr) 3360, 3210, 3060, 1720, 1615, 1370, 1125 cm−1; δH (DMSO-d6/CDCl3, ca. 1:25) 6.415 (1H, d, JC-4-H.C-3-H=9.7 Hz, C-3-H), 7.285 (1H. dd, JC-8-H.C-6-H=2.3 Hz and JC-5-H.C-6-H=8.5 Hz, C6-H), 7.379 (1H, d, JC-6-H.C-8-H=2.2 Hz, C-8-H), 7.508 (2H, br s, D2O exchanged. —NH2) 7.543 (1H. d, JC-6-H.C-5-H=8.4 Hz, C-5-H) and 7.760 (1H, d, JC-3-H.C-4-H=9.7 Hz, C-4-H). MS: m/z (E.I., rel. intensity) 241.0(10), 162.0(97), 134.0(100), 105.0(23). Acc. MS: m/z 241.0068, C9H7NO5S requires 241.0045. Found: C. 44.8; H. 2.89; N. 5.82. C9H7NO5S requires C, 44.81; H. 2.92; N. 5.81%.
Following the above-mentioned general procedure, 7-Hydroxy-4-methylcoumarin (500 mg, 2.753 mmol) gave a crude product (633 mg) which was fractionated on silica (200 g) by gradient elution with chloroform/acetone (8:1, 500 ml; 4:1, 1000 ml, 2:1, 500 ml and then 1:1, 500 ml). Upon evaporation, the second fraction gave a creamy yellow residue (425 mg, 60.5%) which was recrystallised in acetone/chloroform (3:5) to give (14) as colorless rhombic crystals (281 mg).
Analytical data were as follows:
M.p. 165–167° C.; RfS=0.48 (ether), 0.29 (ether/hexane 8:1), 0.26 (chloroform/acetone, 4:1); νmax (KBr) 3320, 3180, 3080, 1700, 1620, 1560, 1380, 1125 cm−1; δH(acetone-d6) 2.507 (3H, s, —CH3), 6.339 (1H. s, C-3-H) 7.299 (2H, m. C-6-H and C-8-H), 7.390 (2H, br s, D2O exchanged, —NH2) and 7.850 (1H, d, JC-6-H.C-5-H=9 Hz, C-5-H). MS: m/z (+ve ion FAB in m-NBA, rel. intensity) 542.2(15), 511.1 [45, (2M+H)+], 461.2(20), 409.1[60, (M+H+NBA)+], 393.3[60, (M+H+NBA-16)+],329.2[10, (M +H+NBA-80)+],256.1[100, (M+H)+]. MS: m/z (−ve ion FAB in m-NBA, rel. intensity) 421.0(20), 407.1[15, (M−H+NBA)−], 335.1(14), 254[100. (M−H)−]175.1[32, (M−H-79−], 121.0(17). Found: C, 47.2; H, 3.56; N, 5.51. C10H9NO5S requires C. 47.06; H, 3.55; N. 5.49%.
Following the above-mentioned general procedure, 7-Hydroxy-3,4,8-trimethylcoumarin (1.0 g, 4.896 mmol) gave a crude product (1.33 g) which upon recrystallisation in hot ethyl acetate yielded 238 mg of starting coumarin. The mother liquor was evaporated and the white residue obtained (1.13 g) was fractionated on silica (200 g) with ether. The second fraction was collected, evaporated and the residue obtained (519 mg, 37.4%) was recrystallised in acetone/hexane (1:2) to give (15) as pale yellow crystals (312 mg).
Analytical data were as follows:
M.p. 197–202° C.; Rfs=0.50 (ether), 0.69 (ethyl acetate); νmax (KBr) 3310, 3040, 1680, 1600 cm−1; δH (acetone-d6) 2.176, 2.383 and 2.458 (9H, three s, 3×CH3) 7.374 (1H, d, JC-5-H.C-6-H=8.8 Hz, C-6-H), 7.390 (2H, br s, D2O exchanged, —NH2) and 7.682 (1H, d, JC-6-H.C-5-H=8.8 Hz, C-5-H). MS: m/z (E.I., rel. intensity) 283.1(10), 204.1(45), 176.1(40), 161.1(22), 69.1(56), 57.1(40), 43.1(100). Acc.
MS: m/z 283.0497, C12H13NO5S requires 283.0514. Found: C, 50.86; H. 4.63; N, 4.97. C12H13NO5S requires C, 50.88; H. 4.63; N. 4.94%.
Following the above-mentioned general procedure. 7-Hydroxy4-(trifluoromethyl)-coumarin (0.90 g, 3.911 mmol) gave a crude product (1.20 g) which was fractionated on silica (200 g) with ether/chloroform (1:4). The residue (392 mg) from the third fraction was further purified by fractionating on silica (100 g) with ether. The first fraction then collected gave a residue (295 mg, 24.4%) which upon recrystallised in ethyl acetate/hexane (1:3) gave (16) as white needle-shaped crystals (160 mg).
Analytical data were as follows:
M.p. 165–168° C.; RfS=0.67 (ether), 0.24 (ether/chloroform, 1:4); νmax (KBr) 3360, 3240, 3100, 1720, 1620, 1380. 1160 cm−1; δH (acetone-d6) 6.995 (1H, s. C-3-H), 7.461 (1H, dd, JC-8-H.C-6-H=2.8 Hz and JC-5-H.C-6-H=8.1 Hz, C-6-H), 7.478 (1H, s, C-8-H), 7.53 (2H, br S. D2O exchanged, —NH2) and 7.89 (1H, m, C5-H). 1H-NMR spectrum of (16) in DMSO-d6/CDCl3 (ca. 1:15) showed partial decomposition to the starting coumarin. MS: m/z (E.I. rel. intensity) 309.0(2.6), 230.0(77), 202.0(100), 183.5(5), 173.0(10), 69.0(33). Acc. MS: m/z 308.9874, C10H6F3NO5S requires 308.9919. Found.; C, 38.8; H. 1.85; N. 4.53. C10H6F3NO5S requires C, 38.84; H. 1.96; N. 4.53%.
To a solution of 7-hydroxy4-methylcoumarin (1.0 g, 5.676 mmol) in dried pyridine (20 ml) under an atmosphere of N2 [
Analytical data were as follows:
M.p. 172–175° C. (dec.); Rfs=0.51 (methanol/ethyl acetate, 1:3), 0.67 (methanol/ether. 1:3); νmax (KBr) 3500 (br), 3080, 1680, 1610, 1560, 1300. 1260, 1050 cm−1; δH (DMSO-d6) 2.407 (3H, s, —CH3), 6.269 (1H, s, C-3-H),7.20 (2H, m, C-6-H and C-8-H), and 7.695 (1H, d, JC-6-H.C-5-H=8.8 Hz, C-5-H). MS: m/z (+ve ion FAB in m-NBA, rel. intensity) 176(100, NBA+Na+). MS: m/z (−ve ion FAB in m-NBA, rel. intensity) 175.1 (14, M-Na+—SO3), 255.0 (100, M-Na+), 408.0 (8. M-Na++NBA), 431.0 (15, M+153), 444.0(20), 533.0(15). 230.0(77). 202.0(100), 183.5(5), 173.0(10), 69.0(33). Acc. MS: m/z (−ve ion FAB in glycerol, rel. intensity) 254.9982(25), C10H7O6S requires 254.9963. Found: C, 40.3; H, 2.92. C10H2O6NaS—H2O requires C, 40.55; H. 3.06%. HPLC [Spherisorb ODS5, 25×4.6 mm; Mobile phase: MeOH/H2O (70:30). Flow rate: 1 ml/min; λmax: 316 nm]: tR=1.5 min, c.f. 7-hydroxy4-methylcoumarin, 3.6 min.
Other data were as follows
Compound 12 is stable in bases such as sodium hydroxide in methanol but not in acidic conditions In addition, incomplete basification of the reaction mixture with sodium hydroxide in methanol (<3 equivalents) leads to decomposition of (12). Two equivalents of sodium hydroxide are required for consuming excess sulphur trioxide-pyridine complex to yield the neutral sodium sulphate. Insufficient amount of sodium hydroxide will therefore lead to the formation of sodium hydrogen sulphate which is acidic. Compound 12 appears labile to high temperature as one experiment has shown complete decomposition to 7-hydroxy-4-methylcoumarin after heating (12) as solid at 90° C. for 4 h.
In vitro tests
The above-mentioned coumarin sulphamates were tested for their ability to inhibit E1-STS activity using intact MCF-7 breast cancer cells or placental microsomes (100,000 g fraction) essentially as previously described.
To examine whether compound (12) could act as a substrate for E1-STS, 100 μg of the compound was incubated for 1 hour with placental microsomes in the absence or presence of EMATE (10 μM). The unconjugated coumarin formed at the end of the incubation was extracted with diethyl ether. After evaporation of solvent, the residue was examined by TLC using ethyl acetate/methanol (80:20) as eluent, in which the coumarin sulphate (12) and 7-hydroxy4-methylcoumarin had Rf values of 0.79 and 0.95 respectively. Only unconjugated 7-hydroxy4-methylcoumarin was detected after incubation of compound (12) with placental microsomes. The inclusion of EMATE in the reaction mixture reduced the hydrolysis of compound (12) by E1-STS, indicating that the coumarin sulphate is indeed a substrate for the sulphatase.
The dose-dependent inhibition of oestrone sulphatase: in intact MCF-7 breast cancer cells by coumarin-7-O-sulphamate (13), 4-methylcoumarin-7-O-sulphamate (14), 3,4,8-trimethyl-coumarin-7-O-sulphamate (15) and 4-(trifuoromethyl)coumarin-7-O-sulphamate (16) can be seen from
The free parent coumarins of all coumarin sulphamates prepared showed little or no E1-STS inhibitory activity when tested up to 10 μM. However. in contrast, all four coumarin sulphamates (compounds 13–16) inhibited oestrone sulphatase inhibitory activity in a dose-dependent manner (
The time- and concentration-dependent inactivation of oestrone sulphatase by 4-methyl-coumarin-7-O-sulphamate (14) can be seen from
As with EMATE, compound 14 inhibited E1-STS activity in a time- and concentration-dependent manner in a biphasic fashion (
Additional experiments revealed that compound 14 inhibited placental microsomal DHA-STS activity by 93.6% at the same concentration.
In Vivo Tests
In order to examine if compound 14 possessed oestrogenic activity and also to test its ability to inhibit E1-STS in vivo, it was administered to rats (1 mg/kg subcutaneously. in propylene glycol for 5 days) 14 days after ovariectomy had been performed.
Administration of compound 14 did not result in any significant increase in the uterine weight in these rats (data not shown), showing that compound 14 showed reduced oestrogenic agonist properties. The E1-STS activity in the uteri obtained from these animals was inhibited by 89.4% compared with the activity in untreated animals.
Preliminary data also demonstrate potent oral activity in rats for compound 14, similar to that observed for EMATE.
In addition to these in vivo results, another series of rats (each weighing approximately 200 g) received 4-methyl coumarin-7-O-sulphamate (compound 14) orally in propylene glycol either as a single dose (SD) or daily for seven days (Multiple Dose, MD).
Inhibition of sulphatase activity was assessed in white blood cells (wbcs) that were collected after a SD or MD. Sulphatase activity was assayed using labelled oestrone sulphate as the substrate and measuring the release of oestrone.
The results are shown in
Similar results were found with liver cells.
Compound 14 therefore demonstrates potent oral activity.
Other modifications of the present invention will be apparent to those skilled in the art. 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 be a mono-sulphamate or a bis-sulphamate. For example. in
The present invention will now be described only by way of example.
The following sulphamate derivatives were synthesised from the following parent compounds:
The formulae are presented in
SYNTHESIS
The sulphamate derivatives were prepared essentially as described previously. In this regard. a solution of the appropriate flavone, isoflavone 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 N2. 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 (MgSO4), filtered and evaporated. The crude product obtained was purified by flash chromatography and recrystallisation to give the corresponding sulfamate.
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 recrystallised in warm acetone/hexane (3:2) to give 2 as creamy rod-shaped crystals (557 mg), m.p. 190–191° C.; Rfs=0.71 (ethyl acetate). 0.51 (ethyl acetatelhexane, 2:1), νmax (KBr) 3260, 3040. 1620. 1600. 1580, 1370. 1180 cm−1; δH (acetone-d6) 6.917 (1H. s. C-3-H), 7.355 (2H, br s. exchanged with D2O, —OSO2NH2), 7.64 (3H, m, C-3′-H, C-4′-H and C-5′-H). 7.75 (1H, dd, JC-7-H.C-8-H=9 Hz and JC-5-H.C-7-H=3 Hz. C-7-H). 7.87 (1H, d, JC-7-H.C-8-H=9 Hz. C-8-H), 8.02 (1H, d, JC-7-H.C-5-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). Acc. MS (E.I.): m/z 317.0296, C5H11NO5S requires 317.0358. Found C. 56.7; H. 3.44; N. 4.31. C15H11NO5S requires C, 56.78; H. 3.49; N. 4.41%.
7-Hydroxyflavone (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.); Rfs=0.78 (ethyl acetate). 0.56 (ethyl acetate/hexane, 4.1); νmax (KBr) 3260. 3100, 1630, 1600, 1400, 1580. 1200, 1150 cm−1; δH (DMSO-d6/CDCl3, ca. 1:20) 6.824 (1H, s. C-3-H), 7.396 (1H, dd, JC-5-H.C-6-H=8.8 Hz and JC-8-H.C-6-H=2.2 Hz, C6-H), 7.47 (2H, br s. exchanged with D2O, —OSO2NH2). 7.55 (3H, m, C-3′-H, C-4′-H and C-5′-H), 7.639 (1H, d, JC-6-H.C-8-H=2.2 Hz, C-8-H). 7.92 (2H, m, C-2′-H and C-6′-H) and 8.220 (1H, d, JC-6-H.C-5-H=8.8 Hz, C-5-H). Found: C, 56.5: H, 3.36: N. 4.19. C15H11NO5S requires C, 56.78; H, 3.49; N, 4.41%.
7-hydroxyflavone (1.0 g. 3.933 mmol) gave crude product (1.13 mg) which was fractionated on silica (200 g) with ethyl acetate. Upon evaporation, the first fraction second fraction gave a yellow residue (324 mg. 24.7%) which was recrystallised in ethyl acetate/hexane (1:1)to give 6 as yellow crystals (213 mg) m.p. 195–200° C. (dec.); Rs=0.21, 0.25 and 0.44 for chloroform/acetone 12:1, 8:1 and 4:1 respectively: νmax (KBr) 3360. 3250. 2925–2850. 1650, 1610. 1380 cm−1, δH(acetone-d6) 6.75. 6.98. 7.17 (3H. three s. C-3-H, C-6-HHC-8-H), 7.63 (2H. br s. exchanged with D2O. —OSO2NH2). 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-6′-H) and 13.0 (1H. br s. exchanged with D2O, C-5-OH) MS: m/Z (+ve ion FAB in 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). Acc. MS (+ve ion FAB in m-NBA): m/z 334.0392. C15H12NO6S requires 334.0385. Found: C, 54.0; H. 3.39; N. 4.21. C15H11NO6S requires C, 54.03; H., 3.33: N. 4.20%.
4′,5,7-Trihydroxyflavanone (1.0 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 mixture of the starting flavanone and product. This mixture 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 acetatelhexane (1:1) gave 8 as white crystals (259 mg), m.p. 211–213° C.; Rf=0.21 (chloroform/acetone. 4:1); νmax (KBr) 3420, 3340, 3260, 3140, 1640, 1510, 1380, 1160 cm−1; δH (acetone-d6) 2.84 (1H, dd. JAB=17.4 Hz and J2x, eq=3.1 Hz. C-3-HB), 3.19 (1H, dd, JBA=16.9 Hz and J2x, 2x =12.8 Hz, C-3-HA) 5.62 (1H. dd, J2x, eq=3.1 Hz and J2x, 2x=12.8 Hz, C-2-H), 5.98 (1H. d, J=2.0 Hz. C-6-H or C-8-H), 6.01 (1H, d. J=2.0 Hz, C-6-H or C-8-H). 7.20 (2H, br s, exchanged with D2O, —OSO2NH2), 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 (1H. br s. C-7-OH) and 12.15 (1H, s. C-5-OH). MS: m/z (+ve ion FAB in 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). Acc. MS (+ve ion FAB in m-NBA): mlz 352.0496, C15H14NO7S requires 352.0491. Found: C. 51.1; H, 3.68; N. 3.98. C15H13NO7S requires C. 51.28. H. 3.73; N. 3.99%.
5.7-Dihydroxy4′-methoxyisoflavone (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 (211 mg), m.p. 184–188° C.; Rfs=0.22 and 0.59 for chloroform/acetone 8:1 and 4:1 respectively; νmax (KBr) 3300–3020, 1660, 1610, 1400 cm−; δH (acetone-d6) 3.86 (3H. s. —OCH3), 6.75 (1H. 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 D2O, —OSO2NH2), 7.58 (2H, d, J=7 Hz, C-2′-H and C-6′-H), 8.41 (1H, s, C-2-H), 13.05 (1H, br s, exchanged with D2O, C-5-OH). MS: m/z (+ve ion FAB in m-NBA, ret. 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). Acc. MS (+ve ion FAB in m-NBA): m/z 364.0494, C16H14NO7S requires 364.0491.
Found: C, 52.8; H, 3.65; N. 3.81. C16H13NO7S requires C, 52.89; H. 3.61; N, 3.85%.
4′.5.7-Trihydroxy isoflavone (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 beize crystals (0.197 g); m.p=>198° C. (dec); Rfs=0.14 and 0.24 for chloroform/acetone 4:1 and 2:1 respectively; νmax (KBr) 3460 (—NH2), 1650 (C═O), 1400 (—SO2N—) cm−1; δH (acetone-d6) 6.78 (1H, d, J=2.2 Hz, C6-H or C-8-H7.03 (1H, d, J=2.2 Hz, C-8-H or C-6-H), 7.4 (4H, br s, exchanged with D2O, C4′-OSO2NH2 and C-7-OSO2NH2), 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 (1H, s, C-2-H) and 12.93 (1H, s. C-5-OH) MS: m/z (+ve ion FAB in m-NBA, rel. intensity) 428.9 [100, (M+H)+, ]350.0 [20, (M+H—SO2NH2)−, ]272.1 [30, (M−H—SO2NH2)−]. MS: m/z (−ve ion FAB in m-NBA. rel. intensity) 426.9 [100, (M−H)−, ]347.9 [95, (M—H—SO2N2H−], 269.0 [30. (M—H—SO2NH2)−]. Acc. MS: m/z (FAB)+429.0083 C15H13N2O9S2 requires 429.0063. Found C, 42.0; H, 2.91; N. 6.45; C15H12N2O9S2 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. Rfs=0.23 and 0.33 for chloroform/acetone 4:1 and 2:1 respectively; νmax (KBr) 3500–3300 (—NH2). 3200 (H—bonded—OH), 1680 (C═O), 1610, 1400 (—SO2N—)cm−1; δH (acetone-d6) 6.32 (1H, d, J=2.2 Hz, C6-H or C-8-H), 6.46 (1H, d, J=2.2 Hz, C-8-H or C-6-H), 7.32 (2H, br s, exchanged with D2O, —SO2NH2), 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 C6′-H or C-3′-H and C-5′-H), 8.31 (1H, s, C-2-H), 9.53 (1H, s, C-7-OH and 12.9 (1H, 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—SO2NH2)+]. MS: m/z (−ve ion FAB in m-NBA. rel. intensity) 347.9 [100, (M−H)−], 269.0 [20, (M−H—SO2NH2)−]. Acc. MS: m/z (FAB) +350.0347 C15H12NO7S requires 350.0335. Found C, 51.0; H. 3.16: N, 3.90; C15H11NO7S requires C, 51.58; H. 3.17; N. 4.01%.
4′,7-Dihydroxy isoflavone (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.); Rfs=0.21 and 0.40 for chloroform/acetone 4:1 and 2:1 respectively; νmax (KBr) 3400 (—NH2), 1640 (C═O), 1360 (—SO2N—) cm−1. δH (DMSO-d6). 7.37 (2H, d, J=8.8 Hz. C-3′-H and C-5′-H or C-2′-H and C6-H, 7.42 (1H, dd, JC-6-H.C-8-H =2.2 Hz. JC-6-H.C-5-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 D2O, —OSO2NH2), 8.24 (1H, d, J=8.8 Hz, C-5-H, 8.36 (2H, br s, exchanged with D2O, —OSO2NH, 8.63 (1H, 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—SO2NH2)+], 255.1 [20, (M+H—SO2NH2)+]. MS: m/z (−ve ion FAB in m-NBA, ret. intensity) 410.9 [100, (M−H)−]332.0 (70, (M−H—SO2NH2)−], 253.0 [30, (M−H—SO2NH2)−]. Acc. MS: m/z (FAB)+413.0119 C15H13N2O8S2 requires 413.0113. Found C. 44.0; H, 2.94; N. 6.62; C15H12N2O8S2 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. Placental microsomes were incubated with 3H E1S, 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. Further placental microsomes (200 μl) were incubated with [1β-3H] 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 VIVO TESTING
Other modifications of the present invention will be apparent to those skilled in the are.
Preparative Methods
The preparation of various compounds in accordance with the present invention is illustrated in
In Vitro Inhibition
The ability of compounds to inhibit oestrone sulphatase activity was assessed using either intact MCF-7 breast cancer cells or placental microsomes as previously described
In this regard, the teachings of that earlier reference are as follows:
Inhibition of Steroid Sulphatase Activity in MCF-7 cells by oestrone-3-sulphamate
Steroid sulphatase is defined as: Steryl Sulphatase EC3.1.6.2.
Steroid sulphatase activity was measured in vitro using intact MCF-7 human breast cancer cells. This hormone dependent cell line is widely used to study the control of human breast cancer cell growth. It possesses significant steroid sulphatase activity (MacIndoe et al. Endocrinology, 123, 1281–1287 (1988); Purohit & Reed, Int. J. Cancer, 50, 901–905 (1992)) and is available in the U.S.A. from the American Type Culture Collection (ATCC) and in the U.K. (e.g. from The Imperial Cancer Research Fund). Cells were maintained in Minimal Essential Medium (MEM) (Flow Laboratories, Irvine, Scotland) containing 20 mM HEPES, 5% foetal bovine serum, 2 mM glutamine, non-essential ammo acids and 0.075% sodium bicarbonate. Up to 30 replicate 25 cm2 tissue culture flasks were seeded with approximately 1×105 cells/flask using the above medium. Cells were grown to 80% confluency and medium was changed every third day.
Intact monolayers of MCF-7 cells in triplicate 25 cm2 tissue culture flasks were washed with Earle's Balanced Salt Solution (EBSS from ICN Flow, High Wycombe, U.K.) and incubated for 3–4 hours at 37° C. with 5 pmol (7×105 dpm) [6,7-3H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear. Boston, Mass. U.S.A.) in serum-free MEM (2.5 ml) together with oestrone-3-sulphamate (11 concentrations: 0; 1 fM; 0.011 pM; 0.1 pM; 1 pM; 0.01 nM; 0.1 nM; 1 nM; 0.01 mM; 0.1 mM; 1 nM). After incubation each flask was cooled and the medium (1 ml) was pipetted into separate tubes containing [14C]oestrone (7×103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, U.K.). The mixture was shaken thoroughly for 30 seconds with toluene (5 ml). Experiments showed that >90% [14C]oestrone and <0.1% [3H]oestrone-3-sulphate was removed from the aqueous phase by this treatment. A portion (2 ml) of the organic phase was removed, evaporated and the 3H and 14C content of the residue determined by scintillation spectrometry. The mass of oestrone-3-sulphate hydrolysed was calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C]oestrone added) and the specific activity of the substrate. Each batch of experiments included incubations of microsomes prepared from a sulphatase-positive human placenta (positive control) and flasks without cells (to assess apparent non-enzymatic hydrolysis of the substrate). The number of cell nuclei per flask was determined using a Coulter Counter after treating the cell monolayers with Zaponin. One flask in each batch was used to assess cell membrane status and viability using the Trypan Blue exclusion method (Phillips, H. J. (1973) In: Tissue culture and applications, [eds: Kruse, D.F. & Patterson, M. K.]; pp. 406–408; Academic Press, New York).
Results for steroid sulphatase activity are expressed as the mean±1 S.D. of the total product (oestrone+oestradiol) formed during the incubation period (20 hours) calculated for 106 cells and, for values showing statistical significance, as a percentage reduction (inhibition) over incubations containing no oestrone-3-sulphamate. Unpaired Student's t-test was used to test the statistical significance of results.
Inhibition of Steroid Sulphatase Activity in Placental Microsomes bv Oestrone-3-sulphamate
Sulphatase-positive human placenta from normal term pregnancies (Obstetric Ward, St. Mary's Hospital, London) were thoroughly minced with scissors and washed once with cold phosphate buffer (pH 7.4, 50 MM) then re-suspended in cold phosphate buffer (5 ml/g tissue). Homogenisation was accomplished with an Ultra-Turrax homogeniser, using three 10 second bursts separated by 2 minute cooling periods in ice. Nuclei and cell debris were removed by centrifuging (4° C.) at 2000 g for 30 minutes and portions (2 ml) of the supernatant were stored at −20° C. The protein concentration of the supernatants was determined by the method of Bradford (Anal. Biochem., 72, 248–254 (1976)).
Incubations (1 ml) were carried out using a protein concentration of 100 mg/ml, substrate concentration of 20 mM [6,7-3H]oestrone-3-sulphate (specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass., U.S.A.) and an incubation time of 20 minutes at 37° C. If necessary eight concentrations of compounds are employed: 0 (i.e. control); 0.05 mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1.0 mM. After incubation each sample was cooled and the medium (1 ml) was pipetted into separate tubes containing [14C]oestrone (7×103 dpm) (specific activity 97 Ci/mmol from Amersham International Radiochemical Centre, Amersham, U.K.). The mixture was shaken thoroughly for 30 seconds with toluene (5 ml). Experiments showed that >90% [14C]oestrone and <0.1% [3H]oestrone-3-sulphate was removed from the aqueous phase by this treatment. A portion (2 ml) of the organic phase was removed, evaporated and the 3H and 14C content of the residue determined by scintillation spectrometry. The mass of oestrone-3-sulphate hydrolysed was calculated from the 3H counts obtained (corrected for the volumes of the medium and organic phase used, and for recovery of [14C]oestrone added) and the specific activity of the substrate.
For the present invention, the percentage inhibition for the series of EMATE analogues tested in either MCF-7 cells or placental microsomes is shown in Table 1
In Vivo Studies
Using 17-deoxy oestrone-3-O-sulphamate (NOMATE,
(i) Inhibition of oestrone sulphatase activity in vivo
NOMATE (0.1 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol). At the end of the study samples of liver tissue were obtained and oestrone sulphatase activity assayed using 3H oestrone sulphate as the substrate as previously described.
As shown in
(ii) Lack of in vivo oestrogenicity
NOMATE (0.1 mg/Kg/day for five days) was administered orally to rats with another group of animals receiving vehicle only (propylene glycol). At the end of the study uteri were obtained and weighed with the results being expressed as uterine weight/whole body weight×100.
As shown in
Other modifications of the present invention will be apparent to those skilled in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 9118478.8 | Aug 1991 | GB | national |
| 9603325.3 | Feb 1996 | GB | national |
| 9604709.7 | Mar 1996 | GB | national |
| 9605725.2 | Mar 1996 | GB | national |
| 9625334.9 | Dec 1996 | GB | national |
This application is a division of U.S. Application Ser. No. 09/579,163, filed May 25, 2000, now U.S. Pat. No. 6,642,397, which in turn was a division of U.S. application Ser. No. 09/238,345, filed Jan. 27, 1999, now U.S. Pat. No. 6,187,766, which in turn was a division of U.S. application Ser. No. 09/111,927, filed Jul. 8, 1998, incorporated herein by reference and now U.S. Pat. No. 6,011,024, which in turn was a continuation-in-part of U.S. application Ser. No. 08/458,352, filed Jun. 2, 1995, now U.S. Pat. No. 5,830,886, which was a division of U.S. application Ser. No. 08/196,192, filed (§102(e) date of) Dec. 27, 1994, now U.S. Pat. No. 5,616,574. U.S. application Ser. No. 08/196,192 was the U.S. National Phase of PCT/GB92/01587, filed Aug. 28, 1992 and designating the U.S, and incorporated herein by reference. U.S. application Ser. No. 08/196,192 has a §371 date of Dec. 27, 1994 and a §102(e) date of Dec. 27, 1994. PCT/GB92/01587 was published as WO93/05064, has a publication date of Mar. 18, 1993, and claims priority from United Kingdom patent application No. 9118478, filed Aug. 29, 1991. U.S. Ser. No. 09/111,927 was also a continuation-in-part of PCT patent application number PCT/GB97/00600, filed Mar. 4, 1997, designating the U.S., and claiming priority from United Kingdom patent applications 9604709.7 and 9605725.2, filed Mar. 5 and 19, 1996, respectively. PCT/GB97/00600 was published as WO 97/32872 on Sep. 12, 1997. U.S. Ser. No. 09/111,927 was also a continuation-in-part of PCT patent application number PCT/GB97/00444, filed Feb. 17, 1997, designating the U.S., and claiming priority from United Kingdom patent application 9603325.3, filed Feb. 16, 1996. PCT/GB97/00444 was published as WO 97/30041 on Aug. 21, 1997. U.S. Ser. No. 09/111,927 was also a continuation-in-part of PCT patent application number PCT/GB97/03352, filed Dec. 4, 1997, designating the U.S., and claiming priority from United Kingdom patent application 9625334.9, filed Dec. 5, 1996. PCT/GB97/03352 was published as WO 98/24802 on Jun. 11, 1998. Each of PCT/GB97/00600 (WO 97/32872), PCT/GB97/00444 (WO 97/30041), and PCT/GB97/03352 (WO 98/24802) is hereby incorporated herein by reference. In addition, the above-mentioned applications, as well as all documents cited herein and documents referenced or cited in documents cited herein, are hereby incorporated herein by reference.
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| 20020128243 A1 | Sep 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09579163 | May 2000 | US |
| Child | 10084235 | US | |
| Parent | 09238345 | Jan 1999 | US |
| Child | 09579163 | US | |
| Parent | 09111927 | Jul 1998 | US |
| Child | 09238345 | US | |
| Parent | 08196192 | US | |
| Child | 08458352 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 08458352 | Jun 1995 | US |
| Child | 09111927 | US | |
| Parent | PCT/GB97/00600 | Mar 1997 | US |
| Child | 08196192 | US | |
| Parent | PCT/GB97/004444 | Feb 1997 | US |
| Child | PCT/GB97/00600 | US | |
| Parent | PCT/GB97/03352 | Dec 1997 | US |
| Child | PCT/GB97/004444 | US |
