WO1995017383A1

WO1995017383A1 - Indole derivatives as anti-estrogens

Info

Publication number: WO1995017383A1
Application number: PCT/EP1994/004250
Authority: WO
Inventors: Ulf Norinder
Original assignee: Karo Bio Ab
Priority date: 1993-12-23
Filing date: 1994-12-23
Publication date: 1995-06-29
Also published as: CA2187296A1; AU1316995A; GB9326332D0; EP0736008A1; JPH09510689A

Abstract

The invention relates to an indole derivative of formula (I), wherein R1 and R2 independently represent hydroxy, methoxy or fluorine, preferably hydroxy; n represents an integer of 9 to 12; and X represents a member of the group consisting of CONR4R5, CSNR4R5, NR4COR5, NR4R5, SO2NR4R5, NR4SO2R5, 1-R4-5-tetrazole, wherein R4 and R5 are independently selected from the group of hydrogen and lower alkyls or R4 and R5 together form a bridge of the formula -(CH2)y- wherein y represents an integer 4 or 5, are disclosed. The invention also relates to a pharmaceutical preparation comprising one or more of said indole derivatives and to the use of said derivatives as a medicament are described. Also disclosed is the use of said indole derivatives for the preparation of a medicament for the therapeutic or prophylactic treatment of an estrogen related disorder or disease, such as in estrogen replacement therapy and/or breast cancer.

Description

INDOLE DERIVATIVES AS ANTI-ESTROGENS

The present invention relates to new indole derivatives and in particular to new indole derivatives which are useful as non-steroidal anti-estrogens.

It is now known that the action of estrogen on bone is direct and that bone cells contain physiological concentrations of estrogen receptors. Those receptors were originally demonstrated in normal human osteoblast-like cells (B.S.Komm et al. , Science 241 (1988) 81 and E.F. Eriksen et al. , Science 241 (1988) 84), but more recently, they have been demonstrated also in osteoclasts (M. J. Ousier et al.. PNAS 88 (1991) 6613). The cellular mechanism of estrogen action on bone is unclear. However, its most important properties include the well-documented ability to antagonize bone resorption by normalizing the resorption depth and decreasing the rate of bone turnover.

It is generally accepted that Estrogen Replacement Therapy (ERT) is effective in the prevention of osteoporosis and fracture. However, it has recently become evident that it may also be valuable in the treatment of established osteoporosis and fractures (C.W. Marx et al. , J. Bone and Mineral Res 11 (1992) 1275 and C. Christiansen et al. in Christiansen (ed) "Hormone replacement and its impact on osteoporosis" , Bailliere Tindall, London (1991) 853). There is evidence that ERT is effective up to the age of 70 years and that termination of postmenopausal ERT results in an accelerated phase of bone loss mimicking that seen at the time of natural menopause: after four to six years, there was little difference between those patients who had terminated ERT and those who had never received it. Thus, some investigators think that it may be necessary to maintain ERT for long periods of time and possibly indefinitely, if it is to be used as the mainstay prophylaxis against postmenopausal bone loss and fracture (H.K. Genant et al. , Am J. Obstet Gynecol 16 (1989) 1842).

However, the serious complications of estrogen therapy include endometrial cancer, breast cancer and venous thrombosis and associated pulmonary embolism.

Presently, co-treatment with a progestin appears to prevent estrogen- induced endometrial cancer.

Transdermal supply of estrogen, which avoids the first hepatic passage occurring with oral formulations, may circumvent conditions favouring thrombosis.

However, no therapeutic manipulation has been found that avoids the increased risk of breast cancer and, in fact, there is evidence that progestins themselves may have carcinogenic properties for the breast. Most disturbing of all, in the light of accumulating evidence that continuous ERT may be needed to prevent lifetime bone loss, is the recently-available evidence indicating that the risk of developing breast cancer increases with the time of exposure to estrogen (D. Grady et al.. Am. Lutern. Med. 117 (1992) 1016). There is clear evidence that not much more than 50% of North

American patients for whom ERT is recommended fulfil their prescriptions for estrogen and that many physicians are unwilling to prescribe estrogen because of fear of complications.

The estrogen receptor antagonist, or anti-estrogen, tamoxifen used particularly today in the treatment of hormone-dependent breast cancer (J. Bowler et al.. Steroids 54 (1989) 71) is one of the safest anti-cancer drugs on the market.

However, tamoxifen has number of disadvantages. First, in long term treatment it has been shown to cause an increased frequency of human endometrial cancers. Second, a very high frequency of liver tumours have been found in rats receiving high doses of tamoxifen. Third, a number of patients treated for hormone-dependent breast cancer do not respond to this treatment (H. Mass et al.. Cancer 46 (1980) 2783). The reason for this failure is not yet understood but may be due to the incomplete antagonism of Tamoxifen. This suggests that tamoxifen is too unspecific to be the estrogen antagonist

of choice for long term adjuvant therapy or treatment of advanced breast cancers.

Thus, an improved anti-estrogen devoid of estrogenic activity would be desirable both as a drug and as a biochemical tool to investigate the mechanisms of action of estrogens.

By "estrogenic activity" we mean that the tested compound gives rise to a cellular response similar to that of estradiol and by "anti-estrogenic activity" we mean that the tested compound affects the cellular response opposite to the activity induced by estradiol.

Numerous compounds, mainly steroids, have been tried with various results. See for instance J. Bowler et al. , Steroids 54 (1989) 71 and Wakeling and

Bowler, J. Steroid. Biochem. 43 (1992) 173, Bowler et al., U.S. Patent 4,659,516; European Patent Application 0138504.

Non-steroidal compounds such as dihydronaphthalenes (Suarez and Jones, U.S. Patent 4,230,862) and benzothiophenes (Jones and Suarez, U.S. Patent

4, 133,814; Jones, European Patent Application 0062503) have also been found to possess anti-estrogenic activity.

Some indoleamine derivatives with shorter (n=4-8 only) aliphatic side chains have been disclosed by von Angerer et al. , J. Med. Chem. 33 (1990) 2635,

US patents no.s 4,943,572 and 5,023,254 and European patent no. 0348341.

Labrie and Merand, have described some indole 6 '-hydroxy derivatives said to possess anti-estrogenic activity (European Patent Application 0367576/ International Patent Application WO93/ 10741 ) .

There is, however, still an urgent need for anti-estrogens, lacking or possessing a minimum of estrogenic activity in breast, since it is believed that estrogenic activity is responsible for the undesirable side effects mentioned previously. Description of the Invention

The present invention provides an indole derivative of the formula

wherein Rl and R2 independently is/are hydroxy, methoxy or fluorine;

n is an integer of between 9 and 12 inclusive;

and X is CONR4R5, CSNR4R5, NR4COR5, NR4R5. SO_:NR4R5. NR4SO₂R5, or 1-

R4-5-tetrazole, wherein R4 and R5 are independently selected from hydrogen and lower alky Is, typically C1-C5, or R4 and R5 together form a bridge of the formula - (CH₂)y- wherein y is 4 or 5 or a salt, preferably a physiologically-acceptable salt, thereof.

These compounds have improved activities compared to prior art compounds. Particularly preferred compounds are recited in claim 3. Of these compounds, compound KB-ER-1 is particularly preferred. Preferably, in addition to lacking or possessing a minimum of estrogenic activity in breast, the indole derivative of the invention also exhibits estrogenic activity in bone and/or in liver and/or lacks or possesses a minimum of estrogenic activity in endometrium.

One or more of the indole derivatives of the invention may be included in a pharmaceutical preparation as an active ingredient together with a pharmaceutically acceptable carrier(s) and/or diluent(s) in accordance with the skill of the average worker.

The indole derivatives of the invention may be used in the treatment, whether therapeutic or prophylactic, of an estrogen-related disorder or disease, such as breast cancer, or in estrogen replacement therapy.

Additionally, the indole derivatives of the invention may be used in the manufacture of a medicament for the therapeutic or prophylactic treatment of an estrogen related disorder or disease comprising administering to an individual in need of such treatment a therapeutically or prophylactically effective amount of such indole derivatives. The medicaments are especially suitable for use in estrogen replacement therapy and/or breast cancer therapy.

The preferred activity profiles make the indole derivative of the invention particularly suitable for use in the long term treatment of patients. This contrasts the worrying evidence emerging for the anti-estrogen Tamoxifen used presently.

The preparation of compounds in accordance with the invention and their testing in comparison with prior art compounds will now be described, by way of example only, with reference to the accompanying drawings Fig.s 1-16 in which:

Figs 1 - 14 illustrate the effect of compounds in accordance with the invention in the ZRAF cell line; a Breast carcinoma cell line; an endometrium carcinoma cell line: and a liver carcinoma cell line; and

Figs 15 and 16 illustrate the effect of prior art compounds ZK 119010 and Tamoxifen on the same cells .

Synthesis of preferred indole derivatives

All structures of the compounds in accordance with the invention were confirmed by NMR analysis and mass-spectroscopy.

Example I

l-(10-(Diethylcarbamoyl)decanyl)-5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole (a) N-methylmorpholine (0.26 g, 2.6 mmole) and isobutylchloroformate (0.38 g, 2.8 mmole) was added to a solution of 11-bromoundecanoic acid (0.53 g, 2.0 mmole) in

CH₂C1₂ (10 ml.) at -10°C. The mixture was stirred at -10°C for 30 minutes before diety lamine (0.2 g, 3 mmole) was added. After 30 minutes at room temperature (RT) the mixture was washed with 1 M HC1 followed by NaHCO₃(sat) and brine. The organic layer was dried using MgSO4) and concentrated. The residue was purified by column chromatography (SiO₂; petroleum ether - ethyl acetate 9: 1) to give 0.48 g

(75%) of ll-bromo-N,N-diethyldecanylamide.

(b) A solution of 5-methoxy-2-(4-methoxyphenyl)-3-methylindol (0.13 g, 0.5 mmole)

(prepared by the method of von Angerer et al, Journal of Medicinal Chemistry, 1984, 27, 1439-1447) in dry dimethyl formanide (DMF) (2 ml.) was added to a stirred mixture of sodium hydride (25 mg, 0.8 mmole) in dry DMF (1 ml.) at 0°C. After stirring for 30 minutes, the amide prepared in (a) (0.26g, 0.8 mmole) dissolved in dry DMF (1 ml.) was added dropwise. The resulting mixture was stirred for 2 hours at

RT and then partitionated between ethylacetate and water. The organic layer was separated, dried (MgSO₄) and concentrated. The residue was purified by column chromatography (SiO₂; petroleum ether - ethyl acetate (8:2) to give 0.18 g (71 %) of 1- (10-diethylcarbamoyl)decanyl)-5-methoxy-2-(4-methoxyphenyl)-3-methylindole.

(c) The above dimethoxy compound (0.2g, 0.4 mmole) was dissolved in CH₂C1₂ (10 ml.) and cooled to -65°C under N₂. Borontribromide (IM in CH₂C1₂, 2 ml. , 2 mmole) was added with a syringe and the temperature was allowed to reach RT. The mixture was stirred overnight and then quenched with NaNCO₃(sat). The mixture was partitionated between ethyl acetate and water. The organic layer was dried (MgSO₄)

and concentrated. The residue was purified by column chromatography (SiO₂; petroleum ether - ethylacetate 4: 1) to give 0.17 g (95 %) of 1-(10-

(diethylcarbamoyl)decanyl)-5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole as a semisolid.

Example II

5-hydroxy-2-(4-hydroxyphenyl)-3-methyl- 1 -( 12-piperidinododecanyl)indole

(a) A solution of 5-methoxy-2-(4-methoxyphenyl)-3-methylindol (2.0 g, 7.5 mmole) (Compare Example 1(b)) in dry DMF (15 ml.) was added to a stirred mixture of sodium hydride (80%, 380 mg. 13 mmole) in dry DMF (3 ml.) at 0°C. The mixture was left for 30 minutes at 0°C and then slowly added to a solution of 1, 12- dibromododecane (3.7 g, 11.3 mmole) in ice-cold, dry DMF (10 ml.). After stirring for an additional 2 hours at RT the reaction mixture was partitionated between water and CH₂C1₂. The organic layer was dried (MgSO₄) and concentrated. The residue was purified by column chromatography (SiO₂; petroleum ether - ethyl acetate 9: 1) to give 1.4 g of l-(12-bromododecanyl)-5-methoxy-2-(4-methoxyphenyl)-3-methylindole.

(b) To the above bromo compound (200 mg. 0.39 mmole) in DMF (3 ml.) was added a solution of piperidin (0.13 g, 1.56 mmole) in DMF (1 ml.) and the mixture was stirred at RT over night. The reaction mixture was concentrated and the residue was partitionated between water and ethyl acetate. The organic layer was separated, dried (MgSO₄) and concentrated. The residue was purified by column chromatography (SiO₂; petroleum ether - ethyl acetate 1:1 saturated with NH₃) to give 0.12 g (60%) of

5-methoxy-2-(4-methoxyphenyl)-3-methyl-l-(12-piperidinododecanyl)indole.

(c) The above dimethoxy compound (0.12 g, 0.23 mmole) was treated with boron tribromide as described in Example 1(c) to give 5-hydroxy-2-(4-hydroxyphenyl)-3- methyl-l-(12-piperidinododecanyl)indole which was purified by column chromatography (Al₂O₃, CH₂C1₂ - MeOH 9:1) yielding 0.10 g (91 %). Found: C, 75.6; H, 9.4; N, 5.1; C₃₂H₄₈N₂O₂ - 0.95 H₂O. Requires: C, 75.7; H, 9.5; N. 5.5%.

Example III

l-(10-N-cyclopropyl-propionylamido)decanyl)-5-hydroxy-2-(4-hydroxyphenyl)-3- methylindole

(a) 5-methoxy-2-(4-methoxyphenyl)-3-methylindole (1.0 g) was alkylated with 1,10- dibromodecane as described in Example 11(a) to give 1.4 g (78%) of 1-(10- bromodecanyl)-5-methoxy-2-(4-methoxyphenyl)-3-methylinodole.

(b) The above bromo compound (1 ,4 g) was reacted with cyclopropylamine as described in Example 11(b) to give 1.0 g (78%) of l-(10-(cyclopropylaminodecanyl)-5- methoxy-2-(4-methoxyphenyl)-3-methylindole. (c) Propionic anhydride (130 mg, 1.0 mmole) was added dropwise to an ice-cold

solution of the above amine (300 mg, 0.66 mmole) and triethylamine (100 mg, 1.0 mmole) in THF (5 ml.). The mixture was stirred at RT overnight and then partitionated between water and ethylacetate. The organic layer was washed, first with 1 M HCl(aq) then NaHCO₃(sat), dried (MgSO₄) and concentrated. The residue was purified by column chromatography (SiO₂; petroleum ether - ethyl acetate 4: 1) to give

310 mg (92%) of l-(10-(N-cyclopropyl-propionylamido)decanyl)-5-methoxy-2-(4- methoxyphenyl)-3-methylindole .

(d) The above dimethoxy compound (0.20 g) was treated with boron tribromide as described in Example 1(c) to give 0.19 g (90%) of l-(10-N-cyclopropyl- propionylamido)decanyl)-5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole. Found: C, 73.3; H, 8.4; N, 5.1; C₃₁H₄₂N₂O₃ - 1.05 H₂O. Requires: C, 73.1 ; H, 8.7; N, 5.5% .

Example IV:

l-(10-(N-cyclopropyl-N-n-propylaminodecanyl)-5-hydroxy-2-(4-hydroxyphenyl)-3- methylindole

(a) A mixture of l-(10-(cyclopropylaminodecanyl)-5-methoxy-2-(4-methoxyphenyl)-3- methylindole (prepared as described in Example IΙI(b) (230 mg, 0.5 mmole), K₂CO₃ (100 mg, 0.75 mmole) and propionyliodide (100 mg, 0.6 mmole) in acetonitrile (5 ml.) was stirred at RT overnight and then partitionated between ethylacetate and water. The organic layer was dried (K₂CO₃) and concentrated. Purification of the residue by column chromatography (SiO₂; petroleum ether - ethyl acetate 1:1) yielded 200 mg (80%) of l-(10-cyclopropyl-n-propylaminodecanyl)-5-methoxy-2-(4-methoxyphenyl)-3- methylindole.

(b) The above dimethoxy compound (0.20 g) was treated with boron tribromide as described in Example 1(c) to give l-(10-(N-cyclopropyl-N-n-propylamino)decanyl)-5- hydroxy-2-(4-hydroxyphenyl)-3-methylindole which was purified by column chromatography (Al₂O₃, CH₂C1₂ - MeOH 9:1) yielding 0.19 g (98%). Found: C, 71.8; H, 8.6; N, 4.9; C^N - 0.6 CH₂C1₂. Requires: C, 71.9; H, 8.6; N, 5.3% .

Example V:

l-(10-tetrazolyldecanyl)-5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole

(a) A solution of sodium cyanide (15 mg, 0.28 mmole) in water (1 ml.) was added to a solution of l-(10-(diethylcarbamoyl)decanyl)-5-methoxy-2-(4-methoxyphenyl)-3- methylindole (prepared as described in Example 1(b) (0.65 g, 0.14 mmole) in DMF (5 ml.) and the resulting mixture was heated at 85°C for two hours. The mixture was diluted with ethylacetate and washed with water, dried (MgSO₄) and concentrated to give 0.60 g of crude l-(10-cyanodecanyl)-5-methoxy-2-(4-methoxyphenyl)-3- methylindole. (b) The above nitrile (230 mg. 0.5 mmole) was dissolved in DMF (5 ml.).

Sodiumazide (163 mg, 2.5 mmole) and ammoniumchloride (135 mg, 2.5 mmole) were added and the mixture was heated for 48 hours. The mixture was acidified with 2 M HC1 and extracted with CH₂C1₂. The organic layer was dried (MgSO₄) and concentrated. The residue was purified by column chromatography (SiO₂: CH₂C1₂ -

MeOH HOAc 90: 10: 1) to give 150 mg (62%) of l-(10-tetrazolyldecanyl(-5-methoxy-2- (4-methoxyphenyl)-3-methylindole.

(c) The above dimethoxy compound (0.24 g, 0.5 mmole) was treated with boron tribromide as described in Example 1(c) to give 0.22 g (98 %) of 1-(10- tetrazolyldecanyl)-5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole.

The compounds prepared in the above Examples I to V are represented by the following formula:

Following the procedures described in the preceding Examples, the following compounds included in the above formula were also prepared. Thus, in the above formula n, Rl, R2 and X are as follows:

COMPOUND n El R2 X

KB - ER-1 11 OH OH CON(CH₃)-i-C₃H₇

KB - ER-13 10 OH OH NH-c-C₃H₅

KB - ER-18 10 OH OH CO-c-NC₅H₁₀

KB - ER-19 11 OH OH c-NC₅H₁₀

KB - ER-20 9 OH OH N(C₂H₅)n-C₄H₉

KB - ER-21 11 OH OH NC(₂H₅)₂

KB - ER-22 11 OH OH CO-c-NC₅H₁₀

KB - ER-26 12 OH OH SO₂-c-NC₅H₁₀

KB - ER-27 11 OH OH CON(C₂H₅)₂ In Vitro Characterization of the Estrogenic/ Antiestrogenic Activity of Indole

Derivatives of the Invention and the Prior Art in Genetically Engineered and

Tissue Specific Estrogen Target Cells.

The compounds of the present invention described above were then tested, as described below, for estrogenic/activity in various cells in comparison to known anti-estrogens ZK 119010 (compound No. 6 described in von Angerer et al (1940) supra and US Patent Nos 5,023,254; and 4,943,572 and European Patent No.0348341) as requiring the lowest dose to exert complete agonism and tamoxifen.

1 Testing of indole compounds in genetically engineered cells:

The estrogen reporter cell line (ZRAF) is an engineered, estrogen receptor (ER) expressing mammalian cell line containing an integrated artificial transcription unit comprising an estrogen response element (ERE) and core promoter sequences fused to a reporter gene encoding a secreted form of alkaline phosphatase. In the absence of estrogenic hormones the cells express only very low levels of the alkaline phosphatase reporter protein. However, following exposure of the ZRAF cells to estrogens, e.g. estradiol, the ER gets activated resulting in transcriptional activation of the alkaline phosphatase reporter gene, mediated through the ERE. The level of estrogen-dependent alkaline phosphatase protein expressed can be determined indirectly by an enzymatic chemiluminescence assay as previously described (Nilsson S.et al. (1993) in Advances in Steroid Analysis '93, Proceedings of the 5th Symposium on the Analysis of Steroids, Ed. Gorδg S. , Published by Akademia Kiadό. Budapest, Hungary, p. 57 - 67).

Since the ZRAF cells show a stringent dependence on the presence of an agonist for expression of the alkaline phosphatase reporter protein the cells have to be stimulated by a low dose of reference agonist (1 nM moxestrol, New England Nuclear) in order to analyse compounds with antagonistic activity.

Using the above described reporter cell line, the synthesized indole derivatives of the invention were tested for their estrogenic/antiestrogenic activity and potency in regulating the expression of the alkaline phosphatase reporter gene, exerted by the human estrogen receptor.

Experimental design

Day 1 ;

The ZRAF cells were cultivated at 37 °C and 5% CO₂ in a humidified incubator under hormone free conditions, suspended in Coon's ^a) (w/o phenolred ) + 10 % FCS ^w (doublestripped from hormone by dextran coated charcoal (2xDCC)) in plastic petridishes (Costar.LabDesign, Sweden) for four days prior to the start of each experiment.

Day 4; ZRAF cells were seeded at a density of 4xl0⁴ cells per well in 96-well microtiterplates

(Costar,LabDesign, Sweden). The cells were cultivated in Coon's (w/o phenolred) +

10 % FCS (2xDCC) and at 37 °C and 5% CO₂ in a humidified incubator.

Day 5;

Change of medium to Coon's (w/o phenolred) + 5 % serum substitute +/- hormone and test substances (see below). The cells were then continued to be cultivated at 37 °C and 5% CO₂ in a humidified incubator.

Day 7;

48 hours post addition of the hormonal/test substances, cell number and cell morphology was examined under the lightmicroscope. The relative levels of alkaline phosphatase expressed were determined by a chemiluminescent assay as follows: a lOμl aliquot of the cell culture medium was mixed with 200μl of assay buffer

(lOmM diethanolamine pH 10. ; lrnM MgCl₂ and 0.5mM AMPPD) in white microtiter plates and incubated at 37 °C for 20 minutes before being transferred to a microplate format luminometer (Luminoskan Labsy stems. Finland ) . The setting of the Luminoskan luminometer was integral measurement with 1 second reading of each well. The alkaline phosphatase activity is expressed in light units (LU). The rate of light emission is directly proportional to the concentration of enzyme present in the sample. 2 Testing of indole compounds in estrogen dependent human breast cancer

cells:

The human breast tumor cell line ZR75-1 (ATCC CRL 1500) is highly dependent on estradiol E₂ for growth in vitro (half maximal proliferative response to Eo is already reached at approximately 50 pM and at a 1 nM concentration of Ε_*2 a maximal response is obtained - in the absence of E these cells become quiescent) and therefore was selected as a tool for evaluation of the agonist/antagonist activity of compounds that mediate their effect through the human estrogen receptor.

Tamoxifen displays a very weak estrogenic activity in the breast tumour cell proliferation assay cells. However, in the presence of 1 nM E^ tamoxifen shows a concentration dependent anti-estrogenic activity. The partial estrogenic/ anti-estrogenic profile of Tamoxifen in the absence or presence of Ei respectively, is known from the literature (Br. J. Cancer 59 (1989) 727 and Cancer Res. 48 (1988) 3693).

The level of cellular ATP was chosen as a measure of cell growth rather than counting the number of cells as it has previously been shown that the level of cellular ATP, indirectly determined by a chemiluminescence assay, is a more reproducible way to study cell growth and cell number than traditional cell counting (Methods in

Enzymology 133 (1986) 27 and Medical Biology 62 (1984) 338).

The effect of the indole derivatives of the present invention was determined on the ZR75-1 breast tumor cell line by monitoring their ability to stimulate or repress cell growth in the absence or presence of 1 nM estradiol, respectively (173-estradiol purchased from Sigma).

Experimental design

Day 0;

The ZR75-1 cells were cultivated under hormone free conditions in Coon's (w/o phenolred ) + 10 % FCS (2xDCC) in plastic petridishes for four days at 37°C and 5% CO₂ in a humidified incubator, prior to start of the experiment.

Day 4;

The cells were trypsinised and seeded at a density of 8 x 10³ cells/well in 96-well microtiterplates. The cells were seeded in Coon's (w/o phenolred) + 10 % FCS (2xDCC) and cultivated at 37 °C and 5% CO, in a humidified incubator.

Day 5;

Exchange of medium to Ham's (w/o phenolred ) + 5 % FCS (2xDCC) +/- hormone and test substances (see below) and cultivated at 37 °C and 5 % CO₂ in a humidified incubator. The cellular ATP pool from one control plate was extracted by 1 % TCA and stored at -20°C.

Day 8;

Spent medium was exchanged for fresh Ham^'s (w/o phenolred ) + 5 % FCS (2xDCC) +/- hormone and test substances (see day 5).

Day 11;

Six days post addition of the hormonal/test substances, cell morphology was examined under the lightmicroscope. All micro-well cultures were then extracted by 1 % TCA for 30 minutes at room temperature and assayed for the level of ATP (ATP monitoring kit, BioOrbit, Finland) according to the supplier's recommendations .

3 Testing of indole compounds on human liver cells:

The liver is another target organ for estrogens/antiestrogens. To enable characterization of the indole derivatives for liver specific effects we have used the human liver cell line HepG2 (ATCC # CRL HB 8065), transformed to express the human estrogen receptor. The gene encoding sex hormone binding globulin (SHBG) is positively regulated by estrogen receptor binding hormones/compounds that have partial or full agonist properties. Therefore the effect of the indole derivatives, in the presence or absence of the reference agonist moxestrol (1 nM), on the transcriptional regulation of the SHGBG gene was used to assess their estrogenic/antiestrogenic activities.

The level of expressed and secreted SHBG was monitored immunologically with a SHBG Delfia assay (Wallac OY, Finland) according to the supplier's recommendations . Experimental design

Day 1;

Approximately 20x10³ HepG2 cells per well in a 96-well plate were seeded in Coon's (w/o phenolred ) supplemented with 2 mM L-glutamine and 1 % FCS

(2xDCC) and cultivated at 37°C and 5% CO₂ in a humidified incubator.

Day 2;

Change of medium to Coon's (w/o phenolred) supplemented with 2 mM L-

glutamine and 1 % FCS (2xDCC) +/- reference agonist and test substances (see below). The cells were then continued to be cultivated at 37 °C and 5% CO₂ in a

humidified incubator.

Day 5; 72 hours post addition of the hormonal/test substances, cell number and cell morphology was examined under the lightmicroscope. An aliqout of the conditioned medium from each well was then immunologically assayed for the levels of SHBG expressed using a SHBG Delfia assay (Wallac OY, Finland) according to the supplier's recommendations.

4 Testing of indole compounds on human endometrial cells:

The uterus is yet another target for effects induced by estrogenic compounds. It is well known that tamoxifen. a potent antiestrogen for treatment of breast cancer, has very high estrogenic activity in endometrial cells. The relatively high agonistic effect of tamoxifen in the endometrium is believed to be one reason for the increased incidence of new endometrial tumors formed during long-term tamoxifen treatment. To avoid the development of new drugs with high estrogenic activity in the endometrium we have characterized the indole compounds also for their agonistic/antagonistic effect on human endometrial cells (Ishikawa cells - Touitou et al Mol.Cell Endocrinol, 66, 231 (1989) and references cited therein).

The endogenous alkaline phosphatase gene was used as the target gene for characterization of the agonistic/antagonistic effect of the indole compounds in the Ishikawa endometrial cell line. Their agonist/antagonist profile is assessed by determining the level of alkaline phosphatase expressed and secreted by the endometrial cells following exposure to the indole compounds in the presence or absence of 0.1 nM of the reference agonist moxestrol.

Experimental design

Day 1; 20x10³ Ishikawa cells per well in a 96-well plate were seeded in Coon's (w/o phenolred) supplemented with 2 mM L-glutamine and 10 % FCS (2xDCC) and cultivated at 37 °C and 5% CO-, in a humidified incubator.

Dav 2; Change of medium to Coon's (w/o phenolred) supplemented with 2 mM L- glutamine and 5 % serum substitute +/- reference agonist and test substances (see below). The cells were continued to be cultivated at 37 °C and 5% CO₂ in a humidified incubator.

Day 5;

72 hours post addition of the hormonal/test substances, cell number and cell morphology was examined under the lightmicroscope. An aliqout of the conditioned

medium from each well was then assayed for the level of alkaline phosphatase expressed as described for the ZRAF cells (see above).

All cell lines used to test the agonistic/antagonistic properties of the indole derivatives were also used in parallel to characterize the agonist/antagonist profile of the Schering compound ZK 119010 (kindly provided by Schering), and tamoxifen (Sigma) for comparison.

Toxicity was monitored by the colorimetric MTS/PMS method (SDS. Sweden) following the supplier's recommendations.

Chemicals:

a) Coon's and Ham's medium (w/o phenolred) were purchased from SVA, Uppsala, Sweden. b) FCS and L-glutamine were purchased from Gibco-BRL. c) Serumsubstitute from Dr. Alan Preston, Med. Vet. supplies limited. Botolth Clayton, Buckingham,

MK 18 2LR, U.K. d) The alkaline phosphatase substrate AMPPD (disodium 3-(4-methoxyspiro

(l,2-dioxetane-3,2+- tricyclo(3.3.1) decan)-4-yl) phenyl phosphate) was purchased from Boule Diagnostics, Sweden.

Hormones/test substances added per well in 96-well mcrotiter plates

For each cell line and each test run a reference against titration curve was performed to monitor the condition of the cells. With all cell lines a dose titration was done for the indole derivatives, the Schering compound ZK 119010 and tamoxifen in the absence or presence of, for each cell line a pre-determined fixed dose of reference agonist (17b-estradiol/moxestrol).

The concentration range of the reference agonist used was IO^"6 - IO"¹¹ M.

The concentration range of the indole derivatives, the Schering compound ZK 119010 and tamoxifen used in the experiments was IO^"5 - IO^"10 M (or as otherwise depicted in the dose response curves) ± 0.1/1 nM reference agonist.

The cellular response to each dose of hormone/compound was performed in triplicate i.e. three wells per dose hormone/compound. Several determinations of the chosen cellular response in the presence of solvent

only i.e. in the absence of any added hormone/compound, was performed on each plate per test run (background level of expressed marker).

The cellular response to hormone/compound is expressed in the accompanying dose response curves as percent of the response elicited by the reference agonist. Background expression of the chosen cellular response was set at 0 % .

Summary:

All cell lines and chosen cellular responses used to characterize the agonist/antagonist function of the indole derivatives displayed a concentration-dependent estrogen effect.

All indole derivatives in accordance with the invention were able to suppress estradiol- dependent growth of the ZR75-1 breast cancer cells and estradiol-mediated transcriptional activation of the endogenous alkaline phosphatase gene in the Ishikawa endometrial cell line.

In comparison to the Schering compound ZK 119010 all indole derivatives except KB

ER-28 displayed a significantly lower growth stimulatory effect of the breast cancer cell line ZR75-1 i.e. they elicited very low or no agonist function. Of those KB ER-1, KB-ER-14, KB ER -16, KB ER -22. KB ER -26 and KB ER -27 also showed a more favourable agonist/antagonist profile in the endometrial cell line than the Schering compound ZK 119010. Compounds KB ER-17, KB ER-18 andKB ER- 21 displayed a degree of agonism similar to ZK 119010 while the other indole derivatives, except KB ER-28, showed only a slightly higher relative agonism.

All indole derivatives including also the the Schering compound ZK 119010 showed lower agonist activity than tamoxifen in both the ZR75-1 breast cancer, and the Ishikawa endometrial cell line.

All of the indole derivatives of the invention showed only anti-estrogenic activity except for KB-ER-13, KB-ER-17 and KB-ER-18 which also displayed very weak estrogenic activity in the absence of E,.

The invention thus provides anti-estrogens lacking or possessing a minium of estrogenic activity in breast.

Claims

1. An indole derivative of the formula

wherein Rl and R2 independently is/are hydroxy, methoxy or fluorine;

n is an integer of 9 to 12 inclusive;

and X is CONR4R5, CSNR4R5, NR4COR5, NR4R5. SO₂NR4R5, NR4SO₂R5, or 1-

R4-5-tetrazole, wherein R4 and R5 are independently selected from hydrogen and lower alky Is, or R4 and R5 together form a bridge of the formula -(CH₂)y- wherein y is 4 or 5 or a salt thereof.

2. An indole derivative as claimed in Claim 1, wherein Rl and/or R2

is hvdroxv. An indole derivative according to claim 2 and in which both Rl and

R2 are hydroxy and n and X are selected from the following combinations:

n X

12 CON(C₂H₅)₂

10 N(c-C₃H₅)-COC₂H₅

11 CON(CH₃)-i-C₃H₇

11 CO-c-NC₅H₁₀

12 SO₂-c-NC₅H₁₀

11 CON(C₂H₅)₂

4. An indole deriviative as claimed in Claim 1,2 or 3 which lacks estrogenic activity in breast tissue.

5. An indole derivative as claimed in any one of Claims 1 to 4, which exhibits estrogenic activity in bone cells and/or in liver cells and/or lacks or possesses a minimum of estrogenic activity in endometrium cells. 6. A pharmaceutical preparation comprising, as an active ingredient. an indole derivative according to any one of Claims 1 to 5, together with a pharmaceutically acceptable carrier and/or diluent.

7. An indole derivative according to any one of Claims 1 to 5, for use in medicine.

8. Use of an indole derivative according to any one of Claims 1 to 6, or a pharmaceutical preparation according to claim 7, for the treatment of an estrogen- related disorder or disease.

9. Use according to Claim 8, wherein said estrogen- related disorder or disease is breast cancer.

10. Use according to Claim 8or 9. wherein said treatment is estrogen replacement therapy.