15beta-substituted estrone derivatives as selective inhibitors of 17beta-hydroxysteoid-dehydrogenases, method of preparation and use thereof
20190330258 ยท 2019-10-31
Assignee
- USTAV ORGANICKE CHEMIE A BIOCHEMIE AV CR, v.v.i. (Praha, CZ)
- USTAV MOLEKULARNI GENETIKY AKADEMIE VED CR, V.V.I. (Praha, CZ)
- Univerzita Palackeho V Olomouci (Olomouc, CZ)
- Helmholtz Zentrum Munchen (Neuherberg, DE)
Inventors
- Martin KOTORA (Praha 5, CZ)
- Eva PRCHALOVA (Praha 9, CZ)
- Jerzy ADAMSKI (Munchen, DE)
- Gabriele MOELLER (Munchen, DE)
- Ondrej Stepanek (Liberec, CZ)
- Petr BARTUNEK (Praha 1, CZ)
- David SEDLAK (Praha 7, CZ)
- Marian Hajduch (Moravsky Beroun, CZ)
- Petr Dzubak (Brodek u Prerova, CZ)
Cpc classification
A61P15/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
15-substituted derivatives of estrone of general formula I wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 are independently selected from the group consisting of: C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 halogenalkyl, halogen, COOR.sup.6 wherein R.sup.6 is C.sub.1-C.sub.4 alkyl; H, OH; optionally, R.sup.3, R.sup.4 and R.sup.5 are each formed by a hydrogen atom, while R.sup.1 and R.sup.2 together form an aryl group, preferably naphthyl, in which the aromatic ring in position C-15 can be mono-, di-, tri-, tetra- and penta-substituted with substituents R.sup.1-R.sup.5. Compounds of the invention may be used for diagnosis and possibly also for the treatment of estrogen-dependent diseases.
Claims
1. 15-substituted derivatives of estrone of general formula I, ##STR00021## wherein: substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 are independently selected from the group consisting of: C.sub.1-C.sub.4 alkyl; C.sub.1-C.sub.4 alkoxy; C.sub.1-C.sub.4 halogenalkyl; halogen; COOR.sup.6, wherein R.sup.6 is C.sub.1-C.sub.4 alkyl; H or OH; or R.sup.1 and R.sup.2 together form an aryl; preferably Wand R.sup.2 together with the phenyl on which they are bound form a naphthyl, in which case R.sup.3, R.sup.4 and R.sup.5 are hydrogen atoms; and wherein the aromatic ring in position C-15 can be mono-, di-, tri-, tetra- and penta-substituted with the R.sup.1 to R.sup.5 substituents.
2. A method for preparing compounds of general formula I according to claim 1, comprising the following steps: a) 3-(t-butyldimethylsilyloxy)-15-vinyl-estra-1.3.5(10)-trien-17-one reacts in a cross metathesis reaction with a second olefin in the presence of a ruthenium catalyst, and preferably in the presence of CuI co-catalyst, at temperature from 40 C. to 70 C. under inert atmosphere to form 3-(t-butyldimethylsilyloxy)-15-vinyl-estra-1.3.5 (10)-trien-17-one terminal vinyl-substituted derivatives; b) t-butyldimethylsilyl protecting group of the product of the cross metathesis reaction from the previous step is removed, preferably using tetrabutylammonium fluoride; c) hydrogenation of the unsaturated deprotected product of the step b) leading to formation of the compound of general formula (I); wherein respective second olefins in step a) are selected from derivatives of styrene, vinylnaphtalene, vinylphenol, vinyl-benzene, either of which can be further substituted with halogen, alkyl, haloalkyl, alkoxy and/or acetoxy group; and wherein the ruthenium catalyst is selected from a group comprising Hoveyda Grubbs catalyst second generation, Hoveyda Grubbs catalyst first generation, Grubbs catalyst second generation, Grubbs catalyst first generation.
3. The method according to claim 2, characterized in that: in the first step, Hoveyda-Grubbs ruthenium catalyst second generation and CuI, respectively, are added to a solution of 3-(t-butyldimethylsilyloxy)-15-vinyl-estra-1.3.5(10)-trien-17-one and the respective second olefin in a solvent mixture of CH.sub.2Cl.sub.2/trifluorotoluene in a volume ratio of 2/1 under an inert atmosphere; the resulting mixture is first stirred at 40-70 C. for 4-12 hr, and, subsequently, further addition of the respective second olefin and the Hoveyda-Grubbs ruthenium catalyst second generation is performed, and the reaction mixture is stirred at the same temperature overnight; then the reaction is quenched by evaporation of solvents, and products of cross-metathesis are obtained by chromatography on silica gel; in the second step, solution of TBAF in THF is successively added dropwise to the metathesis product, dissolved in THF, at room temperature; after 1 h, water is added and the reaction mixture is extracted with CH.sub.2Cl.sub.2 and/or CHCl.sub.3; the combined organic phases are then washed with saturated NaCl solution, dried with MgSO.sub.4; the solvents are removed under reduced pressure and deprotected products are isolated by chromatography on silica gel; and in a third step, the flask with a mixture of deprotected product of metathesis in ethyl acetate and Pd/C catalyst (10 wt. %) is evacuated under vigorous stirring, and then filled with hydrogen gas; the reaction mixture is stirred overnight, then filtered through diatomaceous earth SiO.sub.2, and solvents are removed.
4. Compounds of general formula I according to claim 1 for use as a medical drug.
5. Compounds of general formula I according to claim 1 for use in the diagnosis and/or treatment of estrogen-dependent diseases.
6. Compounds of general formula I for use in the diagnosis and/or treatment of estrogen-dependent diseases according to claim 5, characterized in that the estrogen-dependent diseases and disorders are selected from breast cancer, ovarian cancer, uterine cancer, endometriosis, adenomyosis, menorrhagia, metrorrhagia, dysmenorrhea, uterine fibroids, polycystic ovarian syndrome, fibrocystic disease of the breast, prostate cancer, non-small cell lung cancer (NSCLC), squamous cell carcinoma, colorectal carcinoma, gastric cancer, acne, hirsutism, pseudohermaphroditism, seborrheic dermatitis, androgens induced alopecia, hyperestrogenism.
7. Compounds of general formula I for use in the treatment of infertility, to induce premature menopause, for hormonal castration, or for use as a contraceptive.
8. A pharmaceutical composition comprising at least one of the compounds of general formula I according to claim 1 as the active ingredient and a pharmaceutically acceptable carrier.
9. The pharmaceutical composition according to claim 8, comprising at least one of the compounds of general formula I, for use in the diagnosis and/or treatment of estrogen-dependent diseases and disorders.
10. The pharmaceutical composition for use in the diagnosis and/or treatment of estrogen-dependent diseases and disorders according to claim 9, characterized in that the estrogen-dependent diseases and disorders are selected from breast cancer, ovarian cancer, uterine cancer, endometriosis, adenomyosis, menorrhagia, metrorrhagia, dysmenorrhea, uterine fibroids, polycystic ovarian syndrome, fibrocystic disease of the breast, prostate cancer, non-small cell lung cancer (NSCLC), squamous cell carcinoma, colorectal carcinoma, gastric cancer, acne, hirsutism, pseudohermaphroditism, seborrheic dermatitis, androgens induced alopecia, hyperestrogenism.
11. The pharmaceutical composition according to claim 8, comprising at least one of the compounds of general formula I, for use in the treatment of infertility, to induce premature menopause, for hormonal castration, or for use as a contraceptive.
12. Compounds of general formula I according to claim 1 and/or the method for preparing compounds of general formula I, for use in diagnosis and/or treatment of estrogen-dependent diseases, selected from breast cancer, ovarian cancer, uterine cancer, endometriosis, adenomyosis, menorrhagia, metrorrhagia, dysmenorrhea, uterine fibroids, polycystic ovarian syndrome, fibrocystic disease of the breast, prostate cancer, non-small cell lung cancer (NSCLC), squamous cell carcinoma, colorectal carcinoma, gastric cancer, acne, hirsutism, pseudohermaphroditism, seborrheic dermatitis, androgens induced alopecia, hyperestrogenism; and/or for the treatment of infertility, to induce premature menopause, for hormonal castration, or for use as a contraceptive.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0053]
[0054]
[0055] The
[0056] The
[0057] The
[0058] For
EXAMPLES
List of Abbreviations
[0059] [].sub.D specific rotation [0060] chemical shift [0061] standard deviation [0062] A549 human lung adenocarcinoma [0063] Ac acetyl [0064] AKR aldo-keto reductases [0065] APCI atmospheric pressure chemical ionization [0066] AR androgen receptor [0067] b broad signal in NMR spectrum [0068] BJ human fibroblasts [0069] Bu butyl [0070] cDNA complementary DNA [0071] CEM T-lymphoblastic leukemia [0072] CEM-DNR-bulk T-lymphoblastic leukemia resistant to doxorubicin [0073] CHO cancer of hamster ovarian [0074] d dublet [0075] DHEA dehydroepiandrosteron [0076] DMEM Dulbecco's Modified Eagle's Medium [0077] DMSO dimethyl sulfoxide [0078] DNA deoxyribonucleic acid [0079] E1 estrone [0080] E2 estradiol [0081] E3 estriol [0082] eq equivalent [0083] ER estrogen receptors [0084] ERE estrogen response elements (DNA sequences capable of binding estrogen receptors) [0085] ESI electrospray ionization [0086] EST estrogen sulfotransferase [0087] Et ethyl [0088] FBS fetal bovine serum [0089] HCT116p53 wt human colon cancer, wild-type [0090] HCT116p53/ human colon cancer, mutant p53 [0091] HMPA hexamethylphosphoramide [0092] HPLC high performance liquid chromatography [0093] HR-MS high resolution mass spectrometry [0094] HSD hydroxysteroid dehydrogenases [0095] IC.sub.50 the concentration of compound required for 50% inhibition [0096] IR infrared spectroscopy [0097] J coupling constant [0098] K562 human myeloid leukemia [0099] K562-Tax human myeloid leukemia resistant to taxol [0100] LHRH luteinizing hormonereleasing hormone [0101] m multiplet [0102] MCF-7 cell line derived from a human breast carcinoma [0103] Me methyl [0104] mp melting point [0105] MRC7 human fibroblasts [0106] MTT test colorimetric determination of cell viability [0107] NAD(P) nicotinamide adenine dinucleotide (phosphate) [0108] NMR nuclear magnetic resonance [0109] NSCLC non-small cell lung cancer [0110] P450 cytochrome P450 [0111] p53 tumor suppressor gene [0112] PGF.sub.2 prostaglandine F2 [0113] pGL4 luciferase reporter vector [0114] PgP multidrug resistance protein [0115] PgR progesterone receptor [0116] ppm parts per million [0117] q quadruplet [0118] R.sub.f retarding factor [0119] s singlet [0120] SDR short-chain dehydrogenases/reductases [0121] SEEM selective modulator of steroidogenesis enzymes [0122] SERD selective estrogen receptors deregulator [0123] SERM selective estrogen receptors modulator [0124] SHBG sex hormone binding globulin [0125] SPE solid phase extraction [0126] STS sulfatase [0127] T testosterone [0128] t triplet [0129] T-47D cell line derived from a human breast carcinoma [0130] TBAF tetrabutylammonium fluoride [0131] TBS t-butyldimethylsilyl [0132] TES triethylsilyl [0133] THF tetrahydrofuran [0134] TLC thin layer chromatography [0135] TMS trimethylsilyl [0136] T.sub.R retention time [0137] U2OS human osteosarcoma cells [0138] UV ultra violet rays [0139] wt wild type [0140] wt. % weight percent [0141] .sup.5-diol androstendiol (androst-5-en-3(3,17-diol)
Synthetic Procedures
[0142] .sup.1H NMR spectra were measured at 400.1 and 500.1 MHz at 24 C. on Bruker AVANCE-400 and 500 spectrometers. .sup.13C NMR spectra were measured at 100.8 MHz. For standardization of .sup.1H NMR spectra the internal signal of tetramethylsilane ( 0.0, CDCl.sub.3) or residual signals of deuterochloroform ( 7.26) or residual signals of deuteromethanol ( 3.31) were used. In the case of .sup.13C spectra the residual signal of deuterochloroform ( 77.00) or residual signals of deuteromethanol ( 49.00) were used. The chemical shifts are given in ppm ( scale); the coupling constants J are given in Hz. Signal multiplicities are designated as follows: s singlet, d doublet, t triplet, q quadruplet, m multiplet, b denotes a broad signal. Melting points were measured on a Kofler bench. Optical rotations were measured on Autopol IV polarimeter (Rudolph Research Analytical, Flanders, USA), [].sub.p values are given in 10.sup.1.Math.deg.Math.cm.sup.2.Math.g.sup.1 and were compensated to a standard temperature of 20 C. Infrared spectra were measured in sample solutions or in KBr tablets using a Bruker IFS 55 spectrometer; frequency is given in cm.sup.1. Mass spectra were measured on a ZAB-EQ spectrometer (at 70 eV) or LCQ Classic (Thermo Finnigan). HPLC chromatography was performed on a Waters 600 device with diode array detector PDA 2996. Fluka 60 silica gel was used for column chromatography, aluminium plates coated with a layer of silica gel 60 FB.sub.254B were used for thin layer chromatography (TLC). KMnO.sub.4 and phosphomolybdic acid solutions and UV detection were used to visualize TLC.
Synthesis of the Starting Compound
Example 1: 3-(t-Butyldimethylsilyloxy)-estra-1,3,5(10),15-tetraen-17-one (1)
[0143] ##STR00003##
[0144] Enone 1 was prepared according to a published method (Sakakibara, M.; Uchida, A. O. Biosci. Biotech. Biochchem 1996, 60, 3, 405) by Saegusa oxidation in 75% yield: mp 148 C.; [].sub.D 37.4 (c 0.203; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 0.19 (s, 6H, Si(CH.sub.3).sub.2), 0.98 (s, 9H, (CH.sub.3).sub.3C), 1.11 (s, 3H, H-18), 1.55 (m, 1H, H-7a), 1.66-1.85 (m, 3H, H-8, 11a, 12a), 2.01 (m, 1H, H-12b), 2.18 (dm, 1H, J=12.9 Hz, H-7b), 2.33 (m, 1H, H-9), 2.43 (m, 1H, H-11a), 2.50 (dm, 1H, J=11.6 Hz, H-14), 2.87-2.96 (m, 2H, H-6), 6.08 (dd, 1H, J=6.0; 3.2 Hz, H-16), 6.59 (bd, 1H, J=2.7 Hz, H-4), 6.64 (dd, 1H, J=8.5; 2.7 Hz, H-2), 7.12 (d, 1H, J=8.5 Hz, H-8), 7.63 (dd, 1H, J=6.0; 1.9 Hz, H-15); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 4.41 (Si(CH.sub.3).sub.2), 181.5 (C(CH.sub.3).sub.3), 20.97 (C-18), 25.34 (C-11), 25.67 (C(CH.sub.3).sub.3), 26.68 (C-7), 29.08 and 29.19 (C-6, 12), 35.48 (C-8), 45.15 (C-9), 51.46 (C-13), 56.13 (C-14), 117.33 (C-3), 120.01 (C-4), 125.82 (C-1), 131.87 (C-16), 132.28 (C-10), 137.31 (C-5), 153.58 (C-3), 158.26 (C-15), 213.09 (C-17); IR (CHCl.sub.3) 3076, 3050, 3029, 2960, 2932, 2897, 2860, 1705, 1615, 1607, 1569, 1497, 1472, 1463, 1443, 1436, 1417, 1391, 1371, 1363, 1258, 1186, 1104, 1005, 941, 885, 697, 582, 449 cm.sup.1; HR-MS (ESI) calculated for C.sub.24H.sub.34O.sub.2SiNa [M+Na.sup.+] 405.22203 found 405.22212. R.sub.f (7/1 hexane/EtOAc)=0.6.
Example 2: 3-(t-Butyldimethylsilyloxy)-15-vinyl-estra-1,3,5(10)-trien-17-one (2)
[0145] ##STR00004##
[0146] To our knowledge, the preparation of 15-vinylestrone was published only in WO200834796. We altered this method so that instead of the benzyl protecting group (giving a reaction yield of 24%) a t-butyldimethylsilyl group was now used. We succeeded to significantly increase yields of vinylestrone 2, which (after crystallization from EtOAc) are reproducibly above 90%.
[0147] Mixture of 1 mol.sup..Math.l.sup.1 solution of vinylmagnesium bromide in THF (13 ml, 13.08 mmol), CuI (65 mg, 0.654 mmol) and HMPA (2.8 ml, 15.7 mmol) in CH.sub.2Cl.sub.2 (50 ml) under argon atmosphere was cooled to 78 C. A solution of enone 1 (2.5 g, 6.54 mmol) and TMSCl (1.7 ml, 13.08 mmol) in CH.sub.2Cl.sub.2 (50 ml) was added dropwise to this mixture. The reaction mixture was then slowly warmed to room temperature and stirred overnight. After adding water and dropwise addition of 1 mol.Math.l.sup.1 HCl, the mixture was stirred for 5 minutes, then diluted with CH.sub.2Cl.sub.2 and washed with water. The combined organic phases were washed with saturated NaCl solution, dried over MgSO.sub.4 and concentrated under reduced pressure. After chromatography on silica gel (1/1 hexane/CH.sub.2Cl.sub.2) 2.5 g vinylestrone 2 was obtained in 92% yield as a white crystalline solid: mp 141 C.; [].sub.D+52.7 (c 0.186; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 0.19 (s, 6H, Si(CH.sub.3).sub.2), 0.98 (s, 9H, (CH.sub.3).sub.3C), 1.03 (s, 3H, H-18), 1.41-1.56 (m, 3H, H-7a, 11a, 12a), 1.72-1.83 (m, 2H, H-8, 14), 1.90 (m, 1H, H-12b), 2.15 (m, 1H, H-7b), 2.28 (m, 1H, H-9), 2.36 (m, 1H, H-11a), 2.52 (dd, 1H, J=19.5; 9.0 Hz, H-16b), 2.63 (dd, 1H, J=19.5; 2.2 Hz, H-16a), 2.80-2.92 (m, 2H, H-6), 3.13 (m, 1H, H-15), 5.13 (dt, 1H, J=10.4; 1.6 Hz, H-2b), 5.17 (dt, 1H, J=17.2; 1.6 Hz, H-2a), 6.10 (ddd, 1H, J=17.2; 10.4; 6.7 Hz, H-1), 6.58 (dm, 1H, J=2.7 Hz, H-4), 6.62 (dd, 1H, J=8.4; 2.7 Hz, H-2), 7.11 (dd, 1H, J=8.6; 0.9 Hz, H-1); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 4.40 (Si(CH.sub.3).sub.2), 16.87 (C-18), 18.17 (C(CH.sub.3).sub.3), 25.48 (C-11), 25.70 (C(CH.sub.3).sub.3), 26.31 (C-7), 29.24 (C-6), 33.38 (C-12), 35.94 (C-8), 37.10 (C-15), 41.75 (C-16), 44.50 (C-9), 47.69 (C-13), 52.69 (C-14), 115.96 (C-2), 117.24 (C-2), 119.98 (C-4), 125.85 (C-1), 132.63 (C-10), 137.62 (C-5), 139.00 (C-1), 153.54 (C-3), 220.40 (C-17); IR (CHCl.sub.3) 3080, 2960, 2932, 2895, 2860, 1732, 1637, 1607, 1570, 1497, 1472, 1463, 1442, 1435, 1419, 1404, 1391, 1377, 1362, 1256, 1186, 1159, 1100, 1008, 1000, 941, 922, 883, 841, 806, 697, 586, 447 cm.sup.1; HR-MS (ESI) calculated for C.sub.26H.sub.38O.sub.2SiNa [M+Na.sup.+] 433.25333 found 433.25330. R.sub.f (7/1 hexane/EtOAc)=0.5.
a) General Procedure for Metathesis of Vinylestrone 2 with Various Olefins
[0148] To the solution of vinylestrone 2 (100 mg, 0.244 mmol) and second olefin (0.488 mmol) in the mixture of CH.sub.2Cl.sub.2/trifluorotoluene (21 ml, 2/1), Hoveyda Grubbs second generation catalyst (15 mg, 0.024 mmol) (Sigma Aldrich, catalog number 569755) and CuI (5 mg, 0.024 mmol) were added under argon atmosphere and the resulting mixture was stirred at 40-70 C., preferably 65 C. for 4-12 h, preferably for 4 h. After further addition of second olefin (0.488 mmol) and catalyst (7.5 mg, 0.012 mmol), the mixture was further stirred at the same temperature overnight. Then the solvents were removed under reduced pressure and chromatography of the residue on silica gel (hexane/EtOAc 95/5) yielded the cross metathesis product. Yields of vinylestrone 2 metathesis with various olefins were in the range of 56-98% for majority of the prepared compounds.
b) General Procedure for C-3 TBS Group Deprotection
[0149] A solution of TBAF, 1 mol.sup..Math.l.sup.1 in THF (1 eq) was added dropwise to the solution of metathesis product in THF at room temperature. After 1 h, water was added and the reaction mixture was extracted with CH.sub.2Cl.sub.2 and/or CHCl.sub.3. The combined organic phases were washed with saturated NaCl solution, dried over MgSO.sub.4 and the solvents were removed under reduced pressure. Chromatography on silica gel yielded the deprotected product. The yields of deprotection reactions always exceeded 90%.
c) General Procedure for Reduction of the Double Bond
[0150] Flask with a mixture of deprotected metathesis product and Pd/C (10 wt. %) catalyst in EtOAc was evacuated under vigorous stirring and then filled with hydrogen. The reaction mixture was stirred overnight. The progress of the reaction was monitored by TLC using KMnO.sub.4 solution for selective visualization of the starting material since R.sub.f values of starting material and product were always the same. Additionally, starting material was usually visible under UV light (254 nm), while the reduced product was not visible at the same wavelength. The reaction mixture was then filtered through Celite, solvents were removed under reduced pressure and chromatography on HPLC (MeCN/H.sub.2O) resulted in final colorless crystalline products. The yields were always higher than 90%.
Example 3: 15-Phenethyl-3-hydroxy-estra-1,3,5(10)-trien-17-one (3)
[0151] ##STR00005##
[0152] Compound 3 was prepared according to the above general procedure by reacting vinylestrone 2 with styrene (56 l). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=15 min) yielded 36 mg of colorless solid 3 (numbering of the C-15 side chain in all the following examples is the same as in derivative 3): mp 153 C.; [].sub.D+52.6 (c 0.547; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.03 (s, 3H, H-18), 1.35-1.55 (m, 3H, H-7a, 11a, 12a), 1.62-1.76 (m, 3H, H-8, 14, 1a), 1.86-1.97 (m, 3H, H-7a, 12b, 1b), 2.25 (m, 1H, H-9), 2.30-2.38 (m, 2H, H-11b, 15), 2.40 (dd, 1H, J=19.3; 2.8 Hz, H-16a), 2.49 (dd, 1H, J=19.3; 8.1 Hz, H-16b), 2.54 (ddd, 1H, J=13.6; 9.2; 7.1 Hz, H-2a), 2.75 (ddd, 1H, J=13.6; 9.8; 5.2 Hz, H-2b), 2.80-2.93 (m, 2H, H-6), 4.82 (bs, 1H, OH), 6.59 (dm, 1H, J=2.8 Hz, H-4), 6.63 (ddm, 1H, J=8.4; 2.8 Hz, H-2), 7.13 (dd, 1H, J=8.5; 1.1 Hz, H-1), 7.18 (m, 2H, H-2), 7.21 (m, 1H, H-4), 7.30 (m, 2H, H-3); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.71 (C-18), 25.47 (C-11), 26.57 (C-7), 29.30 (C-6), 33.09 (C-1), 33.73 (C-15), 33.82 (C-12), 35.80 (C-2), 35.91 (C-8), 42.70 (C-16), 44.47 (C-9), 47.19 (C-13), 52.77 (C-14), 112.70 (C-2), 115.22 (C-4), 126.04 (C-4), 126.16 (C-1), 128.41 (C-2), 128.46 (C-3), 132.33 (C-10), 138.00 (C-5), 141.65 (C-1), 153.54 (C-3), 221.37 (C-17); IR (CHCl.sub.3) 3599, 3413, 3086, 3064, 3027, 2929, 2861, 1729, 1603, 1585, 1499, 1454, 1465, 1378, 1261, 1178, 1166, 1151, 1124, 1030, 903, 701, 472 cm.sup.1; HR-MS (APCI) calculated for C.sub.26H.sub.30O.sub.2Na [M+Na.sup.+] 397.21380 found 397.21384. R.sub.f (4/1 hexane/EtOAc)=0.4.
Example 4: 15-(4-(Trifluoromethyl)phenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (4)
[0153] ##STR00006##
[0154] Compound 4 was prepared according to the above general procedure by reacting vinylestrone 2 with 4-(trifluoromethyl)styrene (72 l). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=19 min) yielded 24 mg of colorless solid 4: mp 168 C.; [].sub.D+51.5 (c 0.136; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.03 (s, 3H, H-18), 1.37-1.55 (m, 3H, H-7a, 11a, 12a), 1.60-1.76 (m, 3H, H-8, 14, 1a), 1.83-1.97 (m, 3H, H-7a, 12b, lb), 2.26 (m, 1H, H-9), 2.30-2.38 (m, 2H, H-11b, 15), 2.41 (dd, 1H, J=19.4, 2.5 Hz, H-16a), 2.47 (dd, 1H, J=19.4; 8.5 Hz, H-16b), 2.60 (bddd, 1H, J=13.8; 9.0; 7.3 Hz, 2a), 2.81 (bddd, 1H, J=13.7; 9.8; 5.3 Hz, 2b), 2.82-2.92 (m, 2H, H-6), 4.91 (s, 1H, OH), 6.59 (bd, 1H, J=2.8 Hz, H-4), 6.64 (bdd, 1H, J=8.5; 2.8 Hz, H-2), 7.13 (bd, 1H, J=8.5 Hz, H-1), 7.29 (m, 2H, H-2), 7.56 (m, 1H, H-3); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.72 (C-18), 25.43 (C-11), 26.60 (C-7), 29.24 (C-6), 32.82 (C-1), 33.71 (C-15), 33.80 (C-12), 35.59 (C-2), 35.87 (C-8), 42.54 (C-16), 44.43 (C-9), 47.18 (C-13), 52.70 (C-14), 112.75 (C-2), 115.21 (C-4), 124.24 (q, J.sup.C-F =271.7 Hz, CF.sub.3), 125.40 (q, J.sup.C-F=3.8 Hz, C-3), 126.15 (C-1), 128.46 (q, J.sup.C-F=32.4 Hz, C-4), 128.71 (C-2), 132.21 (C-10), 137.88 (C-5), 145.73 (q, J.sup.C-F=1.3 Hz, C-1), 153.61 (C-3), 220.97 (C-17); .sup.19F NMR (470.3 MHz, CDCl.sub.3) 58.46; IR (CHCl.sub.3) 3598, 3433, 3060, 2938, 2864, 1730, 1618, 1560, 1466, 1453, 1418, 1377, 1326, 1249, 1167, 1128, 1108, 1068, 1095, 1057, 1019, 834, 611, 446 cm.sup.1; HR-MS (APCI) calculated for C.sub.27H.sub.29O.sub.2F.sub.3Na [M+Na.sup.+] 465.20119 found 465.20102. R.sub.f (4/1 hexane/EtOAc)=0.4.
Example 5: 15-(4-Fluorophenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (5)
[0155] ##STR00007##
[0156] Compound 5 was prepared according to the above general procedure by reacting vinylestrone 2 with 4-fluorostyrene (58 l). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=21 min) yielded 26 mg of colorless solid 5: mp 164 C.; [].sub.D+33.8 (c 0.222; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.03 (s, 3H, H-18), 1.34-1.56 (m, 3H, H-7a, 11a, 12a), 1.58-1.77 (m, 3H, H-8, 14, 1a), 1.82-1.96 (m, 3H, H-7a, 12b, 1b), 2.26 (m, 1H, H-9), 2.28-2.38 (m, 2H, H-11b, 15), 2.39 (bdd, 1H, J=19.4, 2.5 Hz, H-16a), 2.45 (bdd, 1H, J=19.4; 8.5 Hz, H-16b), 2.50 (m, 1H, 2a), 2.72 (m, 1H, 2b), 2.78-2.94 (m, 2H, H-6), 5.03 (bs, 1H, OH), 6.60 (bd, 1H, J=2.7 Hz, H-4), 6.64 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 6.99 (m, 1H, H-2), 7.07-7.20 (m, 3H, H-1, 3); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.71 (C-18), 25.44 (C-11), 26.56 (C-7), 29.27 (C-6), 33.16 (C-1), 33.56 (C-15), 33.79 (C-12), 34.90 (C-2), 35.88 (C-8), 42.64 (C-16), 44.43 (C-9), 47.20 (C-13), 52.72 (C-14), 112.73 (C-2), 115.20 (d, J.sup.C-F=21.2 Hz, C-3), 115.22 (C-4), 126.14 (C-1), 129.72 (d, J.sup.C-F=7.8 Hz, C-2), 132.20 (C-10), 137.21 (d, J.sup.C-F=3.3 Hz, C-1), 137.92 (C-5), 153.62 (C-3), 161.32 (d, J.sup.C-F=243.7 Hz, C-4), 221.3 (C-17); .sup.19F NMR (470.3 MHz, CDCl.sub.3) 110.93 (m, 1F); IR (CHCl.sub.3) 3598, 3431, 3028, 2936, 2864, 1729, 1610, 1585, 1502, 1466, 1453, 1440, 1378, 1281, 1248, 1190, 1157, 1095, 1057, 983, 959, 939, 877, 582, 472 cm.sup.1; HR-MS (APCI) calculated for C.sub.26H.sub.29O.sub.2FNa [M+Na.sup.+] 415.20438 found 415.20447. R.sub.f(4/1 hexane/EtOAc)=0.4.
Example 6: 15-(4-Chlorophenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (6)
[0157] ##STR00008##
[0158] Compound 6 was prepared according to the above general procedure by reacting vinylestrone 2 with 4-chlorostyrene (59 l). Chromatography on HPLC (35/65 MeCN/H.sub.2O, t.sub.R=30 min) yielded 34 mg of colorless solid 6: mp 168 C.; [].sub.D+69.3 (c 0.150; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.02 (s, 3H, H-18), 1.36-1.56 (m, 3H, H-7a, 11a, 12a), 1.65 (m, 1H, H-1a), 1.64-1.76 (m, 2H, H-8, 14), 1.84-1.94 (m, 3H, H-1b, 7b, 12b), 2.26 (m, 1H, H-9), 2.26-2.38 (m, 2H, H-11b, 15), 2.40 (dd, 1H, J=19.3, 2.6 Hz, H-16a), 2.44 (dd, 1H, J=19.3; 8.3 Hz, H-16b), 2.50 (ddd, 1H, J=13.8; 8.9; 7.3 Hz, H-1a), 2.72 (ddd, 1H, J=13.8; 9.5; 5.2 Hz, H-1b), 2.81-2.93 (m, 2H, H-6), 4.70 (s, 1H, OH), 6.59 (d, 1H, J=2.8 Hz, H-4), 6.63 (dd, 1H, J=8.4; 2.8 Hz, H-2), 7.10 (m, 2H, H-2), 7.13 (d, 1H, J=8.4 Hz, H-1), 7.21 (m, 2H, H-3); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.73 (C-18), 25.45 (C-11), 26.60 (C-7), 29.27 (C-6), 32.96 (C-1), 33.62 (C-15), 33.82 (C-12), 35.08 (C-2), 35.89 (C-8), 42.58 (C-16), 44.45 (C-9), 47.17 (C-13), 52.73 (C-14), 112.73 (C-2), 115.21 (C-4), 126.17 (C-1), 128.57 (C-3), 129.74 (C-2), 131.79 (C-4), 132.33 (C-10), 137.95 (C-5), 140.04 (C-1), 153.54 (C-3), 220.95 (C-17); IR (KBr) 3372, 1728, 1711, 1618, 1611, 1501, 1492, 1464, 1407, 1375, 1015 cm.sup.1; HR-MS (APCI) calculated for C.sub.26H.sub.30O.sub.2Cl [M+H.sup.+] 409.19288 found 409.19284. R.sub.f (4/1 hexane/EtOAc)=0.4.
Example 7: Methyl 4-(2-(3-hydroxy-estra-1,3,5(10)-trien-17-on-15-yl)ethyl)benzoate (7)
[0159] ##STR00009##
[0160] Compound 7 was prepared according to the above general procedure by reacting vinylestrone 2 with methyl 4-vinylbenzoate (62 l). Chromatography on HPLC (40/60 MeCN/H.sub.2O, t.sub.R=30 min) yielded 17 mg of colorless solid 7: mp 139 C.; [].sub.D+70.2 (c 0.084; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.02 (s, 3H, H-18), 1.34-1.55 (m, 3H, H-7a, 11a, 12a), 1.65-1.76 (m, 3H, H-1a, 8, 14), 1.82-1.98 (m, 3H, H-1b, 7b, 12b), 2.25 (m, 1H, H-9), 2.28-2.38 (m, 2H, H-11b, 15), 2.39 (dd, 1H, J=19.3; 2.6 Hz, H-16a), 2.45 (dd, 1H, J=19.3; 8.4 Hz, H-16b), 2.60 (ddd, 1H, J=13.7; 8.8; 7.2 Hz, H-2a), 2.80 (ddd, 1H, J=13.7; 9.6; 5.3 Hz, H-2b), 2.80-2.93 (m, 2H, H-6), 3.91 (s, 3H, OCH.sub.3), 5.01 (bs, 1H, OH), 6.59 (bd, 1H, J=2.8 Hz, H-4), 6.64 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 7.12 (dd, 1H, J=8.5; 1.0 Hz, H-1), 7.25 (m, 2H, H-2), 7.98 (m, 2H, H-3); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.73 (C-18), 25.43 (C-11), 25.59 (C-7), 29.25 (C-6), 32.68 (C-1), 33.64 (C-15), 33.80 (C-12), 35.73 (C-2), 35.87 (C-8), 42.53 (C-16), 44.43 (C-9), 47.17 (C-13), 52.07 (OCH.sub.3), 52.71 (C-14), 112.74 (C-2), 115.22 (C-4), 126.13 (C-1), 128.05 (C-4), 128.48 (C-2), 129.84 (C-3), 132.19 (C-10), 137.90 (C-5), 147.14 (C-1), 153.65 (C-3), 167.09 (CO), 220.99 (C-17); IR (KBr) 3406, 3020, 1733, 1720, 1700, 1610, 1584, 1502, 1436, 1415, 1351, 1282, 964 cm.sup.1; HR-MS (APCI) calculated for C.sub.28H.sub.33O.sub.4 [M+H.sup.+] 433.23734 found 433.23716. R.sub.f (4/1 hexane/EtOAc)=0.2.
Example 8: 3-Hydroxy-15-(2-(naphtalen-2-yl)ethyl)-estra-1,3,5(10)-trien-17-one (8)
[0161] ##STR00010##
[0162] Compound 8 was prepared according to the above general procedure by reacting vinylestrone 2 with 2-vinylnaphthalene (75 mg). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=38 min) yielded 13 mg of colorless solid 8: mp 201 C.; [].sub.D+77.0 (c 0.309; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.05 (s, 3H, H-18), 1.32-1.55 (m, 3H, H-7a, 11a, 12a), 1.66-1.75 (m, 2H, H-8, 14), 1.76 (dddd, 1H, J=13.6; 11.7; 8.9; 5.3 Hz, H-1a), 1.86-1.95 (m, 2H, H-7b, 12b), 2.02 (dddd, 1H, J=13.6; 9.5; 7.2; 2.1 Hz, H-1b), 2.24 (m, 1H, H-9), 2.31-2.42 (m, 2H, H-11b, 15), 2.40-2.52 (m, 2H, H-16), 2.71 (ddd, 1H, J=15.8; 8.9; 7.2 Hz, H-2a), 2.78-2.91 (m, 2H, H-6), 2.92 (ddd, 1H, J=13.8; 9.5; 5.3 Hz, H-2b), 5.11 (bs, 1H, OH), 6.59 (bd, 1H, J=2.8 Hz, H-4), 6.64 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 7.12 (dd, 1H, J=8.5; 1.1 Hz, H-1), 7.32 (dd, 1H, J=8.4; 1.8 Hz, H-3), 7.44 (bddd, 1H, J=8.0; 6.8; 1.4 Hz, H-6), 7.47 (bddd, 1H, J=8.1; 6.8; 1.5 Hz, H-7), 7.61 (m, 1H, H-1), 7.79 (m, 1H, H-8), 7.80 (bd, 1H, J=8.4 Hz, H-4), 7.82 (m, 1H, H-5); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.74 (C-18), 25.47 (C-11), 26.62 (C-7), 29.27 (C-6), 32.85 (C-1), 33.75 (C-15), 32.85 (C-12), 35.89 (C-2), 35.95 (C-8), 42.71 (C-16), 44.47 (C-9), 47.24 (C-13), 52.82 (C-14), 112.74 (C-2), 115.24 (C-4), 125.30 (C-6), 126.07 (C-1), 126.11 (C-7), 126.51 (C-1), 127.07 (C-3), 127.35 (C-8), 127.64 (C-4), 128.09 (C-5), 132.06 (C-4a), 132.28 (C-10), 133.58 (C-8a), 137.97 (C-5), 139.07 (C-2), 153.65 (C-3), 221.38 (C-17); IR (KBr) 3371, 3052.3018, 1729, 1719, 1610, 1601, 1584, 1502, 1451, 1442 cm.sup.1; HR-MS (APCI) calculated for C.sub.30H.sub.33O.sub.2 [M+H.sup.+] 425.24751 found 425.24746. R.sub.f(4/1 hexane/EtOAc)=0.4.
Example 9: 3-Hydroxy-15-(4-methoxyphenethyl)-estra-1,3,5 (10)-trien-17-one (9)
[0163] ##STR00011##
[0164] Compound 9 was prepared according to the above general procedure by reacting vinylestrone 2 with 4-methoxystyrene (65 l). Chromatography on HPLC (37/63 MeCN/H.sub.2O, t.sub.R=30 min) yielded 24 mg of colorless solid 9: mp 159 C.; [].sub.D+48.0 (c 0.154; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.02 (s, 3H, H-18), 1.35-1.55 (m, 3H, H-7a, 11a, 12a), 1.63 (m, 1H, H-1), 1.65-1.76 (m, 2H, H-8, 14), 1.85-1.94 (m, 3H, H-1b, 7b, 12b), 2.25 (m, 1H, H-9), 2.28-2.36 (m, 2H, H-11b, 15), 2.39 (dd, 1H, J=19.4; 2.7 Hz, H-16a), 2.44 (dd, 1H, J=19.4; 8.7 Hz, H-16b), 2.49 (ddd, 1H, J=13.8; 9.0; 7.2 Hz, H-2a), 2.69 (ddd, 1H, J=13.8; 9.7; 5.3 Hz, H-2b), 2.79-2.93 (m, 2H, H-6), 3.80 (s, 3H, OCH.sub.3), 4.69 (bs, 1H, OH), 6.59 (bd, 1H, J=2.8 Hz, H-4), 6.63 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 6.84 (m, 2H, H-3), 7.09 (m, 2H, H-2), 7.13 (bd, 1H, J=8.4 Hz, H-1); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.71 (C-18), 25.49 (C-11), 26.59 (C-7), 29.32 (C-6), 33.27 (C-1), 33.66 (C-15), 33.83 (C-12), 34.87 (C-2), 35.93 (C-8), 42.17 (C-16), 44.49 (C-9), 47.19 (C-13), 52.79 (C-14), 55.27 (OCH.sub.3), 112.69 (C-2), 113.86 (C-3), 115.22 (C-4), 126.18 (C-1), 129.30 (C-2), 132.39 (C-10), 133.69 (C-1), 130.06 (C-1), 138.03 (C-5), 153.51 (C-3), 157.89 (C-4), 221.31 (C-17); IR (KBr) 3386, 3059, 2835, 1730, 1718, 1619, 1583, 1512, 1452, 1442, 1246, 1035, 965, 708 cm.sup.1; HR-MS (APCI) calculated for C.sub.27H.sub.33O.sub.3 [M+H.sup.+] 405.24242 found 405.24235. R.sub.f (4/1 hexane/EtOAc)=0.3.
Example 10: 3-Hydroxy-15-(3-methoxyphenethyl)-estra-1,3,5(10)-trien-17-one (10)
[0165] ##STR00012##
[0166] Compound 10 was prepared according to the above general procedure by reacting vinylestrone 2 with 3-methoxystyrene (66 l). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=16 min) yielded 52 mg of colorless solid 10: mp 178 C.; [].sub.D+58.3 (c 1.706; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.03 (s, 3H, H-18), 1.36-1.50 (m, 2H, H-7a, 12a), 1.50 (m, 1H, H-11a), 1.61-1.76 (m, 3H, H-1a, 8, 14), 1.86-1.96 (m, 3H, H-1b, 7b, 12b), 2.25 (m, 1H, H-9), 2.23-2.38 (m, 2H, H-11b, 15), 2.40 (dd, 1H, J=19.4; 2.8 Hz, H-16a), 2.46 (dd, 1H, J=19.4; 8.1 Hz, H-16b), 2.51 (ddd, 1H, J=13.8; 9.1; 7.1 Hz, H-2a), 2.73 (ddd, 1H, J=13.7; 9.7; 5.3 Hz, H-2b), 2.80-2.93 (m, 2H, H-6), 3.81 (s, 3H, OCH.sub.3), 5.00 (bs, 1H, OH), 6.59 (bd, 1H, J=2.8 Hz, H-4), 6.64 (ddm, 1H, J=8.4; 2.8 Hz, H-2), 6.73 (bdd, 1H, J=2.6; 1.6 Hz H-2), 6.76 (ddd, 1H, J=8.2; 2.6; 1.0 Hz, H-4), 6.78 (ddd, 1H, J=7.5; 1.6; 1.0 Hz, H-6), 7.13 (dd, 1H, J=8.5; 1.1 Hz, H-1), 7.22 (bt, 1H, J=7.8 Hz, H-5); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.70 (C-18), 25.46 (C-11), 26.58 (C-7), 29.29 (C-6), 32.91 (C-1), 33.72 (C-15), 33.80 (C-12), 35.81 (C-2), 35.90 (C-8), 42.68 (C-16), 44.47 (C-9), 47.21 (C-13), 52.74 (C-14), 55.16 (OCH.sub.3), 111.03 (C-4), 112.70 (C-2), 114.41 (C-2), 115.22 (C-4), 120.83 (C-6), 126.15 (C-1), 129.44 (C-5), 132.24 (C-10), 137.96 (C-5), 143.29 (C-1), 153.59 (C-3), 159.64 (C-3), 221.54 (C-17); IR (CHCl.sub.3) 3598, 1729, 1611, 1602, 1594, 1585, 1500, 1489, 1439, 1281, 1259, 1233, 1191, 1165, 1153, 1017, 616 cm.sup.1; HR-MS (APCI) calculated C.sub.27H.sub.33O.sub.3[M+H.sup.+] 405.24242 found 405.24247. R.sub.f (4/1 hexane/EtOAc)=0.3.
Example 11: 3-Hydroxy-15-(3,4-dimethoxyphenethyl)-estra-1,3,5(10)-trien-17-one (11)
[0167] ##STR00013##
[0168] Compound 11 was prepared according to the above general procedure by reacting vinylestrone 2 with 3,4-dimethoxystyrene (72 l). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=14 min) yielded 17 mg of colorless solid 11: mp 146 C.; [].sub.D+55.3 (c 0.347; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.03 (s, 3H, H-18), 1.31-1.56 (m, 3H, H-7a, 11a, 12a), 1.60-1.77 (m, 3H, H-1a, 8, 14), 1.85-1.95 (m, 3H, H-1b, 7b, 12b), 2.25 (m, 1H, H-9), 2.28-2.38 (m, 2H, H-11b, 15), 2.37-2.52 (m, 3H, H-16, H-2a), 2.71 (ddd, 1H, J=13.8; 9.3; 5.2 Hz, H-2b), 2.78-2.92 (m, 2H, H-6), 3.86 (s, 3H, OCH.sub.3), 3.88 (s, 3H, OCH.sub.3), 5.62 (bs, 1H, OH), 6.60 (bdm, 1H, J=2.8 Hz, H-4), 6.65 (bdd, 1H, J=8.5, 2.8 Hz, H-2), 6.70 (d, 1H, J=2.0 Hz, H-2), 6.72 (dd, 1H, J=8.1; 2.8 Hz, H-6), 6.81 (d, 1H, J=8.1 Hz, H-5), 7.12 (bd, 1H, J=8.5 Hz, H-1); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.69 (C-18), 25.41 (C-11), 26.56 (C-7), 29.25 (C-6), 33.03 (C-1), 33.49 (C-15), 33.75 (C-12), 35.20 (C-2), 35.87 (C-8), 42.64 (C-16), 44.40 (C-9), 47.22 (C-13), 52.67 (C-14), 55.80 (OCH.sub.3), 55.87 (OCH.sub.3), 111.19 (C-5), 111.62 (C-2), 112.73 (C-2), 115.22 (C-4), 120.29 (C-6), 126.06 (C-1), 131.98 (C-10), 134.17 (C-1), 137.79 (C-5), 147.24 (C-4), 148.79 (C-3), 153.79 (C-3), 221.87 (C-17); IR (CHCl.sub.3) 3598, 2839, 1610, 1591, 1516, 1501, 1454, 1442, 1260, 1193, 1155, 1029, 870, 808, 581, 544 cm.sup.1; HR-MS (APCI) calculated C.sub.34H.sub.47O.sub.4Si [M+Si] 547.32381 found 547.32368. R.sub.f(4/1 hexane/EtOAc)=0.25.
Example 12: 15-(4-Ethoxyphenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (12)
[0169] ##STR00014##
[0170] Compound 12 was prepared according to the above general procedure by reacting vinylestrone 2 with 4-ethoxystyrene (73 l). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=17 min) yielded 62 mg of colorless solid 12: mp 173 C.; [].sub.D+62.5 (c 0.208; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.02 (s, 3H, H-18), 1.35-1.49 (m, 2H, H-7a, 12a), 1.41 (t, 3H, J=7.0 Hz, OCH.sub.2CH.sub.3), 1.50 (m, 1H, H-11a), 1.58-1.75 (m, 3H, H-1a, 8, 14), 1.84-1.93 (m, 3H, H-1b, 7b, 12b), 2.25 (m, 1H, H-9), 2.28-2.37 (m, 2H, H-11b, 15), 2.39 (dd, 1H, J=19.4, 2.8 Hz, H-16a), 2.44 (dd, 1H, J=19.4; 8.1 Hz, H-16b), 2.47 (ddd, 1H, J=13.8; 9.0; 7.1 Hz, H-2a), 2.69 (ddd, 1H, J=13.8; 9.6; 5.3 Hz, H-2b), 2.78-2.93 (m, 2H, H-6), 4.02 (q, 2H, OCH.sub.2CH.sub.3), 4.83 (bs, 1H, OH), 6.59 (bdt, 1H, J=2.8; 1.0; 1.0 Hz, H-4), 6.63 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 6.83 (m, 2H, H-3), 7.08 (m, 2H, H-2), 7.13 (dd, 1H, J=8.5; 1.1 Hz, H-1); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 14.87 (OCH.sub.2CH.sub.3), 17.70 (C-18), 25.48 (C-11), 26.57 (C-7), 29.32 (C-6), 33.25 (C-1), 33.63 (C-15), 33.82 (C-12), 34.86 (C-2), 35.91 (C-8), 42.72 (C-16), 44.48 (C-9), 47.20 (C-13), 52.77 (C-14), 63.42 (OCH.sub.2CH.sub.3), 112.69 (C-2), 114.44 (C-3), 115.22 (C-4), 126.16 (C-1), 129.28 (C-2), 132.33 (C-10), 133.55 (C-1), 138.01 (C-5), 153.54 (C-3), 157.23 (C-4), 221.46 (C-17); IR (CHCl.sub.3) 3598, 3098, 3060, 1729, 1611, 1583, 1512, 1502, 1453, 1441, 1245, 1042 cm.sup.1; HR-MS (APCI) calculated C.sub.28H.sub.35O.sub.3 [M+H.sup.+] 419.25807 found 419.25810. R.sub.f (4/1 hexane/EtOAc)=0.3.
Example 13: 15-(4-t-Butoxyphenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (13)
[0171] ##STR00015##
[0172] Compound 13 was prepared according to the above general procedure by reacting vinylestrone 2 with 4-t-butoxystyrene (92 l). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=17 min) yielded 94 mg of colorless solid 13: mp 192 C.; [].sub.D+62.5 (c 0.208; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.02 (s, 3H, H-18), 1.35-1.49 (m, 2H, H-7a, 12a), 1.41 (t, 3H, J=7.0 Hz, OCH.sub.2CH.sub.3), 1.50 (m, 1H, H-11a), 1.58-1.75 (m, 3H, H-1a, 8, 14), 1.84-1.93 (m, 3H, H-1b, 7b, 12b), 2.25 (m, 1H, H-9), 2.28-2.37 (m, 2H, H-11b, 15), 2.39 (dd, 1H, J=19.4; 2.8 Hz, H-16a), 2.44 (dd, 1H, J=19.4; 8.1 Hz, H-16b), 2.47 (ddd, 1H, J=13.8; 9.0; 7.1 Hz, H-2a), 2.69 (ddd, 1H, J=13.8; 9.6; 5.3 Hz, H-2b), 2.78-2.93 (m, 2H, H-6), 4.02 (q, 2H, OCH.sub.2CH.sub.3), 4.83 (bs, 1H, OH), 6.59 (bdt, 1H, J=2.8; 1.0; 1.0 Hz, H-4), 6.63 (bdd, 1H, J=8.4; 2.8 Hz, H-2), 6.83 (m, 2H, H-3), 7.08 (m, 2H, H-2), 7.13 (dd, 1H, J=8.5; 1.1 Hz, H-1); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 14.87 (OCH.sub.2CH.sub.3), 17.70 (C-18), 25.48 (C-11), 26.57 (C-7), 29.32 (C-6), 33.25 (C-1), 33.63 (C-15), 33.82 (C-12), 34.86 (C-2), 35.91 (C-8), 42.72 (C-16), 44.48 (C-9), 47.20 (C-13), 52.77 (C-14), 63.42 (OCH.sub.2CH.sub.3), 112.69 (C-2), 114.44 (C-3), 115.22 (C-4), 126.16 (C-1), 129.28 (C-2), 132.33 (C-10), 133.55 (C-1), 138.01 (C-5), 153.54 (C-3), 157.23 (C-4), 221.46 (C-17); IR (CHCl.sub.3) 3598, 3098, 3060, 1729, 1611, 1583, 1512, 1502, 1453, 1441, 1245, 1042 cm.sup.1; HR-MS (APCI) calculated C.sub.28H.sub.35O.sub.3 [M+H.sup.+] 419.25807 found 419.25810. R.sub.f (4/1 hexane/EtOAc)=0.3.
Example 14: 15-(4-Hydroxyphenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (14)
[0173] ##STR00016##
[0174] Compound 14 was prepared according to the above general procedure by reacting vinylestrone 2 with 4-acetoxystyrene (75 l). Resulting (3-(Hydroxy)-estra-1,3,5(10)-trien-17-one-15-yl)ethenyl)phenyl-acetate was further dissolved in methanol and catalytical amount of freshly prepared solution of 1 mol.Math.l.sup.1 CH.sub.3ONa in methanol was added at room temperature. After 3 h, DOWEX 50W (H+, ion exchange resin) was added until the pH of the mixture reached 5-6. Then the resin was filtered off, solvent was removed under reduced pressure and the residue was purified by chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=12 min) yielded 37 mg of colorless solid 14: mp 169 C.; [].sub.D+79.1 (c 0.283; CH.sub.3OH); .sup.1H NMR (500 MHz, MeOD) 0.99 (s, 3H, H-18), 1.29 (s, 1H, H-7a), 1.34-1.47 (m, 2H, 11a, 12a), 1.57-1.68 (m, 3H, H-1, 8, 14), 1.75-1.89 (m, 3H, H-1b, 7b, 12b), 2.17 (m, 1H, H-9), 2.25-2.33 (m, 2H, H-11b, 15), 2.34-2.40 (m, 2H, H-16), 2.42 (dt, 1H, J=13.6, 7.9 Hz, H-2a), 2.66 (ddd, 1H, J=13.6; 8.5; 5.4 Hz, H-2b), 2.74-2.80 (m, 2H, H-6), 6.49 (dm, 1H, J=2.7 Hz, H-4), 6.53 (bdd, 1H, J=8.5; 2.7 Hz, H-2), 6.71 (m, 2H, H-3), 7.00 (m, 2H, H-2), 7.04 (dd, 1H, J=8.5; 1.1 Hz, H-1); .sup.13C NMR (150.9 MHz, MeOD) 18.21 (C-18), 26.68 (C-11), 27.78 (C-7), 30.38 (C-6), 34.57 (C-15), 34.58 (C-1), 35.09 (C-12), 35.69 (C-2), 37.41 (C-8), 43.64 (C-16), 45.83 (C-9), 48.49 (C-13), 53.92 (C-14), 113.71 (C-2), 116.12 (C-4), 116.14 (C-3), 126.91 (C-1), 130.54 (C-2), 132.33 (C-10), 134.00 (C-1), 138.76 (C-5), 156.11 (C-3), 156.52 (C-4), 224.20 (C-17); IR (KBr) 3370, 2926, 2855, 1716, 1612, 1583, 1514, 1450, 1441, 1376, 1356, 1217, 1170, 1099, 922, 827, 755, 731 cm.sup.1; HR-MS (APCI) calculated C.sub.26H.sub.31O.sub.3 [M+H.sup.+] 391.22677 found 391.22690. R.sub.f(7/1 CHCl.sub.3/MeOH)=0.3.
Example 15: 3-Hydroxy-15-(4-methylphenethyl)-estra-1,3,5(10)-trien-17-one (15)
[0175] ##STR00017##
[0176] Compound 15 was prepared according to the above general procedure by reacting vinylestrone 2 with 4-methylstyrene (64 l). Chromatography on HPLC (35/65 MeCN/H.sub.2O, t.sub.R=36 min) yielded 25 mg of colorless solid 15: mp 159 C.; [].sub.D 13.9 (c 0.170; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.02 (s, 3H, H-18), 1.36-1.55 (m, 3H, H-7a, 11a, 12a), 1.64 (dddd, 1H, J=13.6; 11.7; 9.2; 5.3 Hz, H-1a), 1.66-1.76 (m, 2H, H-8, 14), 1.86-1.95 (m, 3H, H-1b, 7b, 12b), 2.25 (m, 1H, H-9), 2.33 (s, 3H, CH.sub.3-Ph), 2.30-2.34 (m, 2H, H-11b, 15), 2.37-2.46 (m, 2H, H-16), 2.49 (ddd, 1H, J=13.7; 9.2; 6.9 Hz, H-1a), 2.71 (ddd, 1H, J=13.7; 9.8; 5.3 Hz, H-1b), 2.80-2.94 (m, 2H, H-6), 4.68 (s, 1H, OH), 6.59 (dm, 1H, J=2.8 Hz, H-4), 6.63 (dd, 1H, J=8.5; 2.8 Hz, H-2), 7.07 (m, 2H, H-2), 7.11 (m, 2H, H-3), 7.13 (dd, 1H, J=8.5; 1.0 Hz, H-1); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.73 (C-18), 21.01 (CH.sub.3-Ph), 25.51 (C-11), 26.62 (C-7), 29.34 (C-6), 33.22 (C-1), 33.81 (C-15), 33.85 (C-12), 35.40 (C-2), 35.95 (C-8), 42.72 (C-16), 44.52 (C-9), 47.20 (C-13), 52.81 (C-14), 112.71 (C-2), 115.23 (C-4), 126.20 (C-1), 128.29 (C-2), 129.15 (C-3), 132.41 (C-10), 135.54 (C-4), 138.05 (C-5), 138.58 (C-1), 153.51 (C-3), 221.34 (C-17); IR (KBr) 3382, 1734, 1719, 1584, 1514, 1501, 1443, 708 cm.sup.1; HR-MS (APCI) calculated C.sub.27H.sub.33O.sub.2 [M+H.sup.+] 389.24751 found 389.24751. R.sub.f (4/1 hexane/EtOAc)=0.3.
Example 16: 15-(3,4,5-Trifluorophenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (16)
[0177] ##STR00018##
[0178] Compound 16 was prepared according to the above general procedure by reacting vinylestrone 2 with 3,4,5-trifluorostyrene (77 mg). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=21 min) yielded 8 mg of colorless solid 16: mp 182 C.; [].sub.D+48.3 (c 0.118; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.03 (s, 3H, H-18), 1.39-1.56 (m, 3H, H-7a, 11a, 12a), 1.57-1.77 (m, 3H, H-1 a, 8, 14), 1.81-1.96 (m, 3H, H-1b, 7b, 12b), 2.21-2.40 (m, 3H, H-11b, 9, 15), 2.36 (dd, 1H, J=19.3; 2.2 Hz, H-16a), 2.46 (dd, 1H, J=19.3; 8.9 Hz, H-16b), 2.48 (ddd, 1H, J=14.0; 9.3; 7.0 Hz, H-2a), 2.69 (ddd, 1H, J=14.0; 9.7; 5.1 Hz, H-2b), 2.82-2.95 (m, 2H, H-6), 4.86 (bs, 1H, OH), 6.60 (d, 1H, J=2.8 Hz, H-4), 6.64 (dd, 1H, J=8.4; 2.7 Hz, H-2), 6.78 (m, 2H, H-2), 7.13 (bd, 1H, J=8.4 Hz, H-1); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.73 (C-18), 25.41 (C-11), 26.66 (C-7), 29.20 (C-6), 32.60 (C-1), 33.56 (C-15), 33.79 (C-12), 35.08 (C-2), 35.86 (C-8), 42.43 (C-16), 44.42 (C-9), 47.16 (C-13), 52.65 (C-14), 112.19 (dd, J.sup.C-F=15.9; 4.7 Hz, C-2), 112.78 (C-2), 115.23 (C-4), 126.14 (C-1), 132.14 (C-10), 137.77 (C-1), 137.82 (C-5), 138.16 (dt, J.sup.C-F=249.4; 15.2 Hz, C-4), 151.10 (ddd, J.sup.C-F=249.6; 9.8; 3.9 Hz, C-3), 153.65 (C-3), 220.74 (C-17); .sup.19F NMR (470.3 MHz, CDCl.sub.3) 160.47 (t, 1F, J.sup.F-F=20.5 Hz), 131.13 (d, 2F, J.sup.F-F=20.5 Hz); IR (KBr) 3295, 1718, 1620, 1585, 1529, 1501, 1376 cm.sup.1; HR-MS (APCI) calculated C.sub.26H.sub.28O.sub.2F.sub.3 [M+H.sup.+] 429.20259 found 429.20349. R.sub.f (4/1 hexane/EtOAc)=0.3.
Example 17: 15-(2,3,4,5,6-Pentafluorophenethyl)-3-hydroxy-estra-1,3,5(10)-trien-17-one (17)
[0179] ##STR00019##
[0180] Compound 17 was prepared according to the above general procedure by reacting vinylestrone 2 with 2,3,4,5,6-pentafluorstyrene (67 l). Chromatography on HPLC (30/70 MeCN/H.sub.2O, t.sub.R=42 min) yielded 27 mg of colorless solid 17: mp 151 C.; [].sub.D+43.7 (c 0.544; CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) 1.00 (s, 3H, H-18), 1.41-1.55 (m, 3H, H-7a, 11a, 12a), 1.61 (m, 1H, H-1a), 1.69 (m, 1H, H-8), 1.74 (m, 1H, H-14), 1.85-1.95 (m, 3H, H-1b, 7b, 12b), 2.27 (m, 1H, H-9), 2.30-2.39 (m, 2H, H-11b, 15), 2.43 (dd, 1H, J=19.4; 2.4 Hz, H-16a), 2.51 (dd, 1H, J=19.4, 8.6 Hz, H-16b), 2.67 (m, 1H, H-2a), 2.79 (m, 1H, H-2b), 2.83-2.96 (m, 2H, H-6), 4.93 (bs, 1H, OH), 6.60 (bd, 1H, J=2.8 Hz, H-4), 6.64 (dd, 1H, J=8.4; 2.8 Hz, H-2), 7.13 (bd, 1H, J=8.4 Hz, H-1); .sup.13C NMR (150.9 MHz, CDCl.sub.3) 17.63 (C-18), 22.19 (C-2), 25.43 (C-11), 26.56 (C-7), 29.19 (C-6), 30.80 (C-1), 33.74 (C-12), 33.96 (C-15), 35.86 (C-8), 42.34 (C-16), 44.40 (C-9), 47.12 (C-13), 52.61 (C-14), 112.77 (C-2), 114.44 (bt, J.sup.C-F=18.8 Hz, C-1), 115.23 (C-4), 126.16 (C-1), 132.10 (C-10), 137.46 (dm, J.sup.C-F=250.4 Hz, C-3), 137.87 (C-5), 139.68 (dm, J.sup.C-F=251.7 Hz, C-4), 144.97 (dm, J.sup.C-F=243.2 Hz, C-2), 153.63 (C-3), 220.53 (C-17); .sup.19F NMR (470.3 MHz, CDCl.sub.3) 158.78 (m, 2F, F-3), 153.61 (t, 1F, F-4), 141.15 (m, 2F, F-2); IR (CHCl.sub.3) 3599, 1732, 1657, 1611, 1585, 1521, 1504, 1440, 1378, 1122, 1068, 963 cm.sup.1; HR-MS (APCI) calculated C.sub.26H.sub.26O.sub.2F.sub.5 [M+H.sup.+] 465.18475 found 465.18468. R.sub.f (4/1 hexane/EtOAc)=0.3.
Example 18: 3-Hydroxy-15-vinyl-estra-1,3,5(10)-trien-17-one (2a)
[0181] ##STR00020##
[0182] Reaction of protected vinylestrone 2 (55 mg, 0.134 mmol) according to the general procedure yielded vinylestrone 18 (38 mg, 95%) as a colorless solid: mp 162 C.; [].sub.D+79.4 (c 0.175, CHCl.sub.3); .sup.1H NMR (500 MHz, CD.sub.2Cl.sub.2) 1.01 (s, 3H), 1.34-1.61 (m, 3H), 1.72-1.73 (m, 2H), 2.13-2.19 (m, 2H), 2.28 (m, 1H), 2.36 (m, 1H), 2.52 (dd, J=19.5, 9.0 Hz, 1H), 2.63 (dd, J=19.5, 2.2 Hz, 1H), 2.83-2.90 (m, 2H), 3.02-3.26 (m, 1H), 4.69 (s, 1H), 5.02-5.27 (m, 2H), 6.14 (ddd, J=17.2, 10.4, 6.7 Hz, 1H), 6.48-6.69 (m, 2H), 7.14 (d, J=8.4 Hz, 1H); .sup.13C NMR (150.9 MHz, CD.sub.2Cl.sub.2) 16.90, 25.49, 26.33, 29.25, 33.40, 35.96, 37.15, 41.75, 44.50, 47.69, 52.69, 115.96, 117.24, 119.98, 125.85, 132.63, 137.62, 139.00, 156.54, 221.20; IR (CHCl.sub.3) 3599, 3400, 3319, 3082, 2938, 2863, 2843, 1731, 1640, 1611, 1585, 1501, 1440, 1377, 1272, 1248, 1190, 1165, 1101, 998, 918, 690, 577, 587, 445 cm.sup.1; HR-MS (ESI) for C.sub.20H.sub.24O.sub.2Na [M+Na.sup.+] calculated 319.16685, found 319.16703. R.sub.f (4/1 hexane/EtOAc)=0.3.
Biological Tests
Example 19: Inhibition of the Enzyme Activity of 17HSDs by 15-Substituted Estrone Derivatives
[0183] The inhibitory activity of the test substances was determined for individual isoenzymes from the 17HSD family, specifically these were the types 1, 2, 3, 4, 5 and 7. Escherichia coli and mammalian cells expression systems were used for recombinant expression of individual isozymes of human 17HSD. Only in the case of 17HSD5 the enzyme was partially purified and supernatant obtained by centrifugation of the bacterial lysate was used for testing. Systems expressing specific 17HSD type were suspended in a reaction buffer and incubated with tritium-labeled substrates and respective cofactors at 37 C. in two parallel tests: the control arrangement (DMSO without the inhibitor) and the test arrangement (DMSO with the inhibitor). DMSO serves as a negative control in this case. After 20-30% of the substrate was converted by the activity of the enzyme to the product in a control test, both tests were terminated. Substrate and product from the test arrangement were isolated by SPE (solid phase extraction), and then separated by reverse phase HPLC. Substrate conversion was determined by integration of the signals of the substrate and product, and was expressed in %. For the purposes of the subsequent calculation of enzyme inhibition, the conversion of the control test was designated as 0% inhibition. All tests were performed in triplicates. IC.sub.50 values were subsequently determined by a standard method using the One Ligand Binding model of SigmaPlot kinetics module (Schustera et. al. J. Ster. Biochem. Mol. Biol. 2011, 125, 148; Mller et. al. Biioorg. Med. Chem. Lett. 2009, 19, 6740; Mller et. al. PLoS One, 2010, 5, 6, e10969).
[0184] The inhibitory activities of the tested estrone derivatives at various concentrations on human 17HSD type 1 are summarized in Table 1. The tested compounds showed high inhibitory activity against human 17HSD type 1 already at 0.1 mol l.sup.1 concentration. Two of the most effective derivatives, compound 6 and 9, are highlighted in Table 1.
[0185] Many of the substances are effective also on 17HSD type 5 (Table 1). The activities of the other isozymes examinednamely 17HSD type 2, 3, 4 and 7were barely reduced by the presence of the substances, even at concentrations of 10 mol l.sup.1 (inhibition never higher than 50%, data not shown). The tested compounds are therefore selective inhibitors of 17HSD1 and in some cases additionally of 17HSD5.
[0186] Compound 2a inhibits exclusively the 17HSD1 isoenzyme; however, at the same time it shows also a partial estrogenicity.
Example 20: Determination of the Activity of Compounds on Steroid Receptors ER, AR and PR in Cell Luciferase Reporter Assays
[0187] Effect of newly prepared substances on the activity of steroid receptors, estrogen receptor (ER) and androgen receptor (AR) and progesterone receptor (PR) was assessed in vitro by selective luciferase reporter assays based on cell reporter lines for ER, AR and PR in U2OS cells (Sedlk et al. Comb. Chem. High T. Scr. 2011, 14, 248). These cell lines were prepared by introducing an expression vector with coding sequence for a particular human steroid receptor and reporter vector pGL4 (Promega, USA) containing responsive elements for a specific steroid receptor in a promoter regulating the expression of luciferase gene. Cells that stably integrated both vectors into the genome were isolated on selection medium containing hygromycin and G418 (Geneticin aminoglycoside). From these selected cell cultures, clones of cells were further isolated, showing an optimal response in assays with reference ligands for the steroid receptor.
[0188] U2OS reporter lines were grown in DMEM (Thermo Fisher Scientific, catalog number 11880-036), without phenol red supplemented with 10% FBS (Thermo Fisher Scientific, catalog number 10270-106), 2 mmol.sup..Math.l.sup.1 Glutamax-1 (Thermo Fisher Scientific, catalog number 35050061) and a solution of penicillin and streptomycin (Thermo Fisher Scientific, catalog number 15070063). Cells were incubated with 5% CO.sub.2 at 37 C. Two days prior to testing of compounds, growth medium was changed to DMEM without phenol red, supplemented with 2 mmol.sup..Math.l.sup.1 Glutamax and 4% FBS depleted of lipophilic components (including the endogenous ligands of steroid receptors) (Hyclone, GE Healthcare Life Sciences, USA). Two days after the medium replacement, the cells were harvested, counted and resuspended in a medium of the same composition at a concentration of 0.510.sup.6/ml. The cell suspension was transferred in batches into 1536-well white plates adjusted for cultivation of adherent cells (Corning Inc., NY, USA). 4 l of cell suspension equivalent to 2500 cells were transferred to each well. Test compounds were diluted in DMSO and transferred into wells to cells using the contactless acoustic dispenser Echo 520 (Labcyte). Testing was performed in 10 concentration points in the range 10 mol.Math.l.sup.1 to 1 nmol.sup..Math.l.sup.1 in triplicates.
[0189] The experiment was performed in two modes. Agonistic properties of test substances were determined in the agonistic mode; antagonistic properties were detected in the antagonistic mode. In the antagonist mode, 30 minutes after the transfer of the test substances, 0.5 l of a solution of the agonist was added to the test samples. A solution of 1 nmol.sup..Math.l.sup.1 E2 was used for ER, 1 nmol.sup..Math.l.sup.1 dihydrotestosterone (DHT) for AR and 1 nmol.sup..Math.l.sup.1 progesterone for PR. Growth medium for the cells was used to dissolve the compounds.
[0190] In order to distinguish between the specific, steroid hormone response elements-driven regulation of the luciferase expression and the possible effects of test compounds on the luciferase activity, compounds were tested in parallel experiment on a U2OS cell line constitutively expressing luciferase gene.
[0191] Luciferase activity was determined by a commercial kit Britelite plus a luciferase reporter gene assay reagent (Perkin Elmer, USA), 24 h after addition of compounds to cells. The intensity of luminescence was measured on an Envision multimode spectrophotometer (PerkinElmer).
[0192] To the extent of considered therapeutic concentrations, the compounds have no estrogenic effect. Selected compounds exhibit even features of ER/AR antagonists. Compounds 3, 4 and 7 are ER antagonist; Compound 9 is an AR antagonist and Compounds 11, 12 and 13 are antagonists of both types of receptors (ER and AR).
[0193] This cytotoxicity test was performed for all the prepared compounds, and the results showed that none of the derivatives prepared exhibits cytotoxic effect up to a concentration of 20 mol.Math.l.sup.1 of the test compounds.
[0194] Compounds 9 and 12 were further tested in extended concentration range from 100 mol.sup..Math.l.sup.1 to 0.01 nmol.sup..Math.l.sup.1 and data are summarized in Table 2. Both compounds show weak partial agonistic properties starting at 10 mol.sup..Math.l.sup.1 on ER. The agonistic effects are unique to ER and are not observed on other steroid receptors: AR and PR. The activation of ER is only partial and the efficacy is below 40% of the maximal effect produced by E2. At even higher concentrations (10-100 mol.sup..Math.l.sup.1), compounds exhibit antagonistic activities mainly on ER and PR and to a lesser extent on AR too. The antagonist activities are not accompanied with luciferase inhibition or cell toxicity. Considering that compounds effectively inhibit 17HSD1 at 0.01 mol.sup..Math.l.sup.1, interactions with steroid receptors occur at considerably higher concentrations (>10 mol.sup..Math.l.sup.1) and do not interfere with 17HSD mediated effects. Moreover, antagonist activities on ER, PR and AR are desirable in case compounds are used to block proliferation of ER/AR positive breast/prostate cancer cells where the proliferation is driven by steroid receptors.
TABLE-US-00001 TABLE 2 In vitro testing of the compounds in ER, AR and PR cell-based reporter assays (EC.sub.50/IC.sub.50, mol .Math. l.sup.1). Compound AGONIST mode ANTAGONIST mode Luciferase Cell No. ER AR PR ER AR PR activity viability 9 >10 n.a. n.a. 23.46 n.a. 13.60 n.a. >100.00 12 >10 n.a. n.a. 28.97 89.09 13.65 n.a. >100.00 E2 6.0 10.sup.5 DHT 2.0 10.sup.4 P4 3.6 10.sup.3 4-OHT 0.03 10.15 11.98 Enzalutamide 6.68 n.a. 89.43 RU486 <0.003 17.90 47.69 n.a.: not active.
Example 21: Compound-Mediated Inhibition of E1 Induced Proliferation of Triple Positive Breast Cancer Cell Line T47D
[0195] Triple positive breast cancer cell line T47D has several features that make it a unique model for study of the biological function of 17HSD1 and of the clinical potential of 17HSD1 inhibitors. T47D cells express high levels of ER, PR and 17HSD1 and cell growth is estrogen-dependent. In the absence of estrogens, proliferation rate slows down considerably and cells stop dividing eventually. Estrogens can be supplied directly, by adding E2 to the growth medium. E2 promotes T47D cell proliferation by directly activating ER. Alternatively, cell proliferation can be induced by suppling the estrogenic precursor, E1, which is transformed by 17HSD1 expressed by cells, to E2.
[0196] In this study, T47D cells were propagated in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol.sup..Math.l.sup.1 glutaMAX (Thermo Fisher Scientific, MA, USA), and penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) and incubated in a 5% CO2-humidified atmosphere at 37 C. to the amount needed for the experiment. Two days prior to testing of compounds, growth medium was changed to medium without phenol red, supplemented with 2 mmol.sup..Math.l.sup.1 Glutamax and 4% FBS depleted of lipophilic components (including the endogenous estrogenic substances) (Hyclone, GE Healthcare Life Sciences, USA). Two days after the medium replacement, the cells were harvested, counted and resuspended in a medium. Cells were dispensed with liquid dispenser Multidrop (Thermo Fisher Scientific, Waltham, USA), to the cell culture treated, 12-well plates (Corning Inc., NY, USA) at 100 000 cells/well in 1 mL of total media volume. Compounds diluted in the medium were added 24 h later and cells were incubated for 7 more days with or without 1 mol.sup..Math.l.sup.1 E1. Growth medium was changed during the experiment on day 3, and freshly diluted compounds were added to cells again. After 7 days of cell cultivation with compounds, cells were harvested and counted. Cell number was normalized and 100% was attributed to cells cultivated in the presence of 1 mol.sup..Math.l.sup.1 E1 and 0% to the cells cultivated without any added compound.
[0197] Results are summarized in
Example 22: Determination of Cytotoxicity of the Compounds in the U2OS Cell Line
[0198] To separate the antagonist activity of test compounds from the cytotoxic effect on U2OS cells (cell line derived from osteosarcoma), an experiment for determination of cell viability was performed in parallel with the reporter assay. Original, genetically unmodified U2OS cells were grown under the same conditions as the reporter cells. These cells were further treated and incubated with the compounds in a completely identical manner and for the same time as cells in the reporter assay. At the end of the experiment, the amount of ATP was determined using BriteLite luciferase homogeneous assay as a measure of cell viability. The data were then processed together with data from the reporter assays.
[0199] This cytotoxicity test was performed for all the prepared compounds, and the results showed that none of the derivatives prepared exhibits cytotoxic effect up to a concentration of 20 mol.Math.l.sup.1 of the tested compounds.
Example 23: Determination of Cytotoxicity of Compounds on Tumor and Non-Tumor Cells
[0200] Compounds 5, 6, 7, 9, 14 and 15 were used for evaluation of antitumor activity. MTT cytotoxicity assay was used in vitro on cell lines derived from normal tissues and from tumors. Specifically, these were the K562 line (human myeloid leukemia), K562-Tax (human myeloid leukemia resistant to taxol and overexpressing PgP protein for multidrug resistance), CEM (T-lymphoblastic leukemia), CEM-DNR bulk (T-lymphoblastic leukemia resistant doxorubicin, lacking the expression of the target gene for inhibitors of topoisomerase II alpha), A549 line (human lung adenocarcinoma), HCT116p53 wt (human colon cancer), HCT116p53/ (human colon cancer, mutant p53), U2OS line of human osteosarcoma and two fibroblast lines MRC5 and BJ as examples of non-tumor cells. Expression characteristics, profiles of susceptibility to classical antitumor drugs and methodology of the MTT cytotoxicity assay were repeatedly published (e.g. Noskova et al. Neoplasma 2002, 49, 418; Sarek et. al. J. Med. Chem. 2003, 46, 25, 5402).
[0201] Substances in the tests did not show significant cytotoxicity on tumor and non-tumor cell lines of various histogenetic origin, which indicates the absence of off-target antitumor effect. The test results are summarized in Table 3.
TABLE-US-00002 TABLE 3 In vitro cytotoxicity (IC.sub.50, mol .Math. l.sup.1) tested on cell lines of tumor and non-tumor origin. Compound CEM CEM-DNR-bulk K562 K562-Tax A549 No. IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 5 25.16 3.80 28.24 1.79 25.25 4.68 19.31 2.44 35.76 4.48 6 16.34 0.90 24.61 2.29 26.89 4.88 13.21 1.72 35.75 3.48 7 25.56 4.97 32.81 3.78 >50.00 0.00 12.49 2.76 >50.00 0.00 9 21.36 3.53 22.14 3.48 27.70 5.71 9.12 0.96 40.98 3.48 14 25.81 3.63 30.76 2.85 22.60 3.20 18.89 1.59 41.32 3.33 15 21.67 2.82 24.10 1.95 23.80 3.25 13.80 1.73 28.21 3.90 Compound HCT116p53 wt HCT116p53-/- U2OS BJ MRCS No. IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 5 29.19 3.33 30.47 2.73 30.38 3.70 24.26 2.89 18.37 3.11 6 29.34 1.97 30.80 2.13 27.88 3.18 28.81 3.76 21.00 1.83 7 >50.00 0.00 >50.00 0.00 >50.00 0.00 >50.00 0.00 >50.00 0.00 9 29.38 3.33 35.73 0.54 45.32 2.86 >50.00 0.00 >50.00 0.00 14 28.22 2.90 32.57 2.14 44.59 4.38 50.00 0.00 50.00 0.00 15 28.27 1.50 29.35 1.88 28.90 2.34 27.93 1.62 19.55 3.58 : standard deviation.
Example 24: Determination of Long-Term Effects of Compounds on Breast, Prostate and Ovarian Cancer Cell Lines
[0202] Compounds 9 and 12 were tested in the proliferation assay with cell lines derived from the triple negative breast cancer (MDA-MB-231), AR-negative prostate cancer (PC3, DU145) and ovarian cancer (SK-OV-3, CaoV-3). These cell lines do not express biologically significant level of endogenous 17HSD1 and are not responsive to antihormone treatment. The experiment was carried out exactly as in the example 21. The cells were treated with 1 mol.sup..Math.l.sup.1 compounds for 7 days. At the end of the treatment, cells were harvested and counted. Cell number was normalized and 100% was attributed untreated cells, 0% to the sample with no cells.
[0203] Cultured cells show no cytotoxic or pro-proliferative effect when cultivated with 1 mol.sup..Math.l.sup.1 compounds for 7 days. The data prove that compounds have no effect on cells that are not dependent/sensitive to steroid hormones, and especially on the estradiol produced by the conversion of E1 to E2 by 17HSD (Table 4).
TABLE-US-00003 TABLE 4 7 day proliferation assay with cell lines derived from breast, prostate and ovarian cancer. Values represent % of survived cells compared to untreated samples. Compound Breast Prostate Ovary No. MDA-MB-231 PC3 DU145 SK-OV-3 CaOV-3 9 106 96 98 113 94 12 98 98 86 97 88
Example 25: Determination of the 17HSD Inhibition in Cells In Vitro
[0204] The effect of compounds on the inhibition of 17HSD in cell lines derived from hormonally active tumors MCF-7 (human breast carcinoma transfected with human 17HSD1) and CHO (hamster ovarian carcinoma transfected with human 17HSD1) was monitored through cumulation of 17HSD precursor, hormone E1, in the supernatant of cell lines in a time interval of 8 and 24 hours. For this experiment, non-cytotoxic concentrations of estrone derivative (10 mol.Math.l.sup.1) were used. E1 levels were determined by an immuno-enzymatic assay.
[0205] Addition of estrone derivatives to 17HSD1 transfected cell cultures of hormonally active/dependent tumors in vitro led to significantly increased level of E1, which is a substrate to 17HSD enzyme, within 8 hours. This observation is consistent with the inhibition of 17HSD in vitro. Results of the analysis of 17HSD inhibition in cell lines in vitro are summarized in Table 5.
TABLE-US-00004 TABLE 5 Results of the analysis of 17HSD inhibition in cell lines in vitro MCF-7 CHO 8 hr. 24 hr. 8 hr. 24 hr. No. E1 (pg/ml) E1 (pg/ml) E1 (pg/ml) E1 (pg/ml) control 564 543 745 482 5 3373 2236 3194 1576 6 4251 3323 4137 1804 7 1921 1839 2101 1476 9 1300 1182 1171 739 14 4298 2626 3179 1054 15 719 948 750 554 Control: cell lines not transfected with 17HSD1
Example 26: Determination of the Inhibition of Human 17HSD In Vivo
[0206] To evaluate the biological activity of compounds in vivo, measurement of decrease in the formation of E2 in the plasma of mice treated with the candidate substance 9 was used. Female mice of NMRI outbred strain aged 8 weeks were used for this purpose. These animals were treated for 7 or 14 days with compound 9 dissolved in an oil vehicle (olive oil), administered orally with a gastric gavage, once a day, 20 mg/kg of mouse weight in a total volume of 0.1 ml. Parallel control group of animals was treated with the vehicle alone. After 7 or 14 days, blood was collected for the purpose of processing blood plasma and analysis of E2 levels. In parallel, a dissection was performed and macroscopic evaluation of individual organs.
[0207] Administration of the compound was well tolerated by the animals; no significant weight loss was observed and macroscopic analysis of organs did not reveal any obvious pathology. Plasma of both treated and control animals was subjected to the of immunoenzymatic analysis for quantification of E2. This analysis showed highly significant reduction in the levels of the E2 hormone after 14 days of administration, which is consistent with the inhibition of 17HSD in vivo (Table 6).
TABLE-US-00005 TABLE 6 Inhibition of E2 production after administration of compound 9 in vivo in plasma of experimental animals. Vehicle 7 days E2 (pg/ml) Vehicle 14 days E2 (pg/ml) Mouse B_1 4660 Mouse D_1 5447 Mouse B_2 4988 Mouse D_2 5991 Mouse B_3 4570 Mouse D_3 4796 Mouse B_4 6835 Mouse D_4 4973 Mouse B_5 6202 mean value 5302 2416 Mouse B_6 6372 Mouse B_7 4824 mean value 5632 950 Compound 9:7 days Compound 9:14 days 20 mg/kg daily E2 (pg/ml) 20 mg/kg daily E2 (pg/ml) Mouse A_1 4927 Mouse C_1 6020 Mouse A_2 4357 Mouse C_2 2668 Mouse A_3 6020 Mouse C_3 2657 Mouse A_4 7379 Mouse C_4 3566 Mouse A_6 5857 Mouse C_5 4312 Mouse A_7 5617 Mouse C_6 4025 mean value 5693 1034 mean value 3874 1253 p 0.362 p 0.019 P: The value of statistical significance; : standard deviation
Example 27: Study of Efficacy of Compound 9 on Breast Carcinoma Tumors Initiated from T47D Cell Lines
[0208] The preparation of the final formulation of compounds used, the incubation of the eggs, the administration of test compounds, the toxicity analysis and the final statistical analysis were carried out at INOVOTION, La Tronche, 38700, France.
[0209] Fertilized White Leghorn eggs were incubated at 37.5 C. with 50% relative humidity for 9 days. At this time (E9), the chorioallantoic membrane (CAM) was dropped by drilling a small hole through the eggshell into the air sac and a 1 cm.sup.2 window was cut in the eggshell above the CAM. Twenty one eggs were used for each condition.
[0210] T47D cell line was cultivated in RPMI medium with 10% FBS, 1% non-essential amino acids, 1% sodium bicarbonate and 0.1 nmol.sup..Math.l.sup.1 estrone (and 1% penicillin/streptomycin). Cells (at 80% confluency, passage 28) were detached with trypsin, washed with complete medium and suspended in PBS. An inoculum was added onto the CAM of each egg (E9). Eggs were then randomized in 3 groups.
[0211] At day 10 (E10), tumors began to be detectable. They were then treated for 6 days, every day (E11, E12, E13, E14,) by dropping 100 l of vehicle (0.5% DMSO in PBS), or ref compound (4-hydroxytamoxifen), or tested compound 9 at one dose onto the tumor (see Table 7 for concentration).
TABLE-US-00006 TABLE 7 Groups for study Group description Molecule name Concentration Group 1 Negative ctrl DMSO in PBS 0.5% DMSO (vehicle) Group 2 Positive ctrl (ref Tamoxifen 200 mol .Math. l.sup.1 compound) Group 3 Exp. group 1 compound 9 50 mol .Math. l.sup.1
[0212] At day 15 (E15) the upper portion of the CAM was removed, washed in PBS and then directly transferred in PFA (fixation for 48 hrs); the tumors were then carefully cut away from normal CAM tissue and weighted. A one-way ANOVA analysis with post-tests has been used for these data.
[0213] In parallel, a 1 cm.sup.2 portion of the lower CAM was collected to evaluate the number of metastasis cells. Genomic DNA is extracted from the CAM and analyzed by qPCR with specific primers for Alu sequences. Statistical analysis was applied on data from the Bio-Rad CFX Manager 3.1 software.
[0214] The toxicity after 6 days of the treatment was characterized by the number of dead embryos, eventual visible macroscopic abnormalities were evaluated as well.
[0215] The target of this study was to test efficacy of compound 9 on breast carcinoma initiated from T47D cells on INOVOTION model. At day 16 of embryo development, following 4 treatments (at day 2, 3, 4 and 5 after grafting), tumors were collected, fixed, cleaned and weighted: at the dose tested (50 mol.Math.l.sup.1) compound 9 had the same effect as 4-hydoxytamoxifen, used as positive control (200 mol.Math.l.sup.1), showing a 12-13% reduction, see
[0216] Concerning metastasis, both compounds showed a reduction of metastasis, see
[0217] In term of toxicity, the same ratio of dead/alive eggs between groups was observed, even in negative control group, see
INDUSTRIAL APPLICABILITY
[0218] Compounds of the invention may be used for diagnosis and possibly also for the treatment of estrogen dependent diseases, especially estrogen-dependent types of tumors, endometriosis, skin diseases or disorders of sexual maturation. Substances may find application in the treatment of infertility, to induce premature menopause, hormonal castration, or as contraceptives.
[0219] Estrogen-dependent diseases include breast cancer, ovarian cancer, uterine cancer, endometriosis, adenomyosis, menorrhagia, metrorrhagia, dysmenorrhea, uterine fibroids, polycystic ovarian syndrome, fibrocystic breast disease, prostate cancer, non-small cell lung cancer (NSCLC), squamous cell carcinoma, colorectal cancer, gastric cancer, acne, hirsutism, pseudohermaphroditism, seborrheic dermatitis, androgens induced alopecia, hyperestrogenism.