-FSTROGENIC AGFNTS
The present invention relates to new 3-[4-(2-Phenyl-Indole-l-ylmethyl)-Phenyl]-Acrylamide compounds which are useful as estrogenic agents, as well as pharm~eutical compositions and methods of tre~tment utilizing these compounds.
Rack~rollnd of the inventior The use of hormone repl~cem.ont therapy for bone loss prevention in post-menopausal women is well precedented. The normal protocol calls for estrogen supplementation using such formulations containing estrone, estriol, ethynyl estradiol or conjugated estrogens isolated from natural sources (Premarin from Wyeth-Ayerst).
In some patients, therapy may be contraindicated due to the proliferative effects unopposed estrogens (estrogens not given in combination with progestins) have onuterine tissue. This proliferation is associated with increased risk for endometriosis and/or en-lometrial cancer. The effects of unopposed estrogens on breast tissue is less clear, but is of some concern. The need for estrogens which can ~ the bone sparing effect while minimi7ing the proliferative effects in the uterus and breast is evident. Certain nonsteroidal antiestrogens have been shown to .~ bone mass in the ovariectomized rat model as well as in human clinical trials. Tamoxifen, forexample, is a useful palliative for the treatment of breast cancer. It has been demonstrated to exert an estrogen agonist like effect on the bone, in humans.
However, it is also a partial agonist in the uterus and this is cause for some concern.
Raloxifene, a benzothiophene antiestrogen, has been shown to stim~ te uterine growth in the ovariectornized rat to a lesser extent than Tamoxifen while Ill~ illg the ability to spare bone. A suitable review of tissue selective estrogens is: Tissue -Selective Actions Of Estrogen Analogs, Bone Vol. 17, No. 4, October 1995, 181S-19OS.
The use of indoles as estrogen antagonists has been reported by Von Angerer, Ch~mir~l Abstracts, Vol. 99, No. 7 (1983), Abstract No. 53886u. Also, see, J.Med.Chem. 1990, 33, 2635-2640; J.Med.Chem. 1987, 30, 131-136. Also see Ger.
Offen., DE 3821148 Al 891228. Additionally, see WO, A, 93 23374 (Otsuka Pharmaceutical Factory, Inc.). Von Angerer's work is limited to aliphatic chains linked to the indole nitrogen and then linked to the basic amine (or amide) or, benzyl groups not possessing the basic amine. The world patent from Otsuka (Japanese) consists of com~ou-lds related to the present invention except R3 (as shown in formula I) is defined as -SR where R is alkyl. Additionally, there are no chains from the indole nitrogen in their patent with the same structure as the ones given in the present invention, either by claim or example. A related patent WO A 93 10741 describes 5-Hydroxyindoles. WO
5 A 95 17383 (Kar Bio AB) describes aliphatic chain compounds.
nescription of the ~nventiorl Compounds of the general structure type shown in formulas (I) and (II) are 10 estrogen agonists/antagonists useful for the tre~tm~nt of diseases associated with estrogen deficiency. The compounds of the present invention show strong binding to the estrogen receptor. These compounds have proven to be antiestrogens with little intrinsic estrogenicity. In a three-day ovariectimi7Yl rat model, compounds of formula (I) are capable of antagonizing the effects of 17,B- estradiol while showing little uterine 15 stim~ tion when dosed alone.
The present invention includes compounds of formulas (I) or (II), below:
Rl~ R3 R2 ~ R2 R5R~ R~
o wherein:
R~ is selected from H, OH or the Cl-C4 esters or alkyl ethers thereof, or halogen;
R2, R3, R4, Rs, and R6 are independently selected from H, OH or the Cl-C4 25 esters or alkyl ethers thereof, halogen, cyano, Cl-C6 alkyl, or trifluoromethyl, with the proviso that, when Rl is H, R2 is not OH;
X is selected from H, C1-C6 alkyl, cyano, nitro, trifluoromethyl, halogen;
Y is selected from:
a) the moiety:
,1~
wherein R7 and R8 are in~epen~ently select~d from the graup of H, Cl-C6 alkyl, phenyl b) a five-membered saturated, unsaturated or partially unsaturated heterocycle containing up to two heteroatoms selected from the group consisting of -O-, 10 -NH-, -N(ClC4 alkyl)-, -N=, and ~S(O)m~~ wherein m is an integer of from 0-2,optionally substituted with 1-3 substituents independently selected from the group consisting of hydrogen, hydroxyl, halo, Cl-C4 alkyl, trihalomethyl, Cl-C4 alkoxy, trihalomethoxy, Cl-C4 acyloxy, Cl-C4 alkylthio, Cl-C4 alkylsulfinyl, Cl-C4 alkylsulfonyl, hydroxy (Cl-C4)alkyl, -CO2H-, -CN-, -CONHRI-, -NH2-, Cl-C4 15 alkylamino, di(Cl-C4)alkylamino, -NHSO2RI-, -NHCORI-, -NO2-, and phenyl substituted with 1-3 (Cl-C4)alkyl;
c) a six-membered saturated, unsaturated or partially unsaturated heterocycle containing up to two heteroatoms selected from the group consisting of -O-, 20 -NH-, -N(ClC4 alkyl)-, -N=, and ~S(O)m~~ wherein m is an integer of from 0-2,optionally substituted with 1-3 substituents independently selected from the group consisting of hydrogen, hydroxyl, halo, Cl-C4 alkyl, trihalomethyl, Cl-C4 alkoxy, trihalomethoxy, Cl-C4 acyloxy, Cl-C4 alkylthio, Cl-C4 alkylsulfinyl, Cl-C4 alkylsulfonyl, hydroxy (Cl-C4)alkyl, -C02H-, -CN-, -CONHRI-, -NH2-, Cl-C4 25 alkylamino, di(Cl-C4)alkylamino, -NHSO2RI-, -NHCORI-, -NO2, and phenyl substituted with 1-3 (Cl-C4)alkyl;
d) a seven-membered saturated, unsaturated or partially unsaturated heterocycle containing up to two heteroatoms selected from the group30 consisting of -O-, -NH-, -N(ClC4 alkyl)-, -N=, and ~S(O)m~, wherein m is an integer of from 0-2, optionally substituted with 1-3 substituents independently selected from the group consisting of hydrogen, hydroxyl, halo, Cl-C4 alkyl, trihalomethyl, Cl-C4 alkoxy, trihalomethoxy, Cl-C4 acyloxy, Cl-C4 alkylthio, Cl-C4 alkylsulfinyl, Cl-C4 ., .
alkylsulfonyl, hydroxy (Cl-C4)alkyl, -CO2H-, -CN-, -CONHR,-, -NH2-, Cl-C4 aLkylamino, di(Cl-C4)alkylamino, -NHS02R,-, -NHCOR,-, -N02-, and phenyl substituted with (Cl-C4)alkyl; or e) a bicyclic ring system consisting of a five or six-membered heterocyclic ring fused to a phenyl ring, said heterocyclic ring con~inillg up to two he~ uatc,llls sek-cted from the group of -O-, -NH-, -N(ClC4 alkyl)-, and -S(O)m-, wherein m is an integer of from 0-2, optionally substitllt~-l with 1-3 substituents independently selected from the group consisting of hydrogen, hydroxyl, halo, Cl-C4 alkyl, trihalomethyl, Cl-C4 alkoxy, trihalomethoxy, Cl-C4 acyloxy, Cl-C4 alkylthio, Cl-C4 alkylsulfinyl, Cl-C4 alkylsulfonyl, hydroxy (Cl-C4)alkyl, -CO2H-, -CN-, -CONHR,-, -NH2-, Cl-C4 alkylamino, di(Cl-C4)alkylamino, -NHSO2R,-, -NHCORI, -NO2, and phenyl substituted with 1-3 (Cl-C4)alkyl.
The more preferred compounds of this invention are those having the general structures I or II, above, wherein:
R~ is selected from H, OH or the Cl-C4 esters or alkyl ethers thereof, halogen;
R2, R3, R4, R5, and R6 are independently selected from H, OH or the Cl-C4 esters or alkyl ethers thereof, halogen, cyano, Cl-C6 alkyl, or trifluoromethyl, with the proviso that, when Rl is H, R2 is not OH;
X is selected from H, Cl-C6 alkyl, cyano, nitro, trifluoromethyl, halogen;
Y is the moiety R ~l~
R7 and R8 are selected independently from H, Cl-C6 alkyl, or combined by -(CH2)p-, wherein p is an integer of from 2 to 6, so as to form a ring, the ring being optionally substituted by up to three substituents selected from the group of hydrogen, hydroxyl, halo, Cl-C4 alkyl, trihalomethyl, Cl-C4 alkoxy, trihalomethoxy, Cl-C4 alkylthio, Cl-C4 alkylsulfinyl, Cl-C4 alkylsulfonyl, hydroxy (Cl-C4)alkyl, -CO2H, -CN, -CONH(Cl-C4), -NH2, Cl-C4 alkylamino, Cl-C4 dialkylamino, -NHSO2(Cl-C4), -NHCO(Cl-C4), and -NO2.
CA 02203074 199i-04-18 The rings formed by a conc~tçn~ted R7 and Rg, mentioned above, may include, but are not limited to, aziridine, ~7.çti~1in5, pyrrolidine, piperidine, or hçY~m--,thylenç~mine rings.
S It is further preferred that, when R7 and R8 are conraten~t~1 together, the ring so formed is optionally substituted with 1-3 substituents selected from a group containing Cl-C3 alkyl, trifluoromethyl, halogen, hydrogen, phenyl, nitro, -CN.
The compounds of the invention are partial estrogen agonists and display high affinity for the estrogen receptor. Unlike many estrogens, however, these compounds do not cause increases in uterine wet weight. These compounds are antiestrogenic in the uterus and can completely antagonize the trophic effects of estrogen agonists in uterine tissue. These compounds are useful in treating or preventing ".~.""~l disease states or syndromes which are caused or associated with an estrogen deficiency.
The present compounds have the ability to behave like estrogen agonists by lowering cholesterol and preventing bone loss. Therefore, these compounds are useful for treating many maladies including osteoporosis, prostatic hypertrophy, infertility, breast cancer, endometrial cancer, cardiovascular disease, contraception, Alzheimer's disease and melanoma. Additionally, these compounds can be used for hormone repl~em.ont therapy in post-menopausal women or in other estrogen deficiency states where estrogen supplementation would be beneficial.
The compounds of this invention may also be used in methods of treatment for bone loss, which may result from an imh~l~n~e in an individual's formation of new bone tissues and the resorption of older tissues, leading to a net loss of bone. Such bone depletion results in a range of individuals, particularly in post-menopausal women, women who have undergone hysterectomy, those receiving or who have received extended corticosteroid therapies, those experiencing gonadal dysgenesis, and those suffering from Cushing's syndrome. Special needs for bone replacemçnt can also be addressed using these compounds in individuals with bone fractures, defective bone structures, and those receiving bone-related surgeries and/or the implantation of prosthesis. In addition to those problems described above, these compounds can be used in treatments for osteoarthritis, Paget's tli~e~e, osteomalacia, osteohalisteresis, endometrial cancer, multiple myeloma and other forms of cancer having deleterious effects on bone tissues. Methods of treating the mal~flies listed herein are understood to comprise a~ministering to an individual in need of such l~ca~ nt a ph~rm~relltir~lly effective amount of one or more of the compounds of this invention or a ph~ reutir~lly acceptable salt thereof. This invention also includes ph~rm~re~1tir~l 5 con~ositions utili7ing one or more of the present compounds, and/or the ph~.,-~relltir~lly acceptable salts thereof, along with one or more ph~rm:~reutic:~lly acceptable carriers, excipients, etc.
It is understood that the dosage, regimen and mode of ~-lmini~tration of these 10 compounds will vary according to the malady and the individual being treated and will be subjected to the judgment of the medical practitioner involved. It is plcfcllcd that the atlmini~tration of one or more of the compounds herein begins at a low dose and be increased until the desired effects are achieved.
Effective administration of these compounds may be given at a dose of from about 0.1 mg/day to about 1,000 mg/day. Preferably, a~lmini.stration will be from about 50 mg/day to about 600 mg/day in a single dose or in two or more divided doses.
Such doses may be a.lmini~tered in any manner useful in directing the active compounds herein to the recipient's bloodstream, including orally, parenterally 20 (including intravenous, intraperitoneal and subcutaneous injections), and transdermally.
For the purposes of this disclosure, tr~n~derm~l a~lmini~trations are understood to include all a lmini~trations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such a-lmini.ctrations may becarried out using the present compounds, or ph:~rm~reutically acceptable salts thereof, 25 in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).
Oral formulations containing the active compounds of this invention may comprise any conventionally used oral forms, including tablets, capsules, buccal30 forms, troches, lozenges and oral liquids, suspensions or solutions. Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharm~reutically acceptable starches (e.g. corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc. Useful tablet formulations may 35 be made by conventional compression, wet granulation or dry granulation methods and utili~ ph~rm~eutic~lly acceptable diluents, binding agents, lubricants, ~ integrants~
suspending or stabilizing agents, including, but not limited to, m~gn~sillm stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carbo~y~ hylcelllllose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia 5 gum, xanthan gum, sodium citrate, complex sili~-~tes, c~ lm carbonate, glycine, dextrin, sucrose, sorbitol, (3ir~1ci~lm phosphate, calcium sulfate, lactose, kaolin, "wl~ ol, sodium chloride, talc, dry st~rches and powdered sugar. Oral forrnul~tions herein may utilize standard delay or time release formulations to alter the absorption of the active compound(s). Suppository formulations may be made from traditional 10 materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.
Compounds of this invention can be synthesi7ed in a general sense according to 15 Scheme 1.
Scheme 1 R2 X R3 NH2 R4 R1~} R4 2 NaH, I 5/R6 Rz X R3 Br~~Br ~¢~ R4 --R 1-Deprotect if necessary Rl~R4 ~ \ 2- Pd(OAc)2,P(O-T~I)3 o~ R6 ~R7 E~N ~ R5 The initial indole synthesis is accomplished by heating an a~plo~iately 20 substituted aniline (1) with an a~l~liately substituted alpha-bromophenylpropriophenone (2) in a suitably high boiling solvent such as DMF. The , -product is then alkylated with a 4-bromobenzyl bromide to give the substituted indole (3). At this point, deprotection of phenols (if present) is done. Normally, the phenols are ~luL~cL~d as benzyl ethers and can conveniently be cleaved with TMSI. The acrylamides are coupled using Heck reaction conditions in either neat Et3N or Et3N/CH3CN.
Solvents used for reactions were anhydrous Aldrich Sure SealTM without further punfic~tion. Reagents were typically Aldrich and used without further purification. All reactions were carried out under a nitrogen atmosphere.
Chromatography was performed using 230-400 mesh silica gel (Merck Grade 60, Aldrich Chemical Company). Thin layer chromatography was performed with Silica Gel 60 F2s4 plates from EM Science. lH NMR spectra were obtained on a Bruker AM-400 instrument in DMSO and chemical shifts reported in ppm. Melting points were determined on a Thomas-Hoover apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer diffraction grating or Perkin-Elmer 784 spectrophotometers. Mass spectra were recorded on a Kratos MS 50 or Finnigan 8230 mass spectrometers. Fl~m~nt~l analyses were obtained with a Perkin-Elmer 2400 elemental analyzer. Analysis values are within 0.4 % of theoretical.
S-Benzvloxy-2-(4-benzvloxy-Dhenvl)-3-methyl-lH-indole A flask was charged with 4-benzyloxyaniline (45 g, 0.23 mol), 4'-benzyloxy-2-bromophenylpropiophenone (21g, 0.066 mol), and DMF (50 mL). The reaction was heated at reflux for 30 minutes and then cooled to rt and then partitioned between EtOAc (250 mL) and lN HCl (aq) (100 mL). The EtOAc was washed with NaHCO3 (aq) and brine, dried over MgSO4. The solution was concentrated and the residue taken up in CH2Cl2 and hexanes added to precipitate out 25g of a crude solid. The solid was dissolved in CH2Cl2 and evaporated onto silica gel and chromatographed using CH2Cl2/Hexane (1:5) to yield 9.2 g of a tan solid (33%): Mpt = 150-152~C; IH NMR(DMSO) 10.88 (s, 1 H), 7.56 (d, 2 H, J = 8.8 Hz), 7.48 (d, 4 H, J = 7.9 Hz), 7.42-7.29(m,6H),7.21 (d, lH,J=7.0Hz),7.13(d,2H,J=8.8Hz),7.08(d, lH,J=
2.2 Hz), 6.94 (dd, 1 H, J = 8.8, 2.4 Hz), 5.16 (s, 2 H), 5.11 (s, 2 H), 2.33 (s, 3 H);
IR (KBr) 3470, 2880, 2820, 1620 cm~l; MS eI m/z 419.
-~XAMP~ F 2 S-Benzyloxy-2-(4-fluoro-phenyl~-3-metl~yl) -lH-illdole The title compound was prepared similarly to (3): MP=132~C; lH NMR
(DMSO) 11.0 (s, 1 H), 7.68-7.64 (m, 2 H), 7.49-7.47 (m, 2 H), 7.41-7.31 (m, 5 H), 7.23 (d, 1 H, J = 8.8 Hz), 7.10 (d, 1 H, J = 2.4 Hz), 6.82 (dd, 1 H, J = 8.8, 2.4 Hz), 5.11 (s, 2 H), 2.34 (s, 3 H); MS EI m/z 331; CHN calcd for C22Hl8FNO.
5-Benzyloxy-2-(4-benzvloxv-~henvl)-3-methyl)-1-ylmethyl-(4-l~henvlbromide)-indole A solution of 60% NaH (0.17g, 7.1 mmol) in DMF (20 mL) was cooled to 0~C
and treated by dropwise addition of benzyloxyindole 1 (2.5g, 5.94 mmol) in DMF (10 mL). After 15 min, 4'-bromobenzylbromide (1.63g, 6.53 mmol) in DMF (10 mL) was added dropwise. The reaction was stirred for S min at 0~C and then at rt for an additional 20 min. The reaction mixture was diluted with ether (300 mL) and washed with NH4Cl (2x25 mL) then NaHCO3 (lx25 mL), and brine (25 mL). The organic extracts were dried over MgSO4 and concentrated. The residue was cryst~lli7e-l from THF/Hexanes to yield 2.7 g (77%) of 2: Mp=144-146~C; lH NMR (CDCl3) 7.51-7.36 (m,8H),7.34(d,4H,J=8.6Hz),7.20(d,2H,J=8.8Hz),7.15 (d, lH, J=2.4 Hz), 7.03-7.00 (m, 3 H), 6.89 (dd, 1 H, J = 8.8, 2.4 Hz), 6.80 (d, 2 H, J = 8.6 Hz), 5.14 (s, 2 H), 5.12 (s, 2 H), 5.09 (s, 2 H), 2.25 (s, 3 H); IR (KBr) 3400, 3020, 1600 cm~~;MSeIm/z587.
5-Benzyloxy-2-(4-fluoro-vhenvl)-3-methvl)-1-vlmethvl-(4-~henvlbromide)-indole The title compound was prepared similarly to compound 5. Mp=139-139.5~C;
lH NMR (DMSO) 7.49-7.46 (m, 2 H), 7.41-7.37 (m, 6 H), 7.33-7.27 (m, 4 H), 7.24 .
(d, 1 H,J=8.8Hz),7.16(d, 1 H,J=2.2Hz),6.84(dd, 1 H,J=8.8,2.4Hz),6.73 (d, 1 H, J = 8.6 Hz), 5.2 (s, 2 H), 5.12 (s, 2 H), 2.15 (s, 3 H); IR (KBr) 2920, 1630 cm~l; MS eI m/z (499/501, Br present); CHN calcd for C29H23BrFNO.
2-(4-hydroxyphenyl)-3-methyl)-1-ylmethyl-(4-pher~vlbrolnide)-indole-5-ol A solution consisting of 5 (0.5 g, 0.85 mmol) in CH2Cl2 (10 mL) was treated with by dropwise addition of 3.5 eq TMSI (0.47 mL, 3.0 mmol) at rt. After a couple hours, the reaction stopped so an additional 2.2 eq TMSI was added and the reaction heated to reflux for 5 h. The reaction was cooled to 0~C and methanol slowly added to quench the reaction. The reaction was diluted with ether (25 mL) and washed withNaHCO3(25 mL), 10 % Na2SO3 (25 mL), and brine. The ether layer was dried over MgSO4 and concentrated onto silica gel. Chromatography with EtOAc/Hexanes (1 :4 to 1:1) yielded 0.25 g 3 (71%): Mp=83-86~C; IH NMR (CDCl3) 2 H's from phenols broad (> 10),s 7.35 (d, 2 H, J = 9.0 Hz), 7.15 (d, 2 H, J = 8.8 Hz), 7.01 (dd, 1 H, J
=2.4,0.4HZ),6.86(d,2H,J=8.8HZ),6.80(d, 1 H,J=8.6 Hz), 6.72 (dd, 1 H, J = 8.6, 2.4 Hz), 5.10 (s, 2 H), 4.88 (s, 1 H), 4.50 (s, 1 H), 2.21 (s, 3 H); MS eI m/z 407/409 contains Br, IR 3390, 2900, 1600 cm~l; CHN calc'd for C22H~8BrNO2 + 0.25EtOAc.
2-(4-fluoro-phenvl)-3-methyl)-1-vlmethvl-(4-~henvlbromide)-indole-5-ol The title compound was prepared similarly to compound 7 and isolated as a foam. IH NMR (DMSO) 8.79 (s, 1 H), 7.39-7.34 (m, 4 H), 7.32-7.30 (m, 3 H), 7.11 (d, 1 H, J = 8.8 Hz), 6.85 (d, 1 H, J = 2.2 Hz), 6.74 (d, 1 H, J = 2.4 Hz), 6.63 (dd, 1 H, J = 8.6, 2.2 Hz), 5.16 (s, 2 H), 2.11 (s, 3 H); IR (KBr) 3400, 2900, 1630 cm~l; MS eI m/z 409/411 contains Br.
General Procedure For Indole Acrylamides-A solution of example 3 in Et3N is treated with tri-o-tolylphosphine (10 mol%), and acrylamide (1.25 eq) is purged thoroughly with N2 and Pd(OAc)2 (2.5 mol%) S added. The reaction is heated at 100-110~C in a sealed tube until completed by TLC
analysis. The crude reaction product is concçn~t~ down and either cryst~lli7e~
directly or chromatographed on silica gel.
F.XAMPI F 7 (F)-N. N-Diethvl-3-r4-r5-hvdroxy-2-(4-l~ydroxy-Dhenyl)-3--nethyl-indol- l-vlmethvll-~henvl~-acrvlamide Mp=160- 165~C; lH NMR 9.67 (s, 1 H), 8.72 (s, 1 H), 7.50 (d, 2 H, J = 8.1 Hz), 7.37 (d, 1 H, J = 15.4 Hz), 7.17 (d, 2 H, J = 8.3 Hz), 7.06 (d, 1 H, J = 8.8 Hz), 6.97 (d, 2 H, J = 15.4 Hz), 6.86-6.82 (m, 5 H), 6.58 (dd, 1 H, J = 8.6, 2.2Hz), 5.19 (br s, 2 H), 3.47-3.42 (m, 2 H), 3.34-3.30 (m, 2 H), 2.09 (s, 3 H), 1.10 (t, 3 H, J = 7.0 Hz), 1.03 (t, 3 H, J = 7.0 Hz); IR (KBr) 3300, 2950, 1660, 1580 cm~l;
MS (eI) m/z 454; CHN calc'd for C29H3oN2o3+o.l5 CH2C12 + 0-30 H2O-l(E)-N-tert-butyl-3-r4-r5-hydroxv-2-(4-hydroxy-~henyl)-3-methvl-indol- l-ylmethyll-~henyl~-acrylamide Mp=168-170~C; lH NMR 9.66 (s, 1 H), 8.71 (s, 1 H), 7.66 (s, 1 H), 7.34 (d, 2H,J=8.3Hz),7.24(d,1H,J=15.8Hz),7.15(d,2H,J=8.3Hz),7.05(d,1 H, J = 8.6 Hz), 6.85-6.82 (m, 5 H), 6.59-6.56 (m, 1 H), 6.55 (d, 1 H, J = 16.0 Hz), 5.18 (s, 2 H), 2.11 (s, 3 H), 1.28 (s, 9 H); IR (KBr) 3350, 2950, 1660, 1620; MS(eI) m/z 454; CHN calc'd for C29H30N203 + 0.4H20 FxAMP~ F 9 (F,).Pyrrolidino.3.r4.rS.hydroxy-2-(4-hydro~y-~herlvl)-3-~nethyl-irldol-1-ylmethvll-phenyl~-acrylamide Mp=170- 175~C; IH NMR 9.67 (s, 1 H), 8.71 (s, 1 H), 7.49 (d, 2 H, J = 8.1 Hz), 7.35 (d, 1 H, J = 15.4 Hz), 7.16 (d, 2 H, J = 8.6 Hz), 7.05 (d, 1 H, J = 8.8 Hz), 6.88-6.81 (m, 6 H), 6.57 (dd, 1 H, J = 8.6, 2.2 Hz), 5.19 (br s, 2 H), 3.56 (t, 2 H,J=6.6Hz),3.35(m,2H),2.11 (s, 3H), 1.87 (p, 2H, J=7.0Hz), 1.77 (p, 2 H, J = 7.0 Hz); MS m/z 452; CHN calc'd for C29H28N2O3 + 0.1 MeOH + 1-3 H2O.
FXAMP~,F 10 (F).N. N-Dimethyl-3-~4-r5-hvdroxy-2-(4-hydroxy-phenyl)-3-methyl-indol-1-vlmethyll-phenvl~-acrylamide Mp=278-280~C; lH NMR (DMSO) 9.65 (s, 1 H), 8.70 (s, 1 H), 7.50 (d, 2 H, J=8.1Hz),7.33(d, lH,J=15.4Hz),7.15(d,2H,J=8.6Hz), 7.07 (d, 1 H, J=
15.6 Hz), 7.05 (d, 1 H, J = 8.8 Hz), 6.85-6.80 (m, 5 H), 6.57 (dd, 1 H, J = 8.6, 2.4 Hz), 5.19 (s, 2 H), 3.09 (s, 3 H), 2.88 (s, 3 H), 2.11 (s, 3 H); MS eI m/z 426; IR
(KBr) 3410, 3220, 1650, 1580 cm~l; CHN calc'd for C27H26N2O3 + 0.5 H20.
(F)-N. N-Dibutyl-3-~4-r5-hydroxy-2-(4-l~ydroxy-~henyl~-3-methyl-indol-l-ylmethyll-vhenvl~-acrylamide Mp=126-128~C, IH NMR (DMSO) 9.65 (s, 1 H), 8.70 (s, 1 H), 7.48 (d, 2 H, J = 8.3 Hz), 7.36 (d, 1 H, J = 15.2 Hz), 7.16 (d, 2 H, J = 8.6 Hz), 7.05 (d, 1 H, J =
8.6 Hz), 6.97 (d, 1 H, J = 15.2 Hz), 6.86-6.81 (m, 5 H), 6.57 (dd, 1 H, J = 8.8, 2.4 Hz), 5.19 (s, 2 H), 3.39 (t, 2 H, J = 7.0 Hz), 3.29 (t, 2 H, J = 7.2 Hz), 2.11 (S, 3 H), 1.48-1.43 (M, 4 H), 1.29-1.20 (M, 4 H), 0.87 (t, 6 H, J = 7.2 Hz); MS eI m/z510; IR (KBr) 3300, 2920, 2900, 2850, 1650, 1625, 1580 cm~l; CHN calc'd for ~ CA 02203074 1997-04-18 (F,).N.BIltyl~ N'-methyl-3-r4-r5-ll~vdroxv-2-(4-hvdroxy-~henyl)-3-lnethyl-indol- 1-vlmethyll-phenyl~-acryl~mide Mp=240-242~C; lH NMR (DMSO) 9.66 (s, 1 H), 8.70 (s, 1 H), 7.50 (d, 2 H, J = 8.1 Hz), 7.38-7.32 (m, 1 H), 7.16 (d, 2 H, J = 6.8 Hz), 7.06-7.01 (m, 2 H), 6.85-6.81 (m, 5 H), 6.57 (dd, 1 H, J = 8.6, 2.2 Hz), 5.19 (s, 2 H), 3.44, 3.33 (2 t, 2 H,J=7.2Hz),3.06,2.87(2s,3H),2.11(s,3H),1.45(m,2H), 1.24(p,2H,J=
7.5 Hz), 0.87 (t, 3 H, J = 7.2 Hz); Ms eI m/z 468; IR (KBr) 3300, 1660, 1590 cm~l;
CHN calc'd for C30H32N2O3 + 0.2 H2O.
(F)-MorDholino-3-r4-r5-hydroxv-2-(4-hvdroxv-~henvl)-3-metl~vl-indol l-vlmethvll-Dhenvl~-acrvlamide Mp=165-167~C, lH NMR (DMSO) 9.66 (s, 1 H), 8.71 (s, 1 H), 7.52 (d, 2 H, J=8.1Hz),7.39(d, lH,J=15.4Hz),7.15(d,2H,J=8.6Hz),7.12 (d, lH, J=
15.4 Hz), 7.06 (d, 1 H, J = 8.6 Hz), 6.85-6.81 (m, 5 H), 6.57 (dd, 1 H, J = 8.6, 2.2 Hz), 5.19 (s, 2 H), 3.65-3.64 (m, 2 H), 3.59-3.53 (m, 6 H), 2.11 (s, 3 H); IR (KBr) 3330, 1650, 1620, 1580 cm-1; MS (FAB) m/z 469 (M+H+); CHN calc'd for C29H28N2O4 + 0.5 H2O-(F,)-3-~4-rS-hvdroxy-2-(4-hvdroxv-Dhenyl)-3-methvl-indol-1-ylmethyll-Dhenvl~-acrvlamide Mp=161-163~C, lH NMR (DMSO) 9.65 (s, 1 H), 8.70 (s, 1 H), 7.48 (s, 1 H), 7.37 (d, 2 H, J = 8.35 Hz), 7.30 (d, 1 H, J = 15.8 Hz), 7.14 (d, 2 H, J = 8.35 Hz), 7.04 (d, 2 H, J = 8.6 Hz), 6.85-6.81 (m, 5 H), 6.57 (dd, 1 H, J = 8.8, 2.4 Hz), 6.48 (d, 1 H, J = 15.8 Hz), 5.18 (s, 2 H), 2.10 (s, 3 H); IR (KBr) 3320, 3180, 1660, 1580 cm~l; MS (FAB) m/z 399 (M+H ); CHN calc d for C25H22N2O3 + 1-3 H20.
~ CA 02203074 1997-04-18 , .
(F).N. Methvl-3-r4-r5-hydroxy-2-(4-l~vdroxy-~he~v1)-3-methvl~ dol-I -ylmethvll-~henvl~-acrylamide s Mp=155-158~C; lH NMR (DMSO) 9.64 (s, 1 H), 8.70 (s, 1 H), 7.99 (q, 1 H, J = 4.4 Hz), 7.37 (d, 2 H, J = 8.1 Hz), 7.30 (d, 1 H, j = 15.8 Hz), 7.14 (d, 2 H, J =
8.6 Hz), 7.03 (d, 1 H, J = 8.6 Hz), 6.85-6.81 (m, 5 H), 6.57 (dd, 1 H, J = 8.6, 2.4 Hz), 6.48 (d, 1 H, J = 15.8 Hz), 5.18 (s, 2 H), 2.66 (d, 3 H, J = 4.6 Hz), 2.10 (s, 3 H); IR (KBr) 3400, 1660, 1620 cm-1; MS eI m/z 412; CHN calc'd for C26H24N2O3 +
0.4 H2O.
(F)-N, N-Dibutyl-3-r4-r5-hydroxv-2-(4-tluoro-Dhenvl)-3-metl~yl-indol-1-vlmethvll-l~henvl~-acrvlamide Mp=180~C; lH NMR (DMSO) 8.77 (s, 1 H), 7.48 (d, 2 H, J = 8.4 Hz), 7.41-7.38 (m, 3 H), 7.38-7.29 (m, 3 H), 7.13 (d, 1 H, J = 8.8 Hz), 6.97 (d, 1 H, J = 15.4 Hz), 6.85 (d, 1 H, J = 2.4 Hz), 6.80 (d, 2 H, J = 8.1 Hz), 5.2 (s, 2 H), 3.40-3.36 (m, 2 H), 3.30-3.27 (m, 2 H), 2.10 (s, 3 H), 1.50-1.40 (m, 4 H), 1.29-1.21 (m, 4H), 0.86 (t, 6 H, J = 7.2 Hz); IR (KBr) 3180, 2950, 2900, 2850, 1650, 1590 crn~l;
MS eI m/z 512; CHN calcd for C33H37N2O2.
(F)-N-Butvl. N'-Methvl-3-r4-r5-hvdroxv-2-(4-fl~oro-Dhenyl)-3-methvl-indol- 1-ylmethvll-phenvl~-acrvlamide Mp=153-153.5~C; lH NMR (DMSO) 8.77 (s, 1 H), 7.50 (d, 2 H, J = 8.1 Hz), 7.42-7.36 (m, 2 H), 7.35-7.28 (m, 3 H), 7.13 (d, 1 H, J = 8.8 Hz), 7.03 (dd, 1 H, J
= 15.4, 2.6 Hz), 6.84 (d, 1 H, J = 2.4 Hz), 6.80 (d, 2 H, J = 8.1 Hz), 6.62 (dd, 1 H, J = 8.8, 2.4 Hz), 5.21 (s, 2 H), 3.44, 3.41 (2 t, 2 H, J = 7.0 Hz), 3.06, 2.87 (2 s, 3 H), 2.10 (s, 3 H), 1.49-1.42 (m, 2 H), 1.27-1.20 (m, 2 H), 0.86 (t, 3 H); IR (KBr) 3300, 2950, 2860, 1645, 1580 cm; MS eI m/z 470; CHN calcd for C30H3lFN2O2.
. . .
R jolo~ l MethQ5~
In vitro estro~n receDtor bindin~ assay s ReceDtor Dre,Daration CHO cells ovele,~l)r~ssing the estrogen receptor were grown in 150 mm2 dishes in DMEM + 10% dextran coated charcoal, stripped fetal bovine serum. The plates were 10 washed twice with PBS and once with lOmM Tris-HCl, pH 7.4, lmM EDTA. Cells were harvested by scraping the surface and then the cell suspension was placed on ice.
Cells were disrupted with a hand-held motorized tissue grinder using two, 10-second bursts. The crude preparation was centrifuged at 12,000g for 20 minutes followed by a 60 minute spin at lOO,OOOg to produce a ribosome free cytosol. The cytosol was then 15 frozen and stored at -80~C. Protein concentration of the cytosol was estimated using the BCA assay with reference standard protein.
Bindin~ ~say conditions The competition assay was performed in a 96-well plate (polystyrene*) which binds <2.0% of the total input [3H]-17,~-estradiol and each data point was gathered in triplicate. lOOuG/lOOuL of the receptor preparation was aliquoted per well. A
saturating dose of 2.5 nM [3H] 17,~-estradiol + competitor (or buffer) in a 50 uL volume was added in the prelimin~ry competition when lOOx and 500x competitor were ev~ t~l, only 0.8 nM [3H] 17,B-estradiol was used. The plate was incubated at room temperature for 2.5 h. At the end of this incubation period 150 uL of ice-cold dextran coated charcoal (5% activated charcoal coated with 0.05% 69K dextran) was added to each well and the plate was im m~ t~:1y centrifuged at 99g for 5 minutes at 4~C. 200 uL of the supernatant solution was then removed for scintillation counting. Samples were counted to 2% or 10 minutes, whichever occurs first. Because polystyrene absorbs a small amount of [3H] 17~-estradiol, wells containing radioactivity and cytosol, but not processed with charcoal were included to q~ ntit~t~ amounts of available isotope. Also, wells containing radioactivity but no cytosol were processed with charcoal to estimate unremovable DPM of [3Hj 17,~-estradiol. Corning ~25880-96, 96-well plates were used because they have proven to bind the least arnount of estradiol.
Anabsis of res--lts s Counts per minute (CPM) of radioactivity were ~ ic~lly converted to rli~integrated per minute (DPM) by the Bec~m~n LS 7500 Scintill~hon Counter using a set of quenched standards to generate a H# for each sample. To calculate the % of estradiol binding in the presence of 100 or fold 500 fold COIllpel~ the following 10 formula was applied:
((DPM sample-DPM not removed by charcoal) /(DPM estradiol-DPM not removed by charcoal)) x 100% = % of estradiol binding For the generation of IC50 curves, % binding is plotted vs compound. IC50's are generated for compounds that show >30% competition at 500x colll~i[or concentration. For a description of these methods, see Hulme, E.C., ed. 1992.
Receptor-Ligand Interactions: A Practical Approach. IRL Press, New York.(see especially chapter 8).
I~hikawa Cell Alkaline Phos~hatase Assav Cell M~inten~nce and Treatment:
Ishikawa cells were ,~ ined in DMEM/F12 (50%:50%) containing phenol red + 10% fetal bovine serum and the m~Aillm was supplemented with 2 Glutamax, 1% Pen/Strap and 1 mM sodium pyruvate. Five days prior to the beginning of each experiment (tleatlllell~ of cells) the m~Aillm was changed to phenol red-free DMEM/F12 + 10% dextran coated charcoal ~llipped serum. On the day before ll~a~ ellt, cells were harvested using 0.5% trypsinlEDTA and plated at a density of S X
104 cells/well in 96-well tissue culture plates. Test compounds were dosed at 10~, 10-7 and 10-8M in addition to 10~ M (compound) + 10-9 M 17~- estradiol to evaluate the ability of the compounds to function as antiestrogens. Cells were treated for 48 h prior to assay. Each 96-well plate contained a 17~estradiol control. Sample population for at each dose was n=8.
Alk~line Phos~h~qt~e Assay:
At the end of 48h the media is aspirated and cells are washed three times with S phosphate buffered saline (PBS). 50~LL of Iysis buffer (0.1 M Tris-HCl, pH 9.8, 0.2%TritonX-100)isaddedtoeachwell. Platesareplacedat-80~Cfora ,~ "- of 15 minutes. Plates are thawed at 37~C followed by the addition of 15011L of 0.1 M
Tris-HCl, pH 9.8, cc,ntaining 4 mM para-nitrophenylphosphate (pNPP) to each well(final concentration, 3 mM pNPP).
Absorbance and slope calculations were made using the KineticCalc Application program (Bio-Tek Instruments, Inc., Winooski, VT). Results are expressed as the mean +/- S.D. of the rate of enzyme reaction (slope) averaged over the linear portion of the kinetic reaction curve (optical density readings every 5 minutes for 30 minutes 15 absorbance reading). Results for compounds are ~ ;7ed as percent of response related to 1 nM 17,B estradiol.
Various compounds were assayed for estrogenic activity by the alkaline phosphatase method and corresponding ED50 values (95% C.I.) were calculated. The20 four listed in the following were used as reference standards:
17~-estradiol 0.03 nM
17a-estradiol 1.42 nM
estriol 0.13 nM
estrone 0.36 nM
A description of such methods is provided by Holinka, C.F., Hata, H., Kuramoto, H. and Gurpide, E. (1986) Effects of steroid hormones and antisteroids on alkaline phosphatase activity in human endometrial cancer cells (Ishikawa Line).30 Cancer Research, 46:2771-2774, and by Littlefield, B.A., Gurpide, E., Markiewicz, L., McKinley, B. and by Hochberg, R.B. (1990) A simple and sensitive microtiter plate estrogen bioassay based on shm~ on alkaline phosphatase in Ishikawa cells;Estrogen action of D5 adrenal steroids. Endocrinology, 6:2757-2762.
2X VIT FRF Trar ~fection Assav Cell M~intenance and Treatment Chinese Harnster Ovary cells (CHO) which had been stably transfected with the human estrogen receptor were .~ ecl in DMEM + 10% fetal bovine serum (FBS).
48h prior to tre~tm~ont the growth m~lillm was replaced with DMEM lacking phenol red + 10% dextran coated charcoal stripped FBS (treatment m~illm). Cells were plated at a density of 5000 cells/well in 96-well plates cont~ining 200 ~L of medium/well.
Calcium Phoshate Transfection Reporter DNA (Promega plasmid pGL2 cont:~ining two tandem copies of the vitellogenin ERE in front of the minim:3l thymidine kinase promoter driving the luciferase gene) was combined with the B-galactosidase expression plasmid pCHl 10 (Pharmacia) and carrier DNA (pTZl 8U) in the following ratio:
10uG of reporter DNA
5uG of pCHl 10DNA
5 uG of pTZ18U
20 uG of DNA/l mL of transfection solution The DNA (20uG) was dissolved in 500 uL of 250 mM sterile CaCl2 and added dropwise to 500 uL of 2 X HeBS (0.28 M NaCl, 50 mM HEPES, 1.5 mM Na2HPO4, pH 7.05) and incubated at room temperature for 20 minutes. 20 uL of this n~i~ e was added to each well of cells and remained on the cells for 16 h. At the end of this incubation the precipitate was removed, the cells were washed with media, fresh tre~tm.ont media was replaced and the cells were treated with either vehicle, 1 nM 17,B-estradiol, luM compound or 1 uM compound + 1 nM 17~-estradiol (tests for estrogen antagonism). Each ~leatll~-lt condition was performed on 8 wells (n=8) which were incubated for 24 h prior to the luciferase assay.
~ CA 02203074 1997-04-18 . .
cifer~ce Assay After 24h exposure to compounds, the media was removed and each well washed with 2 X with 125 uL of PBS lacking Mg++ and Ca++. After removing the 5 PBS, 25 uL of Promega lysis buffer was added to each well and allowed to stand at room ~Illpe~lu~e for 15 min, followed by 15 min at -80~C and 15 min at 37~C. 20 uL
of lysate was transferred to an opaque 96 well plate for luciferase activity ev~ tion and the rem~ining lysate (5 uL) was used for the B-galactosidase activity ev~lu~tiQn (norm~li7~ transfection). The luciferan substrate (Promega) was added in 100 uL
10 aliquots to each well automatically by the luminometer and the light produced (relative light units) was read 10 seconds after addition.
B-Galactosidase Assay To the remaining 5 uL of lysate 45 uL of PBS was added. Then 50 uL of Promega B-galactosidase 2X assay buffer was added, mixed well and incubated at 37~C for 1 hour. A plate containing a standard curve (0.1 to 1.5 millillnit.c in triplicate) was set up for each ~ .lental run. The plates were analyzed on a Molecular Devices spectrophotometric piate reader at 410 nm. The optical densities for the unknown were converted to milli~lnitc of activity by mathematical extrapolation from the standard curve.
Analysis of Results The luciferase data was generated as relative light units (RLUs) ~cllmlll~t~
during a 10 second mea~u-t;--~llt and automatically transferred to a JMP (SAS Inc) file where background RLUs were subtracted. The B-galactosidase values were aulo~ 11y imported into the file and these values were divided into the RLUs to n()rm~li7e the data. The mean and standard deviations were determined from a n=8 for each treatment. Compounds activity was co-,-pared to 17,B-estradiol for each plate.
Percentage of activity as compared to 17,B-estradiol was calculated using the formula %=((Estradiol-control)/(compound value)) X 100. These techniques are described by T7llk~rm~n, M.T., Esty, A., Santiso-Mere, D., D~ni~ n, P., Parker, M.G., Stein, R.B., Pike, J.W. and McDonnel, D.P. (1994). Human estrogen receptor transactivational capacity was determined by both cellular and promoter context and m~ t~l by two functionally distinct intr~m~l~c~ r regions (see Molecular Endocrinology, 8:21-30).
~t Uterotrophic/AntillterotroDhic Bioassay The estrogenic and antiestrogenic properties of the compounds were dt;lr. ..lil~ed in an imm~tllre rat uleruh~hic assay (4 day) that (as described previously by L.J.Black and R.L.Goode, Life Sciences, 26, 1453 (1980)). Tmm~ re Sprague-Dawley rats (female, 18 days old) were tested in groups of six. The anirnals were treated by daily ip injection with 10 uG compound, 100 uG compound, (100 uG compound + 1 uG
17,B-estradiol) to check antiestrogenicity, and 1 uG 17~-estradiol, with 50%
DMSO/50% saline as the injection vehicle. On day 4 the animals were sacrificed by CO2 asphyxiation and their uteri were removed and stripped of excess lipid, any fluid removed and the wet weight determined. A small section of one horn was submitted15 for histology and the rem~inder used to isolate total RNA in order to evaluate complement component 3 gene expression.
l~iolo~ical Results Fstro~en Receptor Affinity (reDorted ~c RBA: 17,B-estradiol=100) Compound RBA
raloxifene 200 tamoxifen 1.8 Example 10 20 Example 7 42 Example 8 40 Example 9 40 Example 12 114 Example 11 80 Exarnple 13 27 Example 14 32 Example 15 53 Infection Lucifer~e Assav Co~o--nd % Activation % Activation with 1 nl~I
1 7~-estradiol 17~-estradiol 100% N/A
estriol 38% N/A
~lloxil~-l 0% 10%
raloxifene 0% 0%
Example 10 1% 2%
Example 7 4% 8%
Example 8 6% 78%
Example 9 6% 8%
Example 12 13% 24%
Example 11 8% 12%
Example 13 8% 17%
Example 14 19% 57%
Example 15 15% 31%
I~hikaw~ Alkaline Phos~hatase Assav Compound % Activation % Activation (Compound +
1 nM 17,B-estradiol) 17,B-estradiol 100% N/A
tamoxifen 0% 45%
raloxifen 5% 5%
Example 10 6% 19%
Example 7 1% 9%
Example 8 10% 22%
Example9 3% 11%
Example 12 7% 16%
Example 11 6% 11%
Example 13 7% 9%
Example 14 2% 14%
Example 15 0% 5%
3-Dav Ov~riectomi~d Rat Model Co~Do--nd 10~1G lOOIIG
Ta ~ xiren 69.6 mg 71.4 mg Raloxifen 47.5 mg 43.2 mg con~ol = 42.7 mg 1 llG 17~-estradiol = 98.2 Comuo--nd 1011G 10011G 100~G plus l~G_ 1 7~B-F~tradiol Example 7 47.8 mg 64.8 mg 75.4 control = 20.2 mg 1 IlG 17~-estradiol = 80.2 mg Com~ound 10~G 10011G 100~G plus l~G_ 1 7,B-Estradiol Example 12 36.9 mg 49.5 mg 63.1 con~ol = 31.4 mg 1 IlG 17,~-estradiol = 89.0 mg I
Com~ound 1011G 10011G 100~G plus l~,lG
17~ F~tradiol 20 Example 11 39.3 mg 59.8 mg 81.0 mg control = 24.5 mg 1 ~G 17,B-estradiol = 90.8 mg Comvound 10~G 10011G 100,uG plus l~uG_ 1 7~-Fstradiol 25 Example 14 32.5 mg 56.4 mg 79.8 mg Exampe 15 40.4 mg 56.3 mg 69.3 mg control = 29.1 mg l~g 17,B-estradiol = 95.5 mg