CA2203073A1 - Estrogenic agents - Google Patents

Abstract

The present invention relates to new 2-Phenyl-1-[4(amino-1-yl-alk-1-ynyl)-benzyl]-1H-indol-5-ol compounds which are useful as estrogenic agents, as well as pharmaceutical compositions and methods of treatment utilizing these compounds, which have the general structure below.

(see fig. I) or (see fig. II)

Description

' i CA 02203073 1997-04-18 FSTROGFNIC AGFNTS

The present invention relates to new 2-Phenyl-1-[4-(amino-1-yl-alk-1-ynyl)-S benzyl]-lH-indol-5-ol compounds which are useful as estrogenic agents, as well as ph~ eutical compositions and methods of tre~tm.-nt utilizing these compounds.

Rack~round of the inventiorl The use of hormone repl~rçment 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 (i.e. Premarin(~) conjugated estrogens from Wyeth-Ayerst). In some patients, therapy may be contr~in-lic~t~d due 15 to the proliferative effects of unopposed estrogens (estrogens not given in combination with progestins) have on uterine tissue. This proliferation is associated with increased risk for endometriosis and/or endometrial cancer. The effects of unopposed estrogens on breast tissue are less clear, but are of some concern. The need for estrogens which can ~ in the bone sparing effect while minimi7ing the proliferative effects in the~0 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 (sold as Novadex~g) brand tamoxifen citrate by Zeneca Pharmaceuticals,Wilmington, Delaware), for example, is a useful palliative for the treatment of breast cancer and has been demonstrated to exert an estrogen agonist-like effect on the bone, 25 in hllm~ns. 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 ovariectomized rat to a lesser extent than Tamoxifen while...~3i.-l;.i.~il-g the ability to spare bone. A suitable review of tissue selective estrogens is seen in the article "Tissue-Selective Actions Of Estrogen Analogs", Bone Vol. 17, No.
4, October 1995, 181S-190S.

The use of indoles as estrogen antagonists has been reported by Von Angerer, Ch~mic~l Abstracts, Vol. 99, No. 7 (19B3), 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 A1 891228 and WO 96/03375. These prior art compounds share structural ~imil~rities with the present compounds, but are filnrtiQn~lly dirr~ t. For co"~pounds co~ "i-lg a basic amine, there is no phenyl group to rigidify the side chain.

S WO A 95 17383 (Karo Bio AB) describes indole antiestrogens with long straight chains. Another related patent WO A 93 10741 describes S-Hydroxyindoleswith a broad range of side chains. WO 93/23374 (Otsuka Pharm~cellticals, Japan) describes compounds sharing structural similarities with those of the present invention, except with the structure referred to as R3 in the present form~ I and II, below, is defined as thioalkyl and the reference discloses no such compounds having chains from the indole nitrogen having the same structure as the ones provided by the present inventlon.

DescriDtion of the Invention The present invention provides 2-Phenylindoles of the general structures shown in formulas (I) and (II), below, which exhibit strong binding to the estrogen receptor and are estrogen agonists/antagonists useful for the treatment of diseases associated with estrogen deficiency. In vitro assays including an Ishikawa alkaline phoshatase assay and an ERE transfection assay show these compounds are antiestrogens with little to no intrinsic estrogenicity. In a three-day ovariectomized rat model, compounds of formula (I) are capable of antagonizing the effects of 17,~- estradiol while showing little uterine stim~ tion when dosed alone.

The present invention includes compounds of formulas (I) or (II):
(I) (II) or ~R R4 R5~ R5 ~

(CH2)n (CH2)n Y Y

W~ C~
Rl is sele~t~d 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 esters or alkyl ethers thereof, halogen, cyano, Cl-C6 alkyl, or trifluol~lllelllyl~ with the proviso that, when Rl is H, R2 is not OH;
X is selected from H, Cl-C6 alkyl, cyano, nitro, triflouromethyl, halogen;
nis20r3;
Y is selected from:
a) the moiety:
~N/ R7 wherein R7 and R8 are independently selected from the group of H, Cl-C6 alkyl,phenyl;

b) a five-m~mhered saturated, unsaturated or partially unsaturated heterocycle containing up to two heteroatoms selected from the group consisting of -O-, -NH-, -N(CIC4 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-, -CONHRl-, -NH2-, Cl-C4 alkylamino, Cl-C4 dialkylamino, -NHS02R,-, -NHCORl-, -N02-, and phenyl optionally 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-, -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 sele~teA from the group con~icting 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-, -CONHRl-, -NH2-, Cl-C4 aIkylamino, di(Cl-C4)alkylamino, -NHS02R,-, -NHCORI-, -N02, and phenyl optionally substituted with 1-3 (Cl-C4)alkyl;

d) a seven-membered saturated, unsaturated or partially unsaturated heterocycle con~ -g up to two he~lvatollls selected from the group consisting of -O-, -NH-, -N(C1C4 alkyl)-, -N=, and ~S(O)m~~ wherein m is an integer 5 of from 0-2, optionally substituted with 1-3 substitlle.nt~ indepen(lently selected from the group con~i~ting 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-, -CONHR1-, -NH2-, Cl-C4 alkylarnino, di(Cl-C4)alkylamino, -NHSO2R,-, -NHCORl-, -NO2, and phenyl 10 optionally substituted with 1-3 (Cl-C4)alkyl; or e) a bicyclic heterocycle containing from 6-12 carbon atoms either bridged or fused and containing up to two heteroatoms solec~ed from the group consisting of -O-, -NH-, -N(ClC4 alkyl)-, and ~S(O)m~~ wherein m is an integer of 15 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-, -CONHR1-, -NH2-, Cl-C4 alkylamino, di(Cl-C4)alkylamino, -NHSO2Rl-, -NHCORl-, -NO2, and phenyl 20 optionally substituted with 1-3 (Cl-C4)alkyl;
and the pharmaceutically acceptable salts thereof.

The more preferred compounds of this invention are those having the general 25 structures I or II, above, wherein:

Rl is selected from H, OH or the Cl-C4 esters or alkyl ethers thereof, halogen;
R2, R3, R4, R5, and R6 are independently selected firom H, OH or the Cl-C4 esters or alkyl ethers thereof, halogen, cyano, Cl-C6 alkyl, or trifluor~ yl, with the 30 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 ~N~ R7 \8 R7 and R8 are selected independently from H, C1-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 select~ from the group of hydrogen, hy(l~o~yl, halo, Cl-C4 alkyl, trihalomethyl, Cl-C4 alkoxy, trihalomethoxy, Cl-C4S alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, hydroxy (C1-C4)alkyl, -CO2H, -CN, -CONH(C1-C4)alkyl, -NH2, C1-C4 alkylamino, C1-C4 dialkylamino, -NHSO2(C1-C4)alkyl, -NHCO(C1-C4), and -NO2;
and the pharmaceutically acceptable salts thereof.

The rings formed by a concatenated R7 and Rg, mentioned above, may include, but are not limited to, aziridine, azetidine, pyrrolidine, piperidine, or hexamethylene~mine rings.

The most preferred compounds of the present invention are those having the 15 structural formulas I or II, above, wherein R1 is OH; R2 - R6 are as defined above; X is selected from the group of Cl, NO2, CN, CF3, or CH3; and Y is the moiety 20 and R7 and R8 are concatenated together as -(CH2)p-, wherein p is an integer of from 4 to 6, to form a ring 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 (C1-C4)alkyl, -CO2H, -CN, -CONH(Cl-C4), -NH3, C1-C4 alkylamino, C1-C4 25 dialkylamino, -NHSO2(Cl-C4), -NHCO(Cl-C4), and -NO3;
and the pharmaceutically acceptable salts thereof.

It is further plcr~ d that, when R7 and R8 are concatenated together as -~CH2)p-, the ring so formed is optionally substituted with 1-3 substituents selected 30 from a group containing Cl-C3 alkyl, trifluoromethyl, halogen, hydrogen, phenyl, nitro, -CN.

The invention includes acceptable salt forms formed from the addition reaction with either inorganic or organic acids. Inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid useful as well as organic acids such as acetic acid, propionic acid, citric acid, maleic acid, malic 5 acid, tartaric acid, phthalic acid, succinic acid, m~th~neslllfonic acid, toluenesulfonic acid, napthalenesulfonic acid, camphorsulfonic acid, ben7.on~sll1fonic acid are useful.
It is known that compounds possessing a basic nitrogen can be complexed with rnany dirr~ acids (both protic and not protic) and usually it is preferred to a~lminister a cc,~ ound of this invention in the form of an acid addition salt.
Compounds of this invention can be synthesized in a general sense according to Scheme 1.
Scheme 1 R2 X R3 Rt~R2 Ri~x DMF. ~ R4 NH3+CI Br 3 1 R4 2 NaH, ~5 R6 R2 X R3 , ~~Br R 1-Deprotect if necessary ~ R4 5\ ~ 2- PdCI2(PPh3)2 piperidine ~

~5a-c = (CH2)n--N~ lÇ~\~R F15 ~ /CH2)n Rf N~R

The initial indole synthesis may be accomplished by heating an a~ iately substituted aniline (1) with an ~pl~liately substituted alpha-bromophenylalkyl-phenone (2) in a suitably high boiling solvent such as DMF. The product is then alkylated with 4-iodobenzyl bromide to give the substituted indole (3). At this point, 20 deprotection of phenols (if present) is done. Normally, the phenols are protected as benzyl ethers and can conveniently be cleaved with TMSI. The p~ yl~mines can then be coupled to the phenyl iodide. The pr~a,~,yl~mines are typically prepared from an alkynyl bromide or alkynyl tosylate by substitutiQn with the al)plu~liate amine. The substitution reaction is done in situ, without isolating the plu~al~ylarnine. Co,lll)oullds 5 substituted at the 3-position with groups other then alkyl may be prepared by first plcpalillg the indole substituted at the 3- position with -H. The indole can then be electrophilically halogen~te~l, formylated, etc., to give other 3-substituted compounds.

The compounds of the invention are partial estrogen agonists and display high 10 affinity for the estrogen ~ecel)lol. 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 m~mm~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 hy~elLIophy, infertility, breast cancer, endometrial cancer, cardiovascular disease, contraception, ~l7hçimrr~s 20 disease and melanoma. Additionally, these compounds can be used for hormone replacement 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 tre~tmlont for 25 bone loss, which may result from an imb~l~nce in a 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 30 suffering from Cushing's syndrome. Special needs for bone repl~r~mrnt 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 disease, osteomalacia, osteohalisteresis, 35 endometrial cancer, multiple myeloma and other forms of cancer having deleterious effects on bone tissues. Methods of treating the m~ s listed herein are understood to comprise ~(1mini~tering to an individual in need of such lle~ el~ a ph~rm~euti~ lly effective amount of one or more of the compounds of this invention or a ph~rm~ceutically acceptable salt thereof. This invention also includes ph~rm~cel1ti-~l S co~ ositions utili7ing one or more of the present compounds, and/or the ph~rm~euti~:3lly acceptable salts thereof, along with one or more ph~rm~euti-~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 pr~felled that the a~lmini~tration of one or more of the compounds herein begins at a low dose and be increased until the desired effects are achieved.

Effective a~lmini~tration of these compounds may be given at a dose of from about 0.1 mg/day to about 1,000 mg/day. Preferably, a~lminictration will be fromabout 50 mg/day to about 600 mg/day in a single dose or in two or more divided doses.
Such doses may be a lministered 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, transdermal a~lmini~rations are understood to include all ~rlmini~trations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such ~lmini~trations may be carried out using the present compounds, or pharmaceutically acceptable salts thereof, 25 in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).

O~al formulations containing the active compounds of this invention may comprise any conventionally used oral forms, including tablets, capsules, buccal30 forms, troches, lo~nges and oral liquids, suspensions or solutions. Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharmaceutically 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 form~ tions may 35 be made by conventional compression, wet granulation or dry granulation methods and utili~ ph~....A~e,~ltis~lly acceptable ~ ents, binding agents, lubricants, disintegrants, suspending or stabilizing agents, including, but not limited to, m~gntoSillm stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcelllllose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia 5 gum, x~nth~n gum, sodium citrate, complex .sili~tes, calcium carbonate, glycine, dextrin, sucrose, sorbitol, r~ cillm phosphate, c:3lrillm sulfate, lactose, kaolin, nnilol~ sodium chloride, talc, dry starches and powdered sugar. Oral form~ ~ionsherein 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.

Solvents used for the reactions described herein were anhydrous Aldrich Sure 15 SealTM without furtherpurification. 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 perforrned with Silica Gel 60 F2s4 plates from EM Science. lH NMR spectra were obtained on a Bruker AM
20 400 instrument in DMSO and chemical shifts reported in ppm. Melting points were ~eter~minecl on a Thomas-Hoover apparatus and are uncorrecte~ IR spectra were recorded on a Perkin-Elmer diffraction grating or Peri~in-Elrner 781 spectrophotometers. Mass spectra were recorded on a Kratos MS 50 or Finnigan 8230 mass spectrometers. FlPment~l analyses were obtained with a Perkin-Eln~er 2400 25 elemental analyzer. Analysis values for compounds with CHN analysis reported were within 0.4% of theoretical values.

The present invention is further illustrated by the following non-limiting examples.

CA 02203073 l997-04-l8 FX~MP~,F 1 5-Benzvlox,y-2-(4-benzvloxy-Dhenvl~-3-metll,yl-lH-indole A flask was charged with 4-benzyloxyaniline (45 g, 0.23 mol), 4'-benzyloxy-2-b~ phenylpropiophenone (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 CH2CI2 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; lH 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, 6 H), 7.21 (d, 1 H, J = 7.0 Hz), 7.13 (d, 2 H, J = 8.8 Hz), 7.08 (d, 1 H, 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.

5-Benzyloxv-2-(4-benzyloxv-Dhenvl)-3-methvl)-1-vlmethvl-(4-phenv1iodide)-indole A solution of 4 (3.0 g, 7.4 mmol) in DMF (25 mL) was treated with NaH (60%
dispersion, 0.21 g, 8.9 mmol) and stirred at rt for 15 minutes. 4-iodobromobenzyl bromide (2.2 g, 7.4 mmol) was added and the reaction was stirred for 1 hour. Thereaction mixture was poured into water and extracted with EtOAc, dried over MgSO4 and concentrated. Trituration of the crude product with ether afforded 2.2 g of the product as a white solid: Mpt = 153-156~C; lH NMR (DMSO) 7.54 (d, 2 H, J = 8.6 Hz), 7.52-7.45 (m, 4 H), 7.37-7.29 (m, 6 H), 7.27 (d, 2 H, J = 8.8 Hz), 7.17 (d, 1 H,J=9.OHz),7.13(d, lH,J=2.2Hz),7.10(d,2H,J=8.8Hz),6.81 (dd, lH,J
= 8.8, 2.4 Hz), 6.60 (d, 2 H, J = 8.3 Hz), 5.18 (s, 2 H), 5.12 (s, 2 H), 5.11 (s, 2 H), 2.15 (s, 3 H); MS eI m/z 635.

F,X AM P~,F, 3 ,~-(4-hvdroxyDhelv1)-3-~nethvl)-1-ylmethvl-(4-Dherl~vliodide)-indole-5-ol s A solution of 4 (2.2 g, 3.5 mmol) in CHCl3 was treated with Iod~ l~thylsilane (1.04 mL, 7.0 mmol) and the reaction was heated to reflux. After 2 h, an additional 3 eq of Iodotrimethylsilane was added and the reaction was stirred at rt for 18 h. The reaction was quenched by adding MeOH (5 mL). The organic layer waswashed with an aqueous 10% solution of Na2SO3, HCl (lM) and dried over MgSO4.
The solution was concentrated and chromatographed on silica gel EtOAc/hexane (3:7) to yield 4a as a foam (1.2 g): lH NMR 9.65 (s, 1 H), 8.71 (s, 1 H), 7.54 (d, 2 H, J =
8.3 Hz), 7.12 (d, 2 H, J = 8.3 Hz), 7.02 (d, 1 H, J = 8.6 Hz), 6.84-6.80 (m, 3 H), 6.61 (d, 2 H, J = 8.3 Hz), 6.57 (dd, 1 H, J = 6.4 Hz), 5.12 (s, 2 H), 2.09 (s, 3 H);
15 MS eI m/z 455.

General Procedure For Indole Pro~ar~ylamine PreDaration The title compounds of Examples 4-6 were produced using a solution 20 containing a 10 fold molar excess of a secondary amine in DMF cooled to 0~C and treated with plopal~yl bromide (3 eq, 80% solution in toluene). After 1 h at 0~C, the reactions were allowed to rt for 1 h. The indole iodide (4a, 1 eq) was added followed by Cu(I)I (0.1 eq) and Pd(PPh3)2Cl2 (0.035 eq). The reaction mixture was then stirred 1~48 h and worked up by pouring into water and extracting into EtOAc. The EtOAc is 25 concentrated and chromatographed on silica gel using EtOAc~exane as eluting system.

2-(4-Hvdroxv-phenvl)-3-methvl-1-r4-(3-N. N- dimethvl-1-vl-Dro~-1-ynyl)-benzvll-lH-indol-5-ol Mp=173-176~C; IH NMR (DMSO) 9.64 (s, 1 H), 8.70 (s, 1 H), 7.25 (d, 2 H, J=8.1 Hz),7.12 (d, 2H, J= 8.3 Hz), 7.03 (d, 1 H, J= 8.6 Hz), 6.83-6.78 (m, 5 H), 6.57 (dd, 1 H, J = 8.8, 2.4 Hz), 5.17 (s, 2 H), 3.39 (s, 2 H), 2.19 (s, 6 H), 2.10 (s, 3 H); IR (KBr) 3390, 1490 cm~l; MS esI 411 (M+H+).

F.X~MPI,~ 5 ~-~4-Hydro~y-pherl~vl)-3-methvl-1-r4-(3-piperidin-1-yl-vrop-1-ynyl)-5benzvll-lH-indol-5-ol Mp=118-123~C; lH NMR (DMSO) 9.65 (s, 1 H), 8.71 (s, 1 H), 7.24 (d, 2 H,J=8.1 Hz),7.12 (d, 2H, J = 8.6 Hz), 7.02 (d, 1 H, J = 8.6 Hz), 6.83-6.80 (m, 5 H),6.57(dd,1H,J=8.6,2.2Hz),5.17(s,2H),3.39(s,2H),2.41(m,4H),2.10 10(s, 3 H), 1.48 (p, 4 H, J = 5.7 Hz), 1.36-1.33 (m, 2 H); IR (KBr) 3400, 2920, 1620, 1420 cm~l; MS EI m/z 450; CHN calc'd for C30H30N2O2 + 0-25 H2O

152-(4-Hydroxy-phenvl)-3-methvl-1-r4-(3-pvrrolidin-1-yl-prop-1-vnyl)-benzyll-lH-indol-5-ol (5c) Mp=174-176~C; lH NMR (DMSO) 9.64 (s, 1 H), 8.70 (s, 1 H), 7.23 (d, 2 H,J = 8.3 Hz), 7.11 (d, 2 H, J = 8.6 Hz), 7.02 (d, 1 H, J = 8.8 Hz), 6.84 (m, 5 H), 206.57 (dd, 1 H, J = 8.6, 2.2 Hz), 5.17 (s, 2 H), 3.53 (s, 2 H), 2.53-2.51 (m, 4 H), 2.09 (s, 3 H), 1.69-1.66 (m, 4 H); IR (KBr) 3400, 2920, 2900, 1620 cm~l; MS eI
m/z 436; CHN calcd for C29H28N2O2 + 0.7 H2O.

In vitro estro~en receptor bindin~ assay Receptor preparation CHO cells ov~ ssing the estrogen receptor were grown in 150 mm2 dishes in DMEM + 10% dextran coated charcoal, stripped fetal bovine serum. The plates were 30washed twice with PBS and once with lOrnM 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 frozen and stored at -80~C. Protein concentration of the cytosol was e~ rA usingthe BCA assay with reference standard protein.

Rjndin~ av conditions The c~ ~lilion assay was ~ ro~ ed 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 prçlimin~ry competition when lOOx and 500x competitor were evaluated, 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 imm~Ai~tely 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 andcytosol, but not processed with charcoal were included to qu~ntit~to arnounts ofavailable isotope. Also, wells containing radioactivity but no cytosol were processed with charcoal to estim~te unremovable DPM of [3H] 17~-estradiol. Corning #25880-96, 96-well plates were used because they have proven to bind the least amount of estradiol.

Analvsis of results Counts per minute (CPM) of radioactivity were automatically converted to disintegrated per minute (DPM) by the Beckman LS 7500 Scintillation 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 competitor the following formula was applied:

((DPM sample-DPM not removed by charcoal /(DPM estradiol-DPM not removed by charcoal)) x 100% = % of estradiol binding CA 02203073 l997-04-l8 For the generation of IC50 curves, % binding is plotted vs compound. IC50's are ~ ~d for compounds that show >30% co...~ ion at 500x ~
concentration. For a description of these methods, see Hulme, E.C., ed. 1992.
Receptor-Ligand Interactions: A Practical Approach. IRL Press, New York.(see 5 especially chapter 8).

F,~tro~en Receptor Affinity (reported ~ RRA: 17~-estr~diol=100) Compound RBA
Raloxifene 400 Tamoxifen 1.8 Example 4 53 Example 5 23 I~hikawa Cell Alkaline PhosDhatase Assay Cell Maintenance and Treatment:

Ishikawa cells were ~ t~ ed in DMEM/F12 (50%:50%) containing phenol red + 10% fetal bovine serum and the m~ m was supplemented with 2 rr~I
Glutamax, 1% Pen/Strap and 1 mM sodium pyruvate. Five days prior to the beginning of each experiment (treatment of cells) the medium was changed to phenol red-free DMEM/F12 + 10% dextran coated charcoal stripped serum. On the day before ent, cells were harvested using 0.5% trypsin/EDTA and plated at a density of S X104 cells/well in 9~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,B- 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,B estradiol control. Sample population for at each dose was n=8.
Alkaline Phosphatase Assav:

At the end of 48h the media is aspirated and cells are washed three times with phosphate buffered saline (PBS). 50~L of lysis buffer (0.1 M Tris-HCl, pH 9.8, 0.2% Triton X- 100) is added to each well. Plates are placed at -80~C for a ~ i" " " ~ l of 15 minutes. Plates are thawed at 37~C followed by the addition of 15011L of 0.1 M
Tris-HCl, pH 9.8, cont~ining 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 Instrum~nt~, 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 absorbance reading). Results for compounds are ~ n~l;7ed as percent of response related to 1 nM 17~estradiol.

Various compounds were assayed for estrogenic activity by the allcaline phosphatase method and collesl)ollding ED50 values (95% C.I.) were calculated. The four listed in the following were used as as reference standards:
17,~-estradiol 0.03 nM
17cc-estradiol 1.42 nM
estriol 0.13 nM
estrone 0.36 nM
A description of these methods is described 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).Cancer Research, 46:2771-2774, and by Littlefield, B.A., Gurpide, E., Markiewicz, 25 L., McKinley, B. and Hochberg, R.B. (1990) A simple and sensitive microtiter plate estrogen bioassay based on s~iml-l~tion alkaline phosphatase in Ishikawa cells;
Estrogen action of D5 adrenal steroids. Endocrinology, 6:2757-2762.

CA 02203073 l997-04-l8 I~hikaw~ Alk~line Phosphat~e Assa y Co,llpoulld % Activation % Activation (Compound +
1 nM 17~-estradiol) 17,B-estradiol 100% N/A
~ 0% 45%
raloxifen 5% 5%
Example 4 34% 34%
Example 5 27% 23%

2X VIT FRF Transfection Assav s Cell Maintenance and Treatment Chinese Hamster Ovary cells (CHO) which had been stably transfected with the human estrogen receptor were m~int~ined in DMEM + 10% fetal bovine serum (FBS).
10 48h prior to treatment the growth medium was replaced with DMEM lacking phenol red + 10% dextran coated charcoal stripped FBS (tre~tme~t medium). Cells were plated at a density of 5000 cells/well in 96-well plates cont~ining 200 IlL 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~l thymidine kinase pl~"lotel driving the luciferase gene) was combined with the B-galacto~ e expression plasmid pCHl lO
(Pharmacia) and carrier DNA (pTZ18U) in the following ratio:
lOuG of reporter DNA
SuG of pCHl lODNA
5 uG of pTZ18U
20 uG of DNA/l rnL 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 Illi~ule was ~ CA 02203073 1997-04-18 added to each well of cells and rem~ine~l on the cells for 16 h. At the end of this incl1b~tion the precipitate was removed, the cells were washed with media, freshllc~ media was replaced and the cells were treated with either vehicle, 1 nM 17~-estradiol, luM compound or 1 uM colllpound + 1 nM 17~estradiol (tests for estrogen 5 antagonism). Each ~ (lllel~l con-lition was performed on 8 wells (n=8) which were incubated for 24 h prior to the luciferase assay.

~ ucifer~e A ss a y 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 PBS, 25 uL of Promega lysis buffer was added to each well and allowed to stand at room temperature 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 evaluation 15 and the remaining Iysate (5 uL) was used for the B-galactosidase activity evaluation (normalize transfection). The luciferan substrate (Promega) was added in 100 uL
aliquots to each well autom~ti~lly by the lurninometer and the light produced (relative light units) was read 10 seconds after addition.

20 Infection Luciferase Assav Compound % Activation % Activation with 1 nM
17B-estradiol 17~-estradiol 100% N/A
~ll~,~iîen 0% 10%
raloxifene 0% 0%
Example 4 34% 34%
Example 5 17% 19%

B-Galactosidase Assay To the rem~ining 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~ in triplicate) was set up for each ~limental run. The plates were analyzed on a Molecular Devices ~ CA 02203073 1997-04-18 spectropholc "lellic plate reader at 410 nm. The optical den~itiçs for the unknown were converted to millillnit~ of activity by m~th~.m~tic~l extrapolation from the standard curve.

Analysis of Results The luciferase data was generated as relative light units (RLUs) a~c~lm~ te~l during a 10 second mea~u~ ellt and autom~tically transferred to a JMP (SAS Inc) file where background RLUs were subtracted. The B-galactosidase values were 10 ~u~o~ c~lly imported into the file and these values were divided into the RLUs to normalize the data. The mean and standard deviations were deterrnined from a n=8 for each treatment. Compounds activity was compared to 17~-estradiol for each plate.Percentage of activity as compared to 17~-estradiol was calculated using the formula %=((Estradiol-control)/(compound value)) X 100. These techniques are described by 15 Tzukerman, M.T., Esty, A., Santiso-Mere, D., D~nieli~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~ tecl by two functionally distinct intramolecular regions (see Molecular Endocrinology, 8:21-30).
Rat Uterotrophic/Antiuterotrophic Bioassav The estrogenic and antiestrogenic properties of the compounds were deterrnined in an immatllre rat ulelollvphic assay (4 day) that (as described previously by L.J.Black 25 and R.L.Goode, Life Sciences, 26, 1453 (1980)). T~ c Sprague-Dawley rats (female, 18 days old) were tested in groups of six. The animals 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,B-estradiol, with 50%
DMSO/50% saline as the injection vehicle. On day 4 the animals were sacrificed by 30 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 submittedfor histology and the rem~in-ler used to isolate total RNA in order to evaluate complement component 3 gene expression.

3 dav Ov~riecto~ni7ed Rat Model COnlDOIInd 1 O~1(T 1 0011G
T~~ ilell 69.6 mg 71.4 mg S Raloxifen 47.5 43.2 control = 42.7 mg 1 IlG 17~-estradiol = 98.2 COmDOUnd 1OI1(G 100,uG 100~1G + l,uG
1 7B-estradiol Example 4 56.0 mg 84.0 mg 77.6 mg con~ol = 32.1 mg1,uG 17,B-estradiol = 90.2 mg Example 555.6 mg 71.3 mg 66.8 mg control = 21.7 mg1 ~g 17,B-es¢adiol = 82.8 mg

Claims (14)

1. A compound having the structure:

or wherein:
R1 is selected from H, OH or the C1-C4 esters or alkyl ethers thereof, or halogen;
R2, R3, R4, R5, and R6 are independently selected from H, OH or the C1-C4 esters or alkyl ethers thereof, halogen, cyano, C1-C6 alkyl, or trifluoromethyl, with the proviso that, when R1 is H, R2 is not OH;
X is selected from H, C1-C6 alkyl, cyano, nitro, trifluoromethyl, or halogen;
n is 2 or 3;
Y is selected from:
a) the moiety:
wherein R7 and R8 are independently selected from the group of H, C1-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-, -NH-, -N(C1C4 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, C1-C4 alkyl, trihalomethyl, C1-C4 alkoxy, trihalomethoxy, C1-C4 acyloxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, hydroxy (C1-C4)alkyl, -CO2H-, -CN-, -CONHR1-, -NH2-, C1-C4 alkylamino, C1-C4 dialkylamino, -NHSO2R1-, -NHCOR1-, -NO2-, and phenyl optionally substituted with 1-3 (C1-C4)alkyl;

c) a six-membered saturated, unsaturated or partially unsaturated heterocycle containing up to two heteroatoms selected from the group consisting of -O-, -NH-, -N(C1C4 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, C1-C4 alkyl, trihalomethyl, C1-C4 alkoxy, trihalomethoxy, C1-C4 acyloxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, hydroxy (C1-C4)alkyl, -CO2H-, -CN-, -CONHR1-, -NH2-, C1-C4 alkylamino, di(C1-C4)alkylamino, -NHSO2R1-, -NHCOR1-, -NO2, and phenyl optionally substituted with 1-3 (C1-C4)alkyl;

d) a seven-membered saturated, unsaturated or partially unsaturated heterocycle containing up to two heteroatoms selected from the groupconsisting of -O-, -NH-, -N(C1C4 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, C1-C4 alkyl, trihalomethyl, C1-C4 alkoxy, trihalomethoxy, C1-C4 acyloxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, hydroxy (C1-C4)alkyl, -CO2H-, -CN-, -CONHR1-, -NH2-, C1-C4 alkylamino, di(C1-C4)alkylamino, -NHSO2R1-, -NHCOR1-, -NO2, and phenyl optionally substituted with 1-3 (C1-C4)alkyl; or e) a bicyclic heterocycle containing from 6-12 carbon atoms either bridged or fused and containing up to two heteroatoms selected from the group consisting of -O-, -NH-, -N(C1C4 alkyl)-, 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, C1-C4 alkyl, trihalomethyl, C1-C4 alkoxy, trihalomethoxy, C1-C4 acyloxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, hydroxy (C1-C4)alkyl, -CO2H-, -CN-, -CONHR1-, -NH2-, C1-C4 alkylamino, di(C1-C4)alkylamino, -NHSO2R1-, -NHCOR1-, -NO2, and phenyl optionally substituted with 1-3 (C1-C4)alkyl;

or a pharmaceutically acceptable salt thereof.

2. A compound of Claim 1 wherein:
R1 is selected from H, OH or the C1-C4 esters or alkyl ethers thereof, or halogen;
R2, R3, R4, R5, and R6 are independently selected from H, OH or the C1-C4 esters or alkyl ethers thereof, halogen, cyano, C1-C6 alkyl, or trifluoromethyl, with the proviso that, when R1 is H, R2 is not OH;
X is selected from H, C1-C6 alkyl, cyano, nitro, trifloutomethyl, or halogen;
Y is the moiety ;

R7 and R8 are selected independently from H, C1-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, C1-C4 alkyl, trihalomethyl, C1-C4 alkoxy, trihalomethoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, hydroxy (C1-C4)alkyl, -CO2H, -CN, -CONH(C1-C4)alkyl, -NH3, C1-C4 alkylamino, di(C1-C4)alkylamino, -NHSO2(C1-C4)alkyl, -NHCO(C1-C4)alkyl, and -NO2;
or a pharmaceutically acceptable salt thereof.

3. A compound of Claim 1 wherein:
R1 is OH;
R2, R3, R4, R5, and R6 are independently selected from H, OH or the C1-C4 esters or alkyl ethers thereof, halogen, cyano, C1-C6 alkyl, or trifluoromethyl, with the proviso that, when R1 is H, R2 is not OH;
X is selected from the group of Cl, NO2, CN, CF3, or CH3;
Y is the moiety ; and R7 and R8 are concatenated together as -(CH2)p-, wherein p is an integer of from 4 to 6, to form a ring optionally substituted by up to three substituents selected from the group of hydrogen, hydroxyl, halo, C1-C4 alkyl, trihalomethyl, C1-C4 alkoxy, trihalomethoxy, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, hydroxy (C1-C4)alkyl, -CO2H, -CN, -CONH(C1-C4)alkyl, -NH3, C1-C4 alkylamino, C1-C4 dialkylamino, -NHSO2(C1-C4)alkyl, -NHCO(C1-C4)alkyl, and -NO2;
or a pharmaceutically acceptable salt thereof.

4. A compound of Claim 3 wherein R7 and R8 are concatenated together as -(CH2)p- to form a ring, p representing an integer from 4 to 6, the ring being optionally substituted with 1-3 substituents selected from the group of C1-C3 alkyl, trifluoromethyl, halogen, hydrogen, phenyl, nitro, or -CN.

5. A compound of Claim 1 which is 2-(4-Hydroxy-phenyl)-3-methyl-1-[4-(3-N, N- dimethyl-1-yl-prop-1-ynyl)-benzyl]-1H-indol-5-ol or a pharmaceutically acceptable salt thereof.

6. A compound of Claim 1 which is 2-(4-Hydroxy-phenyl)-3-methyl-1-[4-(3-piperidin-1-yl-prop-1-ynyl)-benzyl]-1H-indol-5-ol or a pharmaceutically acceptable salt thereof.

7. A compound of Claim 1 which is 2-(4-Hydroxy-phenyl)-3-methyl-1-[4-(3-pyrrolidin-1-yl-prop-1-ynyl)-benzyl]- 1H-indol-5-ol or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising a compound of Claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical composition of Claim 8 in which the compound is 2-(4-Hydroxy-phenyl)-3-methyl-1-[4-(3-N, N- dimethyl-1-yl-prop-1-ynyl)-benzyl]-1H-indol-5-ol or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition of Claim 8 in which the compound is 2-(4-Hydroxy-phenyl)-3-methyl- 1-[4-(3-piperidin-1-yl-prop- 1 -ynyl)-benzyl]- 1H-indol-5-ol or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical composition of Claim 8 in which the compound is 2-(4-Hydroxy-phenyl)-3-methyl-1-[4-(3-pyrrolidin-1-yl-prop-1-ynyl)-benzyl]-1H-indol-5-ol or a pharmaceutically acceptable salt thereof.

12. A method of treating or preventing bone loss in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a compound of Claim 1, or a pharmaceutically acceptable salt thereof.

13. A method of treating or preventing disease states or syndromes which are caused or associated with an estrogen deficiency in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a compound of Claim 1, or a pharmaceutically acceptable salt thereof.

14. A method of treating or preventing cardiovascular disease in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a compound of Claim 1, or a pharmaceutically acceptable salt thereof.