Synthesis and biological evaluation of 1,1-dichloro-2,3-diarylcyclopropanes as antitubulin and anti-breast cancer agents.

Z-1,1-Dichloro-2,3-diphenylcyclopropane (1) is an effective anti-breast cancer agent in rodents and in cell culture. We recently determined that 1 inhibits tubulin assembly in vitro and causes microtubule loss in breast cancer cells, leading to accumulation in the G2/M portion of the cell cycle. Aryl ring-halogenated, methoxylated and benzyloxylated derivatives of 1, as well as its E-isomer and the dichlorocyclopropyl derivative of diethylstilbestrol (DES), were synthesized and tested for their ability to inhibit, the assembly of tubulin into microtubules. Including 1, 17 cyclopropyl compounds were tested. One (Z-1,1-dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane (12)) was found to be more active than 1. In addition, E-1,1-dichlorocyclopropylDES (17) was more potent than DES. The E-isomer of 1 (16) was inactive. The cytostatic activities of the compounds against MCF-7 and MDA-MB231 human breast cancer cells, and their abilities to perturb microtubules in MCF-7 cells were also evaluated. Z-Dichloro-2-(4-fluorophenyl)-3-phenylcyclopropane (5), Z-1,1-dichloro-2-(4-fluorophenyl)-3-(4-methoxyphenyl)cyclopropane (11), and Z-1,1-dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane (12) were more potent than 1 against the breast cancer cells.

Antitumor mechanism of action of a cyclopropyl antiestrogen (compound 7b) on human breast cancer cells in culture.

Cyclopropyl compound 7b [(Z)-1,1-dichloro-2-[4-[2-(dimethylamino)ethoxy] phenyl]-2-(4-methoxyphenyl)-3-cyclopropane] has been shown to be a pure antiestrogen in mouse uterine tissue. Antitumor activity was examined by evaluating the influence of 7b on the proliferation, estrogen receptor (ER) affinity and cell-surface morphology of ER-positive and ER-negative human breast cancer cells in culture. The antiproliferative potency of 7b was found to be equal to tamoxifen in ER-positive MCF-7 human breast cancer cells. Further, the antiproliferative activities of 7b and tamoxifen were reversed by coadministration of estradiol. Accordingly, the antiproliferative activity of compound 7b appears to be estrogen-mediated since it did not influence the growth of either ER-negative MDA-MB-231 human breast cells or A-549 human lung cancer cells in culture. An ER-dependent mechanism of action is also supported by the specific binding affinity of 7b for ER in MCF-7 cells. Further, a study of cell surface morphology using scanning electron microscopy (SEM) revealed that 7b reduced the density and distribution of microvilli (MV) on MCF-7 cells, which was reversed by coadministration of estradiol. Compound 7b did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7b inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on either ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study confirm an antiestrogenic mechanism of action for 7b as previously observed in vivo and suggest that 7b would be effective in the treatment of estrogen-dependent breast cancer or as a prophylactic treatment for women with a high risk of breast cancer development.

Influence of cyclopropyl antiestrogens on the cell cycle kinetics of MCF-7 human breast cancer cells.

Five cyclopropyl compounds, previously shown to exhibit pure antiestrogenic activity in the mouse uterotropic assay and antiproliferative activity of MCF-7 human breast cancer cells in culture, were examined for their influence on the cell cycle kinetics of MCF-7 cells. The DNA-histogram of a single cell suspension was obtained on Coulter Epics V after fixing the cells in 70 % ethyl alcohol and staining in propidium iodide. Tamoxifen increased the percentage of cells in G1-phase with a concomitant decrease in percentage of cells in S-phase, in an estradiol reversible manner. Cyclopropyl compound 7a increased the percentage of cells in G1-phase, in an estradiol-irreversible manner. Further, compounds 5a, 5c, 7a and 7b decreased the percentage of cells in S-phase and increased percentage of cells in the G2M-phase, in an estradiol-irreversible manner. Of the five cyclopropyl compounds tested, only 4d had no influence on the cytokinetic parameters, even though this compound was found to exhibit antiproliferative activity on MCF-7 cells equal to that of tamoxifen. In conclusion, all of the cyclopropyl compounds, except 4d, altered cell cycle parameters of MCF-7 cells in a manner different than that of tamoxifen. Thus, the results of this study indicate that, although these cyclopropyl compounds are antiestrogenic, they produce antiproliferative activity by a distinct mechanism of action in estrogen receptor positive breast cancer cells.

Synthesis and biological evaluation of basic side chain derivatives of Analog II as pure antiestrogens and antitumor agents.

In an effort to prepare effective non-steroidal antiestrogens without intrinsic estrogenicity and with greater antagonism than those of the triarylethylenes (tamoxifen; TAM) four N-substituted (Z)-1,1-dichloro-2-[4-(2-aminoethoxy)phenyl]-3-phenylcyclopropa ne derivatives of the antiestrogen, Analog II, in which the basic side chains contain cyclic (piperidino and piperazino) and non-cyclic (dimethyl amino and diethyl amino) moieties, were synthesized. These compounds were prepared from an intermediate methanesulfonyloxyethoxy side chain ester of 1,1-dichloro-2,3-cis-diphenylcyclopropane using their respective side chain bases in triethylamine and acetonitrile. The gem-dichloro-cis-diarylcyclopropane derivatives were tested for their ability to inhibit the growth-stimulating effect of estradiol on immature mouse uteri and the growth of estrogen receptor (ER)-positive MCF-7E3, ER-negative MDA-MB-231 and the ER-positive MCF-7LY2 antiestrogen-resistant breast cancer cells in culture. The introduction of the various aminoethoxy side chains into Analog II did not improve its ER-binding affinity. Like Analog II, the derivatives did not exhibit any intrinsic estrogenicity, and compounds 9 and 10 antagonized estradiol action more completely than the parent compound. None of the compounds potentiated the uterine weight gain from the stimulating dose of estradiol (0.03 micrograms). Derivatives 9 (150 micrograms), 10 (150 micrograms) and 11 (150 micrograms) had uterine mean weights significantly below the estradiol-treated group, and were better antagonists than Analog II and MER25 at the same concentrations. All compounds exhibited a statistically significant (P < 0.01) reduction in control growth (antitumor activity) from 0.01 to 10 nM concentration in the MCF-7E3 cells. At 10 nM concentration, 8 (66%) and 9 (64%) had the greater antitumor activity over 10 (58%) and 11 (58%). No activity in this cell line was observed for Analog II, TAM and ICI 182,780. Antitumor action was also demonstrated in the MDA-MB-231 cells for all derivatives at 1.0 microM dose, with 9 having the greatest (27%) inhibition of control growth, followed by 8 (20%), 10 (18%) and 11 (12%). Analog II and ICI 182,780 had no antitumor activity in this cell line, while TAM exhibited only 8% inhibition. In the MCF-7E3 cell line at 1.0 microM, 9 exhibited 86% inhibition of the estradiol-stimulated growth (antiestrogenic activity), followed by 8 (64%), 10 (52%) and 11 (21%).(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of the antitumor activity of two triarylcyclopropyl antiestrogens (compounds 4d and 5c) on human breast cancer cells in culture.

Compound 4d ((E)- and (Z)-1,1-Dichloro-2-[4-(benzyloxy)-phenyl]2,3-bis(4-methoxyphenyl) cyclopropane) and compound 5c ((Z)-1,1-Dichloro-2-[4-(benzyloxy)-phenyl]- 2-(4-methoxyphenyl)-3-phenylcyclopropane) are two members of a novel series of triarylcyclopropyl compounds which have been shown to be pure antiestrogens. In the present study, the antiproliferative activity of 4d and 5c was examined on estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells. Compound 4d inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-13) to 10(-5) M while compound 5c inhibited MCF-7 cell growth in a dose-related manner over a concentration range of 10(-9) to 10(-5) M. Further, neither compound altered the growth of MDA-MB-231 or A-549 cells. Co-administration of estradiol reversed the antiproliferative activity of 4d but not 5c on MCF-7 cells. Both compounds bound specifically to ER in MCF-7 cells; however, the relative binding activity of 4d was five times greater than estradiol and 5000 times greater than 5c. The influence of 4d and 5c on the cell surface morphology of MCF-7 and MDA-MB-231 cells was studied using scanning electron microscopy. Both compounds, at a concentration of 10(-6) M, reduced the density of microvilli on MCF-7 cells, which was reversed by co-administration of estradiol (10 (-8) M). These compounds did not alter the cell surface morphology of ER-negative MDA-MB-231 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular structures and conformational studies of triarylcyclopropyl and related nonsteroidal antiestrogens.

Molecular structures and conformational characteristics of a series of 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs), which were reported previously to be distinctly antiestrogenic and inhibitors of the estrogen-receptor-positive MCF-7 human breast cancer cells in culture, are reported. In addition, structural and conformational features of the DTACs were compared to the first-known nonsteroidal antiestrogen, MER25, and the clinically useful antiestrogen Tamoxifen. The molecular structures of four DTAC compounds were determined by X-ray diffraction. Crystallographic structures show that the DTAC molecules have nearly the same relative conformation for the three aryl rings which is designated as a "nonpropeller" conformation in contrast to the observed "propeller" conformation for the three rings in all known triarylethylenes. Systematic conformational searches were performed to find the conformational preferences of DTACs, MER25, and Tamoxifen using idealized model compounds built from their respective crystal structure. Energy-minimization and conformational-search studies demonstrated that all DTAC molecules have a common, single global minimum energy conformer for their central core containing the dichlorotriarylcyclopropyl system, which is similar to that found in their crystal structures. Conformational search of MER25 showed that the molecule can assume a number of low-energy conformers of which two, one anti (A1) and one gauche (G1A), have about the same energy. The anti conformation is similar to the one observed in its crystal structure and resembles the estrogenic E-isomer of Tamoxifen, while the lowest energy gauche conformer of MER25 resembles more closely the antiestrogenic Z-isomer of Tamoxifen. NMR spectroscopic analysis of MER25 showed that the molecule exists predominantly in the anti conformation in solution. A comparative review of the structural features and bioactivities of Tamoxifen, DTACs, and MER25 provides a possible explanation for their low estrogen receptor binding affinity which is common to these compounds together with their antiestrogenic activity.

(Z)-1,1-dichloro-2-(4-benzyloxyphenyl)-2,3-bis(4-methoxyphenyl)cyclopropane: the synthesis and enantiomeric separation of an antitumor agent.

(Z)-1,1-dichloro-2-(4-benzyloxyphenyl)-2,3-bis(4-methoxyphenyl)cyc lopropane (5), a potential antitumor agent designed to treat breast cancer, was prepared in three steps. A stereospecific palladium-catalyzed cross coupling reaction which provided the intermediate (Z)-triaryl alkene 4 was a crucial step in the synthesis. Makosza phase transfer reaction on 4 gave the enantiomeric (Z)-dichlorocyclopropane derivatives 5 which were resolved by semipreparative HPLC on a chiral stationary phase consisting of amylose tris-3,5-dimethylphenyl carbamate coated on silica gel.

Structure of 1-(4-[2-(diethylamino)ethoxy]phenyl)-2-(4-methoxyphenyl)-1-phenylethan- 1-ol, the non-steroidal antiestrogen MER25.

C27H33NO3, M(r) = 419.6, monoclinic, P2(1)/a, a = 22.833 (6), b = 9.370 (3), c = 11.434 (4) A, beta = 110.71 (8) degrees, V = 2288.2 A3, Z = 4, Dx = 1.22 g cm-3, lambda (Cu K alpha) = 1.54178 A, mu = 5.8 cm-1, F(000) = 904, T = 138 K, R = 0.049 for 3265 observed reflections. The molecule of MER25 assumes an extended conformation with rings alpha' and beta in an antiperiplanar (trans) conformation giving the solid-state conformer a closer resemblance to the estrogenic (E) isomer of tamoxifen than the antiestrogenic (Z) isomer. The geometrical features of the triarylethan-1-ol moiety are comparable to related structures but the orientations of the phenyl rings are different. The O-C-C-N segment in the (diethylamino)ethoxy side chain has the uncommon trans conformation instead of the more commonly observed gauche conformation seen in tamoxifen and many of its derivative structures. The amino N atom forms a hydrogen bond with the hydroxyl group of a neighboring molecule to form an infinite chain along the b axis.

The influence of a novel cyclopropyl antiestrogen (compound 7a) on human breast cancer cells in culture.

Compound 7a ([Z]-1,1,-dichloro-2,3-diphenyl-2-(4-(2- dimethylamino)ethoxy)phenyl) cyclopropane, dihydrogen citrate salt) is a novel cyclopropyl antiestrogen which was shown to be an estrogen antagonist without estrogen agonist activity. The antiproliferative activity of 7a was examined on estrogen receptor (ER) positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells. Compound 7a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-9) to 10(-5) M, but did not alter the growth of MDA-MB-231 or A-549 cells. The antiproliferative activity of 7a (10(-7) M) on MCF-7 cells was reversed by co-administration of estradiol (10(-8) M). An ER-dependent mechanism of action is also supported by the specific ER binding of 7a in MCF-7 cells observed in this study. A study of cell surface morphology using scanning electron microscopy (SEM) revealed that compound 7a at 10(-6) M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10(-8) M). Compound 7a did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7a inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells or A-549 lung cancer cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of 1,1-dichloro-2-(4-methoxyphenyl)-2,3-diphenylcyclopropane.

C22H18Cl2O, M(r) = 369.3, monoclinic, P2(1)/a, a = 16.585(1), b = 17.328(1), c = 13.192(3) A, beta = 107.443(8) degrees, V = 3616.8 A3, Z = 8, Dx = 1.356 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 3.2 cm-1, F(000) = 1536, T = 138 K, R = 0.039 for 5450 observed reflections. The structural features of the two independent molecules are quite similar except in the orientation of the methoxy group. The cyclopropane ring shows the expected bond-length asymmetry with C(2)-C(3) as the longest bond. The two cis-arranged phenyl rings adopt similar conformations as observed in diaryl-cyclopropanes, with one ring in the bisecting position and the other near the perpendicular position. The conformation of the third aryl ring is also near the perpendicular position. The overall conformation of the three aryl rings is different from the helical propeller conformation consistently observed in tamoxifen and all other known tri(tetra)aryl-vinyl systems.

Antiestrogenic effects of Z-1, 1-dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane(5a) on human breast cancer cells in culture.

Compound 5a ([Z]-1, 1-Dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane) is a novel cyclopropyl compound which was shown to be a pure antiestrogen. In the present study, the antiproliferative activity of 5a was examined on estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells using the hemocytometric trypan blue exclusion method. Compound 5a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-9) to 10(-5) M, but did not alter the growth of MDA-MB-231 or A-549 cells. Co-administration of estradiol (10(-8) M) reversed the antiproliferative activity of 5a (10(-7) M) on MCF-7 cells. Further, an ER-dependent mechanism of action is supported by the specific ER binding of 5a in MCF-7 cells observed in this study. The influence of 5a on the cell surface morphology of MCF-7 and MDA-MB-231 cells was studied using scanning electron microscopy (SEM). Compound 5a at 10(-6) M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10(-8) M). This compound did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 5a and tamoxifen inhibited the growth of ER-prositive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells. The results of this study with human breast cancer cells suggest that 5a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.

Synthesis and biological evaluation of a series of gem-dichlorocyclopropanes as antitumor agents.

As part of our continuous effort to produce non-steroidal antiestrogens demonstrating less intrinsic estrogenicity and greater antagonism than those in use, a series of Analog II (1,1-dichloro-2,3-diphenylcyclopropanes) derivatives was synthesized. The compounds were tested for their ability to inhibit the growth-stimulating action of estradiol in the immature mouse uterus and estrogen receptor (ER) (+) MCF-7 human breast cancer cells in vitro. Like Analog II, the derivatives were found to have no intrinsic estrogenicity (except 30) and they antagonized estradiol action less completely than the lead compound. Polarity improved the ER binding affinity of Analog II, but was quite small for all compounds, except 30, for which it was comparable to tamoxifen. Six compounds (8, 10, 14, 23, 29 and 30) demonstrated antiproliferative activity toward MCF-7 cells, in vitro, and the mean inhibition period over 6 days ranged from 20 to 37%. Only compound 30 was reversed by estradiol.

Antiproliferative activity of a series of novel cyclopropyl antiestrogens on MCF-7 human breast cancer cells in culture.

The potential antitumor activity of a series of novel cyclopropyl compounds, which lack estrogen agonist activity, was evaluated in estrogen receptor positive human breast cancer cells (MCF-7) in culture. The compounds were evaluated to determine their antiproliferative activity at a concentration of 1 microM at 2, 4 and 6 days of treatment by hemocytometer using the Trypan Blue exclusion method to count viable cells. Estradiol-induced reversibility of the antiproliferative activity of these compounds was also evaluated. The activity of a series of 19 diaryl- and triarylcyclopropyl compounds was examined. Thirteen compounds inhibited the growth of MCF-7 cells while six were inactive. Five of the 13 active compounds produced antiproliferative activity which was reversed by 0.1 microM estradiol. Thus, several of these novel cyclopropyl compounds may be useful in the treatment of hormone-dependent breast cancer and other estrogen-dependent tumors.

Structural comparison of a gem-dichlorodiarylcyclopropane antiestrogen and three of its derivatives.

The pure antiestrogenic activity of compound (1) gave the impetus to synthesize a series of its derivatives (2)-(4). Structural features of these compounds are compared. Compound (1): 1,1-dichloro-cis-2,3-diphenylcyclopropane, C15H12Cl2, Mr = 263.2, orthorhombic, Pbca, a = 19.627 (7), b = 19.460 (6), c = 6.670 (2) A, V = 2547.5 A3, Z = 8, D chi = 1.372 g cm-3, lambda (MoK alpha) = 0.71069 A, mu (Mo K alpha) = 4.3 cm-1, F(000) = 1088, T = 138 K, R = 0.026 for 1923 observed reflections. Compound (2): 1,1-dichloro-cis-2,3-bis(4-methoxyphenyl)cyclopropane, C17H16Cl2O2, Mr = 323.2, monoclinic, P2(1)/C, a = 16.540(1), b = 7.4749(7), c = 12.333 (3) A, beta = 91.53 (2) degrees, V = 1524.2 A3, Z = 4, D chi = 1.408 g cm-3, lambda (Cu K alpha) = 1.54178 A, mu (Cu K alpha) = 37.0 cm-1, F(000) = 672, T = 163 K, R = 0.031 for 2919 observed reflections. Compound (3): 1,1-dichloro-cis-2-(4-benzyloxyphenyl)-3-phenylcyclopropane, C22H18Cl2O, Mr = 369.3, monoclinic, P2(1)/alpha, a = 21.064 (3), b = 14.749 (2), c = 5.8222 (8) A, beta = 95.48 (2) degrees, V = 1800.5 A3, Z = 4, D chi = 1.362 g cm-3, lambda (Cu K alpha) = 1.54178 A, mu (CuK alpha) = 31.5 cm-1, F(000) = 768, T = 163 K, R = 0.032 for 3256 observed reflections. Compound (4): 1,1-dichloro-trans-2-(4-acetoxyphenyl)-3-phenylcyclopropane, C17H14Cl2O2, Mr = 321.2, monoclinic, P2(1)/n, a = 16.555 (4), b = 12.297 (2), c = 7.439 (1) A, beta = 98.31 (2) degrees, V = 1498.5 A3, Z = 4, D chi = 1.423 g cm-3, lambda (Mo K alpha) = 0.71069 A, mu (Mo K alpha) = 3.8 cm-1, F(000) = 664, T = 163 K, R = 0.034 for 2474 observed reflections. The crystal structure determinations show that the relative conformation of the two aryl rings in all four structures are quite similar. In this conformation one of the phenyl rings is in a bisecting position with respect to the cyclopropane ring, while the other is in a perpendicular position. In each of the four molecules the cyclopropane ring shows significant bond-length asymmetry with d[C(2)-C(3)] greater than d [C(1)-C(3)] greater than d[C(1)-C(2)].(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and biological evaluation of a series of 1,1-dichloro-2,2,3-triarylcyclopropanes as pure antiestrogens.

A series of 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs) was synthesized and evaluated as pure antiestrogens. Addition of 4-methoxy- or 4-(benzyloxy)phenyl Grignard reagents to p-methoxy, p-benzyloxy, or unsubstituted deoxybenzoins, followed by dehydration of the resulting carbinols produced a mixture of E and Z olefins, which were reacted with dichlorocarbene to give O-protected DTACs. The E and Z isomers were separated by fractional crystallization and the central or geminal phenyl ring was deprotected to provide phenolic DTACs. Alkylation with (N,N-dimethylamino)ethyl chloride yielded basic cyclopropanes. Two chlorodiarylindenes were isolated as thermolysis products of the DTACs, and one was converted to a phenol by hydrogenolysis. All DTACs and indenes were competitive inhibitors of [3H]estradiol binding in the immature rat uterine cytosol receptor assay, with relative binding affinities of 0.1-3.6% of estradiol. None of the new compounds were estrogenic in the 3-day immature mouse uterotrophic assay at doses up to 750 micrograms. In the 3-day immature mouse antiuterotrophic assay, five DTACs with either a methoxy (5a), benzyloxy (4d, 5c), or (dimethylamino)ethoxy (7a, 7b) central ring side chain produced significant decreases in uterine weight at doses up to 750 micrograms. One compound, (Z)-1,1-dichloro-2-[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4- methoxyphenyl)-3-phenylcyclopropane (7b), elicited a dose-dependent decrease in vivo comparable to MER 25. These same five compounds, as well as the lead compound Analog II, were active in vitro against the estrogen-dependent MCF-7 human breast tumor cell line in a dose-dependent fashion.

Biodistribution of a novel antiestrogen (Analog II) in the mouse and rat.

The biodistribution of a novel antiestrogen Analog II was determined in the mouse and rat. The tritiated product, [3H]-Analog II was prepared by New England Nuclear and was purified by preparative chromatography using silica gel and petroleum ether/methylene chloride (80:20). The fat tissue had the highest uptake due to the hydrophobic nature of Analog II. The second highest uptake was in the mouse uterine tissue which was greater than that observed in the rat. The differences in biodistribution between the mouse and rat may partially explain the differences in biological activity of Analog II previously observed in these two animal species.

Biological evaluation of novel cyclopropyl analogues of stilbene, stilbenediol, and phenanthrene for estrogenic and antiestrogenic activity.

The triphenylethylene-type antiestrogens, such as tamoxifen, are known to be useful in the treatment of estrogen-dependent tumors. However, these compounds display mixed estrogen agonist/antagonist activity which may limit their therapeutic effectiveness. This problem of mixed activity led to the synthesis and identification of a cyclopropyl derivative of cis-stilbene which we have named Analog I. This compound (1,1-dichloro-cis-2,3-diphenylcyclopropane) displayed only antiestrogenic activity in the mouse. The present study was designed to evaluate cyclopropyl derivatives of Analog II for estrogenic and antiestrogenic activity in the rat using the standard 3-d uterotropic assay and the uterine cytoplasmic estrogen receptor assay. Five compounds (B-F) which are cyclopropyl derivatives of stilbene, stilbenediol, and phenanthrene were evaluated in this study. Three of the compounds (B-D) displayed neither estrogenic nor antiestrogenic activity in the rat. The relative estrogenic activities of E and F were 11.3 and 1.5%, respectively, of diethylstilbestrol in the uterotropic assay, and 39 and 6.2%, respectively, of estradiol in the estrogen receptor assay. Neither E nor F was found to display antiestrogenic activity in the rat. The results indicate that the relative estrogenic and receptor binding activities of E and F are similar to those previously observed in the mouse, while B-D appear to be inactive in both species.

Protein binding characterization of a novel antiestrogen (Analog II) in the rat.

The present study examined the protein binding of a novel antiestrogen, Analog II, in rat serum and amniotic fluid. Binding experiments were conducted both in vitro and in vivo using a tritiated derivative of Analog II to measure total protein binding. Free and bound drugs were separated using the dextran-coated charcoal method. It was determined that 80-90% of Analog II in the circulation is bound to plasma protein in both mature and immature rats. The in vitro study indicated that the binding of Analog II to protein is nonspecific except in amniotic fluid and serum from 10-day old rats, where Analog II binding could be partially reversed by the addition of tamoxifen. The results suggest that Analog II binds to a specific triphenylethylene-associated binding site on neonatal rat serum and amniotic protein.

Methods of extraction and high-performance liquid chromatographic analysis of butylated hydroxytoluene from the tissues and serum of rats.

Butylated hydroxytoluene (BHT) is a phenolic antioxidant which is widely used in foods and has been shown to inhibit chemical carcinogenesis in the mammary gland induced by 7,12-dimethylbenz(a)anthracene. However, its mechanism of action as a tumor inhibitor is unclear. The purpose of this work was first to develop a method for extracting and quantitating BHT and then to determine the amounts that accumulate in the tissues and serum of rats as a starting point for looking at mechanistic possibilities in the inhibition of mammary carcinogenesis. Methodology of extracting BHT from rat tissues and serum was developed using a modified lipid extraction procedure. The sensitive nature of reverse-phase high-performance liquid chromatography proved useful in detecting and quantifying BHT after its extraction from biological tissues. All tissues were taken from animals consuming semipurified diets with and without 0.3% BHT for various periods of time (weeks). BHT was found in much higher levels in mammary tissue than in the liver and serum of rats. The lipid content in mammary tissue appears to be predictive of the amount of BHT found in this tissue, presumably because of the lipophilic character of the antioxidant.

Effects of nonsteroidal antiestrogens, analog II and tamoxifen, on a metastatic transplantable rat mammary tumor.

This study was designed to determine whether treatment with the nonsteroidal antiestrogens analog II and tamoxifen given three times per week, 2 weeks before and 2 weeks after tumor cell transplantation, influenced the metastasis of a transplantable, metastatic rat mammary tumor, DMBA-4. Following transplantation of 2,000 viable tumor cells into the fifth and sixth mammary fat pads of 50-day-old inbred, female WF rats, all rats in all 3 groups displayed primary tumors by 5 weeks post tumor transplant. Analog II delayed the primary tumor development when compared to the time of the primary tumor development in either the control (untreated) or tamoxifen-treated groups. No metastatic tumors were found in the analog II-treated group 5 weeks after tumor transplantation, and only 1 animal in the tamoxifen-treated group had a secondary tumor, whereas 50% of the control animals had metastatic tumors. Six weeks after tumor implantation, palpable secondary tumors had developed in 40% of the analog II-treated group and 80% of the tamoxifen-treated group, whereas 60% of the control animals had developed secondary tumors. By the end of the study (7 wk) no differences existed between primary or secondary tumor incidences or between control and antiestrogen-treated groups. Both antiestrogens were effective in delaying the development of secondary tumors, especially during the time of treatment. Following cessation of treatment, analog II prevented metastatic tumor development for up to a month and tamoxifen, for 3 weeks. Further studies are indicated to determine if continuous treatment can effectively inhibit metastatic tumor development indefinitely.