Estrogen receptor-binding afYfinityof tamoxifen analogs with various side chains and their biologic profile in immature rat uterus Anil K. Agarwal” and Rajeshwar D. Bindalt Divisions of *Endocrinology Lucknow, India

Estrogen

receptor-binding

tamoxifen series

analogs

appears

indicate

that

very slow.

affinity

substituted

to be dependent the dissociation

(Steroids

and tMedicina1 Chemistry,

56:486-489,

and estrogenic

with

various

and antiestrogenic

side chains.

on the presence of these

from

activity activity

the estrogen

have

been evaluated

of the compounds moiety.

The results

receptor-binding

for

of this also

site at 25 C is

1991)

tamoxifen; tamoxifen analog; estrogen receptor; estrogen antagonist activity; steroid

Introduction The clinical application of tamoxifen (tam) in estrogendependent breast cancer’-5 has renewed interest in the study of compounds with a triphenylethylene or triphenylethane parent structure. Antiestrogenic action of these compounds is mediated through the estrogen receptor.6 Tamoxifen has appreciable receptor affinity in vitro7 and high receptor affinity for estrogen receptor in vivo.8 Estrogen antagonistic activity’ of this compound is conferred by the tert-aminoethoxy moiety in the side chain. Since the side chain participates in the antagonistic activity of tamoxifen, it could be presumed that a change in the side chain might intluence the receptor affinity and subsequently the biologic activity of tamoxifen analogs. Accordingly, we selected three tamoxifen analogs having varied side chains-quatemary-tam, piperazinyl-tam, and chloro-tam (see Figure 1 and the Appendix)-and undertook to determine estrogen receptor affinity in vitro and estrogenic/antiestrogenic activity in vivo of this series relative to tamoxifen.

Experimental Female Sprague-Dawley rats (20 to 23 days old) weighing 30 to 35 g from the Institute colony were used Address reprint requests to Dr. Anil K. Agarwal at the National Institute of Immunology, Shahid Jeet Singh Marg. New Delhi-l 10067, India. Received August 9, 1990: accepted March 26, 1991.

Steroids,

Antagonist

of the P-tert-aminoethoxy

compounds

Keywords:

486

Central Drug Research Institute,

1991, vol. 56, September

relative binding affinity; estrogen

agonist activity;

in the present study. [3H]Estradiol ([3H]EJ, 40 to 60 Ci/mmol, 2,5-diphenyloxazole (PPO), and p-bis(2-[5phenyloxazole])benzene (POPOP) were obtained from New England Nuclear Corporation (Boston, MA, USA). Norit and dextran (molecular weight, 70,000) were from Sigma Chemicals (St. Louis, MO, USA). All other chemicals used were of analytic grade. Relative

binding

af$nity

Relative binding affinity (RBA) of each compound for rat uterine estrogen receptor in vitro with reference to estradiol (RBA 100%) was determined by the dextrancoated charcoal (DCC) method.“’ Uteri obtained from immature intact rats were homogenized in TEA buffer, pH 7.4 (10 mM Tris, 1.5 mM EDTA, and 0.02% NaN,), approximately 1 ml per uterus at 0 to 4 C, and the homogenate was spun at 105,000 x g for 60 minutes. The resulting supernatant (cytosolic fraction) was dispensed in 200~~1 aliquots into tubes containing a fixed concentration of [3H]E2 and concentrations of the test compounds (in triplicate) ranging from 10-i’ to 1O-4 M in a total volume of 60 ~1. Test compounds were prepared in assay buffer with dimethyl formamide added to the incubation mixture in an amount not exceeding 5%. Tubes were vortexed for a few seconds and incubated for 20 to 24 hours at 0 to 4 C. At the end of this incubation, 0.1 ml of cold DCC slurry (2.5% Norit and 0.25% dextran in TEA buffer) was added to each tube, 0

1991 Butterworth-Heinemann

RBA and tamoxifen

analogs: Agarwal

and Bindal

line. Autopsy was performed 24 hours after the last administration; the uteri were carefully dissected and blotted to release intraluminal fluid and then weighed on a torsion balance. Control animals received vehicle alone for a similar period. l2 The dose-response curves were constructed. Doses that doubled the uterine weight were taken for the determination of the relative uterotrophic activity, and the responses were expressed as percentage of estradiol.

Antiuterotrophic

Tamoxifen

R = CH2CH2N(CH3)2

Quaternary-tam

R = CH,CH2(N+(CH3)2)CH,CH2N(CH3)2CI

Piperazinyl-tam

R = CH2CH(OH)CH2

Chloro-tam

R = CH2CH2CI

Ns

-C6H5.2HCI

activity

Antiuterotrophic activity was assayed by concomitant administration of the various doses (0.5 to 100 pg) of test compounds and E, (0.5 pg) in the same way as given above. Control rats received vehicle alone or 0.5 pg E,. All animals were killed 24 hours after the last injection. The uteri were removed, and the organ wet weights were recorded as described above.8 Inhibition is expressed using the formula % Inhibition =

1 -

(

2

1:“)

P

x

100

Y

Figure 1 Triphenylbutylene structure with side chain substitution determining tamoxifen and tamoxifen analogs in this report.

where E,, represents the effect of standard and test substance, E, represents the effect of standard substance, and E, represents the effect of vehicle.13

the mixture was vortexed briefly and incubated for a further 15 minutes, and the DCC was separated out by 10 minutes of centrifugation at 800 x g. The supernatant was counted for radioactivity in 10 ml of scintillation cocktail (3.25 g PPO, 0.065 g POPOP, 52 g naphthalene, 150 ml methanol, 250 ml dioxane, and 25 ml toluene). Relative binding affinity was computed from a plot of the log concentration of the competitor versus percent bound radioactivity. For determination of the RBA at 25 C, the incubation time was 4 hours,” after which the tubes were cooled in an ice bath for 15 minutes; thereafter, the procedure followed was the same as described above.

Results

Uterotrophic

activity

Groups of five to six rats were subcutaneously administered different doses of either estradiol or test compounds(rangingfrom0.01 to lOO~g)in0.5mlofpropylene glycol/saline (1 : 1 v/v) for 3 consecutive days. Estradiol (0.5 kg) was administered in 5% ethanol saTable 1

Relative binding affinity of tamoxifen

The receptor affinity of each compound relative to estradiol was determined at 0 C and 25 C; the RBAs are given in Table 1. Measurement of RBA at these temperatures gives an approximation of the kinetics of the interaction with estrogen receptor. A decreased affinity at a higher temperature (25 C) indicates a faster dissociation of the ligand from the binding site, whereas increased affinity suggests slower dissociation. l4 All compounds showed greater receptor-binding affinity at the near-physiologic temperature of 25 C than at 4 C. At either temperature, affinity of all analogs was decreased with respect to tamoxifen. Tamoxifen and its analogs produced suboptimal uterine growth, shown in the dose-response curves (Figure 2). Quaternary-tam and piperazinyl-tam have less agonistic activity than tamoxifen. It is interesting to observe that at higher doses chloro-tam, with the lowest RBA, became more effective in inducing uterine growth and was thus more potent than tamoxifen.

and its various side chain analogs determined

at 0 to 4 C and at 25 C

RBA (% of E2) Ligand

Mean 2 SEM (n) at 0 to 4 C (20 to 24 h)

Tamoxifen Quaternary-tam Chloro-tam Piperazinyl-tam

2.26 ” 0.64 (3) 1.29 2 0.21 (3) 0.03 0.23 + 0.03 (3)

18.23 7.62 3.27 0.65 100.0

100.0

Estradiol Abbreviation:

Mean + SEM (n) at 25 C (4 h)

n, number of independent

k + 2 k

2.11 2.25 0.97 0.20

(3) (4) (3) (3)

determinations.

Steroids,

1991, vol. 56, September

487

Papers Table 2

Antiuterotrophic

activity of tamoxifen

and its analogs in immature

rats % Inhibition”

Dosage of test compound

(pg)

Ratioa of E2 test compound 1 1 1 1 1 1

0.5 1.0 5.0 10.0 50.0 100.0 Symbol: (+ 1, synergistic effect. a Fixed dose of E2 used = 0.5 pg/rat. b Data from three independent experiments

1

Tamoxifen

Cluaternary-tam

Piperazinyl-tam

Chloro-tam

2 10

12.5 27.1 28.8

f+) 9.93 -

18.3 24.4 -

(+I I+)

20 100

42.6 40.5

6.57 -

25.3 -

G

200

45.0

15.70

36.6

c-tr

(five rats per group)

Tamoxifen had the most potent antiuterotrophic activity of all the compounds tested (Table 2). At compound to E, dose ratios of 1: 1 and 1 : 2, piperazinyltam was an antagonist at least as potent as tamoxifen, but with increasing dose ratios exhibited a progressive decrease relative to tamoxifen; nevertheless, it retained considerable antagonistic activity. Quaternarytam was much less potent than tamoxifen at all dose ratios. Chloro-tam was devoid of any antagonistic activity throughout the entire range of dose ratios from 1 : 1 to 1 : 200. Discussion Tamoxifen showed greater affinity for the estrogen receptor than did any of the analogs at temperatures of 0 to 4 C. The several-fold decreased receptor affinity of piperazinyl-tam is likely a consequence of sheer bulk of its side chain. Notwithstanding the size variation in the side chains, all compounds showed stronger binding at 25 C; thus, the fit of these ligands to the receptor, although suboptimal, was in all cases still sufficient to counter increased dissociative tendency at the higher

temperature. In comparison with tamoxifen, the increase in receptor affinity from 4 to 25 C was relatively greater for the short side chain chloro-tam and relatively less for the two analogs with bulky amino substituents on the side chain. Estrogenic activity of these molecules is weak, as evidenced in vivo by suboptimal rodent uterine growth. This is in accord with the results of the in vitro estrogen receptor competitive binding assay. The behavior of chloro-tam, however, is intriguing, in that despite a decreased affinity for the estrogen receptor (RBA < 1% at 25 C), it promotes significant uterine growth at higher doses. It has been reported that removal of the side chain results in expression of full estrogenic activity. I5 It is likely that chloro-tam undergoes transformation in vivo to an active metabolite, which would also explain the slow onset and steeper rise in its apparent activity with increasing concentration. Antagonistic activity of quaternary-tam and piperazinyl-tam is decreased relative to tamoxifen. Chloro-tam is devoid of any antagonistic activity. Partial retention of antagonistic activity by quaternary-tam suggests that a P-rert-nitrogen, although not essential, enhances antagonistic activity. It would be of interest to study other triphenylethylene compounds with primary or secondary amine substituents to elucidate the structural requirements for antagonist activity. Acknowledgments The authors express their thanks to Dr. N. Anand and Dr. V. P. Kamboj for critically reviewing the manuscript. CDRI communication No. 3798. Appendix

I

0.01

qua&tam 0.1

1.0

10.0

dose (wd

Figure 2 Semilogarithimic plot of dose-response curve of tamoxifen and its various side chain analogs in immature rat uterus. Log doses (wg) are plotted on abscissa. The curve for estradiol is provided as a reference.

488

Steroids,

1991, vol. 56, September

Tamoxifen:

(Z)-2-[4-( 1,2-diphenylbut-lenyl)phenoxy]-N,Ndimethylethylamine

Chloro-tam:

E/Z-2-[4( 1,2-diphenylbut-lenyl)phenoxyl-1-chloroethane

Quaternary-tam:

E/Z-2-[[4-( 1,2-Diphenylbut- lenyl)phenoxy]-N,N-

, 100.0

RBA and tamoxifen

dimethylethyll-N-[2-N’N’dimethylaminoethane] ammonium chloride Piperazinyl-tam:

E/Z-Zhydroxy-1-(4phenylpiperazinyl)-3-[4-( diphen ylbut- lenyl)phenoxy]propane,

1,2-

2. 3.

5. 6.

7.

10.

Stoll BA (1980). Clinical experience with tamoxifen in advanced breast cancer. Recent Results Cancer Res 71:207-211. Ward HWC (1973). Antiestrogenic therapy for breast cancer. A trial of tamoxifen at two levels. Br MedJ [C/in Res] 1:13-14. Gasoarini G. Canobbio L. Galliaioni E. Fassio T, Brema F, Crivellari D, Villalta D, DiFronzo G, Talamini R, Monfardini S (1987). Sequential combination of tamoxifen and high dose medroxyprogesterone acetate: therapeutic and endocrine effects in postmenopausal advanced breast cancer patients. Eur J Cancer

4.

9.

2HCl

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11.

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Watkins SM (1988). The value of high dose tamoxifen in postmenopausal breast cancer patients progressing on standard doses! a pilot study. Br J Cancer 57;32&321. _ Colletti RB, Roberts JD, Devlin JT, Copeland KC (1989). Effects of tamoxifen on plasma insulin-like growth factor I in patients with breast cancer. Cancer Res 491882-1884. McGuire WL. Carbone PP. Sears ME. Escher GC (1974). Estrogen receptors in human breast cancers: an overview. In: McGuire WL, Carbone PP, Volmer EP (eds), Estrogen Receptor in Human Breast Cancer. Raven Press, New York, pp. l-7. Durani S, Agarwal AK, Saxena R, Setty BS, Gupta RC, Kole PL, Ray S, Anand N. (1979). Seco-estradiol and some non-

analogs: Aganwal and Bindal

steroidal estrogens: structural correlates of estrogenic action. J Steroid Biochem 11~67-77. Foster AB, Jarman M, Leung T, McLague R, Leclercq G, Devleeschouwer N (1985). Hvdroxv derivatives of tamoxifen. _ J Med Chem 28:1491-1497. . Agarwal AK, Salman M, Ray S, Setty BS, Anand N (1982). Sub-structural contribution in 3,4-diphenylchromans and chromenes for estrogen receptor binding and for their estrogenie profile. In: International Symposium on Hormone Receptor in Growth and Reproduction, Serono Symposium (abstr), Bombay, India. Jhingran AG, Gupta RC, Ray S, Agarwal AK, Singh MM, Anand N (1983). Studies in antifertility agents-part XLI: seco-steroid-X: syntheses of various steroisomers of (+ / - ) 2,6P_diethyl-7ethynyl-3-(phydroxyphenyl) [4.3.0] nonan-7/3-01. Steroids 42~627-634. Bouton MM, Raynaud JP (1978). The relevance of kinetic parameters in the determination of specific binding to the estrogen receptor. J Steroid Biochem 9:9-15. Salman M, Ray S, Agarwal AK, Durani S, Setty BS, Kamboj VP, Anand N (1983). Antifertility agents. Effect of the sience chain and its position on the activity of 3,4-diarylchromans. J Med Chem 26:592-595.

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Hartman RW, Kranzfelder G, Angerer EV, Schonenberger SH (1980). Antiestrogens: synthesis and evaluation of mammary tumor inhibiting activity of 1,1,2,2-tetra-alkyl- 1,2-diphenylethanes. J Med Chem 23:841-848. Bignon E, Pons M, Crastes de Paulet A, Dore JC, Gilbert J, Abecassis J, Miquel JF, OjasooT, Raynaud JP(1989). Effect of triphenylacrylonitrile derivatives on estradiol receptor binding and on human breast cancer cell growth. J Med Chem 32~2092-2103. Jordon VC, Gosden B (1982). Importance of the alkylaminoethoxy side-chain for the estrogenic and antiestrogenic actions of tamoxifen and trioxifene in immature rat uterus. Mol Cell Endocrinol27:291-298.

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