11β-Amidoalkyl estradiols, a new series of pure antiestrogens

J. Steroid Biochem. Molec. Biol. Vol. 41, No. 3-8, pp. 609-614, 1992

0960-0760/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press plc

Printed in Great Britain. All rights reserved

l l/~-AMIDOALKYL

ESTRADIOLS,

PURE

A NEW

SERIES

OF

ANTIESTROGENS

A. CLAUSSNER, L. NI~DI~LEC, F. NIQUE,* D. PH1LIBERT, G. TEUTSCH and P. VAN DE VELDE

Centre de Recherches, Roussel Uclaf, 102-111 Route de Noisy, 93230 RomainviUe, France Summary--In order to find new antiestrogens, devoid of any agonistic activity, a series of I 1fl-amidoalkyl estradiols were prepared. These compounds have been studied in comparison with tamoxifen (TAM): in vitro, for their relative binding affinities (RBA) for mouse and MCF-7 estrogen receptors (ER) and for their antiproliferative effect on MCF-7 (estradiol or EGF/PDGF stimulated) and Ly2 human breast cancer cell lines; in vivo, for their uterotrophic/antiuterotrophic activities in the mouse and for their antitumoral activities on MCF-7 tumors implanted in nude mice. The most representative compounds are N-methyl-N-isopropyl-(3,17fl-dihydroxy-estra1,3,5(10)-trien- 1113-yl)-undecanamide (RU 51625) and its 17~t-ethynyl derivative (RU 53637). They showed good RBAs for ER and a stronger antiproliferative effect than TAM in vitro. Unlike TAM, these compounds inhibited growth factor stimulated MCF-7 proliferation, and the growth of the TAM resistant cell line Ly2. In vivo, they were completely devoid of uterotrophic activity, when given subcutaneously in mice, but exhibited a slight agonistic effect when administered orally. They showed interesting antitumor activities in nude mice by the percutaneous route, but RU 53637 was significantly more potent than RU 51625 when given orally.

INTRODUCTION We showed [1] that 1 lfl-[4-[2-(dimethylamino)ethoxy]phenyl]-estradiol (RU 39411) was a potent antiestrogenic and antiproliferative molecule (Fig. 1), but this compound also displayed a partial estrogenic activity. The introduction of an undecanamide side chain in the 7~ position of the estradiol molecule by another group has given rise to a pure antiestrogenic compound: ICI 164,384 [2]. Owing to our experience in introducing substituents in the 1113 position of the steroid skeleton[3], we prepared a series of estradiol derivatives in which various alkylamide side chains were branched in that position. The biochemical and pharmacological profiles of these compounds were studied in comparison with tamoxifen (TAM). EXPERIMENTAL Synthesis: general procedure

cal rotations [~]D were measured at 20°C on a Roussel-Jouan polarimeter. Silica gel Merck (5-20 #m) was used for all column chromatography. All compounds described showed analytical data (microanalyses, u.v., i.r., [~H]NMR) consistent with the assigned structures. As shown in Scheme 1, the key step of the synthesis was the regiospecific addition [3] of the Grignard reagent derived from the appropriate o g - t e r t - b u t y l d i m e t h y l s i l y l o x y alkyl bromide, in the presence of copper(l) salt, on the aUylic epoxide 1 [4]. The acid hydrolysis of the ketals 2 thus obtained afforded the dienediones 3, in which side chains were oxidized by Jones reagent to the acids 4. Conversion to the amides 5 were done with the appropriate amines, using the mixed anhydride method. Aromatization of the A ring was performed by a classical procedure [3] to lead to the estratrienones 6 which were either reduced by sodium borohydride to estradiol derivatives 7a, b, d or treated with potassium acetylide to give compound %.

Melting points were determined on a Kofler hot stage apparatus and are uncorrected. OptiProceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.

*To whom correspondenceshould be addressed.

1113- [11 -[[dimethyl(1,1- dimethylethyl)silyl]oxy]undecyl]- 5~t - h y d r o x y - estr - 9- en - 3,17- dione - 3(l,2-ethanediyl)cyclic acetal (2a)

Copper(I) chloride (8.32g, 84mmol) was added, in a nitrogen atmosphere, to a cold (0°C) 0.3 M T H F solution of the Grignard reagent 609

610

A. CLAUSSNERet al. "-N /

I

/

o~N~,

H

RU 39411

Tsmoxifen (TAM)

.

OH O

I

Bu ICI 164,384

Fig. 1

derived from 11-[[(tert-butyldimethyl)silyl]oxy]undecyl bromide [2] (2760 ml, 0.83 mol) and the mixture was stirred for 30 min at 0°C. A solution of epoxide 1 [4] (91 g, 0.27mol) in dry THF (470 ml) was added over a 1 h period at -30°C, then the solution was allowed to reach 0°C (1.5 h) and was poured into an aqueous ammonium chloride solution. The crude product, obtained after extraction with ethyl acetate, was chromatographed over silica gel (hexane-ethyl acetate, 7:3) to give pure 2a (108 g, 65%), as a colorless oil.

1 lfl- (11 - hydroxyundecyl)- estra- 4,9- dien - 3,17dione (3a)

Ketal 2a (1 g, 1.62mmol) was dissolved in methanol (20 ml). 2 N hydrochloric acid (5 ml, 10mmol) was added and the solution was stirred for 1.5 h at room temperature. The crude product, obtained by neutralization with sodium bicarbonate and extraction by ethyl acetate, was chromatographed over silica gel (cyclohexane-ethyl acetate, 6:4) to yield pure 3a (670 mg, 94%), as a viscous light yellow oil. X

X

i

i

~k.-O HO

2a,d iii

m

R

R'

a

10

Bu

H

b

10

iPr

H

c

10

iPr

C-=CH

d

12

iPr

H

~

X--CH2OH

3a,d

X = COOH

4a,d

X = CONMeR

5a,b,d

iv

I

I

R,N.90

R-N~. 0

4

HO

or

vii 7a-d

AcO ~ 6a,b,d

Scheme I. (i) TBDMSO-(CH2).-MgBr, CuC1, THF; (ii) HCI, H20, MeOH; (iii) CrO3, H2SO4, acetone; (iv) iBuOCOC1, NMe-morpholine, CH2C12, then RNHMe; (v) Ac20, AcBr, CHzCI:; (vi) NaBH4, MeOH then KOH; and (vii) HC = CH, tBuOK, THF.

Pure antiestrogens

3,17-dio xo-estra -4,9-diene- l l f -undecanoic acid

(4a) Alcohol 3a (10.4 g, 23.6 retool) dissolved in acetone (575 ml) was cooled to 0°C and treated with Jones reagent (20.3 ml, 55 meq). Methanol (4 ml) was carefully added, 15 min after the end of the introduction, followed by baryum carbonate (50 g) and water (500 ml). After 1 h stirring, the solid was filtered off and extracted with acetone. The filtrate was concentrated under reduced pressure and the aqueous layer was extracted with chloroform to give crude acid 4a (l 2.4 g, quantitative yield), as a brown viscous oil.

3,17-dioxo-N-methyl-N- (1-methylethyl)-estra4,9-diene- I I f -undecanamide (Sb) A solution of acid 4a (610 rag, 1.34 mmol) in methylene chloride (12ml) was cooled to - 10°C. N-methyl morpholine (0.55 ml, 4.9 mmol) was added, followed by isobutyl chloroformate (0.64ml, 4.9mmol). After 30min stirring at this temperature, the solution was cooled to - 1 5 ° C and isopropyl methyl amine (0.73 ml, 7.5 mmol) was introduced dropwise. The solution was allowed to warm to room temperature. Forty-five minutes later, the mixture was poured in an aqueous sodium bicarbonate solution. The crude product obtained by extraction with methylene chloride was purified by chromatography over silica gel (methylene chloride-acetone, 9:1) to provide pure 5b (455 rag, 67%) as a light yellow oil. 3-acetoxy-N-methyl-N- (l-methylethyl)- 17- oxoestra- l,3,5(lO)-triene- l lf-undecanamide (lib) A solution of dione 5b (360 rag, 0.7 mmol) in methylene chloride (4 ml) was cooled to 0°C. Acetic anhydride (0.3ml, 3.18mmol) was added, followed by acetyl bromide (0.15 ml, 2 mmol). The solution was allowed to warm to room temperature and was stirred for 1.5 h. After cooling to - 15°C, methanol (0.5 ml) was carefully added and the mixture was poured into an aqueous sodium bicarbonate solution. The product was extracted with methylene chloride and chromatographed over silica gel (methylene chloride-acetone, 95:5) to give pure 6b (310 rag, 80%), as a colorless syrup. 3,17fl- dihydroxy- N- methyl - N- (1 - methylethyl)estra- 1,3,5(10)-triene- lift-undecanamide (Tb), RU 51625 A solution of ketone 6b (272 mg, 0.49 mmol) in methanol (4ml) was cooled to 0°C and treated with sodium borohydride (24 mg,

611

0.63 mmol) for 20 min followed by potassium hydroxide addition (80mg, 1.4mmol). The mixture was diluted with water and acidified by 2 N hydrochloric acid. The product, obtained by ethyl acetate extraction, was purified by chromatography over silica gel (methylene chloride-acetone, 9:1) to afford pure 7b (195 mg, 78%) as a colorless amorphous solid, [~D] + 85° (c = 1% ethanol). The following compounds were prepared by the same procedure, using the appropriate alkyl bromide or amine: N- butyl - 3,17f - dihydroxy - N- methyl - estra 1,3,5(10)-triene-llf-undecanamide (7a), RU 50667,. m.p. 127°C; [~D]+90.5 ° ( C = 1 % ethanol). 3,17f-dihydroxy-N-methyl-N-(1-methylethyl)estra-l,3,5(10)-triene-1 lf-tridecanamide (7d), RU 54485, [~D]- 17° (C = 1% ethanol).

3,17f- dihydroxy-N- methyl-N- (!- methylethyl)19-nor- 17~-pregna- 1,3,5(10)-trien-20-yne- I l f undecanamide (7c), RU 53637 Acetylene was bubbled through a 0.88 M THF solution of potassium tert-butoxide (55ml, 48.4mmol) for 15 min. The suspension thus obtained was diluted with 55 ml of dry THF and introduced into a solution of ketone 6b (5.92 g, 10.7 mmol) in dry THF (54 ml). After stirring the mixture for 30 min, it was poured in a saturated aqueous ammonium chloride solution. The crude product obtained by ethyl acetate extraction was chromatographed over silica gel (methylene chloride-acetone, 9: 1) to yield pure 7c (3.08 g, 54%), as a colorless amorphous solid, [~D] + 40-5° (C = 1% ethanol).

Receptor binding The relative binding affinities (RBA) of test compounds (t.c.) for the estrogen receptors were determined in cytosol from immature mouse uterus or from an MCF-7 cell line. The cytosols were incubated for 5 h at 25°C with 2.5 nM of tritiated estradiol (E2) in the presence of increasing concentrations of cold E2 or t.c. Bound radioactivity was measured by the dextrancoated charcoal adsorption technique [5]. The RBA of E2 was taken arbitrarily as 100. Antiproliferative activities in vitro MCF-7 and Ly2 cells were routinely subpassaged in Dulbecco's modified Eagle's medium supplemented with insulin (50 ng/ml), penicillin (100 U/ml), streptomycin (100 #g/ml) and 7%

612

A. CLAUSSNERet al.

charcoal-stripped serum. Cells were maintained in a 5% CO2 enriched humidified air atmosphere and subpassaged in the same medium without phenol red (1 week) and without insulin (48 h), prior to seeding for growth studies. Cells were seeded in triplicate in 24-well tissue culture dishes at an initial density of 50,000 cells/ml/ well and allowed to attach for 24 h. Then MCF7 cultures were supplemented with 0.1 nM of E2 or 1 ng/ml of P D G F plus 10 ng/ml of E G F and Ly2's were supplemented with 50 ng/ml of insulin. 10/~1 of the appropriate concentrations of the t.c., dissolved in 10% ethanol (EtOH), were added at the same time. The medium was renewed on days 2 and 5 and on day 7 the cell growth was evaluated by a DABA D N A assay derived from Puzas et al. [6]. Uterotrophic and antiuterotrophic activities

Eighteen- to nineteen-day-old female mice of Swiss strain (Iffa-Credo, Les Oncins, France) received the t.c. daily, either orally in suspension in an aqueous solution containing 0.5% of methyl cellulose (M.C.), or subcutaneously in solution in sesame oil containing 5% benzylic alcohol, for three days, either alone or in combination with l0/lg/kg of E2 given subcutaneously. Twentyfour hours after the last administration the animals were sacrificed and the uteri removed and weighed. The estrogenic activity was calculated according to the formula: UWItc/ UWIE2 X 100 ( U W I - - Uterine Weight Increase). The antiestrogenic activity was expressed as the percentage of inhibition of the uterotrophic activity of E2. The doses which inhibited the uterotrophic effect of E2 by 50% were determined. Antitumoral activities in nude mice

One to two millimeter pieces of MCF-7 tumors were implanted subcutaneously at the level of the first right mammary pad in 4- to 5-weekold female nude mice (Balb/ca, Iffa Credo, Les Oncins, France), in groups of 8-10 animals. The tumor growth was stimulated by 100/~g of E2 in l0/~l of EtOH given by percutaneous route once a week for 5 weeks. The animals were then randomized (week 0) according to their tumor volume and received 5/~g of E2 topically once a week _+ 1 mg of t.c., percutaneously in l0 pl of EtOH, or orally in 0.2ml of M.C., twice a week for 5 further weeks. The tumor sizes were measured each week and the volumes calculated according to the formula: length x width2/2. The results were expressed as the ratio:

tumor volume on week n/tumor volume on week 0. RESULTS AND DISCUSSION

In our search for new antiestrogens with potent antitumoral activities, several amidoalkyl side chains were introduced in the l lfl position of the estradiol (or 170t-ethynyl E2) molecule. Compounds 7a-d thus obtained were studied: (1) in vitro for their RBAs for mouse and human estrogen receptors (ER) and for their antiproliferative effect on the human breast cancer cell lines MCF-7 and Ly2, the latter being TAM resistant; and (2) in vivo for their uterotrophic/antiuterotrophic activities in mouse and for their antitumoral activities on MCF-7 tumors implanted in nude mice. In vitro results reported in Table 1 indicate that these compounds displayed high RBAs for the ER. Contrarily to 4-OH-TAM, binding for the human ER proved to be higher than for mouse ER. For a same length of the chain attaching the amide to the steroid nucleus (m = 10), the change of the nitrogen amide substituent from butyl to isopropyl (Tb vs 7a) caused a doubling of the RBA. With R = isopropyl the increase in chain length from m = l0 to m = 12 (7d vs 7b) led to a clear decrease of the RBA. The same tendency was noticed for the strong antiproliferative activities observed on E2 stimulated MCF7 cells. Unlike TAM and 4-OH-TAM, the undecanamide derivatives 7a and 7b were also able to inhibit the insulin stimulated growth of the Ly2 cell line. Moreover, as shown in Fig. 2, they were also strongly antiproliferative on E G F / P D G F stimulated MCF-7 cells, whereas TAM and 4-OH-TAM were inactive, showing even a stimulating effect of the cell growth at 10-SM and l0 -1° M, respectively. Introduction of a 17~t-ethynyl substituent in 7b led to a ........• ......... TAM -----,ll.... 4-OH-TAM RU 50667 (7a) R S1625 Tb ~ RU 53637 (7c)

160" 1,o

....,.,I , 120"

J"

",,

~ 100' ~

80'

,o.

20 L

-

. . . .

1o'.,, ,o"'o 1o-0 ,o"

1;-'

1o~

concentration (M)

Fig. 2. Antiproliferativeactivityon EGF/PDGF stimulated MCF-7 cells.

Pure antiestrogens Table I. In vitro activities

most active on TAM-sensitive tumors either by oral or percutaneous routes, being nearly as active as TAM. The above results show that the introduction of an amidoalkyl side chain in the 11/~ position of the E2 molecule has given rise to a series of compounds which antagonize completely, in the studied models, the biological effects of E2, without having any noticeable agonistic activity. In addition these compounds show an antiproliferative activity independent of the presence of E2, as demonstrated by the inhibition of the growth factor stimulated MCF-7 proliferation. This suggests that they may be of interest for breast cancer therapy, not only in the case of purely estrogeno-dependent tumors, but also in the case of autocrine stimulated tumors. Indeed, autocrine growth factor production could be one of the possible explanations for escape from TAM treatment [7, 8]. In order to confirm, in vivo, the advantages of such compounds, a model of MCF-7 tumor variants, in which growth is stimulated by TAM or by E2, is now under investigation.

IC50 on cell growth R B A for E2 receptor

7a 7b 7¢ 7d

Compounds

Mouse

MCF-7

MCF-7, E2 stim.

Tam 4-OH-TAM R U 50667 R U 51625 R U 53637 R U 54485

0.6 230 27 50 45 12

2 60 125 225 175 30

300 nM 2.6 nM 3 nM 0.9nM 0.75 nM 6nM

613

Ly2,

insulin stim. inactive inactive 0.3 n M 0.2 nM 0.1 n M NT

N T = not tested.

compound, 7e, with a similar in vitro activity profile. In vivo, as shown in Fig. 3(a), all the compounds were completely antiuterotrophic by subcutaneous route, but at a higher dose for 7d, as compared to 7b. Moreover, under the same conditions, these compounds were completely devoid of uterotrophic activity [Fig. 3(b)]. By oral route, they were antiuterotrophic at 10 times higher doses than by the subcutaneous route, but displayed at the same time a very slight uterotrophic activity. Two compounds of this series, 7b and 7e, were tested for their in vivo antitumor activity. Figure 4 shows that 7e is the (a)

tO

..... A-....

R U 50667 (7a) p.o.

.... -o ....

R U 51625 (7b) p.o.

.... -o ....

R U 53637 (7c) p.o. RU RU RU RU

u

50667 51625 53637 54485

(7a) s.c. (7b) s.c. (7c) s.c. (Td) s.c.

50

ll0

l

l0

1130

dose (mg/kg)

(b) 100"

..... ~r....

R U 50667 (7a) p.o.

.... -o ....

R U 51625 (Tb) p.o.

.... -0 ....

RU 53637 (7c) p.o.

80"

•; • • u O

RU RU RU RU

50667 51625 53637 54485

(7a) s.c. (7b) s.c. (7c) s.c. (7d) s.c.

4.0"

20"

" -

":::::::". "

:.:

o

-20

........

•t

,

........

1

, 10

........

, I00

dose (mg/kg) F i g . 3. (a) A n t i u t e r o t r o p h i c activity in mice. (b) U t e r o t r o p h i c activity in m i c e .

614

A. CLAUSSNEReta].

[] E2

[] i

'

E2

[] E2+Tamoxifen

1

i~ilil

;" 0

-

-

•

0

1

3

2

4

5

0

]

2

week

3

4

5

week

oral route

4] D 3t

8] [] ~2

E2

[~'] E2+Tamoxifen [] E2+RU53637 (7c)

i

:!: jili! •

"

1

2

"

2 0

week

1

2

3

4

5

week Fig. 4. Antitumoral activities in rivo,

Acknowledgements--The authors wish to thank J. Br6maud, D. Gottlo, J. Humbert, D. Lucas, J. Scheer and S. Viet (Biology Department), S. Bernard and F. Goubet (Chemistry Department) for their excellent technical assistance and L. Lalouani and L. Martinho (animal maintenance) for helping in the/n vivo experiments.

R E F E R E N C E S

1. N6delec L., Bouton M. M., Nique F., Teutsch G., Van de Velde P. and Philibert D.: l lfl-Aminoalkoxyphenylestradiols, a new series of potent antiestrogens. 9th lnt. Syrup. J. Steroid Biochem. Las Palmas, Spain (1989). Abst. No 34P. 2. Bowler J., Lilley T. J., Pittam J. D. and Wakeling A. E.: Novel steroidal pure antiestrogens. Steroids 54 (1989) 71-99.

3. B61anger A., Philibert D. and Teutsch G.: Regio and stereospecific synthesis of liB-substituted 19-nor-steroids. Steroids 37 (1981) 361-382. 4. Teutsch G., Costerousse G., Philibert D. and Deraedt R.: Steroid derivatives. U.S. Patent US 4,447,424 (08/05/84). C.A. 101 (1984) 130975m. 5. Ojasoo T. and Raynaud J. P.: Unique steroid congeners for receptor studies. Cancer Res. 38 (1978) 4186-4198. 6. Puzas J. E. and Goodman D. B. P.: A rapid assay for cellular deoxyribonucleic acid. Analyt. Biochem. 86 (1978) 50-55. 7. Lippman M. E. and Dickson R. B.: Mechanisms of normal and malignant breast epithelial growth regulation. J. Steroid Biochem. 34 (1989) 107-121. 8. Gibson D. F. C., Gottardis M. M. and Jordan V. C.: Sensitivity and insensitivity of breast cancer to tamoxifen. Z Steroid Biochem. Molec. Biol. 37 (1990) 765-770.