Synthesis, x-ray crystal structure and antiestrogenic activity of 17-methyl-16,17-secoestra-1,3,5(10)-triene derivatives

Article abstract of DOI:10.1135/cccc20050063

Starting from 3-(benzyloxy)-16-(hydroxyimino)-estra-1,3,5(10)-trien-17-one (1) several 17-methyl-16,17-secoestratriene derivatives were synthesized. In the first step of synthesis, hydroxyimino ketone 1 was transformed into 3-(benzyloxy)-16-(hydroxyimino)

Full text of DOI:10.1135/cccc20050063

17-Methyl-16,17-secoestra-1,3,5(10)-triene Derivatives
63
SYNTHESIS, X-RAY CRYSTAL STRUCTURE AND ANTIESTROGENIC
ACTIVITY OF 17-METHYL-16,17-SECOESTRA-1,3,5(10)-TRIENE
DERIVATIVES
a3
Marija N. SAKAČa1,*, Dušan A. MILJKOVIĆa2, Katarina M. PENOV GAŠI
,
b2
Mirjana POPSAVINa4, Olivera R. KLISURIĆb1, Slobodanka M. STANKOVIĆ
,
c1
c2
Silvana ANDRIĆ and Radmila KOVAČEVIĆ
a
Department of Chemistry, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad,
1
Trg Dositeja Obradovića 3, Serbia and Montenegro; e-mail: marijas@ih.ns.ac.yu,
2
3
4
d_milkovic@hotmail.com, sazvon@eunet.yu, popsavin@ih.ns.ac.yu
b
c
Department of Physics, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad,
1
Trg Dositeja Obradovića 4, Serbia and Montenegro; e-mail: olivia@uns.ns.ac.yu,
2
cica@uns.ns.ac.yu
Department of Biology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad,
1
Trg Dositeja Obradovića 2, Serbia and Montenegro; e-mail: sandric@im.ns.ac.yu,
2
radmilak@unsim.in.ns.ac.yu
Received February 5, 2004
Accepted July 1, 2004
Startin g from 3-(ben zyloxy)-16-(h ydroxyim in o)-estra-1,3,5(10)-trien -17-on e (1) several
17-m eth yl-16,17-secoestratrien e derivatives were syn th esized. In th e first step of syn th esis,
h ydroxyim in o keton e 1 was tran sform ed in to 3-(ben zyloxy)-16-(h ydroxyim in o)-17α-m eth yl-
estra-1,3,5(10)-trien -17β-ol (2), th e Beckm an n fragm en tation of wh ich gave 3-(ben zyloxy)-
17-m eth yl-17-oxo-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (3a). Reduction of 3a with so-
dium boroh ydride yielded (17S)-3-(ben zyloxy)-17-h ydroxy-17-m eth yl-16,17-secoestra-
1,3,5(10)-trien e-16-n itrile (4a), wh ose con figuration at th e n ewly form ed ch iral cen ter was
establish ed by X-ray structural an alysis. Catalytic h ydrogen ation of com poun d 3a un der dif-
feren t reaction con dition s yielded 3-h ydroxy-17-m eth yl-17-oxo-16,17-secoestra-1,3,5(10)-
trien e-16-n itrile (3b) an d 16-am in o-17-m eth yl-16,17-secoestra-1,3,5(10)-trien e-3,17-diol (6b).
Biological tests in vivo of com poun ds 3b an d 6b sh owed th eir m oderate an tiestrogen ic ac-
tivity.
Keyw ord s: 17-Substituted 16,17-secosteroids; Steroids; An tiestrogen ic activity; Arom atase in -
h ibition ; X-ray an alysis; Oxim es; Nitriles.
With th e aim of preparin g poten tial an tiestrogen s, we syn th esized in our
previous works1–4 various 17-substituted 16,17-secoestratrien e-16-n itrile de-
rivatives. Som e of th ese com poun ds sh owed a m oderate an tiestrogen ic ac-
tivity in biological in vivo studies2,4. Th us, 3-h ydroxy-17-oxo-16,17-seco-
estra-1,3,5(10)-trien e-16-n itrile exibited 16% of an tiestrogen ic activity an d
Collect. Czech. Chem. Commun. (Vol. 70) (2005)
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Sakač et al.:
correspon din g 3,17-diol exibited 21% of an tagon istic effect2. On th e oth er
h an d, 17-ch loro an d 17-brom o derivatives of 16,17-secoestratrien e-
16-n itrile exibited stron ger an tiestrogen ic effect (33 an d 32%, respec-
tively)4. In th is work we con tin ue our in vestigation s of th e in fluen ce of di-
verse fun ction al groups in th e position s C-16 an d C-17 of 16,17-seco-
estratrien e derivatives on biological respon se of th e m olecule. Accordin gly,
we syn th esized 17-m eth yl-17-oxo-16,17-secoestra-1,3,5(10)-trien e-16-n itrile
as well as 17-h ydroxy an d 16-am in o-17-h ydroxy derivatives. Also,
we wan ted to exam in e th e poten tial in h ibitory activity of 16-cyan o-
17-m eth yl-16,17-secoestratrien e derivative 3b again st arom atase, h avin g
in m in d th at in our previous work5 th e correspon din g 3-oxo-4-en e-, as well
as 3-oxo-1,4-dien e- an d 17-m eth yl-3-oxo-16,17-seco-1,4,6-trien e-16-n itrile
derivatives of an drostan e, sh owed a h igh degree of an tiarom atase activity.
EXPERIMENTAL
Meltin g poin ts were determ in ed usin g a Büch i SMP 20 apparatus an d are un corrected. In fra-
red spectra (waven um bers in cm –1) were recorded in KBr pellets on a Nexus 670 SP-IR spec-
trom eter. NMR spectra were taken on a Bruker AC 250E spectrom eter operatin g at 250 MHz
(1H) an d 62.5 MHz (13C) with tetram eth ylsilan e as th e in tern al stan dard. Ch em ical sh ifts are
given in ppm (δ-scale); couplin g con stan ts (J) are given in Hz. Mass spectra were recorded on
a Fin n igan MAT 8230 in strum en t, usin g ch em ical ion ization (isobutan e) or electron im pact
(70 eV) tech n ique; th e first n um ber den otes th e m/z value, th e ion abun dan ces are given in
paren th eses. All reagen ts used were of an alytical grade.
3-(Ben zyloxy)-16-(h ydroxyim in o)-17α-m eth ylestra-1,3,5(10)-trien -17β-ol (2)
To m eth ylm agn esium iodide, prepared from m eth yl iodide (4.8 m l, 77 m m ol) an d m agn esium
(1.25 g. 51.3 m m ol) in absolute eth er (80 m l), a solution of com poun d 1 (2.0 g, 5.13 m m ol)
in absolute tetrah ydrofuran (80 m l) was added dropwise at room tem perature durin g 1.5 h .
Th e reaction m ixture was stirred vigorously at th e sam e tem perature for an oth er 2 h , th en
poured in to water (500 m l) an d left stan din g for 2–3 days, to allow th e organ ic solven t to
evaporate. Th e cake form ed on th e water surface was separated by filtration an d wash ed
with a sm all volum e of EtOAc. After dryin g, th e crude product was purified by flash ch rom a-
tograph y (ben zen e–EtOAc, 1:1), affordin g pure com poun d 2 (1.45 g, 70%), wh ose recrystal-
lization from m eth an ol gave a product, m .p. 179–180 °C. IR: 3396, 3032, 2978, 1609, 1499,
1282, 1233, 1045, 901. 1H NMR (CDCl3): 0.91 s, 3 H (H-18); 1.42 s, 3 H (CH3-C17); 2.68 dd,
1 H, J(15a,15b) = 18.9, J(15a,14) = 7.3 (H-15a); 2.92 m , 2 H (H-6); 5.06 s, 2 H (OCH2C6H5);
5.42 bs, 2 H (NOH an d C17-OH); 6.72 d, 1 H, J(2,4) = 2.7 (H-4); 6.80 dd, 1 H, J(1,2) = 8.5,
J(2,4) = 2.7 (H-2); 7.25 d, 1 H, J(1,2) = 8.5 (H-1); 7.40 m , 5 H (C6H5CH2). 13C NMR (CDCl3):
13.72 (C-18); 22.96 (CH3-C17); 25.88, 26.65, 27.56, 29.57, 30.84, 38.37, 43.73, 45.22, 46.23
(C-15); 69.84 (CH2-Ph ); 81.41 (C-17); 112.21, 114.78, 126.10, 127.35, 127.77, 128.45 (Ar
CH); 132.40, 137.17, 137.69 (Ar C); 156.73 (C-3); 169.51 (C=NOH). MS: 406 (M+ + 1, 100);
388 (M+ + 1 – H2O, 8). For C26H31NO3·CH3OH (437.6) calculated: 74.14% C, 8.01% H,
3.20% N; foun d: 74.38% C, 7.91% H, 3.12% N.
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17-Methyl-16,17-secoestra-1,3,5(10)-triene Derivatives
65
3-(Ben zyloxy)-17-m eth yl-17-oxo-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (3a)
A solution of com poun d 2 (0.98 g, 2.42 m m ol) an d acetic an h ydride (12 m l, 0.13 m ol) in
absolute pyridin e (24 m l) was stirred at 100 °C for 2 h . Th e reaction m ixture was th en
poured in to water (100 m l) an d its pH adjusted to 1 with 6 M HCl. Th e precipitate form ed
was separated by filtration an d wash ed with water. Dryin g of th e crude product gave 0.83 g
(88.5%) of 3a. Recrystallization from m eth an ol afforded 0.76 g (81%) of wh ite n eedle-like
crystals, m .p. 128–129 °C. IR: 3030, 2909, 2238, 1693, 1604, 1500, 1286, 1027. 1H NMR
(CDCl3): 1.33 s, 3 H (H-18); 2.23 s, 3 H (CH3-C17); 2.53 dd, 1 H, J(15a,15b) = 17.8, J(15a,14) =
5.3 (H-15a); 2.91 m , 2 H (H-6); 5.06 s, 2 H (OCH2C6H5); 6.76 d, 1 H, J(2,4) = 2.7 (H-4);
6.82 dd, 1 H, J(1,2) = 8.5, J(2,4) = 2.7 (H-2); 7.20 d, 1 H, J(1,2) = 8.5 (H-1); 7.30–7.48 m , 5 H
(C6H5CH2). 13C NMR (CDCl3): 15.49 (C-18); 18.09 (C-15); 25.36 (CH3-C17); 25.44, 26.70,
29.74, 36.49, 39.15, 40.43, 42.43, 52.01 (C-13); 69.91 (CH2Ph ); 112.70, 114.49, 119.11 (CN);
126.27, 127.38, 127.84, 128.51, 131.29, 137.11, 137.49, 156.99 (C-3); 213.23 (C=O). MS: 444
(M+ + i-Bu – 1, 100); 388 (M+ + 1, 52). For C26H29NO2 (387.5) calculated: 80.58% C, 7.54% H,
3.61% N; foun d: 80.50% C, 7.49% H, 4.28% N.
3-Hydroxy-17-m eth yl-17-oxo-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (3b)
Com poun d 3a (0.15 g, 0.39 m m ol) was dissolved in a m ixture of m eth an ol an d m eth ylen e
ch loride (1:1, 4 m l). To th e solution was added 10% Pd/C (0.015 g) an d th e suspen sion
stirred in a h ydrogen atm osph ere at room tem perature for 12 h . Th e reaction m ixture was
th en filtered, th e precipitate wash ed with m eth ylen e ch loride an d th e solven t rem oved by
evaporation to dryn ess. Th e crude reaction product was purified by flash ch rom atograph y
(ben zen e–EtOAc, 6:1), affordin g 0.97 g (83%) of com poun d 3b in th e form of wh ite crystals,
wh ich after recrystallization from EtOAc h ad m .p.186–187 °C. IR: 3370, 3018, 2252, 1697,
1609, 1224, 1108, 866. 1H NMR (CDCl3): 1.33 s, 3 H (H-18); 2.23 s, 3 H (CH3-C17); 2.55 dd,
1 H, J(15a,15b) = 17.8, J(15a,14) = 5.3 (H-15a); 2.90 m , 2 H (H-6); 6.59 d, 1 H, J(2,4) = 2.7
(H-4); 6.67 dd, 1 H, J(1,2) = 8.5, J(2,4) = 2.7 (H-2); 7.13 d, 1 H, J(1,2) = 8.5 (H-1). 13C NMR
(CDCl3): 15.51 (C-18); 18.10 (C-15); 25.40 (CH3-C17); 25.46, 26.66, 29.57, 36.49, 39.12,
40.35, 42.39, 52.04, 113.07, 115.10, 126.45 (Ar CH); 119.13 (CN); 130.88 (C-10); 137.65
(C-5); 153.87 (C-3); 213.60 (C=O). MS: 297 (M+, 40); 279 (M+ – H2O, 6); 254 (M+ – CH3CO,
19); 43 (100). For C19H23NO2 (297.4) calculated: 76.73% C, 7.79% H, 4.71% N; foun d:
77.38% C, 7.35% H, 5.19% N.
(17S)-3-(Ben zyloxy)-17-h ydroxy-17-m eth yl-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (4a)
an d (17aR)-3-(Ben zyloxy)-17a-m eth yl-17a-h om o-17-oxaestra-1,3,5(10)-trien -16-on e (5a)
To a solution of com poun d 3a (0.22 g, 0.57 m m ol) in m eth an ol (12 m l) sodium boro-
h ydride (0.25 g, 6.61 m m ol) was added portion wise at room tem perature. Th e reaction
m ixture was stirred at boilin g tem perature for 1.5 h , th en poured in to water (50 m l) an d
extracted with m eth ylen e ch loride (3 × 20 m l). Th e com bin ed extracts were wash ed with
water, dried an d th e solven t was rem oved. Th e rem ain in g m ixture of products was separated
by flash ch rom atograph y (ben zen e–EtOAc, 6:1). Th is gave 0.141 g (64%) of com poun d 4a,
m .p. 113–114 °C an d 0.021 g (9%) of com poun d 5a, m .p. 177–179 °C.
Compound 4a: IR: 3440, 3032, 2241, 1608, 1500, 1236, 1025. 1H NMR (CDCl3): 0.97 s, 3 H
(H-18); 1.26 d, 3 H, J = 6.3 (CH3-C17); 2.46 dd, 1 H, J(15a,15b) = 17.8, J(15a,14) = 3.6
(H-15a); 2.66 dd, 1 H, J(15a,15b) = 17.8, J(15b,14) = 5.3 (H-15b); 2.91 m , 2 H (H-6); 3.80 q,
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Sakač et al.:
1 H, J = 6.3 (H-17); 5.06 s, 2 H (OCH2C6H5); 6.76 d, 1 H, J(2,4) = 2.7 (H-4); 6.82 dd, 1 H,
J(1,2) = 8.5, J(2,4) = 2.7 (H-2); 7.20 d, 1 H, J(1,2) = 8.5 (H-1); 7.30–7.48 m , 5 H (C6H5CH2).
13C NMR (CDCl3): 14.92 (C-15); 16.99 (C-18); 17.76 (CH3-C17); 25.92, 27.09, 29.95, 31.23,
39.52, 40.39, 40.50 (C-13); 42.69, 69.88 (CH2Ph ); 72.41 (C-17); 112.57, 114.34, 126.31,
127.32, 127.79, 128.47 (Ar CH); 120.04 (CN); 132.12, 137.16, 137.57 (Ar C); 156.81 (C-3).
For C26H31NO2 (389.5) calculated: 80.17% C, 8.02% H, 3.60% N; foun d: 80.19% C, 8.28% H,
3.88% N.
Compound 5a: IR: 1720, 1609, 1498, 1246, 1022. 1H NMR (CDCl3): 0.91 s, 3 H (H-18);
1.29 d, 2 H, J = 6.4 (CH3-C17); 2.90 m , 3 H (H-6, H-15a); 4.20 q, 1 H, J = 6.4 (H-17); 5.05 s,
2 H (OCH2C6H5); 6.76 d, 1 H, J(2,4) = 2.7 (H-4); 6.82 dd, 1 H, J(1,2) = 8.5, J(2,4) = 2.7 (H-2);
7.20 d, 1 H, J(1,2) = 8.5 (H-1); 7.30–7.50 m , 5 H (C6H5CH2). 13C NMR (CDCl3): 10.60, 14.80,
17.86, 25.36, 29.66, 31.59, 35.32, 36.55, 39.25, 42.52, 44.79, 69.92 (CH2Ph ); 86.24 (C-17);
112.59, 114.58, 126.13, 127.39, 127.86, 128.52 (Ar CH); 131.74, 137.13, 137.51 (Ar C);
156.94 (C-3); 170.90 (C=O). MS: 390 (M+, 20); 297 (12); 256 (5); 91 (100).
(17S)-3,17-Dih ydroxy-17-m eth yl-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (4b)
From com poun d 4a (0.14 g, 0.36 m m ol) in th e presen ce of 10% Pd/C (0.014 g) un der th e re-
action con dition s given for com poun d 3b, crude 4b (0.113 g) was obtain ed in th e form of
colorless oil. Its purification by flash ch rom atograph y (ben zen e–EtOAc, 4:1) gave 0.067 g
(62%) of wh ite crystals, m .p. 183 °C. IR: 3449, 2246, 1612, 1500, 1287, 1120, 928. 1H NMR
(aceton e-d6): 0.92 s, 3 H (H-18); 1.16 d, 3 H, J(17,1′) = 6.3 (CH3-C17); 2.58 dd, 1 H,
J(15a,15b) = 17.8, J(15a,14) = 4.1 (H-15a); 2.75 dd, 1 H, J(15a,15b) = 17.8, J(15b,14) = 5.0
(H-15b); 2.83 m , 2 H (H-6); 3.73 m , 1 H (H-17); 3.88 d, 1 H, J(17,OH) = 4.9 (C17-OH); 6.55 d,
1 H, J(2,4) = 2.7 (H-4); 6.61 dd, 1 H, J(1,2) = 8.5, J(2,4) = 2.7 (H-2); 7.11 d, 1 H, J(1,2) = 8.5
(H-1); 8.13 s, 1 H (C3-OH). 13C NMR (aceton e-d6): 15.25 (C-15); 17.35 (C-18); 17.87
(CH3-C17); 27.03, 27.94, 28.88, 32.22, 41.00, 41.18, 41.52, 43.62, 72.28 (C-17); 113.75,
115.58, 127.14 (Ar CH); 121.00 (CN); 131.55 (C-10); 138.10 (C-5); 156.00 (C-3). MS: 299
(M+, 9); 284 (M+ – CH3, 5); 214 (20), 43 (100). For C19H25NO2·0.5H2O (308.4) calculated:
73.99% C, 8.50% H, 4.54% N; foun d: 73.59% C, 8.70% H, 5.05% N.
16-Am in o-17-m eth yl-16,17-secoestra-1,3,5(10)-trien e-3,17-diol (6b)
Com poun d 3a (0.2 g, 0.52 m m ol) was dissolved in a m ixture of m eth an ol an d m eth ylen e
ch loride (1:1, 4 m l). To th is solution 10% Pd/C (0.05 g) was added an d th e suspen sion was
stirred in th e h ydrogen atm osph ere at room tem perature for 24 h . After th at th e reaction
m ixture was filtered, th e precipitate wash ed with m eth ylen e ch loride an d th e solven t re-
m oved. Th e resultin g crude m ixture was purified by flash ch rom atograph y (CH2Cl2–MeOH,
1:1), affordin g pure com poun d 6b (0.14 g, 89%) in th e form of wh ite crystals, m .p. 265 °C.
IR: 3431, 2940, 2861, 1585, 1286, 1252, 1126, 870. 1H NMR (DMSO-d6): 0.80 s, 3 H (H-18);
0.90 d, 3 H, J(17,1′) = 6.4 (CH3-C17); 1.82 s, 1 H (OH); 2.37 q, 1 H, J(17,1′) = 6.4 (H-17);
3.16 s, 2 H (NH); 6.42 d, 1 H, J(2,4) = 2.7 (H-4); 6.50 dd, 1 H, J(1,2) = 8.5, J(2,4) = 2.7 (H-2);
7.03 d, 1 H, J(1,2) = 8.5 (H-1). 13C NMR (DMSO-d6): 11.88 (C-18); 14.53 (CH3-C17); 23.44,
25.65, 29.46, 36.17, 42.89, 46.12 (C-16); 48.87, 61.49 (C-17); 112.79, 114.65, 125.89 (Ar
CH); 130.53 (C-10); 137.07 (C-5); 154.97 (C-3). MS: 285 (M+ – H2O, 23); 270 (M+ – H2O –
CH3, 37); 58 (100). For C19H29NO2 (303.4) calculated: 75.15% C, 9.63% H, 4.61% N; foun d:
75.56% C, 9.77% H, 4.95% N.
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17-Methyl-16,17-secoestra-1,3,5(10)-triene Derivatives
67
X-ray Crystal Studies of 4b an d 5a
Diffraction data (Table I) for com poun ds 4b an d 5a were collected on a CCD Bruker Sm art
Apex diffractom eter at 173 an d 110 K, respectively, usin g MoKα radiation (λ = 0.71073 Å).
Both structures were solved by direct m eth ods (SHELXS97)6 an d refin ed by full-m atrix
least-squares procedures (SHELXL97)6. Non -h ydrogen atom s were refin ed an isotropically. All
h ydrogen atom s were iden tified on differen ce electron den sity m aps an d isotropically re-
fin ed. Loren tz an d polarization correction s were applied to th e data. No absorption correc-
tion was m ade.
TABLE I
Crystallograph ic data, data collection an d structure refin em en t for com poun ds 4b an d 5a
Param eter
Com poun d 4b
Com poun d 5a
Form ula
C
19H25NO2
C26H30O3
Mw
299.4
390.5
Crystal size, m m 3
Crystal description
Crystal system , space group
a, Å; α, °
0.51 × 0.50 × 0.81
colorless prism
orth orh om bic, P212121
8.218(2); 90
9.567(3); 90
21.383(6); 90
1681.0(8); 4
1.183
0.20 × 0.30 × 0.50
colorless prism
triclin ic, P1
6.852(2); 91.903(3)
8.744(2); 101.881(3)
9.740(2); 112.525(3)
523.38(8); 1
1.24
b, Å; β, °
c, Å; γ, °
V, Å3; Z
Dc, g/cm –3
F(000), µ(MoKα), m m –1
θ ran ge, °; data com pleten ess, %
Ran ge of h, k, l
647.9; 0.076
2.3–33.1; 100
–12/9, –14/14, –32/32
5813
210.0; 0.079
2.2–33.0; 100
–10/10, –12/12, –14/14
6909
No. of un ique diffraction s
No. of observed diffraction sa
No. of param eters
R, wR for observed diffraction s, %
R, wR for all data, %
GOF for all data
5199
6305
311
382
3.5, 9.4
3.5, 9.4
4.0, 9.8
4.0, 9.7
0.846
1.018
(∆/σ)m ax
0.001
0.001
Residual electron den sity, e Å–3
0.360, –0.161
0.359, –0.167
a
Diffraction s with Fo > 4σ(Fo).
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

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Sakač et al.:
CCDC 244805 (for 4b ) an d 244804 (for 5a ) con tain th e supplem en tary crys-
tallograph ic data for th is paper. Th ese data can be obtain ed free of ch arge via
www.ccdc.cam .ac.uk/con ts/retrievin g.h tm l (or from th e Cam bridge Crystallograph ic
Data Cen tre, 12, Un ion Road, Cam bridge, CB2 1EZ, UK; fax: +44 1223 336033; or
deposit@ccdc.cam .ac.uk).
Biological Tests
Chemicals. An tiestradiol serum No. 244 was kin dly supplied by Dr G. D. Niswen der
(Colorado State Un iversity, CO, U.S.A.); pregn an t m ares serum gon adotroph in (PMSG) was
obtain ed from th e Veterin ary In stitute Subotica (Serbia an d Mon ten egro), [1,2,6,7-3H4]-
estradiol from New En glan d Nuclear (Belgium ), NADPH an d am in ogluteth im ide (AG) from
Sigm a (St Louis, MO, U.S.A.).
Uterotrophic assay. Im m ature Wistar strain fem ale rats (21–23 days old) were ran dom ly di-
vided in to groups of six to eigh t an im als each . Th e an im als were treated by subcutan eous
in jection on ce a day with 0.1 m l of a solution of th e test com poun d in olive oil for 3 days,
eith er solely or in com bin ation with estradiol ben zoate (EB). Th e con trol group obtain ed th e
veh icle on ly. Th e total adm in istered am oun ts of tested com poun ds were 5 or 25 m g/kg body
weigh t, wh ereas th e EB dose was 0.03 m g/kg body weigh t. Th e an im als were killed on th e
fourth day. Th e adh erin g fat was rem oved from uteri an d blotted dry after expulsion of uter-
in e fluid an d th e wet weigh t was recorded.
Per cen t agon ist an d an tagon ist activity in im m ature rat uterin e weigh t assays were calcu-
lated from th e ratio of values recorded in treated an d con trol an im als th us
% agon ism = (C – A) × 100/(B – A)
an d
% an tagon ism = (B – D) × 100/(B – A),
wh ere A, B, C an d D are uterin e wet weigh ts, corrected for differen ces in body weigh t, i.e.
(m g/100 g body weigh t) for th e veh icle alon e, EB, test com poun d alon e, or test com poun d
plus EB groups, respectively.
Animals, treatments and assays. Preparation of den ucleated ovarian fraction from PMSG
pretreated rats an d determ in ation of arom atase activity in ovarian h om ogen ate was carried
out as described previously3,5
.
For prelim in ary assessm en t of poten tial an tiarom atase activity of com poun d 3b, th e com -
poun d was added in th ree con cen tration s (10, 20 an d 50 µM) to th e in cubation m ixture con -
tain in g 500 n M of testosteron e as a substrate. (saturated con cen tration ; th e estim ated Km for
testosteron e was 49 n M an d Vm ax 5.76 pm ol/m in /m g protein ).
RESULTS AND DISCUSSION
Th e startin g com poun d, 16-(h ydroxyim in o) derivative of 3-(ben zyloxy)-
estron e (1; Sch em e 1), was syn th esized in two syn th etic steps, startin g from
estron e2. Regio- an d stereospecific addition of m eth ylm agn esium iodide to
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

17-Methyl-16,17-secoestra-1,3,5(10)-triene Derivatives
69
th e C-17 carbon yl group of h ydroxyim in o keton e 1 yielded th e correspon d-
in g 17β-h ydroxy-17α-m eth yl derivative 2, th e fragm en tation of wh ich with
acetic an h ydride in pyridin e gave 16,17-seco derivative 3a.
O
H3C
OH
N
N
(i)
OH
OH
BnO
BnO
1
2
(ii)
CH3
CH3
CHOH
O
C
(iv)
N
CH2NH2
RO
HO
3
6b
(iii)
CH3
H
CH3
O
In formulae 3 and 4
OH
H
a, R = Bn
b, R = H
(iv)
C
N
O
Bn = Benzyl
RO
BnO
4
5a
(i) CH3MgI, ether, THF, then NH4Cl, H2O, r.t.; (ii) Ac2O, Py, 100 0C;
(iii) NaBH4, MeOH, reflux; (iv) H2, 10% Pd/C, MeOH, CH2Cl2, r.t.
SCHEME 1
Reduction of com poun d 3a with sodium boroh ydride gave th e h ydroxy
derivative 4a as m ain reaction product (yield 64%). Con figuration at th e
n ewly form ed ch iral cen ter C-17 was establish ed by X-ray an alysis of com -
poun d 4b (Fig. 1), wh ich was obtain ed from 4a after deprotection of th e
3-h ydroxy fun ction , un der con dition s of catalytic h ydrogen olysis. As a by-
product of th e reduction of com poun d 3a with NaBH4, lacton e 5a was iso-
lated in a yield of 9%. Th e structure of com poun d 5a was establish ed on th e
basis of spectroscopic data an d X-ray an alysis (Fig. 1).
Reduction of com poun d 3a un der con dition s of catalytic h ydrogen ation
with 10% Pd/C as a catalyst (weigh t ratio 3a:10% Pd/C 4:1) for 24 h , yiel-
ded 3,17-dih ydroxy-16-am in o derivative 6b. Wh en th e ratio 3a:10% Pd/C
was 10:1 an d th e reaction tim e 12 h , on ly rem oval of th e ben zyl group took
place, resultin g in com poun d 3b.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

70
Sakač et al.:
Th e estrogen ic an d an tiestrogen ic effects of com poun ds 3b, 4b an d 6b
were tested on fem ale rats usin g th e uterotroph ic an d an tiuterotroph ic
m eth ods8. Th e differen ces in weigh ts of uteri of treated an d con trol an im als
served for th e calculation of th e agon istic an d an tagon istic effects9 presen -
ted in Table II.
It is eviden t from Table II th at com poun ds 3b an d 6b caused iden tical de-
crease in estrogen ic activity an d a m oderate an tiestrogen ic effect, but th is
activities is stron ger th at in earlier syn th esized com poun ds (see in troduc-
tion part of th e paper).
Also, accordin g to our results, 3b expressed an tiarom atase activity wh en
applied in h igh con cen tration s (20 an d 50 µM). However, th is activity is
m uch lower com pared with our previous data obtain ed with an drostan e de-
rivatives m odified in rin g D 5.
TABLE II
Agon istic an d an tagon istic effects of com poun ds 3b, 4b an d 6b
Dose
m g/kg
Agon istic effect
%, m ean ± SEM
An tagon istic effect
%, m ean ± SEM
Com poun d
n
n
3b
5
25
5
6
7
7
6
6
–4.0 ± 1.31
12.3 ± 1.51
–
8
6
7
7
7
37.6 ± 5.51
32.9 ± 0.95
29.0 ± 4.41
38.1 ± 1.58
39.8 ± 1.16
4b
6b
5
–9.7 ± 0.62
–1.5 ± 0.29
25
O2
O3
O2
O1
N
O3
O1
4b
5a
FIG. 1
Perspective view7 of th e m olecular structure of com poun ds 4b an d 5a
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

17-Methyl-16,17-secoestra-1,3,5(10)-triene Derivatives
71
This research was supported by the Ministry of Science, Technologies, and Development of the
Republic of Serbia (grant No. 1896).
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