Synthesis and biological activity of new 16, 17-secoestrone derivatives

Article abstract of DOI:10.1135/cccc20000077

Starting from estrone 3-benzyloxy-17β-hydroxyestra-1,3,5(10)-trien-16-one oxime (3b) was synthesized, which underwent Beckmann fragmentation giving the 3-benzyloxy-17-oxo-16,17-secoestra-1,3,5(10)-triene-16-nitrile (4b). Sodiumb borohydride reduction of this compound afforded 3-benzyloxy-17-hydroxy-16,17-secoestra-1,3,5(10)-triene-16-nitrile (5b). The deprotection of the 3-hydroxy group was achieved by action of hydrogen upon derivatives 4b and 5b in presence of Pd/C as a catalyst, yielding 3-hydroxy-17-oxo-16,17-secoestra-1,3,5(10)-triene-16-nitrile (4a) and 3,17-dihydroxy-16,17-secoestra-1,3,5(10)-triene-16-nitrile (5a). In biological tests on experimental animals, compounds 4a, 4b, 5a and 5b showed virtually a complete loss of estrogenic activity, whereas compounds 4a, 5a and 5b exhibited moderate antiestrogenic effect.

Full text of DOI:10.1135/cccc20000077

16,17-Secoestrone Derivatives
77
SYNTHESIS AND BIOLOGICAL ACTIVITY OF NEW
16,17-SECOESTRONE DERIVATIVES
b2
Suzana JOVANOVIĆ-ŠANTAa1,*, Silvana ANDRIĆb1, Radmila KOVACEVIĆ
a2
and Vjera PEJANOVIĆ
a
Institute of Chemistry, Faculty of Science, University of Novi Sad, Trg Dositeja Obradovica 3,
YU-21000 Novi Sad, Yugoslavia; e-mail: santas@posejdon.ih.ns.ac.yu, ognjenpc@beotel.yu
Institute of Biology, Faculty of Science, University of Novi Sad, Trg Dositeja Obradovica 2,
1
2
b
1
YU-21000 Novi Sad, Yugoslavia; e-mail: sandric@unsim.in.ns.ac.yu,
2
radmilak@unsim.in.ns.ac.yu
Received February 24, 1999
Accepted Novem ber 8, 1999
Startin g from estron e 3-ben zyloxy-17β-h ydroxyestra-1,3,5(10)-trien -16-on e oxim e (3b) was
syn th esized, wh ich un derwen t Beckm an n fragm en tation givin g th e 3-ben zyloxy-17-oxo-
16,17-secoestra-1,3,5(10)-trien e-16-n itrile (4b). Sodium boroh ydride reduction of th is com -
poun d afforded 3-ben zyloxy-17-h ydroxy-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (5b). Th e
deprotection of th e 3-h ydroxy group was ach ieved by action of h ydrogen upon derivatives
4b an d 5b in presen ce of Pd/C as a catalyst, yieldin g 3-h ydroxy-17-oxo-16,17-secoestra-
1,3,5(10)-trien e-16-n itrile (4a) an d 3,17-dih ydroxy-16,17-secoestra-1,3,5(10)-trien e-16-n itrile
(5a). In biological tests on experim en tal an im als, com poun ds 4a, 4b, 5a an d 5b sh owed vir-
tually a com plete loss of estrogen ic activity, wh ereas com poun ds 4a, 5a an d 5b exh ibited
m oderate an tiestrogen ic effect.
Key w ord s: Steroids; Secosteroids; Horm on es; Estrogen es; Estrogen ic activity; Beckm an n
fragm en tation reaction ; Hydrogen olysis.
In th e fram e of a broader project directed towards obtain in g poten tial an ti-
estrogen s, th e preparation of a series of n ew 16,17-secoestron e derivatives
with m eth oxy fun ction at C-3 h as been reported previously1. Th e aim of
th is paper was th e syn th esis of two n ew D-secoestron e derivatives bearin g a
free C-3 h ydroxy fun ction an d testin g of th eir biological activity on experi-
m en tal an im als.
EXPERIMENTAL
Meltin g poin ts were determ in ed in open capillary tubes on a Büch i SMP apparatus an d are
un corrected. In frared spectra (wave n um bers in cm –1) were recorded in KBr pellets or as film
on a Perkin –Elm er M457 spectroph otom eter. NMR spectra were taken on a Bruker AC 250E
spectrom eter (1H at 250.13 MHz, 13C at 62.5 MHz) an d are reported in ppm down field from
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78
Jovanović-Šanta et al.:
a tetram eth ylsilan e in tern al stan dard; sym bols s, bs, d, dd, q an d m den ote sin glet, broad
sin glet, doublet, double doublet, quartet an d m ultiplet, respectively. Mass spectra were re-
corded on a Fin n igan -MAT 8230 in strum en t, usin g electron im pact (70 eV) or ch em ical ion -
ization (isobutan e) tech n iques; th e first n um ber den otes m/z value, an d th e ion abun dan ces
are given in paren th eses.
3-Ben zyloxyestra-1,3,5(10)-trien -17-on e (1b)
To a solution of estron e (1a; 5.0 g, 18.5 m m ol) in a m ixture of fresh ly distilled m eth an ol
an d aceton e (1 : 1, 250 m l), potassium carbon ate (5.0 g, 36.2 m m ol) an d ben zyl ch loride
(25.0 m l, 217 m m ol) were added an d th e m ixture was refluxed for 8 h . After evaporation of
th e solven t, an excess of ben zyl ch loride was rem oved by steam distillation . Th e precipitate
was colected by filtration , wash ed with water, an d air-dried, givin g crude 1b (6.63 g, 99%).
After crystallization from m eth an ol, 5.40 g (81%) of pure com poun d 1b, m .p. 132–133 °C
(ref.2 m .p. 135–136 °C) was obtain ed. IR: 3 020, 2 920, 1 605, 1 580, 1 500, 1 460, 1 290,
1 260, 1 240, 740, 700. 1H NMR (CDCl3): 0.98 (s, 3 H, 3 × H-18); 5.15 (s, 2 H, O-CH2-C6H5);
6.70–7.40 (group of sign als, 8 H, arom atic proton s). 13C NMR (CDCl3): 13.83 (C-18); 76.49
(O-CH2-C6H5); 156.85 (C-3); 220.86 (C=O). For C25H28O2 (360.5) calculated: 83.29% C,
7.83% H; foun d: 83.50% C, 7.71% H.
3-Ben zyloxyestra-1,3,5(10)-trien e-16,17-dion e 16-Oxim e (2b)
Metallic potassium (0.96 g, 24.6 m m ol) was dissolved in 2-m eth ylpropan -2-ol (40 m l), 1b
(3.0 g, 8.3 m m ol) was added an d th e m ixture was stirred for 15 m in at room tem perature.
Isopen tyl n itrite (4.5 m l, 33 m m ol) was th en in troduced an d th e stirrin g con tin ued for 1 h .
Th e m ixture was left overn igh t an d th en poured in to water (150 m l). Th e solid was filtered
off an d th e filtrate n eutralized with dilute HCl (1 : 4) an d th e wh ite precipitate of crude 2
was collected by filtration , wash ed with water an d air-dried (3.10 g). Th e crude 2b was dis-
solved in a solution of KOH (1.30 g) in 50% aqueous eth an ol (60 m l) an d th e solution n eu-
tralized with diluted HCl (1 : 4). Th e separated crystals were filtered off, wash ed with water
un til n eutral an d dried, yieldin g th e pure 2b (2.90 g, 89.5%), m .p. 196–198 °C (ref.3 m .p.
193.5–195.5 °C). IR: 3 400, 2 910, 1 740, 1 610, 1 580, 1 500, 1 460, 1 390, 1 290, 1 240,
945. 1H NMR (DMSO-d6): 0.95 (s, 3 H, 3 × H-18); 4.85 (s, 2 H, O-CH2-C6H5); 6.58–7.25
(group of sign als, 8 H, arom atic proton s); 12.82 (s, 1 H, C=NOH). 13C NMR (DMSO-d6):
13.82 (C-18); 69.37 (O-CH2-C6H5); 155.16 (C=NOH); 156.32 (C-3); 205.15 (C=O). MS: 389
(4; M+); 149 (93); 91 (76); 57 (91); 43 (100). For C25H27NO3 (389.5) calculated: 77.09% C,
6.99% H, 3.60% N; foun d: 77.17% C, 7.16% H, 4.24% N.
3-Ben zyloxy-17-h ydroxyestra-1,3,5(10)-trien -16-on e Oxim e (3b)
Com poun d 2b (1 g, 2.6 m m ol) was dissolved un der h eatin g in a m ixture of m eth an ol
(20 m l), m eth ylen e ch loride (8 m l) an d 1% aqueous solution of KOH (30 m l). To th e cooled
solution , NaBH4 (0.95 g, 25.1 m m ol) was added portion wise. Th e reaction m ixture was
stirred for 20 m in at room tem perature an d th en refluxed for 40 m in . After coolin g, acetic
acid was added to pH 5 an d th e wh ite precipitate collected an d wash ed th orough ly with wa-
ter (0.98 g, 98%, m .p. 193–195 °C). Recrystallization from m eth an ol afforded an alytically
pure 3b: 0.83 g (83%), m .p. 195–196 °C. IR: 3 600–3 100, 2 920, 1 610, 1 580, 1 500, 1 260,
1 080, 950. 1H NMR (CDCl3): 0.80 (s, 3 H, 3 × H-18); 3.68 (bs, 1 H, OH); 4.20 (s, 1 H, H-17);
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16,17-Secoestrone Derivatives
79
5.13 (s, 2 H, O-CH2-C6H5); 6.72–7.34 (group of sign als, 8 H, arom atic proton s); 8.87 (bs, 1 H,
C=N-OH). 13C NMR (CDCl3): 11.22 (C-18); 69.93 (O-CH2-C6H5); 81.70 (C-17), 156.81 (C-3);
165.59 (C=NOH). MS: 391 (19; M+); 373 (5); 91 (100). For C25H29NO3 (391.5) calculated:
76.70% C, 7.47% H, 3.58% N; foun d: 76.84% C, 7.26% H, 3.73% N.
3-Ben zyloxy-17-oxo-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (4b)
Oxim e 3b (1.0 g, 2.56 m m ol, fin ely groun d an d dried for 3 h at 90 °C) an d 4-m eth yl-
ben zen esulfon yl ch loride (1.53 g, 8.0 m m ol) were dissolved in pyridin e (15 m l). Th e reac-
tion m ixture was kept at room tem perature for 3 h an d th an poured in to an excess of cold
dilute HCl. Th e separeted precipitate of th e crude 4b was collected, wash ed with water an d
dried (0.95 g). Colum n ch rom atograph y on silica gel (70 g, toluen e–eth yl acetate 95 : 5) af-
forded 0.72 g (72%) of pure com poun d 4b, m .p. 137–138 °C. IR: 2 910, 2 220, 1 725, 1 620,
1 580, 1 290, 1 265, 1 020, 760. 1H NMR (CDCl3): 1.18 (s, 3 H, 3 × H-18); 2.95 (d, 2 H, 2 ×
H-15); 5.08 (s, 2 H, O-CH2-C6H5); 6.78–7.35 (group of sign als, 8 H, arom atic proton s); 9.40
(s, 1 H, CHO). 13C NMR (CDCl3): 13.11 (C-18); 69.97 (O-CH2-C6H5); 118.63 (C≡N); 156.94
(C-3); 204.76 (C=O). MS: 373 (19; M+); 91 (100). For C25H27NO2 (373.5) calculated: 80.39% C,
7.29% H, 3.75% N; foun d: 80.38% C, 7.15% H, 3.89% N.
3-Ben zyloxy-17-h ydroxy-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (5b)
Nitrile 4b (1.0 g, 2.7 m m ol) was dissolved un der h eatin g in m eth an ol (35 m l). To th e cooled
solution NaBH4 (0.81 g, 22.2 m m ol) was added portion wise. After stirrin g for 30 m in at
room tem perature an d refluxin g for 20 m in , th e reaction m ixture was diluted with water
(100 m l). Th e wh ite precipitate was filtered off, wash ed with water an d dried, yieldin g 0.98 g
of th e crude 5b. Th e product was purified by ch rom atograph y on a silica gel colum n (100 g,
toluen e–eth yl acetate 2 : 1), wh ereby 0.96 g (96%) of pure 5b, m .p. 135–136 °C, was ob-
tain ed. IR: 3 600–3 200, 2 920, 2 240, 1 610, 1 500, 1 280, 1 240, 1 020. 1H NMR (CDCl3):
0.95 (s, 3 H, 3 × H-18); 2.16 (d, 1 H, OH, J = 13.3); 2.54 (dd, 1 H, H-15a, Jgem = 16.1, J15a,14
=
6.8); 2.68 (dd, 1 H, H-15b, J15b,14 = 7.2); 3.44 (dd, 2 H, H-17, Jgem = 10.0, JH,OH = 12.5); 5.03
(s, 2 H, O-CH2-C6H5); 6.78–7.35 (group of sign als, 8 H, arom atic proton s). 13C NMR
(CDCl3): 15.47 (C-18); 69.87 (O-CH2-C6H5); 70.90 (C-17); 119.94 (C≡N); 156.81 (C-3). MS:
375 (22; M+); 91 (100). For C25H29NO2 (375.5) calculated: 79.96% C, 7.79% H, 3.73% N;
foun d: 79.81% C, 7.92% H, 3.92% N.
3-Hydroxy-17-oxo-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (4a) an d
3,17-Dih ydroxy-16,17-secoestra-1,3,5(10)-trien e-16-n itrile (5a)
To th e solution of th e correspon din g ben zyl eth er 4b an d 5b (1.0 m m ol) in dich loro-
m eth an e–m eth an ol m ixture (1 : 1, 10 m l), 10% Pd/C (0.24 g) was added. Th e suspen sion
was stirred at room tem perature for 12 h , i.e. 48 h un der an atm osph ere of h ydrogen . After
rem ovin g of th e catalyst, th e solven t was evaporated to dryn ess an d th e product isolated by
colum n ch rom atograph y (25 g silica gel, toluen e–eth yl acetate 4 : 1).
Compound 4a: yield 41%, colourless oil. IR: 3 400, 3 300, 2 920, 2 250, 1 720, 1 650, 1 600,
1 505, 1 320, 1 120. 1H NMR (aceton e-d6): 1.18 (s, 3 H, 3 × H-18); 6.62–7.15 (group of sig-
n als, 3 H, arom atic proton s); 8.05 (s, 1 H, OH); 9.45 (s, 1 H, CHO). 13C NMR (aceton e-d6):
13.45 (C-18); 17.87 (C-15); 120.08 (C≡N); 156.23 (C-3); 206.1 (C=O). For C18H21NO2 (283.4)
calculated: 76.28% C, 7.47% H, 4.96% N; foun d: 76.38% C, 7.46% H, 3.93% N.
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Jovanović-Šanta et al.:
Compound 5a: yield 41%, m .p. 198–199 °C. IR: 3 600–3 100, 2 920, 2 250, 1 620, 1 505,
1 230, 1 020. 1H NMR (aceton e-d6): 0.92 (s, 3 H, 3 × H-18); 3.28 (dd, 1 H, H-17a, Jgem = 11.1,
J17a,OH = 5.1); 3.55 (dd, 1 H, H-17b, J17b,OH = 5.1); 6.62–7.12 (group of sign als, 3 H, arom atic
proton s); 8.04 (s, 1 H, OH).13C NMR (aceton e-d6): 15.79 (C-15); 16.44 (C-18); 71.08 (C-17);
120.87 (C≡N); 156.06 (C-3). MS: 343 (68; (M + i-Bu)+); 342 (100; (M + i-Bu–1)+); 303 (17);
286 (87; (M + 1)+); 285 (38; M+); 268 (17; (M + 1 – H2O)+). For C18H23NO2 (285.4) calcu-
lated: 75.75% C, 8.12% H, 4.91% N; foun d: 75.62% C, 8.01% H, 5.08% N.
BIOLOGICAL TESTS
Im m ature Wistar strain fem ale rats (21–23 days old) were ran dom ly divided
in to groups of seven to ten an im als. Th e an im als were treated by subcuta-
n eous in jection on ce a day for 3 days with 0.1 m l of a solution of th e test
com poun d in olive oil, 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 is-
tered am oun ts of com poun ds 4a, 4b, 5a or 5b were 240 µg, wh ereas th e EB
dose was 1.5 µg. Th e an im als were killed on th e fourth day. Th e uteri were
rem oved, dissected free of adh erin g fat an d blotted dry after expulsion of
uterin e fluid an d th e wet weigh t was recorded.
Percen tage of agon ist an d an tagon ist activity in im m ature rat uterin e
weigh t assays was calculated from th e ratio of values recorded in treated
an d con trol an im als th us:
% AGONISM = (C – A) · 100 / (B – A)
% ANTAGONISM = (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 veh icle alon e, EB, test com -
poun d alon e, an d test com poun d plus EB, respectively.
RESULTS AND DISCUSSION
Th e estron e 3-ben zyl eth er 1b (Sch em e 1) was obtain ed in 99% yield (81%
after crystallization ) by treatin g estron e (1a) with ben zyl ch loride in th e
presen ce of potassium carbon ate an d th en con verted in to th e correspon d-
in g 16-oxim in o derivative 2b usin g th e kn own procedure4. In addition to
2b, th e crude reaction m ixture con tain ed a less polar com poun d, presum -
ably th e Z-isom er of 2b. However, all attem pts to isolate th is m in or product
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

16,17-Secoestrone Derivatives
81
failed, probably to its isom erization on silica. Th e reduction of keton e 2b
with sodium boroh ydride in m eth an ol afforded 3-ben zyloxy-17β-h ydroxy-
estra-1,3,5(10)-trien -16-on e oxim e (3b) in a h igh yield. Th e β-con figuration
of OH fun ction at C-17 was proved by NOE experim en t.
O
O
(i)
N
OH
RO
C6H5H2CO
(ii)
2b
1
H
OH
N
OH
In formulae 1,4,5
a, R = H
(v)
b, R = CH2C6H5
C6H5H2CO
(iii)
3b
CHO
C
CH2OH
(iv)
N
C
N
5
RO
RO
4
(i) (CH3)2CHCH2CH2ONO/(CH3)3COK/(CH3)3COH, 89.5%; (ii) NaBH4-1% aq.
KOH/MeOH-CH2Cl2, 98%; (iii) TsCl/pyridine, 72%; (iv) NaBH4/MeOH, 96%;
(v) H2-Pd/C/CH2Cl2-MeOH, 41% 4a, 41% 5a
SCHEME 1
Th e Beckm an n cleavage of oxim in o alcoh ol 3b was carried out un der
sim ilar reaction con dition s as earlier5, yieldin g cyan oaldeh yde 4b in 95%
yield, wh ich was con verted, by sodium boroh ydride reduction , in to th e cor-
respon din g secocyan oalcoh ol 5b, in a h igh yield.
Deprotection of th e 3-h ydroxy fun ction was perform ed by h ydrogen o-
lysis at room tem perature an d low h ydrogen pressure, usin g Pd/C as cata-
lyst, wh ereby h igh yields of 3-h ydroxy-16,17-secoestron e derivatives 4a an d
5a were obtain ed.
Th e estrogen ic an d an tiestrogen ic effects of com poun ds 4a, 4b, 5a an d
5b were tested on experim en tal an im als, usin g th e uterotropic an d an ti-
uterotropic m eth ods6. 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
effects7 presen ted in Table I.
As can be seen from Table I, all th e com poun ds exh ibited an alm ost total
loss of estrogen ic activity an d com poun ds 4b an d 5a even preven ted th e ac-
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

82
Jovanović-Šanta et al.:
tion of en dogen ous estrogen s. On th e oth er h an d, com poun ds 4a, 5a an d
5b partially h in dered th e action of estradiol ben zoate, beh avin g as m oder-
ate an tagon ists.
TABLE I
Agon istic an d an tagon istic effects of com poun ds 4a, 4b, 5a an d 5b
Com poun d
Dose, m g/kg
Agon istic effect, %
An tagon istic effect, %
4a
4b
5a
5b
5
5
5
5
4.13
–10.35
–2.06
0.71
15.96
0.42
21.07
31.47
Furth er derivatization s of com poun ds 5b with an aim of obtain in g com -
poun ds with h igh er an tagon istic action are un der progress.
The authors acknowledge financial support of the Ministry of Science and Technology of the Repub-
lic of Serbia.
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