Synthesis and estrogenic activity screening of some 6,9-disubstituted estradiol derivatives

Article abstract of DOI:10.1135/cccc20050479

Oxidation of estradiol dipropionate (1) with chromium(VI) oxide-3,5-dimethylpyrazole complex yielded 9α-hydroxy-6-oxoestra-1,3,5(10) -triene-3,17β-diyl dipropionate (2) and 6-oxoestra-1,3,5(10)-triene-3, 17β-diyl dipropionate (3). Dehydration of compound 2 with phosphorus(V) oxide or acetic anhydride gave 6-oxoestra-1,3,5(10),9(11)-tetraene-3,17β- diyl dipropionate (5). Reduction of compounds 2 and 5 with sodium borohydride afforded 3,6β,9α-trihydroxyestra-1,3,5(10)-triene-17β-yl propionate (4) and 3,6β-dihydroxyestra-1,3,5(10),9(11)-tetraene-17β-yl propionate (6), respectively. The action of thionyl chloride on compound 2 yielded 6-hydroxyestra-1,3,5(10),6,8-pentaene-3,17β-diyl dipropionate (7). Biological tests in vivo of these compounds showed a moderate antiestrogenic activity of compound 4.

Full text of DOI:10.1135/cccc20050479

6,9-Disubstituted Estradiol Derivatives
479
SYNTHESIS AND ESTROGENIC ACTIVITY SCREENING OF
SOME 6,9-DISUBSTITUTED ESTRADIOL DERIVATIVES
a3
Marija N. SAKAČa1,*, Katarina M. PENOV GAŠIa2, Mirjana POPSAVIN
,
Evgenija A. DJURENDIĆa4, Silvana ANDRIĆ and Radmila M. KOVAČEVIĆ
b1
b2
a
Department of Chemistry, Faculty of Sciences, University of Novi Sad,
21000 Novi Sad, Trg Dositeja Obradovića 3, Serbia and Montenegro;
e-mail: sazvon@eunet.yu, lmg@eunet.yu, mpopsavin@ih.ns.ac.yu, djena@ih.ns.ac.yu
Department of Biology, Faculty of Sciences, University of Novi Sad,
1
2
3
4
b
21000 Novi Sad, Trg Dositeja Obradovića 2, Serbia and Montenegro;
1
2
e-mail: silvana@ib.ns.ac.yu, radmilak@ib.ns.ac.yu
Received Jan uary 11, 2005
Accepted April 3, 2005
Oxidation of estradiol dipropion ate (1) with ch rom ium (VI) oxide-3,5-dim eth ylpyrazole com -
plex yielded 9α-h ydroxy-6-oxoestra-1,3,5(10)-trien e-3,17β-diyl dipropion ate (2) an d
6-oxoestra-1,3,5(10)-trien e-3,17β-diyl dipropion ate (3). Deh ydration of com poun d 2 with
ph osph orus(V) oxide or acetic an h ydride gave 6-oxoestra-1,3,5(10),9(11)-tetraen e-3,17β-diyl
dipropion ate (5). Reduction of com poun ds 2 an d 5 with sodium boroh ydride afforded
3,6β,9α-trih ydroxyestra-1,3,5(10)-trien e-17β-yl propion ate (4) an d 3,6β-dih ydroxyestra-
1,3,5(10),9(11)-tetraen e-17β-yl propion ate (6), respectively. Th e action of th ion yl ch loride
on com poun d 2 yielded 6-h ydroxyestra-1,3,5(10),6,8-pen taen e-3,17β-diyl dipropion ate (7).
Biological tests in vivo of th ese com poun ds sh owed a m oderate an tiestrogen ic activity of
com poun d 4.
Keyw ord s: Steroids; Estradiol derivatives; Equilen in e derivatives; Estrogen ic activity;
An tiestrogen ic activity; Oxidation s.
Breast can cer represen ts on e of th e m ost frequen t can cers in wom en . In
view of th e fin din g th at som e types of breast can cers are estrogen depen d-
en t th eir treatm en t is based on an ti-estrogen s, substan ces com petin g with
en dogen ous estrogen s for th e receptor bin din g sites1. In th e fram e of a
broader project directed towards obtain in g poten tial an ti-estrogen s we h ave
reported th e preparation of a series of estradiole an d estron e derivatives2–8
Som e of th ese com poun ds sh owed a m oderate an tiestrogen ic activity3,5,6
.
.
Th e aim of th is paper was th e syn th esis of th e B-m odified estradiol deriva-
tives an d testin g th eir biological activity on experim en tal an im als.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)
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Sakač et al.:
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 or as film on a NEXUS 670
SP-IR spectrom eter. NMR spectra were taken on a Bruker AC 250E spectrom eter operatin g at
250 MHz (proton ) an d 62.5 MHz (carbon ) an d are reported in ppm (δ-scale) down field from
a tetram eth ylsilan e in tern al stan dard; 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. All reagen ts used were of an alytical grade. All solu-
tion s were dried over an h ydrous sodium sulfate.
9α-Hydroxy-6-oxoestra-1,3,5(10)-trien e-3,17β-diyl Dipropion ate (2) an d
6-Oxoestra-1,3,5(10)-trien e-3,17β-diyl Dipropion ate (3)
To dry m eth ylen e ch loride (160 m l) ch rom ium (VI) oxide (19.3 g, 0.19 m ol) was added an d
th e suspen sion was cooled to 0 °C. To th e cooled suspen sion 3,5-dim eth ylpyrazole (18.3 g,
0.19 m ol) was added at stirrin g, an d estradiol dipropion ate (1; 3.71 g, 9.7 m m ol) 15 m in
later. Th e reaction m ixture was stirred at 0 °C for 5 h , an d th en left at room tem perature for
th e n ext two days. After th at, to th e reaction m ixture previously cooled to 0 °C, 5 M NaOH
(80 m l, 0.4 m ol) was added an d th e con ten t stirred at th e sam e tem perature for 1 h . Th e
m eth ylen e ch loride layer was wash ed sequen tially with 2 M HCl, water an d saturated solu-
tion of NaCl. After dryin g th e solven t was evaporated. Th e brown oily residue (3.28 g) was
ch rom atograph ed on a silica gel colum n (65 g) in toluen e–EtOAc (9:1).
Compound 2. Yield 1.80 g (45%), m .p. 149–150 °C. IR: 3526 (OH); 1760 an d 1730 (C=O,
ester); 1670 (C=O, C-6); 1605 (ArC–C). 1H NMR (CDCl3): 0.82 s, 3 H (3 × H-18); 1.15 t, 3 H,
J = 7.5 (CH3CH2COO at C-3); 1.24 t, 3 H, J = 7.5 (CH3CH2COO at C-17); 1.60 s, 1 H (OH at
C-9); 2.35 q, 2 H, J = 7.5 (CH3CH2COO at C-3); 2.60 q, 2 H, J = 7.5 (CH3CH2COO at C-17);
4.80 t, 1 H, J = 8.2 (H-17); 7.24 dd, 1 H, J2,4 = 2.5, J1,2 = 8.5 (H-2); 7.57 d, 1 H, J1,2 = 8.5
(H-1); 7.67 d, 1 H, J2,4 = 2.5 (H-4). 13C NMR (CDCl3): 8.90 an d 9.14 (2 × CH3CH2COO);
11.08 (C-18); 22.65 (C-15); 27.40 an d 27.55 (2 × CH3CH2COO); 31.97; 32.44; 37.15; 40.95;
42.69; 42.90; 69.02 (C-9); 81.76 (C-17); 120.40, 125.59, 126.99 (Ar C-H); 132.96 an d 144.79
(Ar C); 150.51 (C-3); 172.71 an d 174.47 (2 × CH3CH2COO); 197.21 (CO, C-6). MS, m/z: 414
(M+), 358 (M+ – CH3CHCO), 340 (M+ – H2O – CH3CHCO). For C24H30O6 (414.5) calculated:
69.56% C, 7.25% H; foun d: 69.32% C, 7.45% H.
Compound 3. Yield 0.32 g (10%), m .p. 109–111 °C. IR: 3080 (ArC–H); 1766 an d 1734
(C=O, ester); 1688 (C=O, C-6); 1616 (ArC–C); 1494, 1210, 1088, 900. 1H NMR (CDCl3):
0.84 s, 3 H (3 × H-18); 1.15 t, 3 H, J = 7.5 (CH3CH2COO at C-3); 1.27 t, 3 H, J = 7.5
(CH3CH2COO at C-17); 2.32 q, 2 H, J = 7.5 (CH3CH2COO at C-3); 2.59 q, 2 H, J = 7.5
(CH3CH2COO at C-17); 4.73 t, 1 H, J = 8.2 (H-17); 7.25 dd, 1 H, J2,4 = 2.5, J1,2 = 8.5 (H-2);
7.44 d, 1 H, J1,2 = 8.5 (H-1); 7.73 d, 1 H, J2,4 = 2.5 (H-4). 13C NMR (CDCl3): 8.96 an d 9.17
(2 × CH3CH2COO); 11.79 (C-18); 22.89; 25.22; 27.32; 27.57; 27.68; 36.35; 39.42; 42.88;
43.73; 49.63; 81.81 (C-17); 119.84, 126.60 an d 126.91 (Ar C-H); 133.50 an d 144.08 (Ar C);
149.29 (C-3); 172.79 an d 174.37 (2 × CH3CH2COO); 196.76 (CO, C-6). MS, m/z: 398 (M+),
342 (M+ – CH3CHCO), 286 (M+ – 2 CH3CHCO). For C24H30O5 (398.5) calculated: 72.35% C,
7.53% H; foun d: 72.47% C, 7.30% H.
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6,9-Disubstituted Estradiol Derivatives
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3,6β,9α-Trih ydroxyestra-1,3,5(10)-trien e-17β-yl Propion ate (4)
To th e solution of com poun d 2 (0.1 g, 0.24 m m ol) in m eth an ol (8 m l) sodium boroh ydride
(0.1 g, 2.65 m m ol) was added. Th e reaction m ixture was stirred at room tem perature for 1 h
an d th en poured in to water (10 m l) an d extracted with eth er. After dryin g of th e extract an d
rem oval of th e solven t, th e crude m ixture was separated by flash ch rom atograph y (ben zen e–
EtOAc, 1:1). Th e result was com poun d 4 (0.073 g, 83.4%) in th e form of wh ite crystals, m .p.
177 °C (h exan e–aceton e). IR: 3404 (OH); 1713 (C=O); 1606 (ArC–C); 1213 (C–O); 755.
1H NMR (DMSO-d6): 0.73 s, 3 H (3 × H-18); 1.02 t, 3 H, J = 7.5 (CH3CH2COO); 1.2–1.85
group of sign als, 10 H (H-16, H-12, H-11β, H-8, H-7); 2.0 m , 1 H (H-14); 2.16–2.35 group of
sign als, 3 H (H-11α, CH3CH2COO); 4.24 s, 1 H (OH at C-9); 4.44 m , 1 H (H-6); 4.61 t, 1 H,
J17,16a = 8.2, J17,16b = 8.5 (H-17); 5.20 d, 1 H, J = 7.8 (OH at C-6); 6.57 dd, 1 H, J2,4 = 2.4,
J1,2 = 8.5 (H-2); 6.91 d, 1 H, J2,4 = 2.4 (H-4); 7.20 d, 1 H, J1,2 = 8.5 (H-1); 9.23 bs, 1 H (OH
at C-3). 13C NMR (DMSO-d6): 9.38 (CH3CH2COO); 11.37 (C-18); 22.83 (C-15); 27.29
(CH3CH2COO); 27.43 (C-16); 30.98 (C-7); 32.33 (C-11); 32.73 (C-12); 40.34 (C-8); 41.81
(C-14); 42.68 (C-13); 68.34 (C-6); 68.84 (C-9); 81.95 (C-17); 113.42 (C-4); 114.00 (C-2);
126.34 (C-1); 133.44 (C-10); 142.34 (C-5); 156.34 (C-3); 173.89 (CH3CH2COO). MS, m/z:
342 (M+ – H2O), 324 (M+ – 2 H2O), 235, 208. For C21H28O5 (360.4) calculated: 69.97% C,
7.83% H; foun d: 69.72% C, 7.78% H.
6-Oxoestra-1,3,5(10),9(11)-tetraen e-3,17β-diyl Dipropion ate (5)
Method A. To th e suspen sion of ph osph orus(V) oxide (0.45 g, 3 m m ol) in ben zen e (30 m l)
com poun d 2 (0.11 g, 0.26 m m ol) was added, an d th e reaction m ixture stirred at room tem -
perature for 2 h . After th at th e m ixture was poured to water (30 m l), th e layers were sepa-
rated an d th e organ ic layer was wash ed with a saturated solution of NaHCO3, dried, an d th e
solven t rem oved. Th e resultin g crude product (0.09 g, 87.4%) in th e form of ligh t-yellow
crystals was recrystallized from h exan e, affordin g pure com poun d 5, m .p. 102–103 °C.
Method B. A solution of com poun d 2 (0.10 g, 0.24 m m ol) in acetic an h ydride (5 m l) was
refluxed for 20 h . Th en th e reaction m ixture was cooled to room tem perature an d poured
in to water (50 m l). Th e aqueous solution was n eutralized by carefully addin g solid NaHCO3
an d th e m ixture extracted with CH2Cl2 (3 × 20 m l). Th e com bin ed organ ic extracts were
wash ed with water, dried, an d th e solven t rem oved. Th e raw oily product was purified by
flash ch rom atograph y (toluen e–EtOAc, 12:1), affordin g pure com poun d 5 (0.06 g, 63%).
IR: 1770 an d 1725 (C=O, ester); 1685 (C=O); 1200, 1155. 1H NMR (CDCl3): 0.85 s, 3 H (3 ×
H-18); 1.15 t, 3 H, J = 7.5 (CH3CH2COO at C-3); 1.25 t, 3 H, J = 7.5 (CH3CH2COO at C-17);
2.35 q, 2 H, J = 7.5 (CH3CH2COO at C-3); 2.60 q, 2 H, J = 7.5 (CH3CH2COO at C-17); 4.85 t,
1 H, J = 7.5 (H-17); 6.45 t, 1 H, J = 5.1 (H-11); 7.25 dd, 1 H, J1,2 = 8.5, J2,4 = 2.5 (H-2);
7.70 m , 2 H (H-1, H-4). 13C NMR (CDCl3): 8.99 an d 9.21 (2 × CH3CH2COO); 11.81 (C-18);
23.67; 27.46; 27.63; 27.70 (CH3CH2COO); 38.15; 39.30; 41.30; 43.25; 47.92; 81.87 (C-17);
119.27 (C-11); 125.31, 125.95 an d 127.29 (C-1, C-2 an d C-4); 131.54 (C-9); 132.71 an d
138.24 (C-5 an d C-10); 150.06 (C-3); 172.75 an d 174.40 (2 × CH3CH2COO); 196.50 (C-6).
MS, m/z: 396 (M+), 341, 340, 266. For C24H28O5 (396.5) calculated: 72.70% C, 7.12% H;
foun d: 72.55% C, 6.98% H.
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Sakač et al.:
3,6β-Dih ydroxyestra-1,3,5(10),9(11)-tetraen e-17β-yl Propion ate (6)
To a solution of com poun d 5 (0.10 g, 0.25 m m ol) in m eth an ol (10 m l), sodium boroh ydride
(0.10 g, 2.65 m m ol) was added. Th e reaction m ixture was stirred at room tem perature for 1 h
an d th en poured in to water (70 m l). Th e wh ite precipitate was filtered, wash ed with water,
an d dried, to give 0.078 g (91%) of 6, wh ich after recrystallization from h exan e–aceton e
system m elted at 164–165 °C. IR: 3544 an d 3438 (OH); 3024 (ArC–H); 1731 (C=O); 1606
(ArC–C); 1242 (C–O); 874 an d 813 (C=CH). 1H NMR (CD3COCD3): 0.82 s, 3 H (3 × H-18);
1.09 t, 3 H, J = 7.8 (CH3CH2COO); 2.34 q, 2 H, J = 7.8 (CH3CH2COO); 4.36 d, 1 H, J = 7.2
(OH at C-6); 4.62–4.80 m , 2 H (H-6, H-17); 6.07 bd, 1 H, J = 5.0 (H-11); 6.67 dd, 1 H, J1,2
=
8.7, J2,4 = 2.5 (H-2); 7.46 d, 1 H, J2,4 = 2.5 (H-4); 7.46 d, 1 H, J1,2 = 8.5 (H-1); 8.44 s, 1 H (OH
at C-3). 13C NMR (CDCl3): 9.25 (CH3CH2COO); 12.00 (C-18); 23.95; 27.54; 27.80; 37.07;
38.19; 39.08; 41.13; 46.85; 68.99 (C-6); 82.49 (C-17); 112.54 an d 115.36 (Ar CH); 118.22
(C-11); 125.21 (Ar CH); 126.58 (C-9); 133.86 an d 140.10 (Ar C); 155.33 (C-3); 174.74
(CH3CH2COO). MS, m/z: 342 (M+), 324 (M+ – H2O), 235, 208. For C21H26O4 (342.4) calcu-
lated: 73.66% C, 7.65% H; foun d: 73.31% C, 7.52% H.
6-Hydroxyestra-1,3,5(10),6,8-pen taen e-3,17β-diyl Dipropion ate (7)
Com poun d 2 (0.95 g, 2.3 m m ol) was dissolved in dry pyridin e (5 m l) an d th en a solution
of th ion yl ch loride (0.7 m l, 9.2 m m ol) in pyridin e (3 m l) was added wh ile stirrin g. After
stirrin g at room tem perature for 10 m in , th e reaction m ixture was poured to water (50 m l)
an d extracted with ben zen e (4 × 20 m l). Th e extracts were dried, ben zen e evaporated, an d
th e pyridin e residues were rem oved by co-evaporation with ben zen e. Th e obtain ed yellow
oil was purified by flash ch rom atograph y to yield com poun d 7 (0.3 g, 34%), m .p. 201 °C.
IR: 3450 (OH); 1760 an d 1715 (C=O, ester); 1610 (ArC–C); 1185. 1H NMR (CDCl3): 0.85 s, 3 H
(3 × H-18); 1.15 t, 3 H, J = 7.5 (CH3CH2COO at C-3); 1.25 t, 3 H, J = 7.5 (CH3CH2COO at
C-17); 2.40 q, 2 H, J = 7.5 (CH3CH2COO at C-3); 2.65 q, 2 H, J = 7.5 (CH3CH2COO at C-17);
4.90 t, 1 H, J = 7.5 (H-17); 5.60 s, 1 H (OH); 6.50 s, 1 H (H-7); 7.25 dd, 1 H, J1,2 = 8.5, J2,4
=
2.5 (H-2); 7.85 m , 2 H (H-1, H-4). 13C NMR (CDCl3): 9.21 an d 9.37 (2 × CH3CH2COO); 11.33
(C-18); 23.61; 23.87; 27.91 an d 28.34 (2 × CH3CH2COO); 34.19; 42.62; 46.37; 81.65 (C-17);
108.05, 113.60, 121.34, 122.50, 123.67, 124.82, 131.06 an d 135.54 (Ar C an d CH); 147.14
(C-6); 149.75 (C-3); 173.75 an d 174.87 (2 × CH3CH2COO). MS, m/z: 396 (M+), 341, 340. For
C
24H28O5 (396.5) calculated: 72.70% C, 7.12% H; foun d: 72.44% C, 7.15% H.
11-{[3,17β-Bis(propion yloxy)estra-1,3,5(10),6,8-pen taen -6-yl]oxy}-
N-butyl-N-m eth ylun decan am ide (8)
11-Brom o-N-butyl-N-m eth ylun decan am ide8 (0.93 g, 2.8 m m ol) an d com poun d 7 (0.57 g,
1.4 m m ol) were dissolved in m eth ylen e ch loride (13 m l), an d a solution of NaOH (0.085 g,
2.1 m m ol) an d tetrabutylam m on ium brom ide (0.45 g, 1.4 m m ol) in water (13 m l) was
added. Th e two-ph ase system was stirred vigorously at room tem perature for 10 h . Th e or-
gan ic layer was separated, wash ed with water un til n eutral, dried an d evaporated to dryn ess.
Th e dark brown oily residue was ch rom atograph ed on a silica gel colum n (50 g, petroleum
eth er–aceton e, 9:1), yieldin g 0.215 g (23%) of pure com poun d 8 as a colorless oil. IR: 2940,
2867 (C–H); 1768 an d 1742 (C=O, ester); 1650 (NC=O); 1608 (ArC–C); 1188 (C–O). 1H NMR
(CDCl3): 0.77 s, 3 H (3 × H-18′); 0.95 m , 3 H (CH3 from Bu); 1.19 t, 3 H, J = 7.6
(CH3CH2COO at C-3′); 2.39 q, 2 H, J = 7.6 (CH3CH2COO at C-3′); 2.67 q, 2 H, J = 7.5
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6,9-Disubstituted Estradiol Derivatives
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(CH3CH2COO at C-17′); 2.91 an d 2.97 2 s, 3 H (N-CH3); 3.25 an d 3.36 2 t, 2 H, J = 7.3
(CH2NCO); 4.07 t, 2 H, J = 6.5 (OCH2); 4.90 t, 1 H, J = 7.2 (H-17′); 6.54 s, 1 H (H-7′);
7.24 dd, 1 H, J1′,2′ = 9.1, J2′,4′ = 2.5 (H-2′); 7.89 d, 1 H, J1′,2′ = 9.1 (H-1′); 7.92 d, 1 H, J2′,4′
=
2.5 (H-4′). 13C NMR (CDCl3): 9.19 an d 9.36 (2 × CH3CH2COO); 11.36 (C-18′); 13.94 (CH3
from Bu); 20.02; 20.14; 23.76; 23.90; 25.19; 25.57; 26.26; 27.86; 27.90; 28.37; 29.35; 29.48;
29.54; 29.61; 30.71; 33.07; 33.40; 33.73; 34.22; 35.38; 42.76; 46.75; 47.47; 49.83; 68.19
(OCH2); 81.63 (C-17′); 104.20, 113.68, 121.48, 121.99, 124.62, 125.06, 130.98 an d 135.52
(Ar C an d CH); 147.37 (C-6′); 153.22 (C-3′); 172.94 an d 173.06 (NC=O from two con form -
ers); 173.40 an d 174.71 (2 × CH3CH2COO).
Uterotroph ic an d An tiuterotroph ic Assays
Th e m eth ods used were sim ilar to th ose described in th e literature9,10. Im m ature Wistar
strain fem ale rats (21–23 days old) were ran dom ly divided in to groups of six to eigh t an i-
m als each . Th e an im als were treated by subcutan eous in jection on ce a day for th ree con sec-
utive 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 diben 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 0.5, 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 24 h after th e
last in jection . Th e adh erin g fat was rem oved from uteri 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 were
calculated from th e ratio of values recorded for 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.
RESULTS AND DISCUSION
Th e startin g com poun d in th e syn th esis of B-m odified estradiol derivatives
was estradiol dipropion ate (1; Sch em e 1), wh ich in th e first step was oxi-
dized with th e CrO3–3,5-dim eth ylpyrazole com plex11. Th is gave 9α-h ydroxy-
6-oxoestra-1,3,5(10)-trien e-3,17β-diyl dipropion ate (2) as th e m ain reaction
product (45%) an d 6-oxoestra-1,3,5(10)-trien e-3,17β-diyl dipropion ate (3)
in a yield of 10%. With th e aim of im provin g th e yield of com poun d 2 it
was attem pted to use som e oth er oxidizin g agen ts such as pyridin ium
ch loroch rom ate12, CrO3–pyridin e com plex13 an d tert-butyl ch rom ate14. How-
ever, eith er a large portion of th e startin g com poun d rem ain ed or a com -
plex reaction m ixture was isolated. Com poun d 2 was reduced with NaBH4
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Sakač et al.:
with a sim ultan eous h ydrolysis of th e C-3 ester fun ction , yieldin g 3,6β,9α-
trih ydroxyestra-1,3,5(10)-trien e-17β-yl propion ate (4) in a yield of 83.4%.
On th e oth er h an d, deh ydration of com poun d 2 with P2O5 in ben zen e at
room tem perature durin g 2 h or by h eatin g with Ac2O at reflux for 20 h ,
afforded 6-oxoestra-1,3,5(10),9(11)-tetraen e-3,17β-diyl dipropion ate (5) in a
yield of 87.4 an d 63.0%, respectively. Reduction of th e carbon yl group of
com poun d 5 with NaBH4, wh ich was also accom pan ied by th e h ydrolysis of
th e C-3 ester fun ction , gave 3,6β-dih ydroxyestra-1,3,5(10),9(11)-tetraen e-
17β-yl propion ate (6) in a yield of 91%.
OCOEt
OCOEt
OCOEt
(i)
OH
O
EtCOO
EtCOO
EtCOO
(iv)
2
O
1
3
(ii)
(iii)
OCOEt
OCOEt
OCOEt
OH
OH
HO
EtCOO
EtCOO
4
O
OH
7
5
(ii)
(v)
OCOEt
OCOEt
HO
EtCOO
OH
6
OCH2(CH2)9CONBuMe
8
(i) CrO3-3,5-dimethylpyrazole, CH2Cl2; (ii) NaBH4, MeOH; (iii) P2O5, benzene or Ac2O; (iv) SOCl2, pyridine;
(v) 11-bromo-N-butyl-N-methylundecanamide, tetrabutylammonium bromide, NaOH, CH2Cl2, H2O
SCHEME 1
A stereoch em ical assign m en t of th e h ydroxy groups at C-6 in both com -
poun ds 4 an d 6 was resolved by NOE experim en ts. Th e ball-stick m odel of
both com poun ds in dicates to a possible NOE between th e OH at C-6 an d
H-8, sin ce th e distan ce between th e β-orien ted h ydroxy group an d th e H-8
is less th en 3 Å. In deed, a 2.4% NOE en h an cem en t was observed for th e sig-
n al of H-8 (1.58 ppm ) wh en th e OH (5.20 ppm ) sign al of 4 was irradiated.
Sim ilarly, a stron g NOE (23%) was observed for th e H-8 (2.18 ppm ), after an
irradiation of th e OH (4.36 ppm ) sign al in 6. Sin ce both alcoh ols 4 an d 6
h ave th e sam e con figuration at C-6, it is clear th at th e h ydride delivery dur-
in g th e reduction of both keto groups occurs from th e α-side.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

6,9-Disubstituted Estradiol Derivatives
485
Deh ydration of com poun d 2 with SOCl2, followed by en olization of th e
C-6 keto group, yielded 6-h ydroxy-17α-dih ydroequilen in e derivative 7.
Com poun d 8 was obtain ed by alkylation of th e h ydroxy group of com -
poun d 7 with 11-brom o-N-butyl-N-m eth ylun decan am ide8 (obtain ed from
11-brom oun decan oic acid an d butyl(m eth yl)am in e via correspon din g acyl
ch loride) carried out in th e two-ph ase CH2Cl2–H2O system un der alkalin e
con dition s, usin g tetrabutylam m on ium brom ide as a ph ase-tran sfer catalyst.
Studies of estrogen ic activity by uterotroph ic m eth od9 en com passed com -
poun ds 2–4 an d 6–8, wh ereas com poun ds 2, 4, 6 an d 8 were also tested for
an tiestrogen ic activity usin g an tiuterotroph ic m eth od10. Results are pre-
sen ted in Table I.
It is eviden t from th e table th at com poun d 4 (3,6β,9α-trih ydroxy deriva-
tive) exh ibited th e h igh est percen tage of an tagon ism (49.46%). Com poun d
2 (6-oxo-9α-h ydroxy derivative) in th e lower dose sh owed certain an tago-
TABLE I
Agon istic an d an tagon istic effects of tested com poun ds in im m ature rat uterin e weigh t test
Com -
poun d
Dose
m g/kg
Agon istic effect
%, m ean ± SEM
An tagon istic effect
%, m ean ± SEM
n
n
2
3
4
0.5
5
25
8
8
8
37.68 ± 3.46a
52.99 ± 3.27a
63.19 ± 4.67a
103.42 ± 4.13a
106.46 ± 6.46a
108.57 ± 8.17a
6
6
6
21.79 ± 6.91
11.89 ± 10.88
9.64 ± 7.34
0.5
5
25
8
8
8
–
–
–
0.5
5
15
7
6
7
1.72 ± 2.78
26.36 ± 2.43
40.87 ± 4.83a
–
8
6
48.97 ± 3.44a
49.46 ± 5.47a
6
7
0.5
15
7
6
5.35 ± 4.51
–
73.38 ± 5.66a
7
3.22 ± 5.07
0.5
5
25
8
8
7
56.08 ± 4.59a
75.70 ± 5.52a
106.75 ± 6.91a
–
–
–
8
0.5
5
25
8
6
8
3.02 ± 2.05
13.93 ± 3.76
37.25 ± 2.37a
–
–
8
7
–5.65 ± 7.02
Tam oxifen
5
25
7
8
39.78 ± 3.13
39.36 ± 4.09
–
62.80 ± 2.13
a
Sign ifican ce: p < 0.05 vs correspon din g con trol (Man n –Wh itn ey n on -param etric test).
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

486
Sakač et al.:
n istic activity (21.79%). In view of th e fact th at com poun d 8 sh owed a n eg-
ligible estrogen ic activity it can be supposed th at th e side ch ain at C-6 as-
sum es such a con form ation th at th e bin din g of th e ph en ol OH group at C-3
to th e correspon din g receptor sites is blocked. In our experim en tal con di-
tion , com poun d 4 tested in th e sam e experim en ts with tam oxifen sh owed
sign ifican t an tagon istic effect. Alth ough applied doses of com poun d 4 an d
tam oxifen were differen t (15 vs 25 m g/kg), both agon istic (40.87 ± 4.83 vs
39.36 ± 4.09) as well as an tagon istic (49.46 ± 5.47 vs 62.80 ± 2.13) effects
were com parable with tam oxifen . Also, on th e basic of th e results of th e an ti-
uterotroph ic test it could be con cluded th at th e α-orien ted h ydroxy group
at th e C-9 position (com poun ds 2 an d 4) in creased an tagon istic effect.
This research was supported by the Ministry of Science, Technologies, and Development of the
Republic of Serbia (Grant No. 1896).
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