Synthesis and in vivo evaluation of 11-substituted estradiol derivatives as anti-implantation agents

Article abstract of DOI:10.1016/j.bmcl.2008.05.093

Synthesis of 11-substituted estradiol derivatives (12-17) has been carried out by the Grignard reaction with alkyl, allyl, and benzyl halides on 17β-hydroxy-3-methoxy-11-oxo-estra-1,3,5(10),8(9)-tetraene (10). The novel compounds (10 and 12-17) were evalu

Full text of DOI:10.1016/j.bmcl.2008.05.093

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4102–4105
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vivo evaluation of 11-substituted estradiol derivatives as
anti-implantation agents q
a
a
b
Indra Dwivedy a, Atul Gupta a, , Arvinder Grover , Vandana Srivastava , Man Mohan Singh ,
*
Suprabhat Ray a
a Medicinal and Process Chemistry Division, Central Drug Research Institute, PO Box 173, Lucknow 226001, India
b Endocrinology Division, Central Drug Research Institute, PO Box 173, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of 11-substituted estradiol derivatives (12–17) has been carried out by the Grignard reaction
with alkyl, allyl, and benzyl halides on 17b-hydroxy-3-methoxy-11-oxo-estra-1,3,5(10),8(9)-tetraene
(10). The novel compounds (10 and 12–17) were evaluated for their preliminary post-coital contraceptive
(anti-implantation) activity in Sprague–Dawley rats. The tested compounds were administered orally and
showed significant anti-implantation activity. Compound 13 is the most potent compound in the series
Received 8 March 2008
Revised 27 April 2008
Accepted 23 May 2008
Available online 29 May 2008
which showed 100% contraceptive efficacy at 1.25 mg kgꢀ1
.
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Estrogen receptor
Estrogen agonist
Estrogen antagonists
Antifertility
Anti-implantation
Steroids
Estrogens and progesterone are the hormones which are in-
volved in the development of secondary sexual characteristics in
female body. In reproductive cycle, the release of gonadotrophin-
releasing hormone (GnRH) from pituitary triggered by low level
of estrogen mainly estradiol (1) and progesterone (2) effects ovula-
tion of a mature ovum from ovary (Fig. 1).1 After ovulation, there is
an increase in the level of estradiol (1) followed by that of proges-
terone (2), which stops further ovulation through a negative feed-
back mechanism. A critical estrogen–progesterone balance is
necessary for implantation of the blastocyst and its subsequent
development. Any alteration in this balance may lead to the termi-
nation of pregnancy. Steroidal pills which contain a combination of
estrogen and progestagen create a pseudo-pregnancy situation by
preventing ovulation.2 However, such pills have some serious side
effects such as nausea, pigmentation, breast tenderness and weight
gain, due to creation of a state of pseudo-pregnancy.3
failure. Since this phenomenon happens at the peripheral end of
the reproductive cycle, it does not create any disturbance in the cy-
cle and is therefore devoid of pregnancy associated side effects
(Fig. 2).
Several modifications have been made in the estradiol (1) and
progesterone (2) with a view to get an ideal fertility regulating
agent (contraceptive) with balanced agonistic and antagonistic
activities.4,5 Amongst various modifications, substitution at posi-
tion 11 of an estrane and progestin led to the development of
two important compounds, 3 and 4 (Fig. 1).
Our research program aimed for development of estrogen
receptor modulators for fertility regulation and treatment of other
estrogen dependent disorders, led to the development of first and
only non-steroidal oral contraceptive, Ormeloxifene (centchroman,
5).6 Ormeloxifene (5), is a racemate mixture and marketed under
the trade name of Saheli. It is a weekly pill and acts as an anti-
implantation agent based on its antiestrogenic property.7 The basic
and significant difference between non-steroidal anti-implantation
agents and steroidal pills (combination of estrogen and progest-
agen) is in their mechanism of action.
In our continuous efforts, we have designed and synthesized
11-substituted estradiol derivatives (12–17) as novel anti-implan-
tation agents. The synthesized compounds were evaluated for their
preliminary anti-implantation activity in Sprague–Dawley rat
model.8 The targeted compounds were expected to be orally active
as desired from an ideal contraceptive (a fertility regulating agent).
An alternate approach to the contraception is through the use of
antiestrogens. In reproductive cycle, the level of estrogen rises two
times. The first rise comes after the ovulation as mentioned be-
fore.1 The second rise comes at the time of implantation of blasto-
cyst. The suppression of this estrogen level causes implantation
q
CDRI Communication No. 5322.
* Corresponding author at present address: University du Quebec à Trois-Rivieres,
C.P. 500, Trois-Rivieres, Que., Canada G9A 5H7. Tel.: +91 522 2629558.
E-mail addresses: atisky2001@yahoo.co.in, atul_gupta04@yahoo.co.in (A. Gupta).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.093

I. Dwivedy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4102–4105
4103
O
OH
N
OH
HO
O
HO
O
1
2
3, RU 3941
N
N
OH
O
H3CO
O
5
4, RU 38,486
Figure 1. Some steroidal and non-steroidal fertility regulating agents.
OR
The synthesis of targeted compounds was started from 17b-hy-
droxy-3-methoxy-1,3,5(10)-estratriene 17-acetate (6) (Scheme 1).
"R
R'
The CrO3 oxidation of 6 using reported procedure, gave 9
a,17b-
dihydroxy-3-methoxy-11-oxo-estra-1,3,5(10)-triene 17-acetate
(8) in 50 % yield along with 17b-hydroxy-3-methoxy-9-oxo-9,11-
secoestra-1,3,5(10)-triene-11- oic acid 17-acetate (7) and 17b-hy-
droxy-3-methoxy-6-oxo-estra-1,3,5(10)-triene 17-acetate (9).9
Compound 8, on treatment with perchloric acid in methanol at re-
flux temperature for 1 h, yielded 17b-hydroxy-3-methoxy-11-oxo-
estra-1,3,5(10),8(9)-tetraene (10) in 76 % yield. Grignard reaction
H3CO
18
Figure 2. Designed prototype.
OCOCH
3
H CO
3
6
a
OCOCH
3
OCOCH
O
3
OCOCH
3
O
O
HO
OH
+
+
H CO
3
H CO
3
H CO
3
O
7
8
9
b
OR
OH
HO
R'
O
c
H CO
3
H CO
3
11
10
Scheme 1. Reagents and conditions: (a) CrO3, glacial acetic acid, room temperature (RT); (b) Perchloric acid, MeOH, reflux; (c) Methyl/ethyl/allyl/benzyl halide, Mg, dry Et2O–
THF, room temperature (RT).

4104
I. Dwivedy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4102–4105
was made on compound 10 with alkyl, allyl, and benzyl magne-
sium halides in dry Et2O/THF mixture at room temperature (RT),
and gave the desired product 11 in 60–76% yields.10
tion activity when administered orally in Sprague–Dawley rat
model. The structure–activity relationship (SAR) of these com-
pounds showed that moderately sized and flexible substituents
like ethyl and allyl groups find better adjustment in the ligand-
binding domain (LBD) of the estrogen receptor. This is in agree-
ment with the reported fact in the literature that small to moderate
sized substituents at the 11-position of estrogens are well toler-
ated by the estrogen receptor.11 The ethyl-substituted derivative,
13, is the most potent compound in the series which showed
100% contraceptive efficacy at 1.25 mg kgꢀ1 in rat model. However,
substitution with benzyl group brought a drop in the activity pos-
sibly due to the presence of a bulky rigid ring system (benzene
ring) as compared to allyl group. Further, the low order of anti-
implantation activity of compounds 16 and 17 showed the signifi-
cance of the hydroxyl group at 11 and 17 positions of the steroidal
Furthermore, to evaluate the significance of hydroxyl group at
position 11 of the steroidal nucleus in these compounds, com-
pound 14 was converted to 11-allyl-17b-hydroxy-3-methoxy-
estra-1,3,5(10),8(9),11-pentaene (17) (Scheme 2). For the synthesis
of compound 17, 14 was transformed to the corresponding mono
acetate (16, 17b-acetate) with acetic anhydride in pyridine which
was dehydrated with p-toluenesulfonic acid in benzene at reflux to
give 17 in 35% yield. The synthesized compounds were character-
ized by the use of IR, NMR, mass spectroscopy, and their elemental
analysis.9
Biological activity of the compounds (10 and 12–17) is given in
the Table 1. The novel compounds showed potent anti-implanta-
OH
OH
OCCH3
OH
a
H3CO
H3CO
14
16
OH
H3CO
17
Scheme 2. Reagents and conditions: (a) acetic anhydride, pyridine, room temperature (RT); (b) p-Toluenesulfonic acid, dry benzene, reflux.
Table 1
Anti-implantation activity of compounds
Compound
R
R0
R00
Dose (mg kgꢀ1
body weight/
,
Route
Schedule
No. of
implantation sites
Anti-implantation
activity (%)
(day of treatment)b
No. of animals)a,c
Control
Centchroman
Centchroman
—
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
1–5
10/10
0/10
0/10
0/10
0/10
10/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
5/10
0/10
0/10
0/10
4/10
4/10
0/10
0/10
0/10
All rats pregnant
10.0
1.00
10.0
5.0
2.0
10.0
5.0
2.0
10.0
5.0
100
100
100
100
Inactive
100
100
100
100
100
100
100
50
10
10
10
12
12
12
13
13
13
13
13
14
14
14
14
14
15
16
17
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C@O at C11
C@O at C11
C@O at C11
CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
—
—
—
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
2.5
1.25
1.00
10.0
5.0
2.0
1.5
CH2CH3
CH2@CHCH2
CH2@CHCH2
CH2@CHCH2
CH2@CHCH2
CH2@CHCH2
CH2C6H5
100
100
100
60
1.0
60
H
OH
OH
10.0
10.0
10.0
100
100
100
CH3CO
H
CH2@CHCH2
CH2@CHCH2
Double bond (C11–C12)
Compounds described in the table are based on structure 18 (Fig. 2).
a
Five animals were used for each dose test.
For schedule of treatment, day 1 was counted from day 1 of the pregnancy.
Control rats did not receive any drug, had a mean of 10 implantation sites (visibly thickened uteri).
b
c

I. Dwivedy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4102–4105
4105
acid (2 ml) was refluxed for 1 h. Methanol was removed under vacuum, residue
was dissolved in ethyl acetate and extracted with water. The organic layer was
separated, dried over anhydrous sodium sulfate, and concentrated to oily
residue which gave compound 10 (2.5 g, 76%) after crystallization with
benzene–hexane.
nucleus which seemed to be essential for the biological activity of
these novel compounds.
In conclusions, the newly synthesized 11-substituted estradiol
derivatives (12–17) showed significant oral anti-implantation
activity in Sprague–Dawley rat model. As desired, the target com-
pounds are orally active. Minimum effective dose of the tested
compounds was determined. The potent biological activity (anti-
implantation activity) of these novel compounds showed success-
ful drug design. Further, investigation of these estradiol derivatives
would be helpful in designing of the compounds for fertility regu-
lation as anti-implantation agents as well as estrogen receptor
modulators.
Yield: 76%; mp 140–142 °C; IR (cmꢀ1): 1650 and 1600 (aromatic); mass: (M+)
298; 1H NMR (d, CDCl3): 1.10 (s, 3H, CH3), 1.86–2.05 (m, 2H, CH2), 3.75 (s, 3H,
OCH3), 4.15 (t, 1H, CH), 6.40–6.60 (m, 2H, ArH), 7.80–8.00 (d, 1H, J = 8.00 Hz,
ArH); 13C NMR (d, CDCl3): 197, 159, 158, 137, 129, 128, 124, 113, 111, 80, 55,
49, 47, 47, 33, 30, 29, 28, 20; Anal. Calcd for C19H22O3; C, 76.49; H, 7.43. Found:
C, 76.30; H, 7.40.
Typical procedures for the synthesis of 11-allyl-11,17b-dihydroxy-3-methoxy-
estra-1,3,5(10),8(9)-tetraene (14): Compound 10 (1.0 g, 0.003 mol) in dry
tetrahydrofuran (THF) was added dropwise to
a stirred mixture of allyl
magnesium bromide (0.017 mol) in dry ether (Et2O) under inert atmosphere.
The reaction mixture was further stirred for2 h. On completion of the reaction,
reaction mixture was decomposed with water, extracted with ether, dried over
anhydrous sodium sulfate, and concentrated at room temperature. The
resulting oil (0.8 g, 74%) was filtered through a column of neutral alumina.
The eluted fractions were concentrated under vacuum to give pure compound
14.
Acknowledgments
Atul Gupta thanks CSIR (India) for Senior Research Fellowship.
The authors thank Miss Mohini Chhabra for efficient technical
assistance and the Ministry of Health and Family Welfare, Govern-
ment of India, for financial support.
Yield: 74 %; mp. Oil; IR (cmꢀ1): 1620 and 1500 (aromatic); mass: (M+) 340,
(M+ꢀH2O) 322, (M+ꢀallyl) 299, (M+ꢀallyl+H2O) 281; 1H NMR (d, CDCl3): 1.15
and 1.17 (2s, 3H, CH3), 2.10 (m, 2H, CH2ACH@CH2), 2.80 (br s, 2H, CH2), 3.75 (s,
3H, OCH3), 4.21 (t, 1H, CH), 4.80–5.10 (m, 3H, CH@CH2), 6.40–6.60 (m, 2H,
ArH), 7.80–8.00 (d, 1H, J = 8.00 Hz, CH); Anal. Calcd for C22H28O3; C, 77.61; H,
8.28. Found: C, 77.41; H, 8.18.
References and notes
Synthesis of 11-allyl-11,17b-dihydroxy-3-methoxy-estra-1,3,5(10),8(9)-tetraene
17-acetate (16):
A mixture of compound 14 (0.1 g, 0.0003 mol) in dry
1. (a) Ray, S.; Dwivedy, I. Adv. Drug Res. 1997, 29, 171; (b) Lednicer, D. The
Chemical Control of Fertility; Marcel Dekker: New Yark, 1969.
2. (a) Goldzieher, J. W. Perspect. Biol. Med. 1993, 36, 363; (b) Perone, N. Perspect.
Biol. Med. 1993, 36, 347.
3. (a) Vojta, M. Ceskoslovenska Gynekol. 1971, 36, 273; (b) Plotz, E. J. Geburtshilfe
Frauenheilked 1970, 30, 193.
4. Claussner, A.; Nedelec, L.; Nique, F.; Philibert, D.; Teutsch, G.; Vande Velde, P. J.
Steroid Biochem. Mol. Biol. 1992, 41, 609.
5. Ray, S.; Sharma, I. Phramazie 1987, 42, 656.
6. (a) Salman, M.; Ray, S.; Agarwal, A. K.; Durani, S.; Setty, B. S.; Komboj, V. P.; Anand,
N. J. Med. Chem. 1983, 26, 592; (b) Singh, M. M. Med. Res. Rev. 2001, 21, 302.
7. Ray, S.; Singh, M. M.; Agarwal, A. K.; Komboj, V. P. Contraception 1987, 35, 283.
8. Ghosh, R.; Kamboj, V. P.; Singh, M. M. Contraception 2001, 64, 261.
9. (a) Cambie, R. C.; Manning, T. D. R. J. Chem. Soc. 1968, 21, 2603; (b) Dygos, J. H.;
Chin, L. J. J. Org. Chem. 1975, 40, 685.
pyridine (2 ml) and acetic anhydride (0.2 ml) was allowed to stand for 12 h
at room temperature. Water was added to the reaction mixture and
extracted with ether. The organic layer was separated and dried over
anhydrous sodium sulfate and concentrated to give compound 16 (0.75 g) as
oil.
Yield: 64%; mp. Oil; IR (cmꢀ1): 3400, 1710, 1600 (aromatic); Mass: (M+) 382,
(M+ꢀH2O) 364, (M+ꢀCOCH3) 340; 1H NMR (d, CDCl3): 1.00 (1s, 3H, CH3),
1.80 (1s, 3H, CH3), 2.12 (m, 2H, CH2ACH@CH2), 2.81 (b rs, 2H, CH2), 3.60 (s,
3H, OCH3), 4.21 (t, 1H, CH), 4.50–5.00 (m, 3H, CH@CH2), 6.50–6.70 (m, 2H,
ArH), 7.90 (d, 1H, J = 8.00 Hz, CH); Anal. Calcd for C24H30O4; C, 75.36; H,
7.91. Found: C, 75.50; H, 7.80.
Synthesis of 11-allyl-17b-hydroxy-3-methoxy-estra-1,3,5(10),8(9),11-pentaene
(17): A mixture of compound 16 (0.32 g, 0.001 mol) and p-toluenesulfonic
acid (0.05 g) in dry benzene was refluxed in a Dean–Stark apparatus for
20 min. The benzene was evaporated off; water was added to the reaction
mixture and extracted with ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate and concentrated to give a crude oily residue. The
10. Experimental:
Synthesis of
9a, 17b-dihydroxy-3-methoxy-11-oxo-estra-1,3,5(10)-triene 17-
acetate (8): A solution of chromium trioxide (4.25 g, 0.04 mol) in distilled
water (2 ml) and glacial acetic acid (30 ml, KMnO4 treated) was added
dropwise to a stirred solution of 3,17b-dihydroxy-estra-1,3,5(10)-triene 17-
acetate,3-methyl ether (6) (5.0 g, 0.02 mol) in glacial acetic acid (30 ml) kept at
5–10 °C. After complete addition, stirring was continued for 15 h and methanol
(2 ml) was added to decompose excessive CrO3. The reaction mixture was
diluted with water and extracted with ethyl acetate, dried over anhydrous
sodium sulfate, and concentrated to oily residue. The crude residue was
purified over a column of silica gel and eluted with ethyl acetate–hexane (3:47)
to get pure compound 8.
Yield: 50%, mp 115–117 °C. FABMS: (M+) 358, (M+1) 359, (M+2) 360; IR
(cmꢀ1): 3465, 1732, 1611, 1244; 1H NMR (d, CDCl3): 0.82 (s, 3H, CH3), 1.57 (s,
2H, CH2), 1.57–1.83 (m, 2H, CH2), 2.04 (s, 3H, CH3), 2.08–2.54 (m, 6H, CH and
CH2), 2.79 (br s, 2H, CH2), 4.41 (br s, 1H, OH), 3.77 (s, 3H, OCH3), 4.71 (t, 1H,
CH), 6.70 (br s, 3H, ArH); Anal. Calcd for C21H26O5; C, 70.37; H, 7.31. Found: C,
70.50; H, 7.48.
crude residue was purified on
compound 17 as oil.
a column of silica gel which gave pure
Yield: 35%; mp. Oil; IR (cmꢀ1): 1680, 1580 (aromatic); mass: (M+) 322; 1H
NMR (d, CDCl3): 1.01 (1s, 3H, CH3), 2.65 (m, 2H, CH2ACH@CH2), 2.80 (br s,
2H, CH2), 3.85 (s, 3H, OCH3), 4.23 (t, 1H, CH), 4.80–5.10 (m, 3H, CH@CH2),
5.40 (s, 1H, CH), 7.00–7.60 (m, 3H, ArH); Anal. Calcd For C22H26O2; C, 81.94;
H, 8.10. Found: C, 81.69; H, 8.00.
Anti-implantation screening:8 Anti-implantation activity of the compounds
was studied in sperm positive adult (180–220 g) Sprague–Dawley female
albino rats mated to coeval males of proven fertility. The compounds were
administered orally as a suspension in gum acacia to colony bread adult
mated female rats on days 1–5 post-coitum using five to seven animals in
each group. The animals were examined by laprotomy on day 10 of
pregnancy for the number and status of implantations and corpora lutea.
The results were scored as positive only if implantations were totally absent
in both the uterine horns of each animal.
Synthesis of 3-methoxy-17b-hydroxy-11-oxo-estra-1,3,5(10),8(9)-tetraene (10): A
solution of compound 8 (4.0 g, 0.011 mol) in menthol (60 ml) and perchloric
11. Bindal, R. D.; Carlson, K. E.; Reiner, G. C. A.; Katzenellenbogen, J. A. J. Steroid.
Biochem. 1987, 28, 361.