Effect of side-chain alteration on hormonal activity of nonsteroidal estrogen antagonists

Article abstract of DOI:10.1007/s00044-009-9238-0

Incorporation of novel side-chains in 3,4-diaryl chromans, an established nucleus present in the nonsteroidal estrogen antagonist centchroman, has been carried out. The effect of variation in the nature of the chain on relative binding affinity (RBA) to e

Full text of DOI:10.1007/s00044-009-9238-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:915–924
DOI 10.1007/s00044-009-9238-0
ORIGINAL RESEARCH
Effect of side-chain alteration on hormonal activity
of nonsteroidal estrogen antagonists
•
•
•
Sangita Anila Dwivedi Philip Prathipati
Suprabhat Ray
Received: 9 February 2009 / Accepted: 25 August 2009 / Published online: 30 September 2009
¨
Ó Birkhauser Boston 2009
Abstract Incorporation of novel side-chains in 3,4-diaryl chromans, an estab-
lished nucleus present in the nonsteroidal estrogen antagonist centchroman, has
been carried out. The effect of variation in the nature of the chain on relative
binding affinity (RBA) to estrogen receptor, estrogenicity, and antiestrogenicity has
been studied. Presence of hydrazide residue on cis- and trans-3,4-diaryl chromans
led to relatively higher RBA and estrogenicity as compared with corresponding
acids and esters.
Keywords Centchroman Á SERM Á Estrogenicity Á Antiestrogenicity Á
Enantiomers Á Hydroxychroman
Introduction
The role of the side-chain in nonsteroidal compounds exhibiting estrogen
antagonistic activity is well established in studies with ligand-bound nonsteroidal
estrogen antagonist. It has been shown that the amino-residue-containing basic
chain interacts with particular amino acid residue Asp351 present on estrogen
receptor (Brzozowski et al., 1997). This interaction prevents the ligand binding
pocket of the receptor from closing for initiation of estrogenic activity. Therefore,
the nature of the chain, its dimension, and its orientation in space are crucial for
determining its estrogen antagonistic activity.
Sangita (&) Á A. Dwivedi Á P. Prathipati Á S. Ray
Central Drug Research Institute, Lucknow 226 001, UP, India
e-mail: sangitap123@hotmail.com
Present Address:
S. Ray
Amity Institute of Pharmacy, AUUP, Lucknow Campus, 5-Viraj Khand, Gomti Nagar, Lucknow
226 010, UP, India

916
Med Chem Res (2010) 19:915–924
Recent interest in the development of nonsteroidal estrogen antagonists is due to
the finding of their tissue-selective action, being agonists in some tissues such as
bone, and antagonists in others such as uterine and breast tissues. Such compounds,
termed selective estrogen receptor modulators (SERM), have the potential to be
developed as drugs according to their selective activity profile (Kauffman and
Bryant, 1995; Ray and Sangita, 2004; Jordan, 2003).
However, antiestrogenic activity has resulted in molecules containing other side-
chains such as a,b-unsaturated propionic acid in GW 5638 (Wijayaratne et al.,
1999). In order to study the adaptability of the antiestrogen binding site, compounds
with acid, ester, and hydrazide chains have been prepared and their effect on
biological activity studied. Since it was essential to compare activity in order to
reach a meaningful conclusion, it was necessary to associate these chains with a
nucleus having established estrogenic and antiestrogenic activities. We therefore
substituted the pyrrolidinoethoxy chain of centchroman (Ray et al., 1976) (i), a
well-studied molecule, with acid, ester, and hydrazide chains.
Chemistry
Synthesis of the novel compounds has been carried out by reacting the
hydroxychroman derivatives with ethyl 2-bromopropionate in dry acetone in the
presence of potassium carbonate to give the esters (2a–d). The hydroxy chroman
derivatives used for the purpose are cis-2,2-dimethyl-3-phenyl-4-(p-hydroxy-
phenyl)-7-methoxy chroman (1a) trans-2,2-dimethyl-3-phenyl-4-(p-hydroxy-
phenyl)-7-methoxy chroman (1b), and its resolved l- (1c) and d-enantiomers
(1d). Hydrolysis of the esters under alkaline condition gave the corresponding
carboxylic acid (3a–d). Hydrazides of the compounds (4a–d) were prepared by
treating the corresponding esters with hydrazine hydrate in alcohol as solvent.
The reaction is shown in Scheme 1. Compounds thus prepared are given in
Table 1.
Results and discussion
The biological activity of the compounds is shown in Table 1. The biological
activity of different isomeric forms of centchroman has been reported earlier
(Salman et al., 1986).
It was found that 3,4-trans-diaryl chromans were biologically manyfold more
active than cis-chroman derivatives. In the trans-diaryl chroman the l-enantiomer
(3R,4R) of centchroman showed sevenfold higher relative binding affinity (RBA)
as compared with the d-form, and similar order of difference in their estrogenic
and antiestrogenic activities (Salman et al., 1986). However, its 100-fold lower
RBA and low antiestrogenicity would suggest inferior binding of the hydrazide
chain as compared with pyrrolidinoethoxy on the antiestrogen binding site of the
receptor. It will be of interest to study the role of the hydrazide chain in tissue-
selective action.

Med Chem Res (2010) 19:915–924
917
CH3
OCH2CH2N
O
O
CHCOOH
MeO
O
MeO
(ii)
i
3 (a-d)
CH3
CH3
CHCOOC2H5
OH
O
O
CHCONHNH2
O
O
(i)
(iii)
MeO
MeO
O
MeO
1 (a-d)
4 (a-d)
2 (a-d)
a: 3,4-cis
b: 3,4-trans
c: 3,4-trans, l (3R,4R)
d: 3,4-trans, d (3S,4S)
Scheme 1 Reagents: (i) Ethyl 2-bromopropionate, anh. K2CO3 and dry acetone, (ii) NaOH and alcohol,
(iii) Hydrazine hydrate and absolute alcohol
Estrogenic activity of l-trans hydrazide (4c) was similar to that of l-centchroman
(1c). In the less active d-hydrazide (4d), the estrogenic activity was low and
comparable to that of the corresponding acid (3d).
The nature of the chain had no definite effect on the antiestrogenicity of the
molecule. A study of the three-dimensional (3D) molecular geometry of the dl-trans
hydrazide (4b) and its superimposition on to the dl-trans centchroman (1b) on a
silicon graphics Indy R 4000 workstation employing Molecular Simulations software
(Insight IIÒ 95 Molecular modeling system; Molecular Simulations and DiscoverÒ
95/3.0.0 Forcefield Simulations; Molecular Simulations) was carried out. Structures
were built in the builder module of the Insight II software. The 3D structures were
later optimized for their geometry using the consistent valence forcefield (CVFF)
(Dauber-Osguthorpe et al., 1988) and energy minimization was performed using
steepest-descent conjugate-gradient and Newton–Raphson algorithms in sequence
followed by quasi-Newton–Raphson (va09a) implemented in discover module by
using 0.001 kcal mol-1 energy gradient convergence and maximum number of
iterations of 1,000. Energy-minimized structures were stored in MDL format.
The conformational resemblances of the molecules were assessed using the root-
mean-square (RMS) fitting option of the Search–Compare forcefield (Search_com-
pare 95.0 conformational Searchand Molecular Comparison.; Molecular Simulations)
module. One-to-one correspondences for atoms in the molecules to be superimposed
were specified. The RMS fit between the superimposed molecules was noted.
Figure 1 shows the molecular similarity between the two molecules discussed
above with RMS fit value of 0.007118. This would explain the observed similar

918
Med Chem Res (2010) 19:915–924
Table 1 Biological activity data
Compound no.
RBA, %
of estradiol
Dose
(mg kg-1
Estrogenicity
(% increase in
uterine weight)
Antiestrogenicity
)
(% inhibition in ethynyl
estradiol-induced uterine
weight gain)
2a
3a
4a
2b
3b
4b
2c
3c
4c
2d
3d
4d
1b
1c
1d
1a
Not detected
Not detected
0.011
10
10
10
10
10
10
3
71.58
44.2
3.71
3.8
110.22
43.7
1.07
3.98
10.8
12.14
40.64
10
0.029
Not detected
0.052
21.6
101.91
122.2
149
Not detected
0.037
10
10
10
10
10
3
0.37
155.02
0.19
25.37
14.33
16.09
6.21
60
Not detected
Not detected
0.24
6.88
6.68
5.42
126.83
165
15.7
3
46.11
50
2.10
3
109
\0.001
10
33.6
Fig. 1 Superposition of
dl-trans centchroman with
dl-trans hydrazide
order of estrogenicity and hence binding ability of the ligand to the estrogen
receptor as seen with dl-centchroman.
Experimental section
All reactions were monitored by thin-layer chromatography (TLC) over silica-gel-
coated TLC plates. Spots on TLC plates were visualized by warming plates sprayed

Med Chem Res (2010) 19:915–924
919
with CeSO4 (1% in H2SO4) in an oven at 100°C or in iodine vapors. For column
chromatography, silica gel (60–120 mesh) was used. Infrared (IR) spectra were
recorded on Perkin-Elmer 881 and FTIR-8210 PC Shimadzu spectrophotometers
1
and values are expressed in cm-1, H nuclear magnetic resonance (NMR) spectra
were recorded on Advance DPX 200 FT Bruker Robotics spectrometer using TMS
as an internal reference. Fast atom bombardment (FAB) mass spectra were recorded
on Jeol SX 102/DA 6000 mass spectrometer using argon/xenon (6 kV, 10 mA) as
the FAB gas. Elemental analyses were carried out on Carlo-Erba-1108 instrument.
Optical rotations were determined on Autopol III polarimeter using 1 dm cell at
28°C in methanol as the solvent with concentrations stated in units of g/100 ml.
Ethyl 2-(4-cis-2,2-dimethyl-3-phenyl-7-methoxychroman-4-yl)-phenoxy-
propanoate (2a)
A mixture of 3,4-cis-2,2-dimethyl-3-phenyl-4-[4-hydroxy phenyl]-7-methoxychro-
man (1.5 g, 4.17 mmol), anhydrous potassium carbonate (6 g, 43.4 mmol), and
ethyl 2-bromopropionate (8 ml, 43.4 mmol) in dry acetone (30 ml) was refluxed for
4 h. Potassium carbonate was filtered off and the solvent was distilled off. The
residue was extracted with ethyl acetate, washed with water, dried over anhydrous
sodium sulfate, and concentrated to give an oil.
Yield: 1.3 g (67.83%); IR (neat, cm-1): 1446, 1504, 1582, 1615 (ArH), 1750
1
(ester), 1240 (OMe), 2983 (CH), 1374 (gem-dimethyl), 1662 (C=O); H NMR (d,
CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s, 3H, gem-dimethyl), 3.76 (s, 3H, MeO),
3.1 (d, 1H, monobenzylic H, J = 12 Hz), 4.2 (d, 1H, dibenzylic H, J = 12 Hz), 6.3–
7.2 (m, 12H, ArH), 1.1 (t, 3H, CH2CH3) 4.6 (q, 1H, CHCH3), 4.1 (q, 2H, CH2CH3),
1.5 (d, 3H, CHCH3); MS m/z: 460. Anal.: (C29H32O5), C and H.
2-[4-(cis-3,4-Dihydro-2,2-dimethyl-3-phenyl-7-methoxybezopyran-4-yl)-
phenoxy] propionic acid (3a)
A mixture of ethyl 2-(4-cis-2,2-dimethyl-3-phenyl-7-methoxychroman-4-yl)-phen-
oxy-propanoate (0.75 g, 1.63 mmol), and sodium hydroxide (2.0 g, 50 mmol) in
alcohol (20 ml) was refluxed for 1.5 h. The solvent was distilled off. The reaction
mixture was acidified with 5% HCl solution and extracted with ethyl acetate,
washed with water to pH 7, and dried over anhydrous sodium sulfate, then ethyl
acetate was distilled off to give an oil, which was crystallized from methanol to give
the desired product.
Yield: 0.6 g (85.19 %); m.p.: 150°C; IR (KBr, cm-1): 1459, 1506, 1599 (ArH),
1
1725 (C=O), 1228 (OMe), 2933 (CH), 1360 (gem-dimethyl), 3410 (OH); H NMR
(d, CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s, 3H, gem-dimethyl), 3.76 (s, 3H,
MeO), 3.1 (d, 1H, monobenzylic H, J = 12 Hz), 4.1 (d, 1H, dibenzylic H,
J = 12 Hz), 6.28–7.26 (m, 12H, ArH), 4.3 (q, 1H, CHCH3), 1.5 (d, 3H, CHCH3);
MS m/z: 432. Anal.: (C27H28O5), Cald: C and H.

920
Med Chem Res (2010) 19:915–924
2-[4-(cis-3,4-Dihydro-2,2-dimethyl-3-phenyl-7-methoxybezopyran-4-yl)-
phenoxy] propionic acid hydrazide (4a)
A mixture of ethyl 2-(4-cis-2,2-dimethyl-3-phenyl-7-methoxychroman-4-yl)-phen-
oxy-propanoate (0.15 g, 0.33 mmol) and hydrazine hydrate (2 ml, 64.2 mmol) in
alcohol (15 ml) was refluxed for 7 h. The solvent was distilled off to give an oil.
Yield: 0.136 g (93.51 %); m.p.: 60°C; IR (KBr, cm-1): 1449, 1504, 1617, (ArH),
1677 (C=O), 1225 (OMe), 2929 (CH), 1375 (gem-dimethyl), 3329, 3439 (NH), 3695
(NH2); 1H NMR (d, CDCl3): 1.25 (s, 6H, gem-dimethyl), 3.79 (s, 3H, MeO), 2.9 (d,
1H, monobenzylic H, J = 12 Hz), 4.3 (d, 1H, dibenzylic H, J = 12 Hz), 6.35–7.51
(m, 12H, ArH), 1.5 (d, 3H, CHCH3) 4.6 (q, 1H, CHCH3), 7.26 (s, 1H, NH); MS m/z:
446. Anal.: (C27H30O4N2), C, H, and N.
Ethyl 2-(4-trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-yl)-phenoxy-
propanoate (2b)
A mixture of 3,4-trans-2,2-dimethyl-3-phenyl-4-(4-hydroxy-phenyl)-7-methoxych-
roman (0.2 g, 0.56 mmol), anhydrous potassium carbonate (0.8 g, 5.7 mmol), and
ethyl 2-bromopropionate (0.39 ml, 3.0 mmol) in dry acetone (10 ml) was refluxed
for 4 h. Potassium carbonate was filtered off and the solvent was distilled off. The
residue was extracted with ethyl acetate, washed with water, dried over anhydrous
sodium sulfate, and concentrated to give oil, which was crystallized from methanol.
Yield: 0.180 g, (70.44 %); m.p.: 120°C; IR (KBr, cm-1): 1450, 1506, 1585, 1612
1
(ArH), 1745 (ester), 1207 (OMe), 2985 (CH), 1377 (gem-dimethyl); H NMR (d,
CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s, 3H, gem-dimethyl), 3.76 (s, 3H, MeO),
3.1 (d, 1H, monobenzylic H, J = 12 Hz), 4.5 (d, 1H, dibenzylic H, J = 12 Hz), 6.3–
7.2 (m, 12H, ArH), 1.1 (t, 3H, CH2CH3), 4.1 (q, 2H, CH2CH3), 1.5 (d, 3H, CHCH3),
4.5 (q, 1H, CHCH3); MS m/z: 460. Anal.: (C29H32O5), C, H, and N.
2-[4-(trans-3,4-Dihydro-2,2-dimethyl-3-phenyl-7-methoxybezopyran-4-yl)-
phenoxy] propionic acid (3b)
A mixture of ethyl 2-(4-trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-yl)-
phenoxy-propanoate (0.5 g, 1.09 mmol) and sodium hydroxide (1.0 g, 25 mmol)
in alcohol (20 ml) was refluxed for 1.5 h. The solvent was distilled off. The reaction
mixture was acidified with 5% HCl solution and extracted with ethyl acetate,
washed with water to pH 7, and dried over anhydrous sodium sulfate, then ethyl
acetate was distilled off to give an oil, which was crystallized from methanol to give
the desired product.
Yield: 0.3 g (63.89 %); m.p.:185°C; IR (KBr, cm-1): 1417, 1512, 1593, 1614
(ArH), 1722 (C=O), 1217 (OMe), 3022 (CH), 1373 (gem-dimethyl), 3425 (OH); 1H
NMR (d, CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s, 3H, gem-dimethyl), 3.69 (s, 3H,
MeO), 3.1 (d, 1H, monobenzylic H, J = 12 Hz), 4.1 (d, 1H, dibenzylic H,
J = 12 Hz), 6.3–7.2 (m, 12H, ArH), 4.4 (q, 1H, CHCH3), 1.51 (d, 3H, CHCH3); MS
m/z: 432. Anal.: (C27H28O5), C and H.

Med Chem Res (2010) 19:915–924
921
2-[4-(trans-3,4-Dihydro-2,2-dimethyl-3-phenyl-7-methoxybezopyran-4-yl)-
phenoxy] propionic acid hydrazide (4b)
A mixture of ethyl 2-(4-trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-yl)-
phenoxy-propanoate (0.15 g, 0.33 mmol) and hydrazine hydrate (2 ml, 64.2 mmol)
in absolute alcohol (20 ml) was refluxed for 7 h. The solvent was distilled off to
give an oil, which was crystallized from ethanol to give the desired product.
Yield: 0.1 g (68.76%); m.p.: 195°C; IR (KBr, cm-1): 1417, 1505, 1617, (ArH),
1670 (C=O), 1217 (OMe), 2930 (CH), 1382 (gem-dimethyl), 3290 (NH), 3437
(NH2); 1H NMR (d, CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s, 3H, gem-dimethyl),
3.77 (s, 3H, MeO), 3.11 (d, 1H, monobenzylic H, J = 12 Hz), 4.29 (d, 1H,
dibenzylic H, J = 12 Hz), 6.3–7.2 (m, 12H, ArH), 1.51 (d, 3H, CHCH3) 4.60–4.64
(q, 1H, CHCH3), 7.26 (s, 1H, NH); MS m/z: 446. Anal.: (C27H30O4N2) C, H, and N.
Ethyl 2-(4-l(-)3,4 trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-yl)-
phenoxy-propanoate (2c)
A mixture of l(-)3,4-trans-2,2-dimethyl-3-phenyl-4-[4-hydroxy phenyl]-7-meth-
oxychroman (1.5 g, 4.17 mmol), anhydrous potassium carbonate (6 g, 43.4 mmol),
and ethyl 2-bromopropionate (4.5 ml, 34.6 mmol) in dry acetone (50 ml) was
refluxed for 4 h. Potassium carbonate was filtered off and the solvent was distilled
off. The residue was extracted with ethyl acetate, washed with water, dried over
anhydrous sodium sulfate, and concentrated to give an oil, which was crystallized
from methanol.
Yield: 1.4 g (73.04 %); m.p.: 96°C; [a]2D0: (C = 1, MeOH): –193.52; IR (KBr,
cm-1): 1450, 1506, 1598, 1612 (ArH), 1745 (ester), 1207 (OMe), 2985 (CH), 1382
1
(gem-dimethyl); H NMR (d, CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s, 3H, gem-
dimethyl), 3.77 (s, 3H, MeO), 3.11 (d, 1H, monobenzylic H, J = 12 Hz), 4.5 (d, 1H,
dibenzylic H, J = 12 Hz), 6.3–7.2 (m, 12H, ArH), 1.1 (t, 3H, CH2CH3) 4.58 (q, 1H,
CHCH3), 4.11 (q, 2H, CH2CH3), 1.5 (d, 3H, CHCH3). MS m/z: 460. Anal.:
(C29H32O5), C, H, and N.
2-[4-(l(-)-trans-3,4-Dihydro-2,2-dimethyl-3-phenyl-7-methoxybezopyran-4-yl)-
phenoxy] propionic acid (3c)
A mixture of ethyl 2-(4-l(-)-3,4-trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-
yl)-phenoxy-propanoate (0.75 g, 1.63 mmol) and sodium hydroxide (2.0 g,
50 mmol) in alcohol (20 ml) was refluxed for 1.5 h. The solvent was distilled off.
The reaction mixture was acidified with 5% HCl solution and extracted with ethyl
acetate, washed with water to pH 7, and dried over anhydrous sodium sulfate, and
ethyl acetate was distilled off to give an oil, which was crystallized from methanol
to give the desired product.
Yield: 0.61 g (86.60%); m.p.: 153°C; [a]2D0: (C = 1, MeOH): –204.761; IR (KBr,
cm-1): 1447, 1506, 1585, 1609 (ArH), 1725 (C=O), 1231 (OMe), 2983 (CH), 1350
(gem-dimethyl), 3402 (OH); 1H NMR (d, CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s,
3H, gem-dimethyl), 3.77 (s, 3H, MeO), 3.1 (d, 1H, monobenzylic H, J = 12 Hz),

922
Med Chem Res (2010) 19:915–924
4.1 (d, 1H, dibenzylic H, J = 12 Hz), 6.3–7.2 (m, 12H, ArH), 4.6 (q, 1H, CHCH3),
1.5 (d, 3H, CHCH3); MS m/z: 432. Anal.: (C27H28O5), C and H.
2-[4-(l(-)-trans-3,4-Dihydro-2,2-dimethyl-3-phenyl-7-methoxybezopyran-4-yl)-
phenoxy] propionic acid hydrazide (4c)
A mixture of ethyl 2-(4-l(-)-3,4-trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-
yl)-phenoxy-propanoate (0.15 g, 0.33 mmol) and hydrazine hydrate (2 ml,
64.2 mmol) in alcohol (20 ml) was refluxed for 7 h. The solvent was distilled off
to give an oil, which was crystallized from ethanol to give product.
Yield: 0.123 g (84.57%); m.p.: 125°C; [a]2D0: (C = 1, MeOH): –146.218; IR (KBr,
cm-1): 1507, 1618, (ArH), 1678 (C=O), 1216 (OMe), 2930 (CH), 1382 (gem-
dimethyl), 3442 (NH), 3758 (NH2); 1H NMR (d, CDCl3): 1.2 (s, 3H, gem-dimethyl),
1.3 (s, 3H, gem-dimethyl), 3.77 (s, 3H, MeO), 3.12 (d, 1H, monobenzylic H,
J = 12 Hz), 4.29 (d, 1H, dibenzylic H, J = 12 Hz), 6.35–7.51 (m, 12H, ArH), 1.73
(d, 3H, CHCH3) 4.61 (q, 1H, CHCH3), 7.26 (s, 1H, NH); MS m/z: 446. Anal.:
(C27H30O4N2), C, H, and N.
Ethyl 2-(4-d(?)-trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-yl)-
phenoxy-propanoate (2d)
A mixture of d(?)3,4-trans-2,2-dimethyl-3-phenyl-4-[4-hydroxy phenyl]-7-meth-
oxychroman (0.2 g, 0.56 mmol), anhydrous potassium carbonate (0.8 g, 5.7 mmol),
and ethyl 2-bromopropionate (0.39 ml, 3.0 mmol) in dry acetone (10 ml) was
refluxed for 4 h. Potassium carbonate was filtered off and the solvent was distilled
off. The residue was extracted with ethyl acetate, washed with water, dried over
anhydrous sodium sulfate, and concentrated to give the desired product.
Yield: 0.170 g (66.52%); [a]D20: (C = 1, MeOH): ?190.566; IR (neat, cm-1):
1449, 1505, 1586, 1615 (ArH), 1744 (ester), 1235 (OMe), 2987 (CH), 1377 (gem-
dimethyl); 1H NMR (d, CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s, 3H, gem-
dimethyl), 3.76 (s, 3H, MeO), 3.1 (d, 1H, monobenzylic H, J = 12 Hz), 4.2 (d, 1H,
dibenzylic H, J = 12 Hz), 6.3–7.2 (m, 12H, ArH), 1.1 (t, 3H, CH2CH3), 4.1 (q, 2H,
CH2CH3), 1.5 (d, 3H, CHCH3), 4.6 (q, 1H, CHCH3); MS m/z: 460. Anal.:
(C29H32O5) C and H.
2-[4-(d(?)-trans-3,4-dihydro-2,2-dimethyl-3-phenyl-7-methoxybezopyran-4-
yl)-phenoxy] propionic acid (3d)
A mixture of ethyl 2-(4-d(?)-trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-
yl)-phenoxy-propanoate (0.4 g, 0.87 mmol), and sodium hydroxide (0.8 g,
20 mmol) in alcohol (20 ml) was refluxed for 1.5 h. The solvent was distilled off.
The reaction mixture was acidified with 5% HCl solution and extracted with ethyl
acetate, washed with water to pH 7, dried over anhydrous sodium sulfate, then ethyl
acetate was distilled off to give an oil, which was crystallized from methanol to give
the desired product.

Med Chem Res (2010) 19:915–924
923
Yield: 0.325 g (86.52%); m.p.:150°C; [a]2D0: (C = 1, MeOH): ?200; IR (KBr,
cm-1): 1447, 1506, 1585, 1609 (ArH), 1725 (C=O), 1231 (OMe), 2983 (CH), 1350
(gem-dimethyl), 3402 (OH); 1H NMR (d, CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s,
3H, gem-dimethyl), 3.77 (s, 3H, MeO), 3.1 (d, 1H, monobenzylic H, J = 12 Hz),
4.1 (d, 1H, dibenzylic H, J = 12 Hz), 6.3–7.2 (m, 12H, ArH), 4.6 (q, 1H, CHCH3),
1.5 (d, 3H, CHCH3); MS m/z: 432. Anal.: (C27H28O5), C and H.
2-[4-(d(?)-trans-3,4-Dihydro-2,2-dimethyl-3-phenyl-7-methoxybezopyran-4-
yl)-phenoxy] propionic acid hydrazide (4d)
A mixture of ethyl 2-(d(?)-4-trans-2,2-dimethyl-3-phenyl-7-methoxychroman-4-
yl)-phenoxy-propanoate (0.15 g, 0.33 mmol) and hydrazine hydrate (2 ml,
64.2 mmol) in alcohol (20 ml) was refluxed for 7 h. The solvent was distilled off
to give an oil.
Yield: 0.115 g (79.07%); [a]2D0: (C = 1, MeOH): ?187.40; IR (neat, cm-1): 1452,
1507, 1617 (ArH), 1671 (C=O), 1231 (OMe), 2996 (CH), 1374 (gem-dimethyl),
3327 (NH), 3658 (NH2); 1H NMR (d, CDCl3): 1.2 (s, 3H, gem-dimethyl), 1.3 (s, 3H,
gem-dimethyl), 3.77 (s, 3H, MeO), 3.12 (d, 1H, monobenzylic H, J = 12 Hz), 4.29
(d, 1H, dibenzylic H, J = 12 Hz), 6.35–7.36 (m, 12H, ArH), 1.51 (d, 3H, CHCH3),
4.6 (q, 1H, CHCH3), 7.26 (s, 1H, NH); MS m/z: 446. Anal.: (C27H30O4N2), C and H.
Biochemical and biological methods
Receptor affinity (Katzenellenbogen et al., 1973; Salman et al., 1983)
Relative binding affinities (RBA) for uterine cytosol 17-b-estradiol receptors
obtained from immature Chares Foster rats, 21–25 days old, were determined by
competition assay, employing dextran-coated charcoal (DCC) for separation of
unbound steroids according to the method of Korenman, as modified by
Katzenellenbogen, and are listed in Table 1.
Estrogenic activity (Salman et al., 1983; Kar et al., 1967)
Estrogenic activity of the compounds, reported in Table 1, was evaluated in
immature rats (25–30 g) as assessed by uterine weight gain. The compounds were
administered subcutaneously once daily over a 3-day period in 0.2 ml saline/
propylene glycol (1:1, v/v).
Estrogen antagonistic activity (Kar et al., 1967)
Twenty-one-day-old immature female Sprague–Dawley rats were bilaterally ovari-
ectomized under light ether anesthesia and, after postoperative rest for 7 days,
randomized into different treatment groups. Each rat received the compound of the
invention and 0.02 mg/kg does of 17a-ethynylestradiol in 10% ethanol-distilled water
once daily for 3 consecutive days on days 28–30 of age by oral route. A separate group

924
Med Chem Res (2010) 19:915–924
of animals receiving only 17a-ethynylestradiol (0.02 mg/kg) in 10% ethanol-distilled
water for similar duration were used for comparison. At autopsy on day 31 of age
vaginal smear of each rat was taken and uterus was carefully excised, gently blotted,
weighed, and fixed for histology and histomorphometry using image analysis.
Inhibition of ethynylestradiol-induced cornification of vaginal epithelium, increase in
uterine fresh weight, total uterine and endometrial area, and uterine luminal epithelial
cell height were taken as parameters for evaluation of estrogen antagonistic effect of
the compounds. Compounds exhibiting potent estrogen antagonistic activity in this
assay were identified for further development as anti-implantation agents.
Acknowledgement Authors (Sangita and Philip Prathipati) would like to thank CSIR, New Delhi
(India) for the award of Senior Research Fellowship.
References
Brzozowski A, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstro¨m O, Ohman L, Greene GL,
Gustafsson JA, Carlquist M (1997) Molecular basis of agonism and antagonism in the estrogen
receptor. Nature 389:753–758
Dauber-Osguthorpe P, Roberts VA, Ostguthorpe DJ, Wolff J, Genest M, Hagler AT (1988) Structure and
energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a
drug-receptor system. Proteins Struct Func Genet 4:31–47
Jordan VC (2003) Antiestrogens and selective estrogen receptor modulators as multifunctional medicines.
1. Receptor interactions. J Med Chem 46:883–908
Kar AB, Kamboj VP, Setty BS (1967) Biological properties of 2-phenyl-3- diethylpyrolidinoethoxy-6-
methoxy-benzofuran hydrochloride a new oral antifertility agent. Indian J Exp Biol 5(2):80–84
Katzenellenbogen JA Jr, Johnson HJ, Myers HN (1973) Photoaffinity labels for estrogen binding proteins
of rat uterus. Biochemistry 12(21):4085–4092
Kauffman RF, Bryant HU (1995) Selective estrogen receptor modulators. Drug News Perspect 8:531–539
Ray S, Sangita (2004) The potent triaryl ethylene pharmacophore. Drugs Future 29(2):185–203
Ray S, Grover PK, Kamboj VP, Setty BS, Kar AB, Anand N (1976) Antifertility agents. 12. Structure-
activity relationship of 3,4-diphenylchromenes and chromans. J Med Chem 19:276–279
Salman M, Ray S, Agarwal AK, Durani S, Setty BS, Kamboj VP, Anand N (1983) Antifertility agents.
38. Effect of the side chain and its position on the activity of 3,4-diarylchromans. J Med Chem
26(4):592–595
Salman M, Ray S, Anand N, Agarwal AK, Singh MM, Selty BS, Kamboj VP (1986) Studies in
antifertility agents. 50. Stereoselective binding of d- and l-centchromans to estrogen receptors and
their antifertility activity. J Med Chem 29:1801–1803
Wijayaratne AL, Nagel SC, Paige LA, Christensen DJ, Norris JD, Fowlkes DM, McDonnell DP (1999)
Comparative analyses of mechanistic differences among antiestrogens. Endocrinology 140:5828–
5840