In vivo evaluation of substituted 3-phenyl,7-methoxy-benzopyrans as modified estrogens

Article abstract of DOI:10.1007/s00044-009-9169-9

Substituted 3-phenyl,7-methoxy-benzopyran derivatives and vitamin D ₃ (cholecalciferol, 1) were evaluated for their estrogen agonistic and antagonistic activities in immature female Sprague-Dawley rat model. The benzopyran derivatives 17 and 18

Full text of DOI:10.1007/s00044-009-9169-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:25–32
DOI 10.1007/s00044-009-9169-9
ORIGINAL RESEARCH
In vivo evaluation of substituted 3-phenyl,7-methoxy-
benzopyrans as modified estrogens
Atul Gupta Æ Govind Keshri Æ M. M. Singh Æ
Suprabhat Ray
Received: 21 May 2008 / Accepted: 6 February 2009 / Published online: 3 April 2009
Ó Birkh a¨ user Boston 2009
Abstract Substituted 3-phenyl,7-methoxy-benzopyran derivatives and vitamin D3
(cholecalciferol, 1) were evaluated for their estrogen agonistic and antagonistic activities
in immature female Sprague–Dawley rat model. The benzopyran derivatives 17 and 18,
which were made as hybrids of estrogen and vitamin D (pseudo vitamin D analogs),
3
3
showed significant estrogen agonistic activity (up to 48%) and weak estrogen antago-
nistic activity (up to 6%) at 10 mg/kg, whereas vitamin D showed significant estrogen
agonistic (82%) and antagonistic activities (39%) at 10 mg/kg.
3
Keywords Vitamin D Á Estrogen agonists Á Estrogen antagonist Á
3
Osteoporosis Á Antiosteoporotic agents Á Benzopyran Á Drug research
Introduction
Osteoporosis is a disease characterized by depletion of bone mass and enhanced
bone fragility, leading to increased risk of fractures (Doggrell, 2003). In women,
osteoporosis sets in after menopause when the estrogen level drops. This indicates
that estrogen is related to maintenance of bone mass. It has now been established
that estrogen has a role in osteoporosis directly and also through an indirect
mechanism involving vitamin D (1) (Fig. 1) (Mizwicki et al., 2005). Regulation of
3
calcium is governed mainly by the parathyroid hormone (PTH) and 1,25-(OH)2-
vitamin D (2) (Fig. 2) (Lips, 2006). 1,25-(OH) -vitamin D (2) helps absorption of
3
2
3
calcium in the gut. This active metabolite is formed from vitamin D (2) under the
3
A. Gupta (&) Á S. Ray
Medicinal and Process Chemistry Division, Central Drug Research Institute,
Lucknow 226001, India
e-mail: atisky2001@yahoo.co.in
G. Keshri Á M. M. Singh
Endocrinology Division, Central Drug Research Institute, Lucknow 226001, India

26
Med Chem Res (2010) 19:25–32
O
R2
O
O
OH
HO
HO
R1
HO
OH
1
; R1 = R = H
3
4
2
2; R1 = R = OH
2
R
O
O
OH
OH
R
OR
R
1
R
1
HO
OH
R2
HO
OH
6
5
; R= CH , CH CH
7
R
R
1
1
= H, R
= H, R
2
2
= CH
= CH
2
OH
CH
3
2
3
2
2
OH
R1 = OH, R2 = H
R
R
1
= CH
= CH
2
OH, R
CH OH, R = H
2
2
= H
1
2
2
R1OH
R2
O
OH
O
O
H
3
CO
3
H CO
8
9
Fig. 1 Ligands for estrogen and vitamin D receptors
influence of estradiol (3) (Fig. 1) (Castillo et al., 1977). Also, estrogen is
responsible for adjustment of a setpoint at which lowered level of calcium in
serum triggers release of parathyroid hormone (PTH) to utilize calcium from bone
to maintain calcium level (Christakos and Prince, 2003). Thus, vitamin D (2) and
3
estrogen are both required for maintenance of bone (Holick, 2006).
Furthermore, the study made by Demirpence et al. (1994) showed antiestrogenic
effect of vitamin D (2) as measured by its inhibitory action on estrogen-induced
3
growth of MCF-7 cells (antiproliferative activity). The results of that study
suggested that the ligand binding, dimerization, and ligand-dependent transactivat-
ing domains (DEF domains) of estrogen receptor (ER) were required for
antiestrogenic effect of vitamin D (1). It also suggested that vitamin D receptor
3
(
VDR) may play a part in imparting the binding of the ER to the estrogen response
element (ERE). Therefore, there could be either direct or an indirect interaction
between VDR and ER.

Med Chem Res (2010) 19:25–32
27
3
Fig. 2 Interplay of estrogens and vitamin D in bone maintenance (Ray and Gupta, 2006)
To study the similarities in their biological functions, we reported a review of
literature on structure–function relationship between vitamin D (2) and potent
3
endogenous estrogen, estradiol (3) (Ray and Gupta, 2006).
In our approach to see whether estrogens also cross-react with vitamin D receptor
(
VDR) in terms of biological activities, an attempt was made towards preparation of
compounds as modified estrogen for bone-selective antiproliferative activities. The
designed compounds will have a properly designed estrogen binding subunit along
with a residue similar to that present in vitamin D (2) to encourage cross-reactivity
3
with both estrogen and vitamin D receptors (VDR).
Structurally, vitamin D (2) has highly flexible side-chain as well as triene sterol
3
structure (3) (Fig. 1) (Yamada et al., 2003). Stereochemistry of vitamin D side-
3
chain also plays an important role in its biological responses (Yamada et al., 2003).
Several structural modifications have been made in the side-chain of vitamin D3,
which include flexible to rigid nature of side-chain (4–6) (Fig. 1) (Saito et al., 2004;
Schepens et al., 2004). Other structural modification involves replacement of highly
flexible seco-triene system of vitamin D by a benzene ring, which led to compound
3
7 (Fig. 1), showing affinity for vitamin D receptor (Posner et al., 1995; Kenzler
et al., 1996).
Furthermore, 3-phenyl-benzopyran nucleus, as present in Equol (8), forms the
basis of several selective estrogen receptor modulators (SERMs) such as ormeloxif-
ene, CDRI-85/287, and EM-800 (Ray and Diwedy, 1997). Equol (8) has affinity
towards estrogen receptors (ER-a and ER-b) (Muthyala et al., 2004). In the present
work, owing to their mild estrogenic action and effective affinity with estrogen
receptors, we have chosen this isoflavonoid as basic nucleus, which was incorporated
with modified side-chains similar to that present in vitamin D and its synthetic
3
analogs. The following compounds of type 9 were designed to achieve this objective.
This study presents the in vivo estrogen agonistic and estrogen antagonistic activities
of these 3-phenyl,7-methoxy-benzopyran-based hybrids of estrogen and vitamin D3
analogs, and vitamin D in immature female Sprague–Dawley rat model.
3

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Med Chem Res (2010) 19:25–32
Materials and methods
Chemistry
The target benzopyran derivatives of type 9 were prepared through known
procedure starting with condensation of resorcinol 10 and 4-methoxy phenylacetic
acid (11) in presence of BF -OEt through Friedel–Craft acylation reaction, yielding
3
2
1
-(2,4-dihydroxyphenyl)-2-(4-methoxy-phenyl) ethanone (12) in 62% yield, as
shown in Scheme 1 (Gupta and Ray, 2007). Compound 12 was alkylated with
benzyl bromide in presence of anhydrous potassium carbonate in dry acetone,
giving 1-(4-benzyloxy-2-hydroxy-phenyl)-2-(4-methoxy-phenyl) ethanone (13) in
7
2% yield. Compound 13 was then condensed with paraformaldehyde in aqueous
solution of sodium hydroxide, which gave 7-benzyloxy-3-(4-methoxy-phenyl)-
chroman-4-one (14) in 40% yield. Catalytic reduction of chromanone 14 using 10%
palladium charcoal in methanol at 50 psi pressure afforded corresponding hydroxy
chroman 15. Alkylation of 15 with ethyl-4-bromobutyrate in presence of anhydrous
potassium carbonate in dry acetone at reflux temperature furnished 4-[3-(4-
methoxy-phenyl)-chroman-7-yloxy] butyric acid ethyl ester (16). Grignard reaction
on compound 16 with alkylmagnesium halide reagents in dry THF–OEt system
2
yielded compounds 17 and 18 in 94% yield. All the synthesized compounds were
1
identified by H nuclear magnetic resonance (NMR), fast-atom bombardment
(
FAB) mass spectroscopy (MS), and infrared (IR) spectroscopy.
Biology
Estrogen agonistic activity (Ghosh et al., 2001)
Twenty-one-day-old immature female Sprague–Dawley rats were bilaterally
ovariectomized under light ether anesthesia and, after postoperative rest for 7 days,
were randomized into different treatment groups. Each rat received the compound
of the invention once daily for three consecutive days on days 28–30 of age by oral
route. A separate group of animals received only the vehicle for similar duration,
serving as control. At autopsy, 24 h after the last treatment on day 31 of age, vaginal
smear of each rat was taken and uterus was carefully excised, gently blotted, and
weighed. Increase in uterine fresh weight was taken as parameter for evaluation
of estrogen agonistic activity in comparison with rats of vehicle control group.
Theobjective was to evaluate estrogen agonistic effect of the compounds on the
uterus.
Estrogen antagonistic activity (Ghosh et al., 2001)
Twenty-one-day-old immature female Sprague–Dawley rats were bilaterally
ovariectomized under light ether anesthesia and, after postoperative rest for 7 days,
were randomized into different treatment groups. Each rat received the compound
of the invention and 0.02 mg/kg dose of 17a-ethynylestradiol (EE) in 10% ethanol–

Med Chem Res (2010) 19:25–32
29
OCH3
O
O
a
+
OH
HO
OH
H3CO
HO
OH
1
0
11
12
b
OCH3
OCH3
O
O
O
c
PhH CO
2
OH
13
PhH CO
2
1
4
d
O
O
O
OCH2CH3
O
OH
e
16
H3CO
15
H CO
3
g
f
OH
OH
O
O
O
O
H3CO
17
H3CO
1
8
Scheme 1 (a) BF
CH
Et O–THF
3
–OEt
2
,Heat, (b) C
6
H
5
CH
CO
2
Br, K
, dry acetone, (f) CH
2
CO
3
, dry acetone, (c) HCHO, aq NaOH, (d) Pd/C, H
2
,
3
OH, (e) ethyl-4-bromobutyrate, K
2
3
3
MgI, diethyl ether, (g) CH CH MgI,
2
3
2
distilled water once daily for three consecutive days on days 28–30 of age by oral
route. A separate group of animals receiving only EE (0.02 mg/kg) in 10% ethanol–
distilled water for similar duration, as 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. Inhibition in ethynylestradiol-induced increase in
uterine fresh weight was taken as parameter for evaluation of estrogen antagonistic
effect of the compounds.
Inhibition in uterine weight in case of estrogen antagonistic action has been
calculated in comparison with respective EE per se treated group. For estrogen
agonistic activity, uterine weight gain was calculated with respect to the
corresponding vehicle control group.

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Med Chem Res (2010) 19:25–32
Results and discussion
The above discussion has shown direct or indirect interplay of estrogens and vitamin
D in maintenance of bones, cellular proliferation, and other biological activities.
3
Vitamin D not only acts as anti-bone-resorptive agent, but also helps in bone
3
formation, whereas estrogens are involved in vitamin D metabolism and also act as
3
anti-bone-resorptive agents. It is noteworthy that selective estrogen receptor
modulators (SERMs) are anti-bone-resorptive agents, similar to many steroidal and
nonsteroidal estrogens. To validate our hypothesis of cross-reactivity of vitamin D3
with estrogen as well as vitamin D receptors for bone maintenance and cellular
proliferation, we first time evaluated in vivo estrogen agonistic and estrogen
antagonistic activities of vitamin D (cholecalciferol, 1) in immature female Sprague–
3
Dawley rat model. Vitamin D showed both significant estrogen agonistic (82%) as
3
well as estrogen antagonistic activity (39%), which highlights its ability to interact
with estrogen receptors. Since the aim of this study was to design and synthesize
modified estrogens as bone-selective antiproliferative agents, we therefore evaluated
the synthesized benzopyran derivatives 17 and 18, which were made as hybrids of
estrogen and vitamin D3 (pseudo vitamin D3 analogs) for estrogen agonistic,
antagonistic activities. The designed compounds (17 and 18), which incorporate 3-
phenyl-benzopyran nucleus as present in Equol 8, and several SERMs such as
Ormeloxifene, CDRI-85/287, and EM-800, were expected to have mixed estrogen
agonistic and antagonistic activities through their interaction with estrogen receptor
(
ER) similar to Equol, ormeloxifene, and raloxifene. Estrogen agonistic and
antagonistic activities were determined in vivo using EE as standard, whereas
ormeloxifene and raloxifene, selective estrogen receptor modulators used for
treatment of osteoporosis, were used as standard for comparisons of their estrogen
agonistic/antagonistic activities with newly synthesized compounds 17 and 18. Tested
compounds showed significant estrogen agonistic activity (up to 48%) and weak
estrogen antagonistic activity (up to 6%). Interestingly, during in vivo evaluation of
these compounds in rats, we did not observe any apparent toxicity of these compounds.
The biological activities of the tested compounds are presented in Table 1.
Conclusion
In summary, the newly designed substituted 3-phenyl,7-methoxy-benzopyran deriv-
atives, which were made as hybrids of estrogen and vitamin D (pseudo vitamin D
analogs), showed significant estrogen agonistic activity (up to 48%) and weak
3
3
estrogen antagonistic activity (up to 6%) at 10 mg/kg, whereas vitamin D showed
3
significant estrogen agonistic (82%) and antagonistic activity (39%) at 10 mg/kg. As
anticipated significant in vivo estrogen agonistic and estrogen antagonistic activities
of vitamin D (cholecalciferol, 1) highlight its ability to interact with estrogen
3
receptors. In comparison with vitamin D , raloxifene, and ormeloxifene, the
3
synthesized benzopyran derivatives showed low order of activities, which prevented
our further experimentation. Further investigation of such molecules could be of
interest for the design of drugs for management of bone disease such as osteoporosis.

Med Chem Res (2010) 19:25–32
31

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Med Chem Res (2010) 19:25–32
Acknowledgements Atul Gupta thanks CSIR (INDIA) for Senior Research Fellowship. The authors
thank Ms. Mohini Chhabra for efficient technical assistance and the Ministry of Health and Family
Welfare, Government of India for financial support.
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