Induction of targeted osteogenesis with 3-aryl-2H-benzopyrans and 3-aryl-3H-benzopyrans: Novel osteogenic agents

Article abstract of DOI:10.1016/j.jsbmb.2016.01.010

Development of target oriented chemotherapeutics for treatment of chronic diseases have been considered as an important approach in drug development. Following this approach, in our efforts for exploration of new osteogenic leads, substituted 3-aryl-2H-be

Full text of DOI:10.1016/j.jsbmb.2016.01.010

Accepted Manuscript
Title: Induction of targeted osteogenesis with
3-aryl-2H-benzopyrans and 3-aryl-3H-benzopyrans: Novel
osteogenic agents
Author: Atul Gupta Imran Ahmad Jyoti Kureel Mohammad
Hasanain Praveen Pandey Sarita Singh Aijaz A. John Jayanta
Sarkar Divya Singh
PII:
S0960-0760(16)30010-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jsbmb.2016.01.010
SBMB 4617
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date:
Revised date:
Accepted date:
6-8-2015
1-12-2015
20-1-2016
Please cite this article as: Atul Gupta, Imran Ahmad, Jyoti Kureel, Mohammad
Hasanain, Praveen Pandey, Sarita Singh, Aijaz A.John, Jayanta Sarkar, Divya
Singh, Induction of targeted osteogenesis with 3-aryl-2H-benzopyrans and 3-aryl-3H-
benzopyrans: Novel osteogenic agents, Journal of Steroid Biochemistry and Molecular
Biology http://dx.doi.org/10.1016/j.jsbmb.2016.01.010
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Induction of targeted osteogenesis with 3-aryl-2H-
benzopyrans and 3-aryl-3H-benzopyrans: Novel
osteogenic agents
a
a
Atul Gupta, * Imran Ahmad, Jyoti Kureel,b Mohammad Hasanain,c Praveen
Pandey, c Sarita Singh, a Aijaz A John, b Jayanta Sarkar, c Divya Singhb
aMedicinal Chemistry Department, CSIR-Central Institute of Medicinal and aromatic Plants,
b
c
PO. CIMAP, Kukrail Road, Lucknow-226015, India, Division of Endocrinology and Division
of Biochemistry, CSIR-Central Drug Research Institute, Sector-10, Jankipuram extension,
Lucknow-226031, India
*Atul Gupta, Tel.: 0915222718556, E-mail: atisky2001@yahoo.co.in (A. G.),
1

Graphical abstract
Highlights:
 3-aryl-2H-benzopyran and 3-aryl-3H-benzopyran derivatives as novel osteogenic agents.
 20b, 22a, 27and 32 showed significant osteogenic activity at EC50 values 1.35, 34.5, 407
and 29.5pM.
 20b and 32 significantly increased mineral nodule formation and the transcript levels of
BMP-2, RUNX-2 and osteocalcin at 100 pM.
2

 22b showed potent anticancer activity at IC50 6.5-13.2 µM in human cancer cell lines, by
inducing apoptosis and arresting cell cycle at sub-G0 phase.
Abstract; Development of target oriented chemotherapeutics for treatment of chronic diseases
have been considered as an important approach in drug development. Following this approach, in
our efforts for exploration of new osteogenic leads, substituted 3-aryl-2H-benzopyran and 3-aryl-
3H-benzopyran derivatives (19, 20a-e, 21, 22a-e, 26, 27, 28a-e, 29, 31a-b, 32 and 33) have been
characterized as estrogen Receptor-β selective osteogenic (bone forming) agents. The
synthesized compounds were evaluated for osteogenic activity using mouse calvarial osteoblast
cells. Four compounds viz 20b, 22a, 27and 32 showed significant osteogenic activity at EC50
values 1.35, 34.5, 407 and 29.5pM respectively. Out of these, 20b and 32 were analyzed for
their bone mineralization efficacy and osteogenic gene expression by qPCR. The results showed
that 20b and 32 significantly increased mineral nodule formation and the transcript levels of
BMP-2, RUNX-2 and osteocalcin at 100 pM concentrations respectively. Further mechanistic
studies of 20b and 32 using transiently knocked down expression of ER-α and β in mouse
osteoblast (MOBs) showed that 20b and 32 exerts osteogenic efficacy via activation of estrogen
receptor-β preferentially. Additionally, compounds showed significant anticancer activity in a
panel of cancer cell lines within the range of (IC50) 6.54 to 27.79 µM. The most active molecule,
22b inhibited proliferation of cells by inducing apoptosis and arresting cell cycle at sub-G0 phase
with concomitant decrease in cells at S phase.
Keywords: Estrogen, antiestrogen, SERMs, isoflavone, anticancer, antiosteoporotic.
3

1.0 Introduction:
The skeleton is a highly specialized and dynamic organ that undergoes continuous
regeneration [1,2]. Once the skeleton attains maturity, regeneration continues in the form
of a periodic replacement of old bone with new one at same location [3]. This process is
known as remodelling and is responsible for complete regeneration of adult skeleton
every ten years. The bone remodelling involves two highly synchronized biological
processes namely osteoblastogenesis and osteoclastogenesis through osteoblast and
osteoclast cells respectively [4,5]. Any imbalance in the process of remodelling which
leads to exaggerated bone resorption culminates in a disorder known as osteoporosis [6a-
b]. It is now established that Osteoprotegrin (OPG)/ receptor activator NFκB ligand
(RANKL)/ receptor activator NFκB (RANK) system is a dominant and ultimate mediator
of osteoclastogenesis [7]. Osteoprotegrin, is a member of tumour necrosis factor receptor
superfamily IIB (TNF II B) and is a decoy receptor for the receptor activatror NF κB
ligand (RANKL) on osteoblast/stromal cells [8]. It blocks the RANKL-RANK ligand
interaction between osteoblast/stromal cells and osteoclast precursor cells thus inhibits
differentiation of osteoclast precursor cell into a mature osteoclast cell and eventually
bone resorption.
Furthermore, estrogen has great role in bone remodelling and management by acting as
bone resorption inhibitor as well as osteogenic agent. It has been reported to stimulate
production of Osteoprotegrin (OPG) and to down regulate many cytokines such as
iterlukins IL-1, IL-6, TNF-α and monocyte/macrophage colony stimulating factor (M-
CSF) involved in bone resorption [9,10,11a-b]. The anti-resorptive action of estrogen was
further shown by Parika et.al. who concluded that estradiol treatment significantly
4

decreased the depth of resorption pits by disturbing the metalloproteinases and cysteine
proteinases mediated organic matrix degradation [12]. The osteogenic effect of estrogen is
through their involvement in stimulated differentiation and activity of osteoblast cells
[13]. Osteobalst cells are reported to express both estrogen receptor-alpha (ER-α) and
estrogen receptor-beta (ER-β) and their differential level of expression during human
osteoblast differentiation reveal gradual increase in ER-β mRNA expression [14]. The
observed differential regulation of ER is suggestive for an additional functional role of
ER-β to ER-α in bone maintenance [15].
In elderly persons specially in women after menopause, the decreased level of estrogen
leads to lack of adequate new bone formation but also exaggerate bone resorption leading
to osteoporosis [16,17]. To compensate the deficiency of estrogen at this stage of life
Estrogen replacement or hormone replacement therapies are generally recommended [18].
However, apart from beneficial effects, these options are also associated with some
serious side effects especially in reproductive tissues and offer threats for estrogen
dependent cancers such as breast, uterine and ovarian cancer. With the advance studies of
ER-α and ER-β, it is now established that ER-α is dominant in the adult uterus and
deletion of ER-β does not affect the response of uterus to estrogen [19,20]. Similarly, ER-
α dominates in breast cancer tissue and is the main culprit for excess cell proliferation
while ER-β is less abundant and has antagonising effect in this tissue [19,20]. These
findings suggest that ligands selectively targeting ER-β may be an important therapeutic
strategy for the clinical management of osteoporosis without or minimal threats for
secondary complications such as breast, uterine and ovarian cancer [20]. A deeper insight
5

into the ligand binding pocket (LBP) of the ER-α and ER-β, reveals that although LBP of
ER-α is slightly larger that ER-β but both are lined with nearly identical residues expect
two amino acid differences [19-22]. The first difference is in the residues below the D-
ring pocket of the binding cavity where ER-α- has Met-421 while ER-β has Ile-373 [21].
The second difference is found above the D-ring pocket where ER-α has Leu-384 while
ER-β has Met-336. These differences in LBP among ER-α and ER-β imparts subtle
differences between two receptor binding cavities which can be used for development of
receptor isofrom selective ligands i.e. ER-α vs ER-β selective ligands [19,21,22]. Also,
there is ample scope for structural modification in the region of D-ring pocket of cavity.
A successful story based on this fact is the development of TAS-108 (SR-16,234) and SR
16,137 [23a-d].
Further, it is interesting to note that difference in the size of the binding cavities and the
above mentioned differences in the amino acid residues of both receptors make the ER-α
more selective and accommodative towards three dimensionally bulky molecules such as
17β-estradiol (3) and other steroids while ER-β more towards structurally cylinderical
molecules such as such as daidzein (
4), genistein (5), formononetin (6) and
isoformononetin ( ) and their reductive metabolites such as equol (8-10) [20-24].
7
Based on these observations, in the present study, we planned to develop 3-aryl-2H-
benzopyran and 3-aryl-3H-benzopyran based non-steroidal structurally cylindrical
molecules as novel target oriented osteogenic agent. It was proposed that these molecules
will have an aryl group substituted with tertiary alkylamino group, usually present in
selective estrogen receptor modulators (SERMs) in a region encompassing D-ring of the
17β-estradiol along with a phenolic or its substituent analogous to its phenolic group of
6

present in ring A. To achieve the objective of the project following prototypes (11 and 12
)
were designed and synthesized.
2.0 Chemistry:
The synthetic strategy for designed molecules 11 and 12 required 7-hydroxy-3-(4-
methoxyphenyl)-3H-benzopyran-4-one (18) and 7-methoxy-3-(4-hydroxyphenyl)-2H-
benzopyran-4-one (29) in large quantity. The synthesis of 18 was done through Friedel-
Craft acylation reaction on resorcinol (13) using 4-methoxyphenyl acetic acid (14) in
BF3-OEt2 at 100°C which yielded 15 in 75-80 % yield (Scheme-1) [25]. Compound 15
was subjected to alkylation reaction using benzyl bromide in presence of anhydrous
potassium carbonate in dry acetone at reflux which gave corresponding benzylated
derivative (16) in 78 % yield. Further, cyclization of 16 to get desired 7-benzyloxy-3-(4-
methoxyphenyl)-3H-benzopyran-4-one
(17) using paraformaldehyde in 50% NaOH
solution at 150°C yielded 17 in 85% yield. Controlled catalytic hydrogenation of 17 using
10% palladium charcoal (Pd/C) in Tetrahydrofuran (THF) gave 7-hydroxy-3-(4-
methoxyphenyl)-3H-benzopyran-4-one (18) in 90% yield however, under these reaction
conditions, prolong reduction of 17 yielded 7-hydroxy-3-(4-methoxyphenyl)-3H-
benzopyran (19) in 88% yield. Furthermore, Grignard reaction performed on 18 using
methylmagnesium bromide (CH3MgBr) in THF-OEt2 at reflux gave 21 in 65% yield.
Finally, compounds 19 and 21 were subjected to alkylation reaction using different
chloroethylalkylamines in presence of anhydrous potassium carbonate in dry acetone
which gave target compounds 20(a-e) and 22(a-e) in 77-90% yields.
7

The synthesis of 7-methoxy-3-(4-hydroxyphenyl)-2H-benzopyran-4-one (29) was
attempted following above synthetic methodology used for synthesis of 18, however, this
approach could not yield 29 in good quantity. Therefore, an alternate approach was
adopted for synthesis of 21. In this approach, synthesis of 29 was started from
deoxybenzoin (25), which was synthesized through Friedel-Craft acylation reaction of 3-
methoxyphenol (23) and 4-hydroxyphenylacetic acid (24) in BF3-OEt2 at 100°C in 74%
yield (Scheme-2). Compound 25 was then reacted with dimethylformamide-
dimethylacetal (DMF-DMA) in dimethylformamide (DMF) at room temperature to yield
7-methoxy-3-(4-hydroxyphenyl)-benzopyran-4-one (26) in good yield. Controlled
catalytic hydrogenation with palladium charcoal (Pd/C) in Tetrahydrofuran (THF) gave 7-
methoxy-3-(4-hydroxyphenyl)-2H-benzopyran-4-one (29) in 50% yield along with 27
,
however, under these reaction conditions, prolong reaction time gave 7-methoxy-3-(4-
hydroxyphenyl)-3H-benzopyran (27) in 68% yield. Furthermore, Grignard reaction on 26
using methylmagnesium bromide (CH3MgBr) in THF-OEt2 at reflux gave compound 30
in 68% yield. Finally, compounds 27 and 30 were subjected to alkylation reaction using
different chloroethylalkylamines in presence of anhydrous potassium carbonate in dry
acetone gave target compounds 33(a-e) in 81-92% yields.
2.1 Optical Resolution of 20a:
3-aryl-3H-benzopyran derivatives have one chiral centre at C-3 atom of bezopyran
nucleus which can generates two optically active isomers. The synthetic methodology
used for synthesis of 3-aryl benzopyran derivatives in this study yielded racemic mixture
of products. In order to know the effect of orientation of phenyl group present at C-3 on
8

biological activities of 3-aryl benzopyran derivatives, pure enantiomers of 3-aryl-3H-
benzopyran derivatives were required. For this purpose, one of the active compounds, 20a
was selected for optical resolution. The optical resolution of 20a was achieved through its
salt formation with pure enantiomers of tartaric acid in absolute methanol followed by
repeated crystallization [26].
3.0 Biology
3.1 Assessment of ostegenic potential of compounds using Osteoblast differentiation assay
Generally, alkaline phosphatase (ALP), osteoclacin (OCN) and type-1 collagen are some
important bone markers which are frequently used to follow the differentiation process of
osteoblasts [27]. Among these bone markers, ALP and type-1 collagen are considered as
early markers of osteoblastogenesis. In our study, we have used ALP as a marker of
osteoblast differentiation to assess osteogenic activity in mouse calvaria derived
osteoblast cells. Bone forming activity of 19, 20a-e, 21, 22a-e, 26, 27, 28a-e, 29, 32 and
33 was thus evaluated by measuring ALP activity. The mouse calvarial osteoblast cells
were treated with test compounds and ALP activity was measured spectrophotometrically.
Compounds 20a-e, 22a-e, 26, and 32 showed significant increase in ALP activity
compared to the control untreated cells. Since, Equol (10), an active metabolite of
Daidzein (
affinity for estrogen receptor-β [34, 35, 40a], it was taken as a positive control in this
study. Among the compounds under study, four compounds viz 20b 22a 27, and 32
4), inhibits bone loss apparently without estrogenic activity and has a high
,
,
showed maximum osteogenic efficacy as assessed by ALP activity at concentrations of
EC50 1.35, 34.5, 407 and 29.5pM respectively, compared to the control untreated cells
(Tabe-1). Considering significant ALP activity (osteogenic activity) of tested compounds,
9

two potential compounds, 20b and 32 with lowest EC50 values, were further evaluated for
their bone mineralization and osteogenic gene expression efficacy (discussed below).
Further, it was also determined if these compounds stimulated osteogenesis via estrogen
receptor mediated pathway.
3.2 In-vitro toxicity in healthy osteoblast cells:
Once the bone forming activity of these compounds was determined the next step was to
confirm that these compounds were devoid of any toxicity. To exclude this possibility,
compounds 20a-e, 22a-e, 26, 32 and equol (10) which showed significant ALP activity
were also evaluated for in-vitro cytotoxicity using osteoblast cells in MTT assay. In this
assay, the viable cells convert the tetrazolium dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] into its insoluble formazon, a purple reaction product,
while dead cells remained uncoloured. In general, the toxicity results showed that
compounds 20a-e, 22a-e, 26, 32 and equol (10) did not have any inherent toxicity for
healthy osteoblast cells at doses ranging from 1pM to 1µM concentration. The cytotoxic
activity results of the most active compounds viz 20b and 32 have been presented in
Figure 2a-b. Though 20b at concentration 1nM exhibited a trend towards higher
proliferation but statistically no significance was observed.
3.3 Mineralization efficacy of 20b and 32 in calvarial osteoblast cells:
The process of osteoblstogenesis involves differentiation of preosteoblastic cells into
terminally differentiated osteoblast cells mainly in three stages viz proliferation, matrix
development and maturation and mineralization. The most reliable indicator of
10

differentiation of osteoblastic precursor cells to the terminal osteoblastic phenotype is the
mineralization. Therefore, 20b and 32 which showed potential alkaline phosphatase
(ALP) activity were further evaluated for their osteogenic activity in osteoblast
mineralization assay. To assess the osteoblast mineralization efficacy of the compounds,
calvarial osteoblast cells were cultured for 18 days in differentiation media containing
10mM β-glycerophosphate and 50 μg/ml ascorbic acid in presence or absence of 20b and
32. Cells were then stained with alizarin red-S and dye was extracted to quantify the
extent of osteoblast mineralization. The alizarin dye stains newly formed mineralized
nodes which accumulate calcium. After treatment, optical densitometry was used to
quantitate mineralization of osteoblast cells by measuring alizarin extracted from stained
cultures.
Both compounds exhibited significant increase in mineral nodule formation at
concentrations as low as 100pM and 10nM compared to the control untreated cells
(Figure 3a and b). The biological activity data showed that 20b and 32 significantly
enhanced the matrix mineralization. In fact, 20b was even more potent than the standard
drug. The percent increase in mineralization by 20b at 100pM concentration over equol
was found to be ~40% (Figure 3a-b).
3.4 Study of 20b and 32 mediated Osteoblastogenesis related osteogenic gene expression:
Bone morphogenetic proteins (BMPs) are members of transforming growth factor-β
(TGF-β) superfamily which play an important role in osteoblast differentiation [28].
Among other BMPs, BMP-2 stimulates osteoblast phenotype expression such as increase
in ALP activity and collagen synthesis [29]. Additionally, Runt-related transcription
11

factor-2 (RUNX-2) is a protein which is a transcription factor associated with osteoblast
differentiation and skeletal morphogenesis [30a-b]. Further, osteoclacin (OCN) is an
established late differentiation marker for bone formation [31]. Considering these markers
as important indicators of bone formation, the mRNA expression levels of these markers
were evaluated in presence of 20b and 32 for further confirmation of their bone forming
potential. To determine the effect of 20b and 32 on osteogenic markers viz BMP-2,
RUNX-2, and OCN, osteoblast cells were treated with compounds at 100pM
concentration for 24 and 48h, total RNA was isolated and cDNA was synthesized. cDNA
was used as a template in real time quantitative PCR. The house keeping gene GAPDH
was used as the internal control in our experiments. Results showed that treatment of
osteoblast cells with 20b and 32 at 100pM concentration increased the transcript levels of
BMP-2 (1.7 and 2.5 fold by 20b at 24 and 48h respectively; 1.8 fold and 1.5 fold by 32 at
24 and 48h respectively), RUNX-2 (2.5 and 2.8 fold at 24 and 48 h respectively by 20b; 2
fold and 1.7 fold at 24 and 48h respectively by 32), and osteocalcin (~2 fold at 24 and 48h
by 20b; ~1.8 fold at 24 and 48h by 32) which are the important markers of osteoblast
differentiation (Figure 4a-c). Based on the mineralization and qPCR data, 20b was found
to have the best activity and was further taken up for the mode of action studies.
3.5 Selectivity of estrogen receptor (ER) -α and β in action of compound 20b
Most of the isoflavones such as daidzein (
4), genestein (5), formononetn (6),
isoformononetin ( ) and Equol (10) induce bone formation with inhibition of bone
7
resorption and have affinity for both estrogen receptors (ER-α and β) [32-36]. Structurally
all these molecules have cylindrical core structure. It is established that molecules with
12

cylindrical and nearly flat core nucleus have preferential affinity for ER-β isoform [37,
38]. Therefore; it was interesting to elucidate that whether 20b exhibited its osteogenic
effect through estrogen receptors and had any receptor subtype selective mode of action.
For this purpose, osteoblast cells were treated with compounds for 48h, following which lysates
were prepared, electrophoresced on SDS-PAGE gel and probed with antibodies against ER-α and
ER-β. It was observed that 20b enhanced the protein expression levels of both ER-α and ER-β.
However, 20b showed selectivity towards ER-β significantly. Equol was taken as a positive
control (Figure 5a-b). Based on the above observations, we propose that 20b and its analogues
exhibits osteogenic effect by enhancing osteoblast differentiation and mineralization in an
estrogen receptor dependent manner.
3.6 Validation of estrogen receptor selectivity by gene silencing
In order to further validate the specific role of ER-α and ER-β in osteogenic effect of 20b
,
we performed selective knockdown of ER-α and ER-β in mouse osteoblasts (MOBs).
Small interfering siERα and siERβ efficiently reduced their respective protein levels thus
also confirming the selectivity of knockdown of the siRNA constructs (Figure 6a).
Subsequently, we determined ALP activity in the presence of selective ER siRNAs. As
expected, 20b strongly induced ALP activity and this increase was abrogated in presence
of siERβ but not siERα (Figure 6b). These results showed that 20b stimulates osteoblast
function through activation of ER-β (Figure 6b).
13

3.7 In vitro anticancer activity
In bone remodelling process, the level of RANKL and OPG is an important and most
likely determinant factor. While elevated level of OGP helps in bone formation, RANKL
favours bone resorption. Some cancerous cells especially breast cancer cells are capable
of imitating normal hormonal and local signals through secretion of parathyroid hormone
related protein (PTHrP) which induces increased level of RANKL relative to OPG, thus
promotes bone resorption and consequently increases chances of breast cancer metastasis
to bone. Keeping this fact in consideration, compounds 19, 20a-e, 21, 22a-e, 26, 27, 28a-
e, 29, 31a-b, 32 and 33 were further evaluated for their anticancer activity in a panel of
cancer cell lines viz MCF-7 (ER+ breast adenocarcinoma), DLD1 (colorectal
adenocarcinoma), A549 (lung carcinoma), FaDu (hypopharyngeal carcinoma), DU 145
(prostate carcinoma). These cell lines were procured from American Type Culture
Collection (ATCC), Manassas, VA, USA. Out of twenty four compounds, 20a-e
, 22a-e,
28a-e 31a-b and 33 showed significant anticancer activity in the range of (IC50) 6.54 to
,
27.79 µM concentration. The anticancer activity of 19, 20a-e, 21, 22a-e, 26, 27, 28a-e,
29, 31a-b, 32 and 33 is described by half maximal inhibitory concentration (IC50) value in
Table 2. In these experiments, tamoxifen (TAM) was used as positive control (Table 2).
Compound 22a-e and 31a-b were found to be most active compounds among the series.
The most active molecule 22b was further taken up for cell cycle analyses and other
biological experiments.
14

3.8 Cell division cycle study of 22b
Compound 22b was further studied for detailed biological characterization. For this
purpose, estrogen receptor (ER) positive human breast cancer cell line (MCF-7) was
selected for subsequent biological assays. The effect of 22b on cell division cycle was
studied. After 24 and 48 hrs incubation with the test compound at 9 µM concentration
(near IC50 value), 22b showed time dependent significant accumulation of cells
population at sub-G0 phase compared to the vehicle treated controls (Figure 7a-b). This
was associated with concomitant decrease in cells at S phase and marginal decrease in
cell population at G2/M phase. We further investigated ability of 22b to trigger apoptosis
in MCF-7 cells. In the present study, we monitored cleavage of Poly (ADP-ribose)
polymerase (PARP) which is considered as a marker of apoptosis. Consistent with
microscopic observation, Western blot analyses of MCF-7 cell lysates revealed
proteolysis of PARP in 22b treated cells confirming induction of apoptosis (Figure 8).
These results are in agreement with cytotoxic activity data where significant increase in
apoptotic population at Sub-G0 was observed after 20b treatment. Compound 22b caused
marked fragmentation of MCF-7 cellular nuclei in comparison to the vehicle control
group (Figure 8). In these experiments, doxorubicin (Doxo), a known apoptosis inducer,
was used as positive control.
4.0 Structure activity relationship (SAR) study
In general designed compounds belong to two different sub sets namely 3-aryl-3H-
benzopyran (20a-e and 28a-e) and 3-aryl-2H-benzopyran (22a-e and 31a-b) derivatives.
The biological activity pattern of 3-aryl-3H-benzopyran derivatives (20a-e and 28a-e
)
15

revealed that positions 4’and 7 of 3-aryl-3H-benzopyran core were optimal locations for
substitution of methoxy and tertiaryaminoalkyl groups respectively to elicit bone forming
activity. The reverse situation yielded compounds with devoid of any activity. Almost,
similar trend of biological activity vs structural requirement of compounds was observed
as far as their anticancer activity was concern. Additionally, a comparison of bone
forming activity of 19 and 27 showed that the presence of a hydroxyl group at position 4’
and a methoxy group at position 7 of 3-aryl-3H-benzopyran core (compound 27) was
required for bone forming activity rather reverse substituent pattern which yielded
corresponding inactive compound (19). It is reported that daidzein (4) and genistein (5)
which also incorporate 3-arylbenzopyran core fit into the ligand binding pocket of
estrogen receptor in such a way that their pendent phenyl ring (ring C, figure 1) present at
C-3 of benzopyranone core simulates with ring A of estradiol and benzopyranone (rings
A and B) simulates with rings C and D of estradiol [20,21,37]. The similar observations
were also made during SAR study of a potent 2,3-diarylbenzopyran based anti-estrogenic
drug candidate, CDRI 85/287, wherein 3-arylbenzopyran, a part of the molecule, was
reported to bind with ligand binding pocket as described for daidzein and geinstein [39].
Perhaps, most logistically, compounds based on 3-aryl-3H-benzopyran nucleus would
have followed the same binding pattern within ligand binding pocket of ER. Thus, in case
of compounds substituted with tertiaryaminoalkyl group at position 7 and a methoxy
group at position 4’, such binding pattern would have allowed the bulky
tertiaryaminoalkyl group to be accommodated in the D-ring region of LBP where D-ring
of estradiol fits in. However, in case of reverse substitution pattern, the bulky
tertiaryaminoalkyl group would have been projected in a region where A-ring of estradiol
16

fits in (A-ring region of LBP) which is not a favourable condition as for ligand binding
with ER and therefore compounds could not show any activity (table-1).
Further, to see the effect of orientation of aryl group present at C-3 of 3-aryl-3H-
benzopyran nucleus, resolution of enantiomers of 20a was made. Interestingly, while
levorotatory isomer of 20a
isomer (32) showed potent bone forming activity as compared with racemic mixture
20a). Possibly, the reported enantiospecific estrogenic activity of 3-aryl-3H-benzopyran
(33) presented improved anticancer activity, its dextrorotatory
(
derivative such as S (-)-equol could have been the reason for such observed activities.
[40, 41]. Surprisingly, conversion of 3-aryl-3H-benzopyran core to 3-aryl-2H-benzopyran
led to compounds with significant anticancer and bone forming activities (compounds
22a-e and 31a-b, table 1 and 2). However, as observed in 3-aryl-3H-benzopyran
derivatives, in this case, the impact of substitution of tertiaryaminoalkyl and methoxy
groups in 3-aryl-2H-benzopyran derivatives was negligible. The plausible reason for such
activity pattern could be that introduction of C=C bond in 3-aryl-3H-benzopyran core
incorporated stilbene framework within the molecule which could have improved
molecular affinity to estrogen receptors as reported literature [42].
Further addition of alkyl group, especially methyl in this case, to the newly generated
stilbene framework resulted molecule with equal similarities of rings A and B of
benzopyran with either rings A and B or rings C and D of the estradiol. Therefore, with an
assumption that molecules will tend to accommodate the bulky tertiaryaminoalkyl group
in ample space provided near the D-ring region of LBP, compounds based on 3-aryl-2H-
benzopyran core had equal options to bind with ER by either A ring or C ring of 3-aryl-
2H-benzopyran derivatives and elicited biological activity irrespective of substitution
17

pattern. As discussed above, the preferential ER-β affinity of most active osteogenic
molecule 20b can be ascribed to slender core structure of the molecule as described for 3-
arylbenzopyran based phytoestrogens such as daidzein and equol [25, 37, 40a].
Additionally, the pyrrolidinoethyl group present at position 4’ of 3-aryl-3H-benzopyran
core in 20b would have been well accommodated in the D-ring pocket of LBD of ER-β
rather than ER-α which possibly helped in induction of ER-β selectivity of 20b
.
5.0 Conclusion:
The approved therapies for osteoporosis are mainly focused on use of bone resorption
inhibitors. The recent observations have revealed that bone resorption is accompanied by
inhibited bone formation due to coupling of these two processes which represents main
disadvantages associated with bone resorption inhibitors [42,43]. The attention has now
been focused on osteogenic agents for osteoporosis treatment [44]. In our efforts, we have
logically modified 3-aryl-benzopyran core as in order to develop novel osteogenic agent.
The designed molecules showed significant osteogenic activity through preferential
activation of estrogen receptor-β and were devoid of any inherent cytotoxicity to healthy
osteobalst cells in nano to pico molar concentrations. The mechanistic studies of 20b and
32 showed their ability to increase transcript level of important bone markers such as
BMP-2, RUNX-2 and osteocalcin at 100pM concentrations. The synthesized compounds
(
20a-e, 22a-e, 28a-e, 31a-b and 33) also presented significant anticancer activity in a
panel of cell lines within the range of (IC50) 6.54 to 27.79 µM concentration. Effect of
22b on cell cycle of MCF-7 cells revealed that 22b inhibited cell proliferation by
18

inducing apoptosis and arresting cell cycle at sub-G0 phase with concomitant decrease in
cells at S phase.
Overall, 3-aryl-2H-benzopyran and 3-aryl-3H-benzopyran derivatives have been
identified as novel structural types of potential osteognic and anticancer agents. Taken
together the activity profile of these molecules, it is predictable that such 3-aryl-
benzopyran based molecules could be develop as target oriented potential osteogenic
agents which have minimal or no threat for development of estrogen dependent cancers or
osteogenic sarcoma.
6.0 Experimental Section
Biological methods: Reagents and chemicals
Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA) and
all fine chemicals from Sigma Aldrich (St. Louis, MO). ECL kit was obtained from
Amersham Pharmacia, USA. All antibodies for Western blot analysis were obtained from
Cell Signaling Technologies (Danvers, MA).
Culture of mice calvarial osteoblasts
Mouse calvarial osteoblasts were obtained following our previously published protocol of
sequential digestion. The study was conducted in accordance with current legislation of
animal experiments (Institutional Animal Ethical Committee at Central Drug Research
Institute). Male or female Balb/c mice of 1-2 days old were used for culture. In Brief,
calvaria from neonatal mice (both sexes) were pooled. After surgical isolation from the
skull and removal of sutures and adherent mesenchymal tissues, calvaria were subjected
19

to five sequential (10–15 min) digestions at 37ºC in a solution containing 0.1% dispase
and 0.1% collagenase P. Cells released from the second to fifth digestions were pooled,
centrifuged, resuspended, and plated in T-25 cm2 flasks in a-MEM containing 10% FCS
and 1% penicillin/streptomycin (complete growth medium).
Osteoblast differentiation
For the measurement of alkaline phosphatase (ALP) activity, osteoblasts at ~80%
confluence were trypsinized and 2 x 103 cells per well were seeded in 96-well plates.
Cells were treated with different concentrations of the compounds for 48 h in a-MEM
supplemented with 5% Fetal bovine serum Serum, 10 mM ß glycerophosphate, 50 µg/mL
ascorbic acid and 1% penicillin/streptomycin (osteoblast differentiation medium). At the
end of the incubation period, the total ALP activity was measured using p-
nitrophenylphosphate (PNPP) as a substrate and quantified colorimetrically at 405 nm.
After that we quantified percentage activity of ALP. Then transformed the data using X =
Log [X] and analyzed in non-linear regression (curve fit). Sigmoidal dose response was
obtained by using prism software and calculated the Emax and EC50 values. Emax is the
maximum response achievable from a drug/compound. The concentration of
compound/drug at which effect (E) is 50% of Emax is termed the half maximal effective
concentration and is abbreviated EC50.
MTT assay
For this assay 1 × 104 cells/well were seeded in 100 µL DMEM, supplemented with 10%
FBS in each well of 96-well microculture plates and incubated for 24 h at 37°C in a CO2
20

incubator. Compounds, diluted to the desired concentrations in the culture medium, were
added to the wells with respect to the control. After 48 h of incubation, media were
removed and to each well 100 uL MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl
tetrazolium bromide) (5 mg/mL) was added and plates were further incubated for 4 h.
Supernatant from each well was carefully removed, formazon crystals were dissolved in
100 µL of DMSO and the absorbance at 570 nm wavelength were recorded.
Mineralization of calvarial osteoblast cells
For mineralization studies, calvarial cells were cultured in a-MEM medium,
supplemented with 10% fetal bovine serum, 50 µg/mL ascorbic acid, and 10 mM ß
glycerophosphate. Cells were cultured with and without compounds for 21 day at 370C in
a humidified atmosphere of 5% CO2 and 95% air, and the medium was changed every 48
h. After 21 d, the attached cells were fixed in 4% formaldehyde for 20 min at room
temperature and rinsed once in PBS. After fixation, the specimens were processed for
staining with 40 mM alizarin redS, which stains areas rich in nascent calcium. Equol was
used as a positive control. For quantification of alizarin red-S staining, 800 µL of 10%
(v/v) acetic acid was added to each well, and plates were incubated at room temperature
for 30 min with gentle shaking. Monolayer, now loosely attached to the plate, was then
scraped with a cell scraper and transferred with 10% (v/v) acetic acid to a 1.5-mL tube.
After vortexing for 30 s, the slurry was over laid with 500 µL mineral oil (Sigma–
Aldrich), heated to exactly 850C for 10 min, and transferred to ice for 5 min. The slurry
was then centrifuged at 20,000 x g for 15 min and 500 µL of the supernatant was removed
21

to a new tube. Then 200 µL 10% (v/v) ammonium hydroxide was added to neutralize the
acid. OD (405 nm) of 150 µL aliquots of the supernatant were measured in 96-well plates.
qPCR assay
Total RNA was extracted from the cultured cells using TRIzol (Invitrogen, Carlsbad,
CA). cDNA was synthesized from 2 µg total RNA with the Revert Aid™ H Minus first
strand cDNA synthesis kit (Fermentas, Vilnius, Lithuania). SYBR green chemistry was
used for quantitative determination of the mRNAs for BMP-2, Runx-2, osteocalcin and a
housekeeping gene, GAPDH, following an optimized protocol. The design of sense and
antisense oligonucleotide primers was done using the Universal Probe Library (Roche
Diagnostics, Indianapolis, IN). Primer sequences for BMP-2 (accession number:
NM_007553.2) were 5′-CGG ACT GCG GTC TCC TAA-3′ (forward) and 5′-GGG GAA
GCA GCA ACA CTA GA-3′ (reverse); for Runx2 (accession number: AF053956.1), 5′-
CCC GGG AAC CAA GAA ATC-3′ (forward) and 5′-CAG ATA GGA GGG GTA AGA
CTG G-3′ (reverse); for osteocalcin (accession number: L24429.1), 5′-TAA GTG GCC
AGA GCC CTT AG-3′ (forward) and 5′-CAG AAA CCA CCC CAG CAC-3′ (reverse)
and for GAPDH (accession number: NM_008084.2), 5′-AGC TTG TCA TCA ACG GGA
AG-3′ (forward) and 5′-TTT GAT GTT AGT GGG GTC TCG-3′ (reverse).
For real-time PCR, the cDNA was amplified with Light Cycler 480 (Roche Diagnostics
Pvt Ltd, Indianapolis, IN). The double-stranded DNA-specific dye SYBR Green I was
incorporated into the PCR buffer provided in the Light Cycler 480 SYBER Green I
Master (Roche Diagnostics Pvt. Ltd, Indianapolis, IN) to allow for quantitative detection
of the PCR product in a 20 µL reaction volume. The temperature profile of the reaction
22

was 95 °C for 5 min, 40 cycles of denaturation at 94 °C for 2 min, and annealing and
extension at 62 °C for 30 s extension at 72 °C for 30 s. GAPDH was used to normalize
differences in RNA expression.
Western blotting
Cells were grown to 60–70% confluence following which they were treated with and
without compound for 48 h. Cells were lysed with cell lysis buffer (Sigma-Aldrich) with
protease inhibitor cocktail (Sigma-Aldrich). Cell lysate was centrifuged at 12000 g for 15
min and supernatant was collected. Estimation of protein concentration was determined
by Bradford assay. Thirty micrograms of total protein was then resolved by 10% SDS-
PAGE gel. After electrophoresis, proteins were transferred onto PVDF membranes
(Immobilon-P, Millipore, Billerica, MA, USA). The membranes were probed with ER-a,
ER-b, PARP and b-Actin antibodies (Cell Signaling Technology, Danvers, MA, USA)
and then incubated with secondary antibodies conjugated with HRP (Cell Signaling
Technology). Immunodetection was done using an enhanced chemiluminescence kit (GE
Healthcare, Little Chalfont, UK) using Image Quant LAS 4000 (GE Healthcare).
Densitometry of blots was done using quantity 1D analysis software and gel doc imaging
system.
SiRNA transfection
Twenty four hours before transfection, mice calvarial osteoblasts (MOBs) were plated in
96 well and six well plates for evaluation of ALP activity and protein expression studies
respectively. The cells were then transfected with 25 nM siC, siERα or siERβ using
Dharmafect I transfection reagent (Dharmacon) in reduced serum media (Opti-MEM)
23

following manufacturer's instructions. For ALP assay and western blotting assay, 16 h
following transfection cells were treated with compound 20b and Equol for 48 h. At the
end of the incubation period, the total ALP activity was measured using p-
nitrophenylphosphate (PNPP) as a substrate and quantified colorimetrically at 405 nm.
For western blotting analysis, cells were lysed with cell lysis buffer (Sigma-Aldrich) with
protease inhibitor cocktail (Sigma-Aldrich). Thirty micrograms of total protein was then
resolved by 12% SDS-PAGE gel. After electrophoresis, proteins were transferred onto
PVDF membranes and probed with ER-α and ER-β antibodies.
Immunodetection was done using an enhanced chemiluminescence kit (GE Healthcare,
Little Chalfont, UK) using Image Quant LAS 4000 (GE Healthcare).
Anticancer assays and other protocols:
SRB assay
In vitro anticancer potential of test molecules was determined by sulphorhidamine B
(SRB) assay. Human cancer cell lines were seeded onto a 96-welled plate (@10,000
cells/well) and grown overnight at 37 °C in 5% CO2 concentration. Serial dilutions of test
compounds were added to the wells and incubated further for 48 h. Cells were then
stained with SRB (0.4% w/v in 1% acetic acid, 50 µL per well) after fixing with 50% ice-
cold Tri-chloroacetic acid. After a brief wash, bound dyes were dissolved in 10 mM Tris
base (150 µL per well) and plates were read at 540 nm. The cancer cell lines were
procured from American Type Culture Collection (ATCC), Manassas, VA, USA.
24

Statistical analysis
Data are expressed as mean ± S.E.M. Cell cycle data was analyzed by student’s t test. The
data obtained from remaining experiments with multiple treatments were subjected to
one-way ANOVA followed by Newman–Keuls test of significance using Prism version
3.0 software.
Acknowledgements
A.G. acknowledges CSIR India for financial support. I.A. acknowledges CSIR, New Delhi, India
for senior research fellowship. Support received from Director, CSIR-CIMAP and CSIR-CDRI
India is dully acknowledged.
25

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