Design and synthesis of 3-arylbenzopyran based non-steroidal vitamin-D3 mimics as osteogenic agents

Article abstract of DOI:10.1039/c6md00469e

3-Arylbenzopyran based non-steroidal osteogenic agents have been explored as structural templates of estrogen and vitamin-D₃. The target molecules 18a-c, 19a-c, 26a-c, 27a-c and intermediates 17a-c and 25a-c were studied for their osteogenic activity in an osteoblast differentiation assay in vitro using mouse calvarial osteoblast cells. Compounds 25c, 26b, 27b and 27c effectively increased ALP activity at 1 pM concentration compared to the untreated cells. The active compounds were devoid of inherent toxicity at 1 pM concentration in osteoblast cells. The most active compound, 27b, was studied for mineralization of osteoblast cells and expression of marker genes, viz. BMP-2, RUNX-2 & Osx, involved in osteogenesis. Molecular docking analysis performed for 27b showed its possible interactions with estrogen receptor-α and -β (ER-α and ER-β) as well as the vitamin-D receptor (VDR).

Full text of DOI:10.1039/c6md00469e

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: I. Ahmad, D. S.
Raghuvanshi, S. Singh, A. A. John, R. PRAKASH, K. S. NAINAWAT, D. Singh, S. Tripathi, A. Sharma and A.
Gupta, Med. Chem. Commun., 2016, DOI: 10.1039/C6MD00469E.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

Page 1 of 15
MedChemComm
Dynamic Article Links ►
MedChemComm
View Article Online
DOI: 10.1039/C6MD00469E
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/medchemcomm
PAPER
Design and synthesis of 3ꢀarylbenzopyran based nonꢀsteroidal vitaminꢀ
D3 mimics as osteogenic agents†
Imran Ahmad,a‡ Dushyant Singh Raghuvanshi,a‡ Sarita Singh,a Aiaiz A Johan,c Ravi Prakash,c Kripa
Shankar Nainawat,a Divya Singh,c Shubhandra Tripathi,b Ashok Sharma,b Atul Gupta*#a
5
3ꢀArylbenzopyran based nonꢀsteroidal osteogenic agents have been explored as a structural template of
estrogen and vitaminꢀD3. The target molecules 18aꢀc, 19aꢀc, 26aꢀc, 27aꢀc and intermediates (17aꢀc and
25aꢀc) were studied for their osteogenic activity in osteoblast differentiation assay in-vitro using mouse
calvarial osteoblast cells. Compounds 25c, 26b, 27b and 27c effectively increased ALP activity at 1pM
10 concentration compared to the untreated cells. The active compounds were devoid of inherent toxicity at
1pM concentration in osteoblast cells. The most active compound, 27b, was studied for mineralization of
osteoblast cells, expression of marker genes viz BMPꢀ2 and RUNXꢀ2 & Osx, involved in osteogenesis.
Molecular docking analysis performed for 27b showed its possible interactions with estrogen receptorꢀα
and β (ERꢀα and ERꢀβ) as well as vitaminꢀD receptor (VDR).
15 Introduction
lithocholic acid (10) and its derivatives, (iii) nonꢀsteroidal
Osteoporosis is an ailment of increasing concern among elderly
women and men.1,2 It is a systemic skeletal disease characterized
by low bone mass and reduced bone strength which leads to
increased fracture risks.3ꢀ5 About 70% of the bone constituents
20 are minerals such as CaPO4, CaCO3, CaF2, and MgCl2 and 30%
of bone is organic matrix called osteoid matrix.6,7 The strength of
bone is greatly reduced by two concurrent processes viz
demineralization of bone to maintain normal level of calcium in
plasma and ablation or slow pace of osteogenesis.8,9 1,2ꢀ
25 dihydroxyvitaminꢀD3 [1,25ꢀ(OH)2D3, 1], an active metabolite of
vitaminꢀD3, plays a vital role in calcium homeostasis, bone
metabolism, cell differentiation and proliferation (Figure 1).10
Chemically, 1,2ꢀdihydroxyvitaminꢀD3 [1,25ꢀ(OH)2D3, 1] is a ring
molecules such as compounds 4ꢀ9.14,15
Furthermore, estrogens e.g. 17βꢀestradiol (11) also play a vital
55 role in bone maintenance (Figure 2).16 These are tetracyclic
steroidal molecules which exert their action through activation of
estrogen receptors α and β (ERꢀα & ERꢀβ).17 Like VDR, estrogen
receptor (ER) is also ligand activated transcription factor and
belongs to nuclear receptor super family.17b Despite its tetracyclic
60 steroidal ligand, ER can also accommodate structurally flexible to
rigid molecules belonging to steroids, secoꢀsteroids and nonꢀ
steroids class of compounds.17
65
70
75
80
B
seco steroid. Structurally, [1,25ꢀ(OH)2D3, 1] bears
30 conformationally flexible side chain connected with
a
geometrically flexible bicyclicꢀtriene core.11 Different structural
modifications have been made in vitaminꢀD3 either by
replacement of flexible triene system or through modification of
its side chain as exemplified by compounds 2ꢀ9 (Figure 1).12ꢀ13
35 These modifications indicated the importance of side chain in
vitaminꢀD3 molecule for its biological activities. Interestingly,
despite the molecular flexibility of vitaminꢀD3, its prime
biological target i.e. vitamin D receptor (VDR) is able to
accommodate (i) structurally flexible secoꢀsteroids, such as
40 vitaminꢀD3 and its analogues, (ii) tetracyclic steroids such as
aMedicinal Chemistry Department, &
Biotechnology Division, CSIR-
b
Central Institute of Medicinal and aromatic Plants, Lucknow-226015,
India Tel.: 915222718556, Email:atisky2001@yahoo.co.in (A. G.),
45 bDivision of Endocrinology, CSIR-Central Drug Research Institute, Sec-
10, Jankipuram Extension, Lucknow, India,
‡Both author contributed equally,
†The authors declare no competing interests.
Electronic
Supplementary
Information
(ESI)
available:See
85
50 DOI: 10.1039/c000000x/#CIMAP communication No.
This journal is © The Royal Society of Chemistry [2015]
MedChemComm, 2015, XXX, 01–08 | 1

MedChemComm
Page 2 of 15
View Article Online
DOI: 10.1039/C6MD00469E
Fig. 1 VitaminꢀD3 and its modified analogues
yield. Subsequently, 16 was alkylated using different long chain
alkylbromo esters which gave corresponding ester derivatives 17
60 in quantitative yields. Finally, Grignard reaction on 17 was
performed in diethyl etherꢀtetrahydrofuran (Et2OꢀTHF) mixture
to incorporate tertiary alcohol moiety as present in the vitaminꢀD3
side chain. Grignard reaction on 17 yielded compound 18 and 19
in 76ꢀ93 % yields.
It is well established that the major biological role of vitaminꢀD3
in bone is calcium homeostasis whereas estrogen has antiꢀbone
resorptive activity through stimulation of osteoprotegrerin (OPG)
and suppression of RANKEL and TNFα which are involved in
osteoclastogenesis. Estrogen also has impact on vitaminꢀD
metabolism.10b, 18ꢀ20
5
In the present communication, considering the above facts, we
designed substituted 3ꢀarylbenzopyran derivatives in anticipation
65 Similarly, synthesis of prototypeꢀII was done following the
synthetic methodology used for synthesis of prototypeꢀI. Briefly,
FriedelꢀCrafts acylation on 2,4ꢀdihydroxyphenol (20) by 4ꢀ
methoxyphenylacetic acid (21) gave 22 in 72% yield (Scheme 2).
Subsequently, 22 was transformed to formononetin (7ꢀhydroxyꢀ3ꢀ
70 (4ꢀmethoxyphenyl)ꢀbenzopyranꢀ4ꢀone, 23), another naturally
occurring isoflavone, using dimethylformamideꢀdimethylacetal
(DMFꢀDMA) in dry dichloromethane (DCM) at room
temperature in 83% yield. Compound 23 on catalytic
hydrogenation using 5% PdꢀC in THF yielded 3ꢀ(4ꢀ
75 methoxyphenyl)ꢀ7ꢀhydroxyꢀ3Hꢀbenzopyran (24) in 55% yield.
Further, alkylation of 24 using different long chain alkylbromo
esters gave corresponding esters 25, in good yields. Grignard
reaction on 25 was performed to introduce a tertiary alcohol
moiety as present in the vitaminꢀD3 side chain. Grignard reaction
80 on 25 yielded target compounds 26 and 27 in 87ꢀ92 % yields.
10 of osteogenic (bone forming) activity. These molecules were
designed to have 3ꢀarylbenzoyran core for its possible
interactions with estrogen receptor, and a modified side chain as
present in 1,2ꢀdihydroxyvitaminꢀD3 [1,25ꢀ(OH)2D3, 1] and its
analogues.
15
20
25 Fig. 2 17βꢀEstradiol, vitaminꢀD3 and designed prototypes
Results and Discussion
The synthesis of designed prototypeꢀI was started with Friedelꢀ
Crafts acylation on 3ꢀmethoxyphenol (12) by 4ꢀ
hydroxyphenylacetic acid (13) which gave 14 in 72% yield,
30 (Scheme 1). Compound 14 was then converted to
isoformononetin (7ꢀmethoxyꢀ3ꢀ(4ꢀhydroxyphenyl)ꢀbenzopyranꢀ4ꢀ
one, 15),
dimethylformamideꢀdimethylacetal
a
naturally occurring isoflavone, using
(DMFꢀDMA) in dry
dichloromethane (DCM) at room temperature in 75% yield.
35 Compound 15 was subjected to catalytic hydrogenation using 5%
PdꢀC in THF which yielded 3ꢀ(4ꢀhydroxyphenyl)ꢀ7ꢀmethoxyꢀ3Hꢀ
benzopyran (16) in 80%
40
Scheme 2; Reaction conditions and reagents; (a) BF3ꢀOEt2, reflux (b)
DMFꢀDMA in dry DCM at room temperature (c) 5% PdꢀC in THF, 40ºC
85 (d) bromoalkyl ester, anhy. K2CO3, dry acetone (e) C2H5MgI, Et2OꢀTHF,
40ºC (f) CH3MgI, Et2OꢀTHF, 40ºC.
All the synthesized compounds were characterized by the use of
IR, 1H NMR, 13C NMR and mass spectrometry.
90 The synthesized molecules 17aꢀc, 18aꢀc, 19aꢀc, 25aꢀc, 26aꢀc and
27aꢀc were studied for their osteogenic activity in osteoblast
differentiation assay in-vitro using mouse calvarial osteoblast
cells by measuring alkaline phosphatase (ALP) activity of cells.
The active compounds were evaluated for their inherent toxicity
95 in osteoblast cells. Further, the most active compound was
studied for mineralization of osteoblast cells, expression of
marker genes involved in osteogenesis and possible interactions
with etsrogen receptors. The biological activity results are
described below.
45
50
Scheme 1; Reaction conditions and reagents; (a) BF3ꢀOEt2, reflux (b)
55 DMFꢀDMA in dry DCM at room temperature (c) 5% PdꢀC in THF, 40ºC
(d) bromoalkyl ester, anhy. K2CO3, dry acetone (e) C2H5MgI, Et2OꢀTHF,
40ºC (f) CH3MgI, Et2OꢀTHF, 40ºC.
100
2
| MedChemComm, 2016, XXX, 01–08
This journal is © The Royal Society of Chemistry [2016]

Page 3 of 15
MedChemComm
View Article Online
DOI: 10.1039/C6MD00469E
Osteoblast differentiation
(PNPP) as substrate and quantitated colorimetrically at 405 nm.
20 Out of eighteen compounds, four compounds viz 25c, 26b, 27b
and 27c effectively increased in ALP activity at 1pM
concentration compared to the untreated cells (Figure 3).
Interestingly, the increase in ALP activity by these compounds
was less pronounced at higher concentrations which might be due
25 to their biphasic level of activities as reported for other
flavonoids.21 Compound 27b was identified as the most
promising osteogenic agent and therefore 27b (with more than
98% purity) was selected for further studies.
In general, the osteogenic activity depends on the osteoblast
differentiation and is related to the number osteoblast cells.
Osteoblast differentiation can be indentified and quantified by
measuring the level of certain markers of osteoblastogenesis.
Amongst others, Alkaline phosphatase is one of the early marker
of osteoblastogenesis. In our study to evaluate the osteogenic
potential of 17aꢀc, 18aꢀc, 19aꢀc, 26aꢀc, 25aꢀc and 27aꢀc, we have
measured the Alkaline phosphatase (ALP) in mouse calvaria
10 derived osteoblast cells as marker of osteoblast differentiation.
Thus, obtained osteoblast cells were treated with compounds and
the ALP activity of test compounds was measured
spectrophotometrically. Briefly, osteoblast cells were treated with
test compounds at different concentrations for 48 h in αꢀMEM
15 supplemented with 5% FCS, 10 mM βꢀglycerophosphate, 50
ꢀg/mL ascorbic acid and 1% penicillin/streptomycin (osteoblast
differentiation medium). At the end of incubation period, total
ALP activity was measured using pꢀnitrophenylphosphate
5
In-vitro toxicity in healthy osteoblast cells
30 Before further biological evaluation of compounds which had
promising ALP activity, it was imperative to assess their inherent
toxicity. To exclude this possibility, 25c, 26b, 27b and 27c were
evaluated for their toxicity in healthy osteoblast cells in in-vitro
using MTT assay.
35
40
50
45
55
60
65
70
75
Figure 3. Osteoblast differentiation efficacy of test compounds: MCOs were seeded in 96 well plates and exposed to 25c, 26b, 27b and 27c at different
concentrations (ranging from 1pM to 1nM) for 48 h and ALP activity was determined spectrophotometrically at 405 nm. 17βꢀEstradiol (11 at 100 pM)
was used as a positive control. Data shown as mean ± SEM; n=8; (*) P < 0.05, (**)P < 0.01, (***)P<0.001 compared with untreated cells taken as control.
Mineralization efficacy
80 In this assay, the viable cells convert the tetrazolium dye MTT [3ꢀ
Mineralization process is
a most reliable indicator for
(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide] into
insoluble formazan, a purple reaction product, while dead cells
remained uncoloured. The toxicity results showed that 25c, 26b,
27b and 27c did not have any inherent toxicity for healthy
85 osteoblast cells at concentrations ranging from 1 pM to1 ꢁM
(Figure 4).
90 differentiation of preostoblastic cells into mature osteoblast cells.
Therefore, 27b which increased maximum ALP activity at 1pM
concentration was selected for assessment of its mineralization
efficacy at 1pM and 100pM concentrations. For osteoblast
mineralization efficacy evaluation, mouse calvarial osteoblast
95 cells were cultured for 18 days in differentiation media
This journal is © The Royal Society of Chemistry [2016]
MedChemComm, 2016, XXX, 01–08 | 3

MedChemComm
Page 4 of 15
View Article Online
DOI: 10.1039/C6MD00469E
15
20
25
5
10
30
35
40
Figure 4. In-vitro toxicity of 25c, 26b, 27b and 27c in healthy osteoblast cells. Oteoblast Cells were cultured in osteoblast differentiation medium and
45 treated with 25c, 26b, 27b and 27c at different concentrations (ranging from 1 pM to1 ꢁM) for 48 h, and cell viability was assessed by MTT assay. The
percent of viable cells were calculated in comparision with control untreated cells. Data represent the mean ± SEM from three independent experiments.
containing 10mM βꢀglycerophosphate and 50 ꢁg/mL ascorbic
50 acid in presence or absence of 27b. Subsequently, osteoblasts
were stained with alizarin redꢀS. Dye was then extracted to
quantify the extent of osteoblast mineralization. The newly
formed mineralized nodes which accumulated nascent calcium
80
55 were stained by alizarin dye. After treatment, optical
densitometry (OD) was measured to quantify the mineralization
of osteoblast cells by measuring alizarin extracted from stained
cultures.
85
Result of mineralization assay showed that 27b exhibited
60 significant increase in mineral nodule formation at concentrations
as low as 1pM compared to the control untreated cells (Figure 3a
and b). The biological activity data showed that 27b significantly
90
enhanced the matrix mineralization. The percent increase in
mineralization by 27b at 1pM concentration over control
65 untreated cells was found to be ~20% (Figure 5).
95 Figure 5: Mouse calvarial osteoblasts (MCOs) were seeded in 12 well
plates at a density of 2X103 cells per well in differentiation medium and
treated with and without 27b (1pM and 100pM) for 18 days. At the end of
the incubation, cells were fixed and stained with alizarin redꢀS. Stain was
70
extracted, and optical density was measured colorimetrically. Data shown
100 as mean ±S.E.M; n=3; ***p<0.001.
75
4
| MedChemComm, 2016, XXX, 01–08
This journal is © The Royal Society of Chemistry [2016]

Page 5 of 15
MedChemComm
View Article Online
DOI: 10.1039/C6MD00469E
presence of 27b for further confirmation of their bone forming
15 potential. In order to determine the impact of 27b on BMPꢀ2,
RUNXꢀ2, and Osx, osteoblast cells were treated with 27b at 1pM
and 100pM concentrations for 48h. The total RNA was isolated
and cDNA was synthesized which was used as a template in real
time quantitative PCR. The house keeping gene GAPDH was
20 used as the internal control in these experiments. The biological
results showed that 27b at 1pM and 100pM concentrations
significantly increased the transcript levels of BMPꢀ2, RUNXꢀ2
and Osterix (Osx) which are the important markers of osteoblast
differentiation (Figure 6). The ALP activity, mineralization and
25 qPCR data showed that 27b possess best activity and was further
taken up for the mode of action studies.
Study of osteogenic gene expression related to
Osteoblastogenesis
In osteoblast differentiation process, expression of some
biomarker genes such as Bone morphogenetic proteins (BMPs),
Runtꢀrelated transcription factorꢀ2 (RUNXꢀ2), and osteoblastꢀ
specific transcription factor Osterix (Osx) greatly affect
osteoblast phenotype related expression such as increase in ALP
activity, collagen synthesis and skeletal morphogenesis.22ꢀ23
While BMPꢀ2 and RUNXꢀ2 are considered as early markers of
10 osteoblast differentiation, osterix, is essential for osteoblast
differentiation and bone formation.
Considering importance of these markers in bone formation, the
mRNA expression levels of these markers were evaluated in
5
30
35
40
45
50
55
Figure 6: qPCR analysis showing increase in mRNA expression of osteogenic marker genes. Mouse calvarial Osteoblast cells were treated with 27b for
48 h. Expression of mRNA level of BMPꢀ2, RUNX and Osterix (Osx) have been checked compared with control. qPCR data of the indicated genes
represent the mean ± SEM from three independent experiments. (*)P < 0.05, (**)P < 0.01, (***)P<0.001
60
bones might be predominantly mediated by ERꢀβ whereas
Study of estrogen receptorꢀα and ꢀβ in action of 27b
remodelling of cortical bones might be through ERꢀα.25
It is well established that there is differential mRNA expression
of ERꢀα and ERꢀβ during osteoblast differentiation and
Therefore, compounds which can modulate ERꢀβ preferentially
could be helpful in maintenance of cancellous bones which are
75 more susceptible for degeneration under osteoporotic conditions.
In view of these observations, it was worthwhile to evaluate the
65 expression of ERꢀβ increases gradually throughout culture.24
Further, it is also known that ERꢀα is highly expressed in cortical
bones than cancellous (trabecular) bone. Contrary to this, ERꢀβ is
most evident in cancellous bones than cortical bones which
suggests their different functions in bones. It is also indicative
70 that estrogen dependent regulation of remodelling of cancellous
possible interactions of 27b with
ERꢀα and ERꢀβ. For this
purpose, mouse calvarial osteoblasts (MCOs) were treated with
27b for 48h at 1pM and 100pM concentrations. This was
80 followed by preparation of lysates which were then
This journal is © The Royal Society of Chemistry [2016]
MedChemComm, 2016, XXX, 01–08 | 5

MedChemComm
Page 6 of 15
View Article Online
DOI: 10.1039/C6MD00469E
Molecular docking analysis
electrophoresced on SDSꢀPAGE gel and probed with antibodies
against ERꢀα and ERꢀβ. Results showed that 27b enhanced
protein expression levels of both ERꢀα and ERꢀβ. However, 27b
showed selectivity towards ERꢀβ (Figure 7). From these
observations, it appeared that 27b and its analogues exhibit
osteogenic effect via increased osteoblast differentiation followed
by mineralization of osteoblast cells in an estrogen receptor
dependent manner.
In order to evaluate the possible interactions of 27b with estrogen
15 receptorꢀα and ꢀβ (ERꢀα and ERꢀβ) as well as vitaminꢀD receptor
(VDR), we performed molecular docking analysis for 27b using
these receptors. The structure of protein receptors were taken
from PDB database, ERꢀα (PDB ID:3ERT), ERꢀβ (PDB
ID:1QKM) and VDR receptor (PDB ID:3AZ3). Molecular
20 docking experiments for synthesised molecules with receptor
proteins were performed using Autodock 4.2 package.26ꢀ27
5
10
25
30
35
40
45
50
55
Figure 7: Western blot analysis of ERꢀα and ERꢀβ. Mouse osteoblast cells were treated with or without compounds 27b (1pM and 100pM) for 48 h time
point. Protein lysates were collected and transblotted onto a PVDF membrane and probed with primary antibodies followed by the corresponding HRP
linked secondary antibodies and these were normalized with βꢀactin. Graph shows the densitometric analysis (fold change) of the observed change in
60 expression of the proteins.
Molecular docking study of 27b with ERꢀα showed that it had 75 8b).
65 good binding affinity with ERꢀα. 17βꢀestradiol (11), a native
ligand of estrogen receptor, was selected as standard molecule for
compression purpose. 27b had good binding energy (ꢀ9.37 kcal
molꢀ1) and is comparable to the binding energy of 17βꢀestradiol
Further docking experiments showed that 27b had interaction
with VitaminꢀD receptor (VDR) with good binding energy (ꢀ
10.75 kcal molꢀ1) and is comparable to the binding energy of
vitaminꢀD3 (1, ꢀ11.61kcal molꢀ1) (Table 3). The binding energy of
(11, ꢀ9.28 kcal molꢀ1) (Table 1). The binding energy of 27b with 80 27b with VDR is shown below (Figure 8c).
70 ERꢀα is depicted below (Figure 8a).
These docking experiments and predicted results had correlation
Similarly, 27b had good binding affinity for ERꢀβ with
significant binding energy (ꢀ8.78 kcal molꢀ1) in comparison to the
binding energy of 17βꢀestradiol (11, ꢀ10.12 kcal molꢀ1) (Table 2).
The binding energy of 27b with ERꢀβ is presented below (Figure
with experimental results (experimental value) which further
validated our assumptions
85
6
| MedChemComm, 2016, XXX, 01–08
This journal is © The Royal Society of Chemistry [2016]

Page 7 of 15
MedChemComm
View Article Online
DOI: 10.1039/C6MD00469E
Table 1: Molecular docking results of Estrogen Receptorꢀα (ERꢀα)
Binding
energy (kcal
molꢀ1)
Inhibitory constant No. of conformations in
Number of
hydrogen bonds
Compound
Residues in Hꢀbonding & bond length (Å)
(nM)
top cluster
27b3:HꢀGLY420:O (1.73)
GLU423:HNꢀCmp3:O (1.79)
27b
ꢀ9.37
ꢀ9.28
136.18 nM
157.2 nM
28
2
1
17βꢀEstradiol
200
Estardiol:HꢀGLY521:O (2.09)
Table 2: Molecular docking result of Estrogen Receptor ꢀ β (ERꢀβ)
Binding energy Inhibitory constant No. of conformations in
Number of
hydrogen bonds
Compound
Residues in Hꢀbonding & bond length (Å)
(kcal molꢀ1)
(nM)
top cluster
27b:HꢀGLY472:O (1.85)
HIS475:HD1ꢀ27b:O (1.86)
Estr:HꢀGLY472:O (2.20)
Estr:HꢀGLU305:O (2.15)
HIS475:HD1ꢀEstr:O (1.89)
27b
ꢀ8.78
364.89 nM
38.18 nM
20
2
3
17βꢀEstradiol
ꢀ10.12
200
5
Table 3: Molecular docking result of VitaminꢀD Receptor (VDR)
No. of
conformations in
top cluster
Binding energy
(kcal molꢀ1)
Inhibitory constant
(nM)
Number of
hydrogen bonds
Compound
Residues in Hꢀbonding & bond length (Å)
ASP144:HNꢀ27b:O (2.13)
SER278:HGꢀ27b:O (2.01)
VitD:HꢀSER278:OG (2.16)
HIS305:HE2ꢀVitD:O (2.20)
VitD:HꢀILE271:O (1.90)
HIS497:HE2ꢀVitD:O (2.05)
SER237:HGꢀVitD:O (2.24)
27b
ꢀ10.75
ꢀ11.61
13.28 nM
2.26 nM
189
2
5
Vitamin D3
104
10
(a)
(b)
(c)
Figure 8: Binding energy of 27b with (a) Estrogen Receptorꢀα (ERꢀα), (b) Estrogen Receptorꢀβ (ERꢀβ), and (c) Vitamin D receptor (VDR).
This journal is © The Royal Society of Chemistry [2016] MedChemComm, 2016, XXX, 01–08 | 7

MedChemComm
Page 8 of 15
View Article Online
DOI: 10.1039/C6MD00469E
55 was carried out on silica gel (100ꢀ200 and 230ꢀ400 mesh). The
melting points were determined on Buchi melting point M560
apparatus in open capillaries and are uncorrected. H (300 MHz)
Structure activity relationship (SAR) study
1
Structurally, designed compounds belong to two different sub
sets namely 3ꢀarylbenzopyran substituted at position 4’ (17aꢀc,
18aꢀc, 19aꢀc) and 3ꢀarylbenzopyran substituted at position 7
(25aꢀc, 26aꢀc and 27aꢀc) derivatives. Interestingly, the biological
activity results showed that compounds bearing substitution at
position 7 of 3ꢀarylbenzopyran core, (25c, 26aꢀc and 27aꢀc), were
superior than corresponding 3ꢀarylbenzopyran derivatives
substituted at position 4’ (17aꢀc, 18aꢀc, 19aꢀc). This indicated
10 that as far as bone forming activity is concern, position 7 of 3ꢀ
arylbenzopyran core was optimal location for substitution than
position 4’. As reported for different 3ꢀarylbenzopyran based
estrogen receptor modulators, in this case also, it is quite possible
that the pendent phenyl group present at position C3 of 3ꢀ
15 arylbenzopyran core, might simulate with ring A of 17βꢀestradiol
and remaining benzopyran nucleus with rings C and D of 17βꢀ
estradiol.28ꢀ30 Additionally, perhaps the stereochemistry of this
pendent phenyl group present at C3 would have been more
beneficial for interaction of compounds with receptors since the
20 reverse situation yielded compounds with devoid of any activity.
Thus, in case of 25c, 26aꢀc and 27aꢀc which had substitution at
position 7 of 3ꢀarylbenzopyran core, the side chain would have
well correlated with any modification in the ring D of 17βꢀ
estradiol and side chain of vitaminꢀD3 (Figure 2).
and 13C NMR (75 MHz) spectra were recorded on a Bruker WMꢀ
300. Chemical shift are reported in parts per million (ppm) shift
60 (δꢀvalue) based on the peak of the solvent. Signal patterns are
indicated as s, singlet; bs, broad singlet; d, doublet; dd, double
doublet; t, triplet; m, multiplet. Coupling constants (J) are given
in Hertz. Infrared (IR) spectra were recorded on a PerkinꢀElmer
AXꢀ1 spectrophotometer in KBr disc and reported in wave
65 number (cmꢀ1). ESIꢀMS mass spectra were recorded on Shimadzu
LCꢀMS and/or LCꢀMSꢀMS APC3000 (Applied Biosystem).
5
General procedure for the synthesis of compounds
1ꢀ(2ꢀHydroxyꢀ4ꢀmethoxyphenyl)ꢀ2ꢀ(4ꢀ
hydroxyphenyl)ethanone (14)
70 Compounds 12 (2.0 mL, 6.5 mmol) and 13 (1.0 g, 6.5 mmol)
were taken in round bottomed flask and boron trifluoride diethyl
etherate (BF3ꢀOEt2, 15 mL) was added to it. The reaction mixture
was heated at 100oC. The progress of reaction was monitored
using TLC (30% ethyl acetate:hexane). After completion of
75 reaction, the reaction mixture was poured on ice and extracted by
ethyl acetate. The organic layer was washed with sodium
bicarbonate solution. Organic layer was then separated and dried
over anhydrous sodium sulphate (Na2SO4) and concentrated. The
crude material was purified by column chromatography using
80 silicagel (100ꢀ200 mesh size) and ethyl acetate:hexane which
yielded pure compound 14 as white solid.
25 Conclusion:
In summary, 3ꢀarylꢀbenzopyran core as present in various
isoflavones and many synthetic estrogen agonists/antagonists has
been logically modified to develop 3ꢀarylbenzopyran based nonꢀ
steroidal osteogenic agents as a structural template of estrogen
30 and vitaminꢀD3. The target molecules 18aꢀc, 19aꢀc, 26aꢀc, 27aꢀc
and intermediates (17aꢀc and 25aꢀc) were studied for their
osteogenic activity in osteoblast differentiation assay in-vitro
using mouse calvarial osteoblast cells. 25c, 26b, 27b and 27c
effectively increased ALP activity at 1pM concentration through
35 activation of estrogen receptorꢀβ and were devoid of any inherent
cytotoxicity to healthy osteobalst cells in nano to pico molar
concentrations. Further, 27b had ability to increase transcript
level of important bone markers such as BMPꢀ2, RUNXꢀ2 and
Osterix (Osx). Molecular docking analysis performed for 27b
40 showed its possible interactions with estrogen receptorꢀα and ꢀβ
(ERꢀα and ERꢀβ) as well as vitaminꢀD receptor (VDR).
Yield: 72% yield; mp: 144ꢀ145oC; Rf: 0.52 (30% Ethyl
acetate/Hexane); 1H NMR (acetone d6, 300 MHz, δ ppm): 3.77 (s,
3H, OCH3), 4.06 (s, 2H, CH2), 6.23ꢀ6.38 (m, 2H, ArH), 6.65 (d, J
85 = 8.4 Hz, 2H, ArH), 7.02 (d, J = 8.1 Hz, 2H, ArH), 7.85 (d, J =
8.7 Hz, 1H, ArH), 8.15 (s, 1H, ArH), 12.65 (s, 1H, OH); 13C
NMR (acetone d6, 75 MHz, δ ppm): 43.9, 55.5, 101.2, 107.6,
115.7 (2xC), 126.1, 130.7 (2xC), 133.3, 156.7, 166.2, 166.6,
203.6; MS (C15H14O4): m/z= 259 [M+H]+ 281 [M+Na]+.
,
90 3ꢀ(4ꢀHydroxyphenyl)ꢀ7ꢀmethoxyꢀ4Hꢀchromenꢀ4ꢀone (15)
In a round bottom flask, 14 (2.0 g, 7.8 mmol) was dissolved in
dry dichloromethane (DCM, 30 mL) and tetrahydrofuran (THF,
10 mL). To this solution, dimethylformadideꢀdimethylacetal
(DMFꢀDMA, 6.4 mL) was added at room temperature (RT, 35ꢀ
95 40°C). The reaction mixture was allowed to stir at RT for 2h. The
progress of reaction was monitored by TLC (30% ethyl acetateꢀ
hexane). On completion of the reaction, DCM was evaporated
and reaction mixture was worked up using ethyl acetate and
water. The organic layer was separated, dried over anhydrous
100 sodium sulphate (Na2SO4) and concentrated. The crude was
purified by column chromatography on silica gel (100ꢀ200 mesh
size) using acetoneꢀhexane (12:88) as eluent which yielded pure
compound 15 as off white solid.
Overall, 3ꢀarylꢀ3Hꢀbenzopyran derivative 27b has been identified
as osteognic agents. Taken together the activity profile of these
molecules, it is assumed that such 3ꢀarylꢀbenzopyran based
45 molecules could be developed as potential bone forming agents.
However, further structural refinement of this prototype and
detailed biological characterization is warranted.
Experimental Section
Yield: 75%; mp: 222ꢀ223°C; Rf: 0.25 (30% Ethyl acetate/Hexane,
105 elution three times); IR (KBr, νmax/cmꢀ1): 3215, 1622, 1586, 1440,
1255, 831; 1H NMR (CDCl3, 300 MHz, δ ppm): 3.94 (s, 3H,
OCH3), 6.87 (d, J = 8.4 Hz, 2H, ArH), 7.00ꢀ7.04 (m, 2H, ArH),
7.41 (d, J = 8.4 Hz, 2H, ArH), 8.00ꢀ8.13 (m, 2H, CH and ArH),
9.35 (bs, 1H, OH); 13C NMR (CDCl3, 75 MHz, δ ppm): 54.4,
110 98.8, 113.1, 113.8 (2xC), 116.7, 121.3, 123.2, 125.8, 128.7 (2xC),
General
50 All the reagents and solvents used in experimentation were of
analytical or laboratory grade and used without further
purification unless stated otherwise. The reactions were
monitored on Merck aluminium silica gel thin layer
chromatography (TLC, UV254nm) plates. Column chromatography
8
| MedChemComm, 2016, XXX, 01–08
This journal is © The Royal Society of Chemistry [2016]

Page 9 of 15
MedChemComm
View Article Online
DOI: 10.1039/C6MD00469E
Ethyl
(17b)
5ꢀ(4ꢀ(7ꢀmethoxychromanꢀ3ꢀyl)phenoxy)pentanoate
151.0, 156.3, 156.6, 162.7, 174.4; MS (C16H12O4): m/z= 269
[M+H]+ 270 [M+2H]+.
,
Yield: 90%; mp: 80ꢀ81°C; Rf: 0.48, 20% Ethyl acetate/Hexane);
IR (KBr, νmax/cmꢀ1): 2928, 1736, 1510, 1256, 1178, 1033, 830; 1H
60 NMR (acetoneꢀd6, 300 MHz, δ ppm): 1.22 (t, J = 7.0 Hz, 3H,
CH3), 1.79 (bs, 4H, 2xCH2), 2.06ꢀ2.10 (m, 1H, CH), 2.36 (bs, 2H,
CH2), 2.95 (bs,1H, CH), 3.10 (bs, 1H, CH), 3.73 (s, 3H, OCH3),
3.92ꢀ3.99 (m, 3H, CH of CH2 and OCH2), 4.05ꢀ4.12 (m, 2H,
OCH2), 4.21ꢀ4.24 (m, 1H, CH of CH2), 6.34 (bs, 1H, ArH), 6.42ꢀ
65 6.44 (m, 1H, ArH), 6.89 (d, J = 8.4 Hz, 2H, ArH), 6.96 (d, J =
8.4 Hz, 1H, ArH), 7.20 (d, J = 8.4 Hz, 2H, ArH); 13C NMR
(acetoneꢀd6, 75 MHz, δ ppm): 19.4, 27.1, 34.8, 37.2, 39.1, 43.3,
60.3, 65.3, 72.8, 76.3, 106.8, 112.4, 119.7, 120.2 (2xC), 133.8
(2xC), 135.6, 139.0, 160.6, 163.6, 164.8, 178.3; MS (C23H28O5):
70 m/z = 408 [M+H+Na]+, 424 [M+H+K]+.
4ꢀ(7ꢀMethoxychromanꢀ3ꢀyl)phenol (16)
In a round bottom flask, 15 (4.0 g, 11.01 mmol) was dissolved in
tetrahydrofuran (THF, 50 mL). Nitrogen gas was flushed in the
reaction mixture to displace dissolved oxygen and palladium
charcoal (PdꢀC, 5% wt, 0.4 g) was added to this solution.
Following this, hydrogen gas was passed to reaction mixture for
12h. The progress of reaction was monitored by TLC (30% ethyl
10 acetateꢀhexane). On completion of the reaction, PdꢀC was filtered
out and reaction mixture was worked up using ethyl acetate and
water. The organic layer was separated, dried over anhydrous
sodium sulphate (Na2SO4) and concentrated. The crude was
purified by column chromatography on silica gel (100ꢀ200 mesh
15 size) using ethyl acetateꢀhexane (10:90) as eluent which yielded
pure compound 16 as off white solid.
5
Ethyl 6ꢀ(4ꢀ(7ꢀmethoxychromanꢀ3ꢀyl)phenoxy)hexanoate (17c)
Yield: 80%; mp: 150ꢀ151°C; Rf: 0.59 (30% Ethyl
acetate/Hexane); IR (KBr, νmax/cmꢀ1): 3394, 2926, 1616, 1507,
1116, 1023, 836; 1H NMR (CDCl3, 300 MHz, δ ppm): 2.61(d, J =
20 7.8 Hz, 2H, CH2), 2.89 (bs, 1H, CH), 3.46 (s, 3H, OCH3), 3.64 (t,
J = 10.3 Hz, 1H, CH), 3.96 (d, J = 10.5 Hz, CH), 6.08 (d, J = 2.4
Hz, 1H, ArH), 6.15 (dd, J = 2.4 and 2.1 Hz, 1H, ArH), 6.51 (d, J
= 8.4 Hz, 2H, ArH), 6.67 (d, J = 8.1 Hz, 1H, ArH), 6.72 (d, J =
8.4 Hz, 2H, ArH), 8.66 (bs, 1H, OH); 13C NMR (CDCl3, 75 MHz,
25 δ ppm): 30.9, 36.8, 54.4, 70.2, 100.5, 106.1, 113.5, 114.9 (2xC),
127.3 (2xC), 129.3, 130.9, 154.1, 155.4, 158.1; MS (C16H16O3):
m/z= 257[M+H]+ 279 [M+Na]+ 295 [M+K]+.
Yield: 87%; mp: 68ꢀ69°C; Rf: 0.8, 30% Ethyl acetate/Hexane);
IR (KBr, νmax/cmꢀ1): 2944, 1733, 1509, 1251, 1162, 1031, 832; 1H
NMR (CDCl3, 300 MHz, δ ppm): 1.24 (t, J = 7.2 Hz, 3H, CH3),
75 1.46ꢀ1.53 (m, 2H, CH2), 1.64ꢀ1.80 (m, 4H, 2xCH2), 2.31 (t, J =
7.2 Hz, 2H, CH2), 2.91 (d, J = 8.4 Hz, 2H, CH2), 3.36 (bs, 1H,
CH), 3.75 (s, 3H, OCH3), 3.91ꢀ3.98 (m, 3H, CH and CH2), 4.11
(q, J = 10.8 Hz, 2H, CH2), 4.28 (dd, J = 3.0 and 3.0 Hz, 1H, CH),
6.40 (d, J = 2.4 Hz, 1H, ArH), 6.46 (dd, J = 2.4 and 2.7 Hz, 1H,
80 ArH), 6.85 (d, J = 8.7 Hz, 2H, ArH), 6.96 (d, J = 8.1 Hz, 1H,
ArH), 7.12 (t, J = 8.4 Hz, 2H, ArH); 13C NMR (CDCl3, 75 MHz,
δ ppm): 14.6, 25.1, 26.0, 29.3, 32.2, 34.6, 38.3, 55.7, 60.6, 68.1,
71.5, 101.8, 107.6, 114.6, 115.1 (2xC), 128.6 (2xC), 130.5, 133.7,
155.4, 158.5, 159.5, 174.0; MS (C24H30O5): m/z = 421 [M+Na]+ ,
85 437 [M+K]+.
,
,
Ethyl 4ꢀ(4ꢀ(7ꢀmethoxychromanꢀ3ꢀyl)phenoxy)butanoate (17a)
In a round bottom flask, 16 (1.0 g, 3.90 mmol) was dissolved in
30 dry acetone. To this solution, anhydrous potassium carbonate
(K2CO3; 1.6 g, 11.71 mmol) and ethyl 4ꢀbromobutyrate (0.62 mL,
4.3mmol) were added. The reaction mixture was refluxed to 2ꢀ3h.
The progress of reaction was monitored by TLC (20% ethyl
acetateꢀhexane). On completion of the reaction, acetone was
35 distilled off and crude was worked up using ethyl acetate and
water. The organic layer was separated, dried over anhydrous
sodium sulphate (Na2SO4) and concentrated. The crude was
purified by flash column chromatography over silicagel (100ꢀ200
mesh) using acetoneꢀhexane (5:95) as eluent to yield pure 17a.
40 Yield: 74%; mp: 87ꢀ88°C; Rf: 0.5, 20% Ethyl acetate/Hexane);
IR (KBr, νmax/cmꢀ1): 2934, 1724, 1509, 1256, 1175, 1028, 935,
825; 1H NMR (acetoneꢀd6, 300 MHz, δ ppm): 1.13 (t, J = 7.0 Hz,
3H, CH3), 1.93ꢀ2.00 (m, 3H, CH2 & CH of CH2), 2.38ꢀ2.43 (m,
2H, CH2), 2.81ꢀ2.84 (m, 1H, CH), 3.02ꢀ3.03 (m, 1H, CH of
45 CH2), 3.65 (s, 3H, OCH3), 3.89ꢀ4.06 (m, 5H, 2xCH2 and CH of
CH2), 4.12ꢀ4.17 (m, 1H, CH of CH2), 6.27 (d, J = 2.4 Hz, 1H,
ArH), 6.38 (dd, J = 2.4 and 2.4 Hz, 1H, ArH), 6.82ꢀ6.91 (m, 3H,
ArH), 7.14ꢀ7.18 (m, 2H, ArH); 13C NMR (CDCl3, 75 MHz, δ
ppm): 14.0, 25.0, 30.6, 32.0, 38.1, 54.9, 60.1, 67.0, 71.0, 101.5,
50 107.2, 114.6, 115.0 (2xC), 128.7 (2xC), 130.5, 134.1, 155.5,
158.4, 159.6, 172.8; MS (C22H26O5): m/z = 393 [M+Na]+ , 409
[M+K]+.
3ꢀEthylꢀ6ꢀ(4ꢀ(7ꢀmethoxychromanꢀ3ꢀyl)phenoxy)hexanꢀ3ꢀol
(18a)
Under inert condition, in a three neck round bottom flask fitted
with double walled condenser, magnesium turnings (Mg, 1.2 g,
90 81.0 mmol) were activated by heating. Dry diethyl ether was
added to the flask which was followed by slow addition of ethyl
iodide (CH3CH2I, 6.51 mL, 81.0 mmol) with stirring to get
Grignard reagent (CH3 CH2MgI). A vigorous reaction occurred
with the formation of reagent. The temperature of reaction
95 content was brought to 0°C over iceꢀbath for about 20 minutes.
Compound 17a (3.0 g, 8.1 mmol) dissolved in dry THF was
added to this mixture. Subsequently, reaction was transferred to
room temperature. The progress of reaction was monitored by
TLC (30% ethyl acetateꢀhexane). On completion, the reaction
100 mixture was treated with dil. HCl and worked up using ethyl
acetate and water. The organic layer was separated, dried over
anhydrous sodium sulphate (Na2SO4) and concentrated. The
crude material was purified by column chromatography using
silica gel (100ꢀ200 mesh size) and ethyl acetateꢀhexane (15:85) to
105 yielded pure compound 18a.
Yield: 81%; mp: 91ꢀ92°C; Rf: 0.23, 20% Ethyl acetate/Hexane);
1
IR (KBr, νmax/cmꢀ1): 3420, 2934, 1256, 1033, 947, 833; H NMR
Compounds 17b and 17c were synthesized from 16 using ethyl 5ꢀ
bromovalerate and ethyl 6ꢀbromohexanoate respectively
55 following the synthetic procedure used for 17a.
(CDCl3, 500 MHz, δ ppm): 0.88 (t, J = 7.5 Hz, 6H, 2xCH3), 1.50
(q, J = 7.5 Hz, 4H, 2xCH2), 1.57ꢀ1.60 (m, 2H, CH2), 1.78ꢀ1.82
110 (m, 2H, CH2), 2.92ꢀ2.94 (m, 2H, CH2), 3.16ꢀ3.18 (m, 1H, CH of
CH2), 3.80 (s, 3H, OCH3), 3.92ꢀ3.98 (m, 3H, OCH2 and CH of
This journal is © The Royal Society of Chemistry [2016]
MedChemComm, 2016, XXX, 01–08 | 9

MedChemComm
Page 10 of 15
View Article Online
DOI: 10.1039/C6MD00469E
CH2), 4.28ꢀ4.31 (m, 1H, CH of OCH2), 6.41 (d, J = 2.5 Hz, 1H,
ArH), 6.47 (dd, J = 2.5 and 2.5 Hz, 1H, ArH), 6.89 (dd, J = 2.0
and 2.0 Hz, 2H, ArH), 6.96 (d, J = 8.5 Hz, 1H, ArH), 7.16 (d, J =
8.5 Hz, 2H, ArH); 13C NMR (CDCl3, 125 MHz, δ ppm): 7.8
(2xC), 23.4, 31.0 (2xC), 31.9, 34.6, 37.9, 55.3, 68.5, 71.1, 74.3,
102.0, 107.8, 114.1 (3xC), 128.3 (2xC), 130.1, 133.4, 154.9,
158.5, 158.6; MS (C24H32O4): m/z = 421 [M+Na]+ , 437 [M+K]+.
32.5, 32.9 (2xC), 38.3, 42.9, 55.7, 68.2, 71.0, 71.5, 101.7, 107.6,
114.5, 115.1 (2xC), 128.7 (2xC), 130.5, 133.8, 155.4, 158.4,
159.5; MS (C22H28O4): m/z = 393 [M+Na]+, 409 [M+K]+.
6ꢀ(4ꢀ(7ꢀmethoxychromanꢀ3ꢀyl)phenoxy)ꢀ2ꢀmethylhexanꢀ2ꢀol
60 (19b)
5
Yield: 93%; mp: 86ꢀ87°C; Rf: 0.13, 20% Ethyl acetate/Hexane);
1
IR (KBr, νmax/cmꢀ1): 3365, 2939, 1510, 1255, 1033, 912, 832; H
NMR (CDCl3, 300 MHz, δ ppm): 1.24 (s, 6H, 2xCH3), 1.57 (bs,
4H, 2xCH2), 1.83 (bs, 2H, CH2), 2.96 (d, J = 8.4 Hz, 2H, CH2),
65 3.15ꢀ3.24 (m, 1H, CH), 3.79 (s, 3H, OCH3), 3.96ꢀ4.03 (m, 3H,
OCH2 and CH of CH2), 4.31ꢀ4.35 (m, 1H, CH of CH2), 6.45 (d, J
= 2.4 Hz, 1H, ArH), 6.58 (dd, J = 2.7 and 2.4 Hz, 1H, ArH), 6.90
(d, J = 8.7 Hz, 2H, ArH), 7.00 (d, J = 8.4 Hz, 1H, ArH), 7.17 (d, J
= 8.7 Hz, 2H, ArH); 13C NMR (CDCl3, 75 MHz, δ ppm): 21.3,
Compounds 18b and 18c were synthesized from 17b and 17c
10 respectively following the synthetic procedure used for 18a.
3ꢀEthylꢀ7ꢀ(4ꢀ(7ꢀmethoxychromanꢀ3ꢀyl)phenoxy)heptanꢀ3ꢀol
(18b)
Yield: 93%; mp: 79ꢀ80°C; Rf: 0.25, 20% Ethyl acetate/Hexane);
1
IR (KBr, νmax/cmꢀ1): 3420, 2934, 1256, 1033, 947, 833; H NMR
15 (CDCl3, 300 MHz, δ ppm): 0.87 (t, J = 7.3 Hz, 6H, 2xCH3), 1.45ꢀ 70 27.6 (2xC), 30.1, 32.2, 38.3, 43.9, 55.7, 68.2, 71.3, 71.5, 101.8,
1.52 (m, 8H, 4xCH2), 1.79 (bs, 2H, CH2), 2.94 (d, J = 8.4 Hz, 2H,
CH2), 3.12ꢀ3.22 (m, 1H, CH), 3.77 (s, 3H, OCH3), 3.93ꢀ4.00 (m,
3H, OCH2 and CH of CH2), 4.31 (dd, J = 2.7 and 2.4 Hz, 1H, CH
of OCH2), 6.43 (d, J = 2.4 Hz, 1H, ArH), 6.48 (dd, J = 2.4 and
20 2.1 Hz, 1H, ArH), 6.88 (d, J = 8.4 Hz, 2H, ArH), 6.98 (d, J = 8.4
Hz, 1H, ArH), 7.15 (d, J = 8.4 Hz, 2H, ArH); 13C NMR (CDCl3,
75 MHz, δ ppm): 8.1 (2xC), 20.4, 30.2, 31.4 (2xC), 32.2, 38.3
(2xC), 55.7, 68.2, 71.5, 74.9, 101.8, 107.6, 114.6, 115.2 (2xC),
128.6 (2xC), 130.5, 133.7, 155.4, 158.5, 159.5; MS (C25H34O4):
25 m/z = 421 [M+Na]+ , 437 [M+K]+.
107.6, 114.6, 115.2 (2xC), 128.6 (2xC), 130.5, 132.7, 155.4,
158.5, 159.5; MS (C23H30O4): m/z = 393 [M+Na]+ , 409 [M+K]+.
7ꢀ(4ꢀ(7ꢀMethoxychromanꢀ3ꢀyl)phenoxy)ꢀ2ꢀmethylheptanꢀ2ꢀol
(19c)
75 Yield: 76%; mp: 76ꢀ77°C; Rf: 0.42, 20% Ethyl acetate/Hexane);
IR (KBr, νmax/cmꢀ1): 3348, 2927, 1737, 1650, 1236, 1033, 975,
832, 804; 1H NMR (CDCl3, 300 MHz, δ ppm): 1.25 (s, 6H,
2xCH3), 1.40 (bs, 6H, 3xCH2), 1.79ꢀ1.86 (m, 2H, CH2), 2.96 (d, J
= 8.4 Hz, 2H, CH2), 3.16ꢀ3.22 (m, 1H, CH), 3.80 (s, 3H, OCH3),
80 3.96ꢀ4.03 (m, 3H, OCH2 and CH of CH2), 4.33 (dd, J = 2.7 and
2.7 Hz, 1H, CH of OCH2), 6.46 (d, J = 2.4 Hz, 1H, ArH), 6.51
(dd, J = 2.4 and 2.4 Hz, 1H, ArH), 6.89 (d, J = 8.7 Hz, 2H, ArH),
3ꢀEthylꢀ8ꢀ(4ꢀ(7ꢀmethoxychromanꢀ3ꢀyl)phenoxy)octanꢀ3ꢀol
(18c)
Yield: 90 %; mp: 63ꢀ64°C; Rf: 0.50, 30% Ethyl acetate/Hexane);
7.01 (d, J = 8.4 Hz, 1H, ArH), 7.18 (d, J = 8.4 Hz, 2H, ArH); 13
NMR (CDCl3, 75 MHz, δ ppm): 24.5, 27.0, 29.6 (2xC), 32.2,
C
IR (KBr, νmax/cmꢀ1): 3348, 2927, 1737, 1650, 1236, 1033, 975,
1
30 832, 804; H NMR (CDCl3, 300 MHz, δ ppm): 0.89 (t, J = 7.5 85 38.3, 44.2, 55.7, 68.3 (2xC), 71.3, 71.5, 101.8, 107.6, 114.6,
Hz, 6H, 2xCH3), 1.29ꢀ1.54 (m, 10H, 5xCH2), 1.79ꢀ1.86 (m, 2H,
CH2), 2.96 (d, J = 8.4 Hz, 2H, CH2), 3.16ꢀ3.22 (m, 1H, CH), 3.80
(s, 3H, OCH3), 3.96ꢀ4.03 (m, 3H, OCH2 and CH of OCH2), 4.31ꢀ
4.36 (m, 1H, CH of OCH2), 6.46 (d, J = 2.7 Hz, 1H, ArH), 6.47
35 (dd, J = 8.4 Hz, 1H, ArH), 6.91 (d, J = 8.4 Hz, 2H, ArH), 7.01 (d,
J = 8.1 Hz, 1H, ArH), 7.17 (d, J = 8.7 Hz, 2H, ArH); 13C NMR
(CDCl3, 75 MHz, δ ppm): 8.1 (2xC), 25.5, 27.1, 29.7, 31.4 (2xC),
32.2, 38.3, 38.5, 55.7, 68.4, 71.5, 74.9, 101.8, 107.6, 114.6, 115.2
(2xC), 128.6 (2xC), 130.5, 133.6, 155.4, 158.5, 159.5; MS
40 (C26H36O4): m/z = 435 [M+Na]+ .
115.2 (2xC), 128.6 (2xC), 130.5, 133.6, 155.4, 158.5, 159.5; MS
(C24H32O4): m/z = 459 [M+H]+, 481 [M+Na]+ , 482 [M+K]+.
1ꢀ(2,4ꢀdihydroxyphenyl)ꢀ2ꢀ(4ꢀmethoxyphenyl)ethanone (22)
90 Compound 22 was synthesized from 20 and 21 following the
synthetic procedure used for 14.
Yield: 72% yield; mp: 149ꢀ150oC; Rf: 0.27 (20% Ethyl
acetate/Hexane); IR (KBr, νmax/cmꢀ1): 3359, 1617, 1509, 1440,
1353, 1242, 1180, 1127, 995, 852, 793; 1H NMR (acetone d6, 300
95 MHz, δ ppm): 3.74 (s, 3H, OCH3), 4.19 (s, 2H, CH2), 6.32 (d, J =
2.4 Hz, 1H, ArH), 6.43 (dd, J = 2.4 and 2.4 Hz, 1H, ArH), 6.88
(d, J = 8.7 Hz, 2H, ArH), 7.24 (d, J = 8.7 Hz, 2H, ArH), 7.93 (d, J
= 9.0 Hz, 1H, ArH); 13C NMR (acetone d6, 75 MHz, δ ppm):
43.7, 55.9, 103.1, 108.4, 114.2, 114.8 (2xC), 127.5, 130.7 (2xC),
100 133.7, 159.1, 165.3, 166.2, 203.1; MS (C15H14O4): m/z= 259
[M+H]+.
General synthesis of compounds 19aꢀc
Compounds 19aꢀc were synthesized from 17aꢀc respectively
using methyl magnesium iodide (CH3MgI) in place of
CH3CH2MgI, following the synthetic procedure used for 18aꢀc.
45 5ꢀ(4ꢀ(7ꢀMethoxychromanꢀ3ꢀyl)phenoxy)ꢀ2ꢀmethylpentanꢀ2ꢀol
(19a)
3ꢀ(4ꢀMethoxyphenyl)ꢀ7ꢀhydroxyꢀ4H-chromenꢀ4ꢀone (23)
Yield: 80%; mp: 90ꢀ91°C; Rf: 0.50, 30% Ethyl acetate/Hexane);
1
IR (KBr, νmax/cmꢀ1): 3433, 2929, 1510, 1036, 925, 834; H NMR
Compound 23 was synthesized from 22 following the synthetic
procedure used for 15.
(CDCl3, 300 MHz, δ ppm): 1.62 (s, 6H, 2xCH3), 1.90ꢀ1.94 (m,
50 2H, CH2), 1.97ꢀ2.01 (m, 2H, CH2), 2.93ꢀ2.95 (m, 2H, CH2), 3.16
(m, 1H, CH of CH2), 3.77 (s, 3H, OCH3), 3.92ꢀ4.00 (m, 3H,
OCH2 and CH of CH2), 4.29ꢀ4.32 (m, 1H, CH of CH2), 6.42 (d, J
= 2.5 Hz, 1H, ArH), 6.48 (dd, J = 2.5 and 3.0 Hz, 1H, ArH), 6.88
(d, J = 8.5 Hz, 2H, ArH), 6.98 (d, J = 8.5 Hz, 1H, ArH), 7.14ꢀ
55 7.16 (m, 2H, ArH); 13C NMR (CDCl3, 75 MHz, δ ppm): 25.6,
105 Yield: 83%; m.p. 247ꢀ248°C; Rf: 0.30 (30% Ethyl
acetate/Hexane, elution three times); 1H NMR (CDCl3, 300 MHz,
δ ppm): 3.85 (s, 3H, OCH3), 6.70 (d, J = 8.4 Hz, 2H, ArH), 7.00ꢀ
7.06 (m, 2H, ArH), 7.41 (d, J = 8.4 Hz, 2H, ArH), 8.00ꢀ8.13 (m,
3H, CH and ArH), 9.35 (bs, 1H, OH); 13C NMR (CDCl3, 75
110 MHz, δ ppm): 54.4, 98.8, 113.1, 113.8 (2xC), 116.7, 121.3,
10 | MedChemComm, 2016, XXX, 01–08
This journal is © The Royal Society of Chemistry [2016]

Page 11 of 15
MedChemComm
View Article Online
DOI: 10.1039/C6MD00469E
123.2, 125.8, 128.7 (2xC), 125.8, 128.7 (2xC), 151.0, 156.3, 55 1170, 1107, 1033, 829; 1H NMR (CDCl3, 300 MHz, δ ppm): 1.26
156.62, 162.8, 174.4; MS (C16H12O4): m/z = 269 [M+H]+
(t, J = 8.4 Hz, 3H, CH3), 1.47ꢀ1.53 (m, 2H, CH2), 1.63ꢀ1.81 (m,
4H, 2xCH2), 2.33 (t, J = 7.5 Hz, 2H, CH2), 2.93 (d, J = 8.4 Hz,
2H, CH2), 3.16 (m, 1H, CH), 3.80 (s, 3H, OCH3), 3.90ꢀ4.00 (m,
3H, CH of CH2 and OCH2), 4.13 (q, J = 7.2 Hz, 2H, OCH2), 4.29
60 (dd, J = 3.0 and 3.0 Hz, 1H, CH of OCH2), 6.40 (d, J = 2.4 Hz,
1H, ArH), 6.46 (dd, J = 2.4 and 2.4 Hz, 1H, ArH), 6.89 (d, J = 8.7
Hz, 2H, ArH), 6.96 (d, J = 8.4 Hz, 1H, ArH), 7.16 (d, J = 8.4 Hz,
2H, ArH); 13C NMR (CDCl3, 75 MHz, δ ppm): 14.6, 25.1, 26.0,
29.3, 32.2, 34.6, 38.3, 55.6, 60.6, 68.1, 71.5, 102.4, 108.2, 114.4,
65 114.5 (2xC), 128.6 (2xC), 130.5, 133.8, 155.3, 159.0, 159.0,
174.0; MS (C24H30O9): m/z = 459 [M+H]+, 481 [M+Na]+ , 482
[M+K]+.
.
3ꢀ(4ꢀMethoxyphenyl) chromanꢀ7ꢀol (24)
Compound 24 was synthesized from 23 following the synthetic
procedure used for 16.
5
Yield: 55%; m.p. 150ꢀ152°C; Rf: 0.75 (20% Ethyl
1
acetate/Hexane); H NMR (acetoneꢀd6, 300 MHz, δ ppm): 2.80ꢀ
2.96 (m, 2H, CH2), 3.11ꢀ3.16 (m, 1H, CH), 3.78 (s, 3H, OCH3),
3.95 (t, J = 10.5 Hz, 1H, CH of CH2), 4.18ꢀ4.23 (m, 1H, CH of
10 CH2), 6.31 (d, J = 2.4 Hz, 1H, ArH), 6.39 (dd, J = 2.4 and 2.4 Hz,
1H, ArH), 6.89ꢀ6.97 (m, 3H, ArH), 7.24 (d, J = 8.7 Hz, 2H, ArH),
8.15 (s, 1H, OH); 13C NMR (acetoneꢀd6, 75 MHz, δ ppm): 30.1,
38.2, 55.0 (OCH3), 70.9, 103.2, 108.4, 113.5, 114.3 (2xC), 128.7
(2xC), 130.5, 134.1, 155.4, 157.1, 159.1; MS (C16H16O3): m/z=
15 257 [M+H]+.
General synthesis of compounds 26aꢀc
Compounds 26aꢀc were synthesized from 25aꢀc respectively
70 using ethyl magnesium iodide (CH3CH2MgI) following the
synthetic procedure used for 18aꢀc.
General synthesis of compounds 25aꢀc
Compounds 25aꢀc were synthesized from 24 following the
synthetic procedure used for 17aꢀc.
3ꢀEthylꢀ6ꢀ(3ꢀ(4ꢀmethoxyphenyl)chromanꢀ7ꢀyloxy)hexanꢀ3ꢀol
(26a)
Ethyl
20 (25a)
4ꢀ(3ꢀ(4ꢀmethoxyphenyl)chromanꢀ7ꢀyloxy)butanoate
Yield: 88%; mp: 101ꢀ102°C; Rf: 0.25, 20% Ethyl
75 acetate/Hexane); IR (KBr, νmax/cmꢀ1): 3417, 2953, 1509, 1257,
1171, 1035, 830; 1H NMR (acetoneꢀd6, 300 MHz, δ ppm): 0.78 (t,
J = 7.5 Hz, 6H, 2xCH3), 1.40 (q, J = 7.5 Hz, 4H, 2xCH2), 1.45ꢀ
1.50 (m, 2H, CH2), 1.67ꢀ1.73 (m, 2H, CH2), 2.80ꢀ2.89 (m, 2H,
CH2), 2.95ꢀ3.02 (m, 1H, CH), 3.69 (s, 3H, OCH3), 3.83ꢀ3.92 (m,
80 3H, CH of CH2 and OCH2), 4.12ꢀ4.13 (m, 1H, CH of CH2), 6.27
(d, J = 2.4 Hz, 1H, ArH), 6.37 (dd, J = 2.4 and 2.4 Hz, 1H, ArH),
6.81ꢀ6.90 (m, 3H, ArH), 7.16 (d, J = 8.4 Hz, 2H, ArH); 13C NMR
(acetoneꢀd6, 75 MHz, δ ppm): 7.6 (2xC), 23.8, 31.2 (2xC), 32.0,
34.7, 38.1, 55.0, 68.8, 71.0, 73.3, 102.2, 107.8, 114.4 (2xC),
85 114.4, 128.7 (2xC), 130.4, 134.0, 155.4, 159.1 (2xC); MS
(C24H32O4): m/z = 407 [M+Na]+, 423 [M+K]+.
Yield: 96%; mp:72ꢀ73°C; Rf: 0.44, 20% Ethyl acetate/Hexane);
IR (KBr, νmax/cmꢀ1): 3348, 2927, 1737, 1650, 1236, 1033, 975,
832, 804; 1H NMR (acetoneꢀd6, 300 MHz, δ ppm): 1.24 (t, J = 7.2
Hz, 3H, CH3), 2.01ꢀ2.10 (m, 2H, CH2), 2.50 (t, J = 7.5 Hz, 2H,
25 CH2), 2.90ꢀ2.95 (m, 2H, CH2), 3.10ꢀ3.15 (m, 1H, CH), 3.80 (s,
3H, OCH3), 3.98ꢀ4.09 (m, 3H, OCH2 and CH of CH2), 4.12 (q, J
= 7.2 Hz, 2H, OCH2), 4.22ꢀ4.27 (m, 1H, CH), 6.38 (d, J = 2.4
Hz, 1H, ArH), 6.47 (dd, J = 2.4 and 2.4 Hz, 1H, ArH), 6.93 (d, J
= 8.4 Hz, 2H, ArH), 6.99 (d, J = 8.4 Hz, 1H, ArH), 7.27 (d, J =
30 8.4 Hz, 2H, ArH); 13C NMR (acetoneꢀd6, 75 MHz, δ ppm): 14.0,
25.0, 30.7, 32.0, 38.1, 55.0, 60.1, 67.0, 71.0, 102.3, 107.8, 114.3
(2xC), 114.7, 128.7 (2xC), 130.5, 134.0, 155.5, 158.8, 159.1,
172.8; MS (C22H26O5): m/z = 371 [M+H]+, 393 [M+Na]+ , 409
[M+K]+.
3ꢀEthylꢀ7ꢀ(3ꢀ(4ꢀmethoxyphenyl)chromanꢀ7ꢀyloxy)heptanꢀ3ꢀol
(26b)
Yield: 92%; mp: 79ꢀ80°C; Rf: 0.34, 20% Ethyl acetate/Hexane);
90 IR (KBr, νmax/cmꢀ1): 3442, 2944, 1613, 1509, 1258, 1169, 1036,
830; 1H NMR (CDCl3, 300 MHz, δ ppm): 1.07 (t, J = 7.3 Hz, 6H,
2xCH3), 1.65 (bs, 8H, 4xCH2), 1.98 (bs, 2H, CH2), 3.13 (d, J =
6.9 Hz, 2H, CH2), 3.32ꢀ3.41 (m, 1H, CH), 4.00 (s, 3H, OCH3),
4.12ꢀ4.21 (m, 3H, CH of OCH2 and OCH2), 4.42ꢀ4.52 (m, 1H,
95 CH of OCH2), 6.61 (d, J = 2.4 Hz, 1H, ArH), 6.69 (dd, J = 2.4
and 2.4 Hz, 1H, ArH), 7.09 (d, J = 8.7 Hz, 2H, ArH), 7.16 (d, J =
8.4 Hz, 1H, ArH), 7.36 (d, J = 8.4 Hz, 2H, ArH); 13C NMR
(CDCl3, 75 MHz, δ ppm): 8.2 (2xC), 20.4, 30.2, 31.4 (2xC), 32.2,
38.3 (2xC), 55.6, 68.2, 71.5, 74.9, 102.4, 108.3, 114.4, 114.5
100 (2xC), 128.7 (2xC), 130.5, 133.8, 155.3, 159.0 (2xC); MS
(C25H34O4): m/z = 421 [M+Na]+, 437 [M+K]+.
35 Ethyl
(25b)
5ꢀ(3ꢀ(4ꢀmethoxyphenyl)chromanꢀ7ꢀyloxy)pentanoate
Yield: 88%; mp: 73ꢀ74°C; Rf: 0.53, 20% Ethyl acetate/Hexane);
IR (KBr, νmax/cmꢀ1): 2914, 1732, 1613, 1581, 1507, 1245, 1168,
1110, 1027, 834; 1H NMR (CDCl3, 300 MHz, δ ppm): 1.26 (t, J =
40 7.2 Hz, 3H, CH3), 1.79ꢀ1.83 (m, 4H, 2xCH2), 2.38 (bs, 2H, CH2),
2.93 (d, J = 8.4 Hz, 2H, CH2), 3.12ꢀ3.21 (m, 1H, CH), 3.80 (s,
3H, OCH3), 3.93ꢀ4.04 (m, 3H, OCH2 and CH of OCH2), 4.13 (q,
J = 7.2 Hz, 2H, OCH2), 4.29 (dd, J = 3.0 and 3.0 Hz, 1H, CH of
OCH2), 6.40 (d, J = 2.4 Hz, 1H, ArH), 6.46 (dd, J = 2.4 and 2.4
45 Hz, 1H, ArH), 6.89 (d, J = 8.4 Hz, 2H, ArH), 6.96 (d, J = 8.4 Hz,
1H, ArH), 7.16 (d, J = 8.4 Hz, 2H, ArH); 13C NMR (CDCl3, 75
MHz, δ ppm): 14.6, 22.0, 29.0, 32.2, 34.3, 38.3, 55.6, 60.6, 67.8,
71.5, 102.4, 108.2, 114.5, 114.5 (2xC), 128.6 (2xC), 130.5, 133.8,
155.3, 158.9, 159.0, 173.8; MS (C23H28O5): m/z = 459 [M+H]+,
50 481 [M+Na]+ , 482 [M+K]+.
3ꢀEthylꢀ8ꢀ(3ꢀ(4ꢀmethoxyphenyl)chromanꢀ7ꢀyloxy)octanꢀ3ꢀol
(26c)
Yield: 87%; mp: 69ꢀ70°C; Rf: 0.40, 30% Ethyl acetate/Hexane);
105 IR (KBr, νmax/cmꢀ1): 3369, 2935, 1614, 1511, 1258, 1171, 1036,
831; 1H NMR (CDCl3, 300 MHz, δ ppm): 0.86 (t, J = 7.5 Hz, 6H,
2xCH3), 1.42ꢀ1.50 (m, 10H, 5xCH2), 1.74ꢀ1.80 (bs, 2H, CH2),
2.92 (d, J = 7.1 Hz, 2H, CH2), 3.11ꢀ3.20 (m, 1H, CH), 3.79 (s,
3H, OCH3), 3.89ꢀ3.99 (m, 3H, CH of OCH2 and OCH2), 4.27ꢀ
Ethyl
(25c)
6ꢀ(3ꢀ(4ꢀmethoxyphenyl)chromanꢀ7ꢀyloxy)hexanoate
Yield: 93%; mp: 80ꢀ81°C; Rf: 0.60, 30% Ethyl acetate/Hexane);
IR (KBr, νmax/cmꢀ1): 2944, 2874, 1730, 1613, 1582, 1509, 1256,
This journal is © The Royal Society of Chemistry [2016]
MedChemComm, 2016, XXX, 01–08 | 11

MedChemComm
Page 12 of 15
View Article Online
DOI: 10.1039/C6MD00469E
Biological methods: Reagents and chemicals
4.31 (m, 1H, CH of OCH2), 6.41 (d, J = 2.1 Hz, 1H, ArH), 6.46
(d, J = 8.4 Hz, 1H, ArH), 6.88 (d, J = 8.7 Hz, 2H, ArH), 6.96 (d, J
= 8.7 Hz, 1H, ArH), 7.16 (d, J = 8.7 Hz, 2H, ArH); 13C NMR
(CDCl3, 75 MHz, δ ppm): 8.1 (2xC), 23.6, 27.1, 29.6, 31.4 (2xC),
32.2, 38.3, 38.5, 55.6, 68.4, 71.5, 75.0, 102.4, 108.3, 114.4, 114.5
(2xC), 128.7 (2xC), 130.5, 133.9, 155.3, 159.0 (2xC); MS
(C26H36O4): m/z = 435 [M+Na]+ , 451 [M+K]+.
Cell culture media and supplements were purchased from
Invitrogen (Carlsbad, CA) and all fine chemicals from Sigma
Aldrich (St. Louis, MO). Enhanced chemiluminescence (ECL) kit
60 was obtained from Amersham Pharmacia, USA. All antibodies
for Western blot analysis were obtained from Cell Signaling
Technologies (Danvers, MA).
5
General synthesis of compounds 27aꢀc
Culture of mice calvarial osteoblasts
Compounds 27aꢀc were synthesized from 25aꢀc respectively
10 using methyl magnesium iodide (CH3MgI) in place of
CH3CH2MgI, following the synthetic procedure used for 18aꢀc.
Mouse calvarial osteoblasts were obtained following our
65 previously published protocol of sequential digestion. In Brief,
calvaria from 1 to 2 day old mice (both sexes) were pooled. After
surgical isolation from the skull and removal of sutures and
adherent mesenchymal tissues, calvaria were subjected to five
sequential (10–15 min) digestions at 37ºC in a solution containing
70 0.1% dispase and 0.1% collagenase P. Cells released from the
second to fifth digestions were pooled, centrifuged, resuspended,
and plated in Tꢀ25 cm2 flasks in aꢀMEM containing 10% FCS
and 1% penicillin/streptomycin (complete growth medium).
5ꢀ(3ꢀ(4ꢀMethoxyphenyl)chromanꢀ7ꢀyloxy)ꢀ2ꢀmethylpentanꢀ2ꢀ
ol (27a)
Yield: 92%; mp: 104ꢀ105°C; Rf: 0.17, 15% Ethyl
15 acetate/Hexane); IR (KBr, νmax/cmꢀ1): 3404, 2938, 1510, 1252,
1
1168, 1034, 831; H NMR (CDCl3, 300 MHz, δ ppm): 1.22 (bs,
6H, 2xCH3), 1.58ꢀ1.64 (m, 2H, CH2), 1.82ꢀ1.89 (m, 2H, CH2),
2.91ꢀ2.94 (m, 1H, CH), 3.08ꢀ3.15 (m, 1H, CH), 3.80 (s, 3H,
OCH3), 3.93ꢀ4.02 (m, 3H, CH of OCH2 and OCH2), 4.22ꢀ4.26 (m,
20 1H, CH of OCH2), 6.38 (d, J = 2.4 Hz, 1H, ArH), 6.47 (dd, J =
2.7 and 2.7 Hz, 1H, ArH), 6.91ꢀ7.00 (m, 3H, ArH), 7.26 (d, J =
8.7 Hz, 2H, ArH); 13C NMR (CDCl3, 75 MHz, δ ppm): 24.6, 29.2
(2xC), 32.0, 38.1, 40.3, 55.0, 68.7, 69.4, 71.0, 102.2, 107.8,
114.4, 114.4 (2xC), 128.7 (2xC), 130.4, 134.0, 155.4, 159.1
25 (2xC); MS (C22H28O4): m/z = 379 [M+Na]+ , 395 [M+K]+.
Osteoblast differentiation
75 For the measurement of alkaline phosphatase (ALP) activity,
osteoblasts at ~80% confluence were trypsinized and 2 x 103 cells
per well were seeded in 96ꢀwell plates. Cells were treated with
different concentrations of the compounds for 48 h in aꢀMEM
supplemented with 5% Fetal bovine serum, 10 mM βꢀ
80 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 pꢀnitrophenylphosphate (PNPP) as a substrate
and quantified colorimetrically at 405 nm.
6ꢀ(3ꢀ(4ꢀMethoxyphenyl)chromanꢀ7ꢀyloxy)ꢀ2ꢀmethylhexanꢀ2ꢀol
(27b)
Yield: 91%; mp: 118ꢀ119°C; Rf: 0.33, 20% Ethyl
acetate/Hexane); IR (KBr, νmax/cmꢀ1): 3481, 2941, 1615, 1058,
30 1247, 1162, 1032, 825; 1H NMR (CDCl3, 300 MHz, δ ppm): 1.23
(s, 6H, 2xCH3), 1.42 (bs, 4H, 2xCH2), 1.78 (bs, 2H, CH2), 2.94
(d, J = 8.4 Hz, 2H, CH2), 3.16ꢀ3.19 (m, 1H, CH), 3.80 (s, 3H,
OCH3), 3.92ꢀ4.00 (m, 3H, CH of OCH2 and OCH2), 4.30 (d, J =
3.0 and 3.0 Hz, 1H, CH of OCH2), 6.42 (d, J = 2.4 Hz, 1H, ArH),
35 6.48 (dd, J = 2.7 and 2.4 Hz, 1H, ArH), 6.89 (d, J = 8.4 Hz, 2H,
ArH), 6.97 (d, J = 8.4 Hz, 1H, ArH), 7.16 (d, J = 8.4 Hz, 2H,
ArH); 13C NMR (CDCl3, 75 MHz, δ ppm): 21.3, 29.6 (2xC),
30.1, 32.2, 38.2, 44.0, 55.6, 68.3, 71.3, 71.5, 102.4, 108.3, 114.4,
114.5 (2xC), 128.6 (2xC), 130.5, 133.9, 155.3, 159.0 (2xC); MS
40 (C23H30O4): m/z = 393 [M+Na]+.
85 In-vitro toxicity using MTT assay
For this assay 1 × 104 cells/well were seeded in 100 µL DMEM,
supplemented with 10% FBS in each well of 96ꢀwell
microculture plates and incubated for 24 h at 37°C in a CO2
incubator. Compounds, diluted to the desired concentrations in
90 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 µL MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyl tetrazolium bromide) (5 mg/mL) was added and plates
were further incubated for 4 h. Supernatant from each well was
95 carefully removed, formazan crystals were dissolved in 100 µL of
DMSO and the absorbance at 570 nm wavelength were recorded.
7ꢀ(3ꢀ(4ꢀMethoxyphenyl)chromanꢀ7ꢀyloxy)ꢀ2ꢀmethylheptanꢀ2ꢀ
ol (27c)
Mineralization of calvarial osteoblast cells
Yield: 87%; mp: 89ꢀ90°C; Rf: 0.20, 20% Ethyl acetate/Hexane);
IR (KBr, νmax/cmꢀ1): 3394, 2935, 1616, 1509, 1248, 1166, 1033,
For mineralization studies, calvarial cells were cultured in aꢀ
MEM medium, supplemented with 10% fetal bovine serum, 50
100 µg/mL ascorbic acid, and 10 mM βꢀglycerophosphate. Cells were
cultured with and without compounds for 21 day at 37°C in a
humidified atmosphere of 5% CO2 and 95% air, and the medium
was changed every 48 h. After 21 days, the attached cells were
fixed in 4% formaldehyde for 20 min at room temperature and
105 rinsed once in PBS. After fixation, the specimens were processed
for staining with 40 mM alizarin redꢀS, which stains areas rich in
nascent calcium. For quantification of alizarin redꢀS 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
1
45 831; H NMR (CDCl3, 300 MHz, δ ppm): 1.21 (s, 6H, 2xCH3),
1.35ꢀ1.49 (m, 6H, 3xCH2), 1.74ꢀ1.80 (m, 2H, CH2), 2.92 (d, J =
8.4 Hz, 2H, CH2), 3.11ꢀ3.21 (m, 1H, CH), 3.79 (m, 3H, OCH3),
3.90ꢀ4.03 (m, 3H, CH of OCH2 and OCH2), 4.29 (dd, J = 3.0 and
3.0 Hz, 1H, CH), 6.41 (d, J = 2.1 Hz, 1H, ArH), 6.46 (dd, J = 2.4
50 and 2.4 Hz, 1H, ArH), 6.88 (d, J = 8.4 Hz, 2H, ArH), 6.96 (d, J =
8.4 Hz, 1H, ArH), 7.15 (d, J = 8.7 Hz, 2H, ArH); 13C NMR
(CDCl3, 75 MHz, δ ppm): 24.5, 27.0, 29.6 (2xC), 32.2, 38.3,
44.2, 55.6, 68.3, 71.3, 71.5, 102.4, 108.2, 114.4, 114.5 (2xC),
128.7 (2xC), 130.5, 133.5, 133.8, 155.3, 159.0; MS (C24H32O4):
55 m/z = 485 [M+H]+, 407 [M+Na]+.
12 | MedChemComm, 2016, XXX, 01–08
This journal is © The Royal Society of Chemistry [2016]

Page 13 of 15
MedChemComm
View Article Online
DOI: 10.1039/C6MD00469E
Molecular docking studies
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 1.5ꢀmL tube. After vortexing for 30 s, the slurry was
over laid with 500 µL mineral oil (Sigma–Aldrich), heated to
exactly 85ºC for 10 min, and transferred to ice for 5 min. The
slurry was then centrifuged at 20000 x g for 15 min and 500 µL
of the supernatant was removed 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
The structure of protein receptors were taken from PDB database,
ERꢀα (PDB ID:3ERT), ERꢀβ (PDB ID:1QKM) and VDR receptor
(PDB ID:3AZ3). Missing residues and side chains of crystal
60 structures were modelled using Modeller software package.27
Geometry of ligand molecules were optimised using Avogadro
software. Molecular docking of test molecules with receptor
proteins was performed using Autodock 4.2 package. Ligand
binding conformations were evaluated using semiemperical free
65 energy force field. Receptor protein and ligand parameter files
were generated using AutodockToolsꢀ1.4.5. A grid surrounding
the protein receptor binding site was created with a grid spacing
of 0.375 for each protein receptor. Lamarkian Genetic Algorithm
(LGA) was used to generate the binding poses. Global
70 optimization with 200 randomly positioned poses, maximum
energy evaluations of 2.5 X 107, and a maximum of 2.7 X 107
generations were used to perform molecular docking. The
mutation rate of .02, crossover rate of .80, and elitism parameter
of 1 were used to perform molecular docking. For each docking,
75 200 docked poses were generated and binding poses were
clustered using root mean square deviation (RMSD) tolerance of
1 Å. Best docked conformation was obtained on the basis of
clustering and binding energy. Top cluster shows cluster of ligand
conformations having highest binding energy. Binding free
80 energy, inhibitory constant and number of poses in cluster of
docked conformation was reported. Docked complex images
were generated using PyMol.26
5
10 96ꢀwell 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
15 synthesis kit (Fermentas, Vilnius, Lithuania). SYBR green
chemistry was used for quantitative determination of the mRNAs
for BMPꢀ2, Runxꢀ2, Osx and a housekeeping gene, GAPDH,
following an optimized protocol. The design of sense and
antisense oligonucleotide primers was done using the Universal
20 Probe Library (Roche Diagnostics, Indianapolis, IN). For realꢀ
time PCR, the cDNA was amplified with Light Cycler 480
(Roche Diagnostics Pvt Ltd, Indianapolis, IN). The doubleꢀ
stranded DNAꢀspecific dye SYBR Green I was incorporated into
the PCR buffer provided in the Light Cycler 480 SYBER Green I
25 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 was 95 °C for 5
min, 40 cycles of denaturation at 94 °C for 2 min, and annealing
and extension at 62°C for 30s extension at 72°C for 30s. GAPDH
30 was used to normalize differences in RNA expression.
Conflicts of interest
85 There is no conflict of interest.
Acknowledgements
Western blotting
Cells were grown to 60ꢀ70% confluence following which they
were treated with and without compound for 48 h. Cells were
lysed with cell lysis buffer (SigmaꢀAldrich) with protease
35 inhibitor cocktail (SigmaꢀAldrich). 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% SDSꢀ
PAGE gel. After electrophoresis, proteins were transferred onto
40 PVDF membranes (ImmobilonꢀP, Millipore, Billerica, MA,
USA). The membranes were probed with ERꢀα, ERꢀβ and βꢀactin
antibodies (Cell Signaling Technology, Danvers, MA, USA) and
then incubated with secondary antibodies conjugated with HRP
(Cell Signaling Technology). Immunodetection was done using
45 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.
Authors are thankful to Director CSIRꢀCIMAP, CSIRꢀCDRI,
Lucknow and BSCꢀ203 project for financial support. DSR
acknowledged SERB, DST New Delhi, India for financial
90 support as DST Inspire Faculty Fellowship.
References
1. Y. Ishimi, J. Nutr. Sci. Vitaminol., 2015, 61, S139.
2. B. C. Lupsa, K. Insogna, Endocrinol. Metab. Clin. North Am., 2015,
44, 517.
95 3 .Doggrell, S. A. Drugs Today, 2003, 39, 633.
4. N. Harvey, E. Dennison, C. Cooper, J. Bone. Miner. Res., 2014,
29, 1917.
5. P. Kiberstis, O. Smith, C. Norman, Science, 2000, 1497.
6. R. F. Service, Science, 2000, 1498.
100 7. B. Clarke, J. Am. Soc. Nephrol., 2008, 3, S131.
8. N. S. Soysa, N. Alles, Biochem. Biophys. Res. Comm., 2016, 476, 115.
9. S.C. Manolagas, Endocr. Rev., 2000, 21, 115.
10. (a) Lips, P. Prog. Biophy. Mol. Biol., 2006, 92, 4. (b) Y. Xue, J. C.
Fleet, Gastroentrology, 2009, 136, 1317.
105 11. (a) S. Ray, A. Gupta, Drugs Fut., 2006, 31, 65. (b) M. T. Mizwicki, J.
E. Bishop, A. W. Norman, Steroid, 2005, 70, 464.
Statistical analysis
12. (a) S. Yamada, M. Shimizu, K. Yamamoto, Med. Res. Rev., 2003, 23,
89. (b) A. M. Deberardinis, U. Banerjee, M. K. Hadden, ACS. Med,
Chem. Lett., 2013, 4, 590. (c) G. H. Posner, Z. Li, M. C. White, V.
50 Data are expressed as mean ± SEM and were analyzed by
student’s t test. The data obtained from remaining experiments
with multiple treatments were subjected to oneꢀway ANOVA
followed by Newman–Keuls test of significance using Prism
version 3.0 software.
110
Vinader, K. Takeuchi, S. E. Guggino, P. Dolan, T. W. Kensler, J.
Med. Chem., 1995, 38, 4529. (d) M. F. Boehm, P. Fitzgerald, A. Zou,
M. G. Elgort, E. D. Bischoff, L. Mere, D. E. Mais, R. P. Bissonnette,
R. A. Heyman, A. M. Nadzan, M. R. Elizabeth A. Allegretto, Chem.
Biol., 1999, 6, 265.
55
This journal is © The Royal Society of Chemistry [2016]
MedChemComm, 2016, XXX, 01–08 | 13

MedChemComm
Page 14 of 15
View Article Online
DOI: 10.1039/C6MD00469E
13. (a) F. Ciesielski, Y. Sato, Y. Chebaro, D. Moras, A. Dejaegere, N.
Rochel, J. Med. Chem., 2012, 55, 8440. (b) S. Fujii, H. Masuno, Y.
Taoda, A. Kano, A. Wongmayura, M. Nakabayashi, N. Ito, M.
Shimizu, E. Kawachi, T.Hirano, Y. Endo, A. Tanatani, H. Kagechika,
J. Am.Chem. Soc., 2012, 133, 20933.
23. (a) J. M. Wozney, V. Rosen, A. J. Celeste, L. M. Mitsock, M. J.
Whitters, R. W. Kriz, R. M. Hewick, E. A. Wang, Science, 1988,
242, 1528. (b) W. Huang, S. Yang, J. Shao, Y. P. Li, Front. Biosci.
Landmark, 2007, 12, 3068. (b) W. Tang, Y. Li, L. Osimiri, C.
Zhang, J. Biol. Chem., 2011, 286, 32995.
24. (a) Y. Onoe, C. Miyaura, H. Ohita, S. Nozawa, T. Suda,
Endocrinology, 1997, 138, 4509. (b) J. Arts, G. G. J. M. Kuiper, J.
M. M. F. Janssen, J. A. Gustafsson, C. W. G. M. Lӧwik, H. A. P.
Pols, J. P. T. M. Vanꢀ Leeuwen, Endocrinology, 1997, 138, 5067.
45 25. S. Bord, A. Horner, S. Beavan, J. Compston, J. Clin Endocrinol.
Metab., 2001, 86, 2309.
26. DeLano, W. The PyMol Molecular Graphics System, Version 0.99
San Carlos CA: DeLano Scientific, 2002.
5
10
15
20
25
30
35
40
14. (a) S. Yamada, M. Makishima, Trends Pharmacol. Sci., 2015, 35,
324. (b) Z. Jin, X. Li, Y. Wan, Mol. Endocrinol., 2015, 29, 172.
15. M. Ishizawa, M. Matsunawa, R. Adachi, S. Uno,
K. Ikeda,
H. Masuno, M. Shimizu, K. Iwasaki, S. Yamada, M. Makishima, J.
Lipid. Res., 2008, 49, 763.
16. (a) S. Khosla, M. J. Oursler, D. G. Trends Endocr. Metab., 2012, 23,
576. (b) J. A. Cauley, Steroids, 2015, 99, 11.
17. (a) A. B. Khalid, S. A. Krum, Bone, 2016, 87, 130. (b) S, Nilsson, S,
Mäkelä, E. Treuter, M. Tujague, J. Thomsen, G. Andersson, E.
Enmark, K. Pettersson, M. Warner, J. A. Gustafsson, Physiol. Rev.,
2001, 81, 1535.
18. L. C. Hofbauer, S. Khosla, C. R. Dunstan, D. L. Lacey, T. C.
Spelsberg, B. L. Riggs, Endocrinology, 2013, 140, 4367.
19. (a) M. Saika, D. Inoue, S. Kido, T. Matsumoto, Endocrinology, 2001,
27. N. Eswar, B. Webb, M. A. MartiꢀRenom, M. S. Madhusudhan, D.
50
Eramian, M. Shen, A. Sali, Comparative Protein Structure Modeling
Using Modeller. In A. Bateman, W. R. Pearson, L. D. Stein, G. D.
Stormo, & J. R. Yates (Eds.), Current Protocols in Bioinformatics
(pp. 5.6.1–5.6.30). Hoboken, NJ, USA: John Wiley & Sons, Inc.,
2006. http://doi.wiley.com/10.1002/0471250953.bi0506s15.
142, 2205. (b) V. Parikka, P. Lehenkari, M. L. Sassi, J. Halleen, J. 55 28. (a) A. Gupta, I. Ahmad, J. Kureel, M. Hasanain, P. Pandey, S. Singh,
Risteli, P. Härkönen, H. K. Väänänen, Endocrinology, 2001, 142,
5371.
A. A. John, J. Sarkar, D. Singh, J. Steroid Biochem. Mol. Biol., 2016,
158, 63. (b) D. K. Yadav, A. K. Gautam, J. Kureel, K. Srivastava, M.
Sahai, D. Singh, N. Chattopadhyay, R. Maurya, Bioorg. Med. Chem.
Lett., 2011, 21, 677. (c) A. L. Strong, Q. Jiang, Q. Zhang, S. Zheng,
S. M. Boue, S. Elliott, M. E. Burow, B. A. Bunnell, G. Wang, ACS
Med. Chem. Lett., 2014, 5, 143.
20. (a) R. L. Jilka, G. Hangoc, G. Girasole, G. Passeri, D. C. Williams, J.
S. Abrams, B. Boyce, H. Broxmeyer, S. C. Manolagas, Science,
1992, 257, 88. (b) G. Girasole, R. L. Jilka, G. Passeri, S. Boswell, G.
Boder, D. C. Williams, S. C. Manolagas, J. Clin. Invest., 1992, 89,
883.
60
65
70
29. (a) M. K. Hussain, M. I. Ansari, N. Yadav, P. K. Gupta, A. K. Gupta,
R. Saxena, I. Fatima, M. Manohar, P. Kushwaha, V. Khedgikar, J.
Gautam, R. Kant, P. R. Maulik, R. Trivedi, A. Dwivedi, K. R.
Kumar, A. K. Saxena, K. Hajela, RSC Adv., 2014, 4, 8828. (b) C. H.
Tseng, Y. L. Chen, C. M. Lu, C. K. Wang, Y. T. Tsai, R. W. Lin, C.
F. Chen, Y. F. Chang, G. J. Wang, M. L. Ho, C. C. Tzeng, Eur. J.
Med. Chem., 2009, 44, 3621.
21. (a) S. Lamy, P. L. Moldovan, A. B. Saad, B. Annabi, Biochim.
Biophys. Acta., 2015, 1853, 126. (b)
E Middleton, Jr., C.
Kandaswami, T Theoharides, Pharmacol. Rev., 2000, 52, 673.
22. (a) G. SánchezꢀDuffhues, C. Hiepen, P. Knaus, P. ten Dijke, Bone,
2015, 80, 43ꢀ59. (b) D. Chen, M. Zhao, G. R. Mundy, Growth
Factors, 2004, 22, 233. (c) Y. Takuwa, C. Ohse, E. A. Wang, J. M.
Wozney, K. Yamashita, Biochem. Biophys. Res. Comm., 1991, 174,
96.
30. G. J. M. Kuiper, J. G. Lemmen, B. O. Carlsson, J. C. Corton, S. H.
Safe, P. T. Van Der Saag, B. Van Der Burg, J. A. Gustafsson,
Endocrinology,
1998,
139,
4252.
14 | MedChemComm, 2016, XXX, 01–08
This journal is © The Royal Society of Chemistry [2016]

Page 15 of 15
MedChemComm
View Article Online
DOI: 10.1039/C6MD00469E
MedChemComm
Graphical Abstract:
Design and synthesis of 3-arylbenzopyran based non-steroidal vitamin-D3 mimics as osteogenic agents
Imran Ahmad, Dushyant singh Raghuvanshi, Sarita Singh, Aiaiz A Johan, Ravi Prakash, Kripa Shankar Nainawat,
Divya Singh, Shubhandra Tripathi, Ashok Sharma, Atul Gupta*
3ꢀArylbenzopyran based nonꢀsteroidal osteogenic agents have been explored as a structural template of estrogen and vitaminꢀD3. The target
molecules 18a-c, 19a-c, 26a-c, 27a-c and intermediates (17a-c and 25a-c) were studied for their osteogenic activity in osteoblast differentiation
assay inꢀvitro using mouse calvarial osteoblast cells. Compounds 25c, 26b, 27b and 27c effectively increased in ALP activity at 1pM
concentration compared to the untreated cells. The active compounds were devoid of inherent toxicity at 1pM concentration in osteoblast cells.
The most active compound, 27b, was studied for mineralization of osteoblast cells, expression of marker genes involved in osteogenesis.
Molecular docking analysis performed for 27b showed its possible interactions with estrogen receptorꢀα and β (ERꢀα and ERꢀβ) as well as
vitaminꢀD receptor (VDR).
27b enhanced osteoblast differentiation at 1pM in mouse calvarial osteoblast cells without inherent toxicity