Synthetic analogs of daidzein, having more potent osteoblast stimulating effect

Article abstract of DOI:10.1016/j.bmcl.2010.12.008

A series of didzein derivatives were synthesized and assessed for stimulation of osteoblast differentiation using primary cultures of rat calvarial osteoblasts. Data suggested that three synthetic analogs, 1c, 3a and 3c were several folds more potent than daidzein in stimulating differentiation and mineralization of osteoblasts. Further, these three compounds did not show any estrogen agonistic activity, however had mild estrogen antagonistic effect. Out of the three compounds, 3c was found to maximally increase the mineralization of bone marrow osteoprogenitor cells. Compound 3c also robustly increased the mRNA levels of osteogenic genes including bone morphogenetic protein-2 and osteocalcin in osteoblasts. Unlike daidzein, 3c did not inhibit osteoclastogenesis. Collectively, we demonstrate osteogenic activity of daidzein analogs at significantly lower concentrations than daidzein.

Full text of DOI:10.1016/j.bmcl.2010.12.008

Bioorganic & Medicinal Chemistry Letters 21 (2011) 677–681
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthetic analogs of daidzein, having more potent osteoblast
stimulating effect q
Dinesh K. Yadav a, Abnish K. Gautam b, Jyoti Kureel b, Kamini Srivastava b, Mahendra Sahai c,
Divya Singh b, Naibedya Chattopadhyay b, Rakesh Maurya a,
⇑
a Medicinal and Process Chemistry Division, Central Drug Research Institute (Council of Scientific and Industrial Research), Lucknow 226 001, India
b Endocrinology Division, Central Drug Research Institute (Council of Scientific and Industrial Research), Lucknow 226 001, India
c Department of Medicinal Chemistry, Institute of medical Sciences, Banaras Hindu University, Varanasi 221 005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of didzein derivatives were synthesized and assessed for stimulation of osteoblast differentiation
using primary cultures of rat calvarial osteoblasts. Data suggested that three synthetic analogs, 1c, 3a and
3c were several folds more potent than daidzein in stimulating differentiation and mineralization of oste-
oblasts. Further, these three compounds did not show any estrogen agonistic activity, however had mild
estrogen antagonistic effect. Out of the three compounds, 3c was found to maximally increase the min-
eralization of bone marrow osteoprogenitor cells. Compound 3c also robustly increased the mRNA levels
of osteogenic genes including bone morphogenetic protein-2 and osteocalcin in osteoblasts. Unlike daidz-
ein, 3c did not inhibit osteoclastogenesis. Collectively, we demonstrate osteogenic activity of daidzein
analogs at significantly lower concentrations than daidzein.
Received 24 September 2010
Revised 8 November 2010
Accepted 2 December 2010
Available online 8 December 2010
Keywords:
Phytoestrogen
Daidzein
Deoxybenzoin
Formylation
Ó 2010 Elsevier Ltd. All rights reserved.
Mineralization
Osteogenic
Osteoblast proliferation
With the discontinuation of hormone replacement therapy for
bone maintenance after menopause, many postmenopausal wo-
men are looking for prophylactic alternatives, particularly from
natural source.1,2 As a result, there is a growing interest in assess-
ing the role of plants and plant-derived compounds in prevention
of postmenopausal osteoporosis.3 Many bioactive compounds have
been identified from plants including a large class of flavonoids, the
so called phytoestrogens, which exhibit antioxidant properties and
may act as estrogen receptor (ER) agonists with beneficial out-
comes in postmenopausal osteoporosis.4
Daidzein is an extensively studied phytoestrogen with respect to
its skeletal effects. Daidzein affords bone-protective action by stim-
ulation of osteoblast5–7 and inhibition of osteoclast functions8
through the ERs. Besides its ER mediated effect, daidzein has non-
genomic effect on bone wherein it stimulates protein synthesis in
osteoblastsbyactivating aminoacyltRNA synthetase.9 Daidzeinalso
enhances bone morphogenetic protein-2 production in osteoblast.5
Ten weeks of daily injection of daidzein at 16.6 mg kgꢀ1 dose to
growing ovariectomized (Ovx) rats exhibited significant bone form-
ing effect.10 In adult OVx mice on high calcium diet, daidzein at
100 mg kgꢀ1 dayꢀ1 oral dose for 12 weeks favorably influenced both
trabecularand corticalbone.11 Besides, highdietaryintakeofisoflav-
ones like daidzein and genistein has been reported to increase BMD
in lumbar spine of Japanese12, Chinese13 and American14 postmeno-
pausal women. However, uterine estrogenic action of daidzein
precludes its osteoprotective use in postmenopausal women. The
uterine estrogenic effect of daidzein is partly contributed by its
highly estrogenic metabolite, equol.15,16 Because of its intestinal
biotransformation to equol, daidzein has low oral bioavailability.
From these reports, it appear that daidzein could be a suitable
therapeutic ‘lead molecule’ for postmenopausal osteoporosis if is-
sues pertaining to its metabolism, estrogenicity, and most impor-
tantly improvement of its potency in promoting osteoblast
function are addressed.
Here, we focused on modifying the structure of daidzein to
achieve improved osteogenic effect and eliminate uterine estroge-
nicity. To that effect, we synthesized novel daidzein derivatives
and assessed osteoblast functions (differentiation and mineraliza-
tion) in vitro using rat calvarial and bone marrow osteoblasts.
We also studied uterine estrogenicity of active compounds.
Daidzein and methoxylated daidzein derivatives were prepared
by reacting appropriate substituted phenyl acetic acid with
properly substituted resorcinols in the presence of BF3ꢁetherate
produced deoxybenzoins, deoxybenzoins on formylation gave iso-
flavones (1–4)17 Scheme 1.
q
CDRI Communication No. 8000.
⇑
Corresponding author. Tel.: +91 (522) 2612411 18x4235; fax: +91 (522) 2623
405/2623938/2629504.
E-mail address: mauryarakesh@rediffmail.com (R. Maurya).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.008

678
D. K. Yadav et al. / Bioorg. Med. Chem. Lett. 21 (2011) 677–681
The compounds 1–4 on Williamson type O-alkylation reaction
osteoblast mineralization, calvarial osteoblasts and bone marrow
cells were cultured for 18–21 days in differentiation media con-
taining 10 mM b-glycerophosphate and 50 lg/ml ascorbic acid in
with different 1-(2-chloro-alkyl) substituted amine hydrochloride
in the presence of potassium carbonate in acetone/DMF gave alkyl
substituted amino compounds 1a–d,g, 2a–d, 3a–d and 4a–d in
good yield. The compounds 1–4 on reaction with different haloalk-
yls (allylbromide, isopropylbromide, 2-bromopropane, epichlrohy-
drine, 1,2-dibromoethane and 1,3-dibromopropane, methyliodide)
afforded compounds 1e,f,h–j, 2e–j, 3e–h, 4e–h.18 The compounds
2i and 3g with NaH in dry DMF afforded 2j and 3i in very good
yield. All the synthetic products gave satisfactory analytical and
spectroscopic data, which were in the full accordance with their
formulated structures.
All compounds were tested for osteogenic activity using pri-
mary cultures of rat osteoblasts following previously described
protocol from our laboratory.19–21 Production of alkaline phospha-
tase (ALP) serves as a differentiation marker of osteoblasts.22,23 We
used osteoblast ALP assay to screen the activity of various synthe-
sized compounds following our previously published protocol. For
presence or absence of compounds. Cells were then stained with
alizarin red-S and dye was extracted to quantitate the extent of
osteoblast mineralization.19,24 Transcript levels of osteogenic
genes (bone morphogenetic protein-2 and osteocalcin) were
determined by real time RT-PCR (qPCR) following our previously
published protocols.25 Primer pairs used were; for BMP-2,
50-CGG ACT GCG GTC TCC TAA-30 (sense); 50-GGG GAA GCA GCA
ACA CTA GA-50 (antisense); for osteocalcin, 50-GGA CAT TAC TGA
CCG CTC C-30 (sense), 50-TTT TCA GTG TCT GCC GTG AG-30 (anti-
sense); for GAPDH, 50-CAG CAA GGA TAC TGA GAG CAA GAG-30
(sense), 50-GGA TGG AAT TGT GAG GGA GAT G-30 (antisense). Data
are expressed as mean SEM. The data obtained in experiments
with multiple treatments were subjected to one-way ANOVA fol-
lowed by post hoc Tukey’ test of significance using MINITAB
13.1 software.
R1O
OH
R3
.
BF3 Et2O
R1O
OH
O
HOOC
+
80-100oC, 6-8 hrs
OR2
R3
OR2
.
1. BF3 Et2O/DMF
00C
R1O
O
O
2. CH3SO2Cl,
100oC, 2hrs
R3
OR2
1: R1=R2=R3=H
2: R1=CH3, R2=R3=H
3: R2=CH3, R1=R3=H
4: R1=H, R2=CH3, R3=OCH3
1a: R1=R2=-CH2CH2pyrrolidine, R3=H
1b: R1=R2=-CH2CH2piperidine, R3=H
1c: R1=R2=-CH2CH2N(C2H5)2, R3=H
1d: R1=R2=-CH2CH2morpholine, R3=H
1e: R1=R2=Allyl, R3=H
2a: R1=CH3, R2=-CH2CH2pyrrolidine, R3=H
2b: R1=CH3,R2=-CH2CH2piperidine, R3=H
2c: R1=CH3,R2=-CH2CH2N(C2H5)2, R3=H
2d: R1=CH3,R2=-CH2CH2morpholine, R3=H
2e: R1=CH3,R2=Allyl, R3=H
2f: R1=CH3,R2=Isopropyl, R3=H
2g: R1=CH3,R2=Isobutyl, R3=H
2h: R1=CH3,R2=-CH2oxiran, R3=H
2i: R1=CH3,R2=-CH2CH2Br, R3=H
2j: R1=CH3,R2=-CH=CH2, R3=H
1f: R1=R2=Isopropyl, R3=H
1g: R1=-CH2CH2N(C2H5)2, R2=R3=H
1h: R1=R2=-CH2oxiran, R3=H
1i: R1=-CH2CH2Br, R2=R3=H
1j: R1=R2=-CH3, R3=H
3a: R1=-CH2CH2pyrrolidine, R2=CH3, R3=H
3b: R1=-CH2CH2piperidine, R2=CH3, R3=H
3c: R1=-CH2CH2N(C2H5)2, R2=CH3, R3=H
3d: R1=-CH2CH2morpholine, R2=CH3, R3=H
3e: R1=Isopropyl, R2=CH3, R3=H
4a: R1=-CH2CH2pyrrolidine, R2=CH3, R3=OCH3
4b: R1=-CH2CH2piperidine, R2=CH3, R3=OCH3
4c: R1=-CH2CH2N(C2H5)2, R2=CH3, R3=OCH3
4d: R1=-CH2CH2morpholine, R2=CH3, R3=OCH3
4e: R1=Allyl, R2=CH3, R3=OCH3
3f: R1=-CH2oxiran, R2=CH3, R3=H
4f: R1=Isopropyl, R2=CH3, R3=OCH3
3g: R1=-CH2CH2Cl, R2=CH3, R3=H
3h: R1=-CH2CH2CH2Br, R2=CH3, R3=H
3i: R1=-CH=CH2, R2=CH3, R3=H
4g: R1=-CH2oxiran, R2=CH3, R3=OCH3
4h: R1=-CH2CH2Br, R2=CH3, R3=OCH3
Scheme 1. Synthesis of daidzein analogs.

D. K. Yadav et al. / Bioorg. Med. Chem. Lett. 21 (2011) 677–681
679
Thirty seven synthetic compounds were screened using
osteoblast ALP assay (osteoblast differentiation). As shown in
Figure 1A–E, five of out of 37 compounds significantly increased
osteoblast ALP activity. These were 1c, 2g, 2i, 3a, and 3c. At concen-
trations ranging from 1.0 pM to 10.0 nM, each of these compounds
stimulated osteoblast ALP activity. At these low concentrations,
daidzein did not stimulate ALP activity (data not shown), and in-
in BMCs treated with 10.0 nM 1c or 3c. As 3c has greater degree
of statistical significance than 1c, we conclude that 3c is the most
effective among the three active compounds in stimulating miner-
alization of BMCs.
We next studied the effect of 3c on the expression of osteogenic
genes (osteocalcin and BMP-2) in rat calvarial osteoblasts. qRT-PCR
data show that 10.0 nM 3c robustly stimulated mRNA levels of
both osteocalcin (10-fold) and BMP-2 (six-fold) in osteoblasts
(Fig. 4).
As daidzein is known to have uterine estrogenicity, we studied
the effect of 1c, 3a and 3c in rat uterus. Twenty-one days old rats
(n = 4) were ovariectomized and oral administration of 1c, 3a and
3c was given at 10 mg kgꢀ1 dayꢀ1 dose for three consecutive days
with or without E2. Raloxifene, a SERM in clinical use was also used
as control. Table 1 shows that as expected E2 treatment resulted in
a robust increase in uterine weight compared with vehicle treated
rats. Raloxifene had modest but significant estrogenic effect. Com-
pounds 1c and 3a had no effect on uterine weight compared with
vehicle, suggesting no estrogenic effect. Compound 3c however,
modestly but significantly reduced uterine weight compared to
vehicle, suggesting anti-estrogenic effect. In anti-estrogenicity
stead required 1.0 lM to significantly stimulate ALP activity in
osteoblasts (Fig 1 A–E). From these data, it appears that 1c, 2g,
2i, 3a, and 3c are ꢂ100-fold more potent than daidzein in stimulat-
ing osteoblast differentiation.
As 1c, 3a and 3c were more active than 2g and 2i in stimulating
osteoblast ALP activity, we next assessed effect of these three com-
pounds (1c, 3a and 3c) in stimulating osteoblast mineralization
in vitro, using calvarial osteoblasts. Compounds 1c, 3a and 3c en-
hanced formation of mineralized nodules at 10.0 nM concentration
(Fig 2).
BMCs were cultured in osteoblast differentiation medium for
18 days in presence or absence of compounds (1c, 3a and 3c).
Mineralized nodules were stained by alizarin red-S. As shown in
Figure 3, number of mineralized nodules was significantly more
Figure 1. Effects of daidzein analogs on osteoblast differentiation. 2 ꢃ 103 rat calvarial osteoblasts were seeded in 96-well plate and exposed to various concentrations of
compounds 1c, 2i, 2g, 3a and 3c for 48 h. ALP activity was determined spectrophotometrically at 405 nm. Daidzein at 1
mean SD; n = 6; ⁄P <0.05, ⁄⁄P <0.01 compared with vehicle treated cells.
lM was used as a positive control. Data shown as

680
D. K. Yadav et al. / Bioorg. Med. Chem. Lett. 21 (2011) 677–681
Figure 2. Effects of 1c, 3a and 3c on mineralization of rat calvarial osteoblasts. 2 ꢃ 103 osteoblasts were seeded in 12-well plates and exposed to 10.0 nM 1c, 3a and 3c for
seven days. At the end of the incubation, cells were fixed and stained with alizarin red-S. Stain was extracted and O.D. measured colorimetrically as described in material and
method. Data shown as mean SD; n = 4; ⁄P <0.05, ⁄⁄P <0.01 compared with vehicle treated cells.
Figure 3. Effect of 1c, 3a and 3c on mineralization of BMCs. BMCs (2 ꢃ 105 cells) were seeded in 12-well plates and incubated with 10.0 nM 1c, 3a and 3c for 18 days. At the
end of the incubation, cells were fixed and stained with alizarin red-S. Stain was extracted and O.D. measured colorimetrically as described in material and method. Data
shown as mean SD; n = 4; ⁄⁄P <0.01, ⁄⁄⁄P <0.001 compared with vehicle treated cells.
assay (Table 1), all three compounds inhibited E2-induced increase
of uterine weight, suggesting their anti-estrogenic effect.
Daidzein is an extensively studied phytoestrogen with respect
to its skeletal effects. It promotes osteoblast functions and inhibits
osteoclast functions in vitro, albeit at micromolar concentrations,
that is, unlikely to translate in vivo.4 Also, daidzein has been re-
ported to exhibit estrogenicity at the uterine level. Therefore, we
synthesized analogs of daidzein with the aim to improve its osteo-
genic potency and eliminate uterine estrogenicity. In the present
work, a total of thirty seven compounds structurally related to
daidzein (1a–j, 2a–j, 3a–i, 4a–h) were evaluated for in vitro osteo-
genic action in the primary cultures of osteoblasts derived from
calvaria and BM. In the initial screening using calvarial osteoblasts
Figure 4. Effects of 3c on mRNA expression of osteocalcin and BMP-2. Osteoblast
cells were cultured with or without 3c for 24 h. qPCR for osteocalcin and BMP-2 was
performed as described in materials and methods. Compound 3c at 10 nM
where ALP production (osteoblast differentiation marker) was
measured, five compounds (1c, 2g, 2i, 3a and 3c) significantly in-
creased ALP activity over control. Daidzein was required at several
folds higher concentration than these five compounds, suggesting
increased osteogenic potency of our synthetic compounds over
dadizein.
increased the osteocalcin mRNA expression (ꢂ10-fold) and BMP-2 expression
(ꢂsix-fold) when compared to vehicle control group. Data shown as mean SEM;
n = 4; ⁄P <0.05, ⁄⁄P <0.01 compared with vehicle treated cells.
As 1c, 3a and 3c were more active (⁄⁄P <0.01 vs control) at
10.0 nM than 2g and 2i (⁄P <0.05 vs control) in osteoblast ALP
assay, we next tested the effect of 1c, 3a and 3c on in vitro miner-
alization of calvarial and BM osteoblasts. Our data show that
although these three compounds had comparable effects on calvar-
ial mineralization however, in the BM, 3c was most active in stim-
ulating mineralization (⁄⁄⁄P <0.001 for 3c vs ⁄⁄P <0.01 for 1c). We
further confirmed the osteogenic effect of 3c in calvarial osteo-
blasts by quantifying osteogenic gene expression. By qRT-PCR,
we demonstrated that 3c increased mRNA levels of BMP-2 and
OCN over control. Collectively, these data indicate that 3c is a po-
tent osteogenic compound in vitro.
Table 1
Estrogen agonistic and antagonistic activities of compounds in OVx immature
Sprague Dawley rats
Treatment
Dose
(mg/kg)
Estrogenic activity
(uterine weight
in mg)
Anti-estrogenic
activity (uterine
weight in mg)
Vehicle
Ethynyl estradiol
Raloxifene
1c
3a
3c
—
0.01
3
10
10
10
35.0 0.6
82.3b 3.4
42.3a 1.3
31.3 1.5
31.3 4.3
28.0a 1.5
67* 4.1
57.3** 2.3
59.3* 5.6
63.0* 3.8
Values are mean SD.
Daidzein is a ‘mixed’ agent as it could stimulate osteoblast
a
P <0.05 versus corresponding vehicle control.
P <0.001 versus corresponding vehicle control.
P <0.05 versus corresponding ethynyl estradiol group.
P <0.01 versus corresponding ethynyl estradiol group.
b
activity and inhibit osteoclast differentiation, both at l
M range.5,8
*
Interestingly, unlike daidzein, 3c did not inhibit osteoclastogenic
differentiation of the BM cells in the presence of RANKL and
**

D. K. Yadav et al. / Bioorg. Med. Chem. Lett. 21 (2011) 677–681
681
M-CSF (data not shown). From these data, it appears that 3c is a
Supplementary data
‘pure’ osteogenic compound, that is, much more potent than daidz-
ein and is devoid of uterine estrogenicity. However, significant
anti-estrogenic effect of 3c may find effective use of 3c against
breast cancer (study in progress).
Supplementary data (experimental procedures, biological eval-
uation methods and NMR data of all synthesized compounds) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.12.008.
Structure–activity relationship (SAR) studies reveal that the
substitution at C-7 and C-40 appear essential for better in vitro
osteogenic activity and diminish uterine estrogenicity of daidz-
ein.19 Keeping this aspect in mind several derivatives of daidzein
have been synthesized and screened for in vitro osteogenic activ-
ity. SAR has been discussed based on the results obtained. Our
study reveals that the presence of free hydroxyl at C-40 with com-
binations at C-7 of 2-diethylamino-ethoxy and 2-bromo-ethoxy
does not appear essential for osteogenic activity, as compounds
1g and 1i were found to be inactive in osteogenic assay. In search
of most suitable combination for lead optimization some more
molecules were designed and synthesized. It appears that methoxy
substituent at C-40 with combinations at C-7 of 2-pyrrolidin-1-yl-
ethoxy and 2-diethylamino-1-yl-ethoxy (3a, 3c) led to more potent
osteogenic activity than dadizein. Compounds 3b, 3d–3i, 4a–4h
possessed methoxy substituent at C-40 moiety, but were inactive
in ALP assay. This could be due to the presence of other substitu-
ents. In addition, presence of methoxy group at C-30 (4a–4h) and
vinylic, allyloxy, oxiranylmethoxy, heloethoxy, morpholin-ethoxy,
piperidin-ethoxy and pyrrolidin-ethoxy moieties at C-7 and C-40
positions or vice–versa also seem non-essential for osteogenic
activity since 1a, 1b, 1d–1j, 2a–2f, 2h, 2j, 3b, 3d–3i, 4a–4h had
no significant osteogenic effect. Together, these activity data sup-
port that the combination of groups at C-7 and C-40 (1c, 2g, 2i,
3a, 3c) in daidzein skeleton are critical determinants of enhanced
osteogenic activity and to eliminate uterine estrogenicity. En-
hanced osteogenic activity as exemplified by increased ALP activ-
ity, mineralization and transcript levels of osteogenic markers by
most active analog designed as, 7-(2-diethylamino-ethoxy)-3-(4-
methoxy -phenyl)-4H-chromen-4-one (3c) led us to conclude that
the 7,40-dihydroxy-isoflavon or 7-hydroxy-3-(4-hydroxy-phenyl)-
chromen-4-one or daidzein skeleton is an important one, which
could provide some useful clue for further design of an effective
bone anabolic agent.
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Authors gratefully acknowledge grant-in-aid from the Ministry
of Health and Family Welfare, Government of India. Research
fellowship grants from the Council of Scientific and Industrial
Research (D.K.Y.) and University Grants Commission (A.K.G.) are
acknowledged. The authors are thankful to SAIF Division, Central
Drug Research Institute for recording spectral data.