Design, synthesis, and osteogenic activity of daidzein analogs on human mesenchymal stem cells

Article abstract of DOI:10.1021/ml400397k

Osteoporosis is caused by an overstimulation of osteoclast activity and the destruction of the bone extracellular matrix. Without the normal architecture, osteoblast cells are unable to rebuild phenotypically normal bone. Hormone replacement therapy with estrogen has been effective in increasing osteoblast activity but also has resulted in the increased incidence of breast and uterine cancer. In this study we designed and synthesized a series of daidzein analogs to investigate their osteogenic induction potentials. Human bone marrow derived mesenchymal stem cells (MSCs) from three different donors were treated with daidzein analogs and demonstrated enhanced osteogenesis when compared to daidzein treatment. The enhanced osteogenic potential of these daidzein analogs resulted in increased osterix (Sp7), alkaline phosphatase (ALP), osteopontin (OPN), and insulin-like growth factor 1 (IGF-1), which are osteogenic transcription factors that regulate the maturation of osteogenic progenitor cells into mature osteoblast cells.

Full text of DOI:10.1021/ml400397k

Letter
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Osteogenic Activity of Daidzein Analogs on
Human Mesenchymal Stem Cells
Amy L. Strong,† Quan Jiang,‡ Qiang Zhang,‡ Shilong Zheng,‡ Stephen M. Boue,§ Steven Elliott,∥
Matthew E. Burow,∥ Bruce A. Bunnell,†,* and Guangdi Wang‡,*
†Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112,
United States
‡Department of Chemistry and RCMI Cancer Research Program, Xavier University, New Orleans, Louisiana 70125, United States
§Southern Regional Research Center, U.S. Department of Agriculture, New Orleans, Louisiana 70130, United States
∥Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112, United States
S
* Supporting Information
ABSTRACT: Osteoporosis is caused by an overstimulation of
osteoclast activity and the destruction of the bone extracellular
matrix. Without the normal architecture, osteoblast cells are
unable to rebuild phenotypically normal bone. Hormone
replacement therapy with estrogen has been effective in
increasing osteoblast activity but also has resulted in the
increased incidence of breast and uterine cancer. In this study
we designed and synthesized a series of daidzein analogs to
investigate their osteogenic induction potentials. Human bone
marrow derived mesenchymal stem cells (MSCs) from three
different donors were treated with daidzein analogs and
demonstrated enhanced osteogenesis when compared to
daidzein treatment. The enhanced osteogenic potential of
these daidzein analogs resulted in increased osterix (Sp7), alkaline phosphatase (ALP), osteopontin (OPN), and insulin-like
growth factor 1 (IGF-1), which are osteogenic transcription factors that regulate the maturation of osteogenic progenitor cells
into mature osteoblast cells.
KEYWORDS: Daidzein analogs, mesenchymal stem cells, BMSCs, osteogenesis
accelerates after menopause.5,6 Estrogen therapy has been
steoporosis, characterized by the loss of bone mass and
Ostrength that leads to fragility fractures, is a major clinical shown to prevent the early phases of bone loss and decrease the
incidence of subsequent osteoporosis-related fractures by about
50%.5 However, the incidence of endometrial cancer is
increased with estrogen therapy and outweighs the benefits of
reducing osteoporosis.6 As alternatives, calcium, vitamin D,
calcitonin, and bisphosphates have been investigated. The use
of bisphosphates has shown significant improvement in bone
density relative to controls, while higher concentrations and
longer delivery period of calcium, vitamin D, and calcitonin
only demonstrated modest improvements.7 These bisphos-
phate compounds are carbon-substituted analogs of pyrophos-
phate, an endogenous physiologic inhibitor of bone mineraliza-
tion, and are thus potent inhibitors of bone resorption.7,8
Nevertheless, concomitant delivery of drugs capable of
increasing the number of osteoblasts or enhancing osteoblastic
activity without increased cancer risk continues to be an
attractive addition to bisphosphate therapy. Phytoestrogenic
concern due to significant morbidity and mortality in
postmenopausal women.1 The prevalence of osteoporosis in
the United States is estimated to increase from 10 million to 14
million people in 2020, increasing the economic burden from
$17 billion to $25 billion.2,3 The broad impact of osteoporosis
across age, sex, race, ethnicity, and fracture site underscores the
need for increased awareness of the disease and the
development of novel therapeutic interventions for patients.2
The loss of bone mass results from an imbalance of two
fundamental processes related to bone turnover: (1) excessive
bone resorption, resulting in decreased bone mass and
architectural deterioration of bone, and/or (2) inadequate
formation in response to increased resorption during bone
remodeling.4 Excessive resorption without adequate bone
formation can result in complete loss of trabecular structure,
destroying the template for future bone formation. Inability to
form bone during remodeling also contributes to the
pathogenesis of osteoporosis. Estrogen deficiency during
aging is likely to be the most important risk factor in the
pathogenesis of bone fragility in women, as bone loss
Received: October 9, 2013
Accepted: December 10, 2013
Published: December 10, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
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molecules, which mimic the effects of estrogen, have received a
great deal of attention due to their potential to prevent
hormone-related cancers and bone loss.9−12 Isoflavones are
found in abundance in soybeans and their derivative foods. In
vitro studies have shown that daidzein and genistein have
stimulatory effects on protein synthesis and on alkaline
phosphatase (ALP) released by various types of osteoblast
cells.11 Recent studies have demonstrated that ovariectomy-
induced bone loss in rats is rescued with the delivery of the
isoflavones daidzein and genistein.9,10 Epidemiological studies
in humans have shown that high dietary phytoestrogen intake is
associated with higher bone mineral density in postmenopausal
women.12
Scheme 1. Synthesis of Daidzein Derivatives and Analogs
Daidzein is a potent estrogenic compound that has a
beneficial effect on bone health,11,12 but its clinical potential is
limited by its low bioavailability, unfavorable metabolism, and
uterine estrogenicity. A recent study reported improved
functions of isoformononetin, a naturally occurring methox-
ydaidzein for the bone anabolic effect.13 At the most effective
osteogenic dose of isoformononetin, plasma and bone marrow
levels were 90% isoformononetin and 10% daidzein. Also under
these conditions, isoformononetin induced mesenchymal stem
cell (MSC) mineralization and osteogenic gene expression in
the calvaria of neonatal rats without causing uterine patho-
genesis. More recently, Yadav et al. reported a series of
synthetic daidzein analogs with both 7-OH and 4′-OH
modified with various substitutions to exhibit a stronger
osteoblast stimulating effect than daidzein.14 Interestingly, a
related isoflavone, genistein, which is a stronger estrogen than
daidzein, was not as effective a bone loss inhibitor as daidzein,10
suggesting that daidzein may be a better lead compound for
pharmacophoric optimization of potential osteogenic therapeu-
tic agents.
Previous studies found that replacing the 7-OH of daidzein
with alkoxy groups attenuated the estrogenic potency to various
degrees, with longer and bulkier substitutions having greater
effects.15 Moreover, some structural modifications on the 7-O
position conferred a dramatic reversal from estrogenic to
antiestrogenic property, suggesting the versatility of the
daidzein structural motif could offer additional pharmacological
functions that require an optimal balance of hormonal activities
of the compounds. In this study, we investigate the utility of a
series of daidzein analogs in promoting bone formation. The
effects of structural variations of daidzein on the osteogenic
induction of human bone marrow derived MSCs, which
differentiate into osteoblasts under appropriate stimulation,
were explored. Structural variations at the 7-OH position and
the central daidzein moiety were made to test how the
osteogenic activities varied as a result of such changes in
substitution and the daidzein skeleton. Because it has been
shown recently that equol, the metabolic product of daidzein,
may be responsible for its superior bone-healing property
compared to genistein and other isoflavones,10 several racemic
equol analogs were also synthesized and tested for potential
gain of activity. We show that daidzein analogs can be potent
stimulators of osteogenesis in MSCs in a dose-dependent
manner. To understand how these daidzein analogs exert
enhanced osteogenic potential in the MSCs, a relevant gene
expression panel was analyzed and results were discussed for
mechanistic interpretations.
diethoxy-3-methylbut-2-ene with picoline as the base under
microwave irradiation. Further, the hydrogenation of 3 using
10% Pd/C resulted in the formation of derivative 4 or 5 as the
major product, respectively, depending on the use of ethyl
acetate or methanol as solvent. The derivative 7 was prepared
by the successive Claisen rearrangement and cyclization of 6 in
refluxing N,N-diethylaniline with CsF as the catalyst. Reacting
with trifluoromethanesulfonic anhydride, 3 was converted into
its triflate, which was the starting material for the synthesis of
derivative 8 through the PdCl2(dppf)-catalyzed borylation
using the diboron reagent in the presence of KOAc. O-
Butylation on the 7-hydroxyl group of equol gave the derivative
9. The derivative 10 was obtained by the reaction of equol and
1,1-diethoxy-3-methylbut-2-ene in refluxing xylene with pico-
line as the base under microwave irradiation.
Scheme 2 shows the synthetic route for compounds 11 and
12a−b. The condensation of 2,4-dimethoxybenzoic acid and 4-
anisidine or 4-((tetrahydro-2H-pyran-2-yl)oxy)aniline with
DCC and DMAP as activating agents gave 12a or 2,4-
dimethoxy-N-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl) ben-
zamide, respectively. Compound 11 was obtained by the
deprotection of the intermediate benzamide in methanol with
4-toluenesulfonic acid. Finally, O-isobutylation of 11 in DMSO
afforded compound 12b in the presence of K2CO3.
Characterization of MSCs. MSCs undergo osteogenic
differentiation under appropriate stimulation. MSCs were
isolated from bone marrow aspirates from three donors and
expanded separately prior to characterization. The demographic
information for the three donors is provided in Supporting
Information Table 1S. Each MSC donor (n = 3) was analyzed
for the expression of cell surface markers and was positive for
CD44, CD90, CD105, and CD166 and negative for CD34,
CD45, and CD11b, determined with flow cytometry
As shown in Scheme 1, selective O-alkylation on the 7-
hydroxyl group of daidzein (1) gave derivatives 2a−l and 6.15
The derivative 3 was obtained by the reaction of 1 and 1, 1-
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Scheme 2. Synthesis of 11, 12a, and 12b
Reagents and conditions: (a) 4-methoxy- or 4-THPO-aniline, DCC,
DMAP, DMF, 0 °C to rt; (b) 4-toluenesulfonic acid, MeOH, reflux;
(c) K2CO3, i-butylbromide, DMSO, rt.
(Supporting Information Figure S1). All MSCs were able to
generate colony-forming units and undergo osteogenesis and
adipogenesis (Supporting Information Figure S1). No differ-
ences were observed among the three MSC donors in cell
surface marker profile, differentiation, or self-renewal capacity,
as defined by colony forming units.
Estrogen and Daidzein Enhance Osteogenic Differ-
entiation of MSCs. To investigate the osteogenic potential of
estrogen, daidzein, and genistein, MSCs from three donors
were individually treated with vehicle, estrogen, daidzein, or
genistein, stained with Alizarin Red, and destained to quantify
the amount of stain for each treatment group. Alizarin red is a
good indicator of osteogenic differentiation, as it stains calcified
extracellular matrix associated with bone formation. Estrogen
enhanced osteogenic differentiation of MSCs by a fold of 1.5
Figure 1. Phytoestrogens enhance osteogenesis of MSC. MSCs from
three donors were plated and allowed to grow to 70% confluence
before being induced to differentiate and being simultaneously
exposed to estrogen (10 nM), daidzein (1 μM), and genistein (1
μM). MSCs induced to undergo osteogenesis for 14 days were stained
with Alizarin Red S to detect calcium deposits, destained with
cetylpyridinium chloride, and measured at 590 nm to quantify the
amount of osteogenic differentiation. (A) Representative images of
Alizarin Red-stained MSCs treated with vehicle, estrogen, daidzein, or
genistein. Images were acquired at 4× magnification. (B) Bone
differentiation was determined relative to vehicle-treated cells
normalized to 1.0. *, P < 0.05; **, P < 0.01.
0.09, 1.5
0.18, and 1.6
0.09 relative to vehicle-treated
MSCs from Donor 1, Donor 2, and Donor 3, respectively (P <
0.05; Figure 1), and daidzein enhanced the osteogenic
differentiation of MSCs by 1.7 0.19, 1.7 0.01, and 1.6
0.15 in MSCs from Donors 1 to 3, respectively (P < 0.05;
Figure 1). Consistent with previous studies, MSCs can be
induced to undergo osteogenic differentiation; in the presence
of estrogen, daidzein, or genistein, the overall potential to
undergo differentiation is enhanced.9−12 Only estrogen and
daidzein consistently enhanced osteogenic differentiation of
MSCs in all three donors. Genistein enhanced the osteogenic
differentiation of MSCs by (1.6 0.19)-fold and (1.7 0.02)-
fold relative to vehicle-treated MSCs from Donor 1 and Donor
2, respectively (P < 0.05; Figure 1), but not in MSCs from
Donor 3 (1.0 0.15; P > 0.05; Figure 1). Our model based on
the use of MSCs has thus verified the known osteogenic
activities of estrogen, daidzein, and genistein, and will be next
used to investigate the osteogenic potential of the synthetic
daidzein analogs.
Daidzein Analogs Enhanced Osteogenic Differentia-
tion of MSCs with Greater Efficacy than Estrogen or
Daidzein. To investigate the efficacy of the daidzein analogs
on osteogenic differentiation of MSCs, 23 daidzein analogs
were screened to determine their ability to enhance osteogenic
differentiation. Initially, MSCs from one donor (age: 21 yrs old;
BMI: 22.7; race: Caucasian, gender: female) were treated with
estrogen, daidzein, or daidzein analog in osteogenic differ-
entiation media (ODM). After 14 days, estrogen and daidzein
enhanced osteogenic differentiation of MSCs 1.7 0.2 (P <
0.05; Figure 2A) and 1.7 0.3 (P < 0.05; Figure 2A) relative to
vehicle-treated MSCs in ODM, respectively. The effect of the
daidzein analogs on osteogenic differentiation can be divided
into three groups: no induction, low induction, and high
induction. Sixteen of the 23 daidzein analogs enhanced
osteogenic differentiation (P < 0.05; Figure 2A). Nine of the
23 daidzein analogs fall into the low induction category and
induce osteogenic differentiation 1.6-fold to 2.0-fold relative to
vehicle: 2d, 2b, 2a, 4, 7, 6, 10, 2g, and 12b, in ascending order.
Seven of the daidzein analogs were high inducers of osteogenic
differentiation (as high as >2.4-fold increase relative to vehicle):
2l, 2f, 12a, 2i, 2c, 2h, 2e, in ascending order. When tested
alongside the daidzein analogs, estrogen and daidzein were only
low inducers of osteogenic differentiation.
To identify the most potent osteogenic daidzein analogs and
determine the robustness of the daidzein analogs in inducing
osteogenesis, the 10 osteogenic daidzein analogs that resulted
in the highest levels of differentiation in Donor 1 were further
examined after 14 days of differentiation in a total of three
donors (mean age: 22.4 years old; mean BMI: 21.5; race:
Caucasian; gender: female). Of the ten compounds inves-
tigated, 2i and 2d were cytotoxic to MSCs from Donor 2 and
Donor 3 (Figure 2B). The other 8 compounds, 2c, 2f, 2g, 2e,
2h, 2l, 10, 11, and 12a, enhanced osteogenesis in all three
donors, increasing osteogenesis by 1.3−5.0-fold relative to
vehicle-treated MSCs (P < 0.05). Additionally, it was observed
that several daidzein analogs enhanced osteogenic differ-
entiation significantly more in Donor 3, than in Donor 1 or
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Figure 4. Enhanced osteogenic potential by the daidzein analogs is
dose dependent. MSCs from three donors were induced to undergo
osteogenic differentiation and simultaneously exposed to daidzein
analogs 2c, 2f, or 2l at 1 nM, 10 nM, 100 nM, or 1 μM concentrations
at every medium change.
Figure 2. Screening of daidzein analogs identifies several molecules
that show enhanced osteogenesis of MSCs compared to daidzein.
MSCs from (A) Donor 1, (B) Donor 2, and Donor 3 were induced to
undergo osteogenic differentiation and simultaneously exposed to
vehicle (DMSO), estrogen (10 nM), daidzein (1 μM), or daidzein
analogs (1 μM) at every medium change. After 14 days in osteogenic
differentiation media, bone differentiation potential was determined
variability adds to the uncertainty in EC50 values, the order of
osteogenesis potency for the three analogs in different donors
appears to be consistent. For example, the EC50 for analog 2f
for Donor 1, Donor 2, and Donor 3 was 65.8 nM, 65.7 nM, and
1.38 μM, respectively, representing the least potent analog. In
contrast, the EC50 for analog 2c was 2.2 nM and 2.1 nM in the
MSCs from Donor 1 and Donor 3, and 170 nM in Donor 2
cells, representing the most potent analog among the three (P
< 0.05; Figure 4).
#
relative to vehicle-treated cells normalized to 1.0. *, P < 0.05; , P <
0.01; **, P < 0.001.
2. While no significant differences were observed through
differentiation, self-renewal, or cell surface marker profile,
Donors 1 and 2 are between the ages of 21 and 22, while
Donor 3 is 33-years-old. It is possible that the enhanced
osteogenic differentiation could be an age-related effect, but
additional studies are necessary to determine the cause of the
enhanced osteogenic differentiation in Donor 3.
Selected Daidzein Analogs Exhibit Low EC50 Con-
centrations in Stimulating Bone Differentiation. To
further determine the dose-dependence of the daidzein analogs
in osteogenic activity, the degree of bone differentiation of the
MSCs was determined following treatment with varying
concentrations of three selected daidzein analogs: 2c, 2f, 2g,
and 2l. These analogs demonstrated enhanced differentiation at
day 7 and more robust osteogenic stimulation (Figure 3). To
test the dose response of these compounds on osteogenesis,
cells were treated with 1 nM to 1 μM concentration of the
daidzein analog to assess the EC50. As shown in Figure 4, the
three analogs appear to have EC50 values in the low nanomolar
to low micromolar range, depending on donor. While donor
Daidzein Analogs Enhance Osteogenic Transcription
Factor Expression in MSCs. To investigate the mechanism
by which daidzein analogs enhance osteogenic differentiation of
MSCs, cells treated with 2c as a supplement to ODM for 14
days were compared to cells treated with vehicle in ODM. Cells
treated with ODM containing 2c expressed higher concen-
trations of ALP (2.45), Sp7 (20.08), OP (2.68), IGF1 (26.96),
and Cola1 (8.00) relative to control (P < 0.05; Figure 5), while
no significant differences were observed in cbfa-1, cFOS, Dlx5,
or ON (Supporting Information Figure S2). Sp7 is one of the
earlier genes necessary to commit progenitor stem cells or
Figure 5. Enhanced osteogenic potential correlates with enhanced
expression of osteogenic transcription factors. MSCs from three
donors were pooled together, induced to undergo osteogenic
differentiation, and simultaneously exposed to vehicle or daidzein
analog 2c (1 μM) at every medium change. After 14 days, cells were
harvested, mRNA was isolated, and cDNA was synthesized from each
sample. Real time RT-PCR of transcript levels of genes involved in (A)
early, (B) middle, and (C) late osteogenesis was analyzed. *, P < 0.05;
**, P < 0.01; ***, P < 0.001.
Figure 3. Daidzein analogs enhance osteogenesis of MSCs as early as
Day 7. MSCs from three donors were induced to undergo osteogenic
differentiation and simultaneously exposed to vehicle (DMSO),
estrogen (10 nM), or daidzein analogs (1 μM) at every medium
change for 7 days. The mean bone differentiation potential of the three
#
donors is shown. *, P < 0.05; , P < 0.01.
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MSCs into the osteoblast lineage.16,17 Following lineage
commitment, osteoprogenitors undergo a proliferative stage
and subsequently express genes, such as alkaline phosphatase
(ALP), bone sialoprotein, and collagen type I, as they produce
the osteogenic extracellular matrix. In the final stages of
maturation, these osteoprogenitor cells mineralize the extrac-
ellular matrix and produce osteopontin (OP), osteocalcin, and
collagen type 1 (Cola1). Once mature bone is formed, insulin-
like growth factor 1 (IGF-1) regulates bone density to allow for
strength and durability.18,19 This highly regulated program of
gene expression and cellular differentiation is governed by the
expression and activity of a myriad of transcription factors.
These factors act in conjunction with other transcription factors
in an integrated and specific manner to allow for successful
generation of bone.
Increased expression of Sp7, ALP, OP, IGF-1, and Cola1 in
human MSCs treated with daidzein analog 2c supporst
increased bone formation observed phenotypically. Recent
studies have described similar increases in ALP and IGF-1
expression in mouse osteoblast cells exposed to estrogen.20,21
Additional studies on daidzein have investigated the upregula-
tion of downstream receptors, such as estrogen receptor α
(ERα) and estrogen receptor β (ERβ). These studies have
demonstrated that daidzein increases not only ERα and ERβ
expression but also vitamin D receptor synthesis, suggesting
that daidzein promotes bone formation and maturation through
these estrogen dependent pathways.22,23 To determine the
estrogenic or antiestrogenic activities that these daidzein
analogs may have toward ER, we performed the ERE luciferase
assays for 11 selected compounds (Supporting Information
Table 2S). Most of the osteogenic analogs showed some level
of diminished estrogenic activity compared to daidzein. Their
antagonistic properties were also low to modest. Additional
assays using the HEK293 cells transfected with ERα or ERβ
revealed that these analogs were not selective for either
estrogen receptor (Supporting Information Table 3S). While
further mechanistic studies are necessary to delineate the exact
mode of action and pathways involved, results obtained thus far
suggest that these daidzein analogs are likely to be more
efficient activators of the estrogen pathway, increasing the
synthesis and maturation of bone.
Structure−Activity Relationships. Of the twelve 2 series
daidzein analogs, 2a and 2b showed modest osteogenic activity
comparable to estrogen and daidzein, while 2d exhibited
toxicity in two donor cells and modest osteogenesis in the third.
Thus, methyl, ethyl, and n-propyl substitutions for the hydroxyl
hydrogen in daidzein failed to bring about significantly
enhanced osteogenic potential. When the substitution group
was isopropyl (2c), n-butyl (2e), 2-butyl (2f), allyl (2l), n-hexyl
(2h), or cyclopentyl (2g), marked enhancement of osteogenic
potentials was observed. In fact, these analogs represent the
most potent compounds in promoting bone formation in the
stem cells of all three donors. Interestingly, analog 6, in which
the substitution is a propyl-1-yne, showed only modest
osteogenic potential. When comparing the activities of 6, 2d,
and 2l, it is clear that the optimal structure for a three-carbon
substitution is one containing a double bond, not a triple bond,
nor a saturated σ bond. Also, in the 2 series, polar substitutions
containing nitrogen (2i, 2j, and 2k) showed either little
osteogenic effect (2j and 2k), or toxicity (2i), indicating that
increased polarity on the 7-O position may not be desirable for
retaining osteogenic activity. Interestingly, in the reported work
of Yadav et al.,14 polar substitutions of the 7-O position,
accompanied by transformation of the 4′-OH into a methoxy
group, showed a strong osteoblast-stimulating effect in vitro,
suggesting the important role of the 4′-O substitution in
conferring the overall osteogenic activity.
Other structural modifications involving cyclization on the
7,8 position did not yield significant osteogenic potential. For
example, of the three cyclic analogs (3, 4, and 7), analog 3 did
not exhibit any osteogenic activity while 4 and 7 showed
modest potential in promoting bone regeneration. When the
daidzein structural moiety in the center was modified to that of
racemic equol, a daidzein metabolite, there appeared to be a
significant loss of activity. For example, the analog 2e is one of
the most potent compounds, promoting over 2-fold increase of
bone formation in 14 days. Change of the central moiety of the
molecule to that of equol (i.e., reductive removal of the
carbonyl and the double bond on the central six-membered
ring) results in analog 9, which has no osteogenic activity in any
of the three donor stem cells. In another comparable pair, the
daidzein analog 4 showed modest osteogenic potential while
the equol analog 5 had no discernible osteogenic activity.
In conclusion, we have designed and synthesized 23 daidzein
analogs as potential osteogenesis agents, of which at least 16
showed significant bone differentiation activities in MSCs.
Treatment with daidzein analogs resulted in increased osterix
(Sp7), alkaline phosphatase (ALP), osteopontin (OPN), and
insulin-like growth factor 1 (IGF-1), which are osteogenic
transcription factors that regulate the maturation of osteogenic
progenitor cells into mature osteoblast cells. These daidzein
analogs thus represent novel organic chemical entities that
could be further explored as promising therapeutic lead
compounds for treatment of osteoporosis and for promoting
bone formation.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for synthesis and characterization of
the daidzein analogs, cell culture, and MSC characterization.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*B.A.B.: E-mail: bbunnell@tulane.edu.
*G.W.: E-mail: gwang@xula.edu.
Funding
This work was supported by the NIMHD RCMI program
through Grant 8G12MD007595; the Louisiana Cancer
Research Consortium (LCRC); Department of Agriculture
Grant 58-6435-7-019; and Office of Naval Research Grant
N00014-99-1-0763.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ALP, alkaline phosphatase; BMI, body mass index; cbfa-1, runt-
related transcription factor 2; cFOS, c-FBJ murine osteosacro-
ma viral onogenic homologue; DCC, N,N′-dicyclohexylcarbo-
diimide; dppf, bis(diphenylphosphino)ferrocenepalladiuM(II)-
dichloride; Dlx5, distal-less homeobox 5; DMAP, 4-dimethyla-
minopyridine; ER, estrogen receptor; IGF-1, insulin-like growth
factor 1; MSC, mesenchymal stem cells; ODM, osteogenic
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and density. J. Clin. Invest. 2002, 110 (6), 771−781.
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J.; Frassica, F.; Zhang, L.; Rodriguez, J. P.; Jia, X.; Yakar, S.; Xuan, S.;
Efstratiadis, A.; Wan, M.; Cao, X. Matrix IGF-1 regulates bone mass by
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differentiation media; ON, osteonectin; OP, osteopontin; Sp7,
osterix; THP, tetrahydropyranyl
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