Synthesis, optical resolution, absolute configuration, and osteogenic activity of cis-pterocarpans

Article abstract of DOI:10.1039/c2ob25722j

A convenient synthesis of natural and synthetic pterocarpans was achieved in three steps. Optical resolution of the respective enantiomers was accomplished by analytical and semi-preparative HPLC on a chiral stationary phase. For medicarpin and its synthetic derivative 9-demethoxymedicarpin, the absolute configuration was confirmed by a combination of experimental LC-ECD coupling and quantum-chemical ECD calculations. (-)-Medicarpin and (-)-9-demethoxymedicarpin are both 6aR,11aR-configured, and consequently the corresponding enantiomers, (+)-medicarpin and (+)-9-demethoxymedicarpin, possess the 6aS,11aS-configuration. A comparative mechanism study for osteogenic (bone forming) activity of medicarpin (racemic versus enantiomerically pure material) revealed that (+)-(6aS,11aS)-medicarpin (6a) significantly increased the bone morphogenetic protein-2 (BMP2) expression and the level of the bone-specific transcription factor Runx-2 mRNA, while the effect was opposite for the other enantiomer, (-)-(6aR,11aR)-medicarpin (6a), and for the racemate, (±)-medicarpin, the combined effect of both the enantiomers on transcription levels was observed. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25722j

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Volume 10 | Number 48 | 28 December 2012 | Pages 9509–9752
ISSN 1477-0520
PAPER
Atul Goel, Gerhard Bringmann et al.
Synthesis, optical resolution, absolute configuration, and osteogenic activity of
cis-pterocarpans

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PAPER
Synthesis, optical resolution, absolute configuration, and osteogenic activity of
cis-pterocarpans†
Atul Goel,*a Amit Kumar,a Yasmin Hemberger,b Ashutosh Raghuvanshi,a Ram Jeet,a Govind Tiwari,a
Michael Knauer,b Jyoti Kureel,c Anuj K. Singh,c Abnish Gautam,c Ritu Trivedi,c Divya Singhc and
Gerhard Bringmann*b
Received 4th June 2012, Accepted 16th August 2012
DOI: 10.1039/c2ob25722j
A convenient synthesis of natural and synthetic pterocarpans was achieved in three steps. Optical
resolution of the respective enantiomers was accomplished by analytical and semi-preparative HPLC on a
chiral stationary phase. For medicarpin and its synthetic derivative 9-demethoxymedicarpin, the absolute
configuration was confirmed by a combination of experimental LC-ECD coupling and quantum-chemical
ECD calculations. (−)-Medicarpin and (−)-9-demethoxymedicarpin are both 6aR,11aR-configured, and
consequently the corresponding enantiomers, (+)-medicarpin and (+)-9-demethoxymedicarpin, possess
the 6aS,11aS-configuration. A comparative mechanism study for osteogenic (bone forming) activity of
medicarpin (racemic versus enantiomerically pure material) revealed that (+)-(6aS,11aS)-medicarpin (6a)
significantly increased the bone morphogenetic protein-2 (BMP2) expression and the level of the bone-
specific transcription factor Runx-2 mRNA, while the effect was opposite for the other enantiomer,
(−)-(6aR,11aR)-medicarpin (6a), and for the racemate, ( )-medicarpin, the combined effect of both the
enantiomers on transcription levels was observed.
Very few de novo synthetic methods to build up medicarpin
have been reported in the literature. They mainly include reduc-
Introduction
Pterocarpans, apart from the isoflavones, represent the second-
largest group of natural isoflavonoids, with medicarpin (6a) and
maackiain occurring almost ubiquitously.1 They are potent
phytoalexins, i.e., defensive substances produced by plants in
response to biotic and abiotic elicitors.2 For example, cell sus-
pensions of the model legume Medicago truncatula accumulated
the isoflavonoid phytoalexin medicarpin in response to yeast
elicitor or methyl jasmonate.3,4 Depending on the producing
plant, medicarpin (6a) was isolated either as a racemate, e.g.,
from Dalbergia odorifera,5 or in enantiomerically pure form
(+)-(6aS,11aS)-medicarpin [(+)-6a] from Arachis hypogea6 and
(−)-(6aR,11aR)-medicarpin [(−)-6a] from Medicago sativa.7
tive cyclisation of the corresponding isoflavone with NaBH4 in
THF·EtOH.8,9 These procedures require multi-step pathways,8
long reaction times, and often lead to poor yields.9 Among the
variety of synthetic routes to ( )-pterocarpans, the most common
approaches involve the reduction and cyclisation of the corres-
ponding 2′-hydroxyisoflavanones,10 1,3-Michael–Claisen annu-
lation,11 cycloaddition reaction of 2H-chromenes with 2-alkoxy-
1,4-benzoquinones,12 Heck arylation of 2H-chromenes with
o-chloromercuriphenol using lithium tetrachloropalladate as a
catalyst,13 or the oxidative rearrangement of chalcones with
thallium(III) nitrate.14,15 Literature procedures for obtaining enan-
tiomerically pure pterocarpans include resolution of racemic
compounds followed by sequential functional-group modifi-
cations and/or ring transformations.16–24 Herein we report a con-
venient synthesis of racemic pterocarpans, the optical resolution,
and confirmation of the absolute configuration by a combination
of experimental electronic circular dichroism (ECD) investi-
gations and quantum-chemical ECD calculations, and an as yet
unknown bone-forming activity of enantiomerically pure natural
and synthetic pterocarpans.
aDivision of Medicinal and Process Chemistry, CSIR – Central Drug
Research Institute, Lucknow 226001, India. E-mail: atul_goel@cdri.res.in;
Fax: +99 522 2623405
bInstitute of Organic Chemistry, University of Würzburg, Am Hubland,
97074 Würzburg, Germany. E-mail: bringman@chemie.uni-wuerzburg.de;
Fax: +49 931 318 4755
cDivision of Endocrinology, CSIR – Central Drug Research Institute,
Lucknow 226001, India
†Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra of all new compounds, Cartesian coordinates of 6a
and 6c; HPLC profile and ALP activity of 6a and 6c. See DOI:
10.1039/c2ob25722j
Results and discussion
Our synthetic approach to the class of pterocarpan is depicted in
Scheme 1. The substituted deoxybenzoins 3a–e were synthesized
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Scheme 1 Synthesis of the natural pterocarpans 6a and 6b, and of the synthetic analogues 6c–e.
in situ by Friedel–Crafts acylation of the resorcinol derivatives
1a–c and the substituted phenylacetic acids 2a and 2b, using
BF3·OEt2 complex solution as the reagent. The resulting reaction
mixture was further treated with mesyl chloride in DMF, which
led to the formation of the desired 7-hydroxy-chromen-4-one
4a–e in good yield.10b Selective O-demethylation of 4a–e to the
2′-hydroxyisoflavones 5a–e was carried out successfully by
AlCl3 in CH3CN.9 Finally, pterocarpans 6a–e were synthesized
by reductive cyclisation using NaBH4 in dry ethanol (see
Scheme 1). The structures of the naturally occurring pterocar-
pans 6a and 6b, and of the synthetic ones 6c–e were confirmed
by spectroscopic analysis and/or by comparison with literature
data.
Fig. 1 Two possible configurations (left) and their two strain-free con-
formations of the backbone (right) of cis-medicarpin (6a).
In Nature only the cis-fused B/C ring system has so far been
found, as present in the pterocarpans. It is energetically more
favourable than the trans-fused ring system as demonstrated by
theoretical studies.25,26 This thermodynamical preference led to
the formation of only one of the possible diastereomers of
( )-medicarpin, with its two enantiomeric forms cis-(6aR,11aR)-
and cis-(6aS,11aS)-6a. For each of the enantiomers, two strain-
free conformations of the backbone may exist; of these, the struc-
ture in which 6a-H is oriented anti relative to one of the hydro-
gen atoms at C-6 (Hanti, ΔE = 0.00 kcal mol−1) is energetically
favoured as compared to that with 6a-H anti to the ring oxygen
(O-5) and, thus, gauche to both hydrogens at C-6 (Oanti, ΔE =
1.88 kcal mol−1).27 The calculated two main minimum confor-
mers of the Hanti and of the Oanti backbone of cis-(6aS,11aS)-6a
and cis-(6aR,11aR)-6a are shown in Fig. 1. The rapid intercon-
version of these two possible backbone structures at room tem-
perature was already evidenced for related pterocarpans by
Alagano and Ghio.27
chemical ECD calculations as a tool for the stereochemical
assignment of numerous natural and synthetic chiral compounds.
The first successful enantiomeric resolution of racemic pterocar-
pans (medicarpin and related compounds) by chiral HPLC was
described by Antus et al.20 and Szarvas et al.18 During their
experiments using methanol on Chiralpack OT(+) columns, they
observed that some polymeric impurities eluted with the com-
pounds leading to disturbances in ECD and UV measurements.18
Therefore we developed an improved method for the resolution
of the enantiomers, exemplarily elaborated for racemic medi-
carpin (6a) and for its synthetic analogue 9-demethoxymedicarpin
(6c), to investigate the absolute configuration of synthetic and
natural pterocarpans. After several attempts with different con-
ditions on chiral stationary HPLC phases, good resolution of the
enantiomers was achieved with Lux cellulose-1 (Phenomenex;
25 cm × 0.46 cm) and cellulose tris(3,5-dimethylphenylcarba-
mate) as the stationary phase, and a mixture of acetonitrile–water
(50 : 50) as the eluent with a constant flow rate of 1 mL min−1 at
room temperature.
Some of us28,29 have developed an online HPLC-ECD
method in combination with modern high-level quantum-
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Full LC-ECD spectra were recorded in the stopped-flow
mode, leading to opposite ECD curves for the two peaks, thus
confirming the assumption that these peaks indeed represented
the two enantiomers of the cis-configured compound (6a). The
ECD bands of the first-eluted compound showed a positive
Cotton effect in the range of 210–260 nm for the 1La band and a
effects, did not permit an assignment of the absolute configur-
ation due to an only ambiguous UV correction, which is usually
an indication that the excited states are not properly calculated
(not shown). Improved results were achieved using the
TDB2GP-PLYP35,36 functional with the larger Ahlrichs’ TZVP37
basis set and furthermore by a combined approach of DFT
(B3LYP/SVP) and MRCI (CAS 12,12) methods.38 It has already
been proven that double-hybrid density functionals show a
higher robustness and, most importantly, provide more accurate
excitation energies, since excited states with charge-transfer and
Rydberg-type character are described in a more balanced way as
compared to the conventional density functional like B3LYP.36
The single CD spectra calculated for the two backbone types
(Hanti and Oanti) exhibited the expected difference in the CD
effect, but interestingly also the orientation of the OMe function
revealed a surprisingly high effect on the CD spectrum, while
the arrangement of the OH group had no real impact (see the
ESI; Fig. S1†). This again showed the importance of a complete
analysis. All single spectra obtained were energetically weighted
and summed up to give the overall UV and CD spectra. After a
now unambiguous UV shift, the predicted ECD spectra were
compared with the experimental ECD curves of the two resolved
enantiomers.39 Due to the strong similarity of the experimental
ECD curves of the corresponding peaks of 6a and 6c with the
calculated ones, only the calculated curves of 6a are shown in
Fig. 2, for reasons of clarity (see the ESI; Fig. S2†).
The comparison revealed good agreement between the ECD
spectrum simulated for (6aS,11aS)-6a and the experimental one
of Peak 1 (see Fig. 2b, left) and between the curve computed for
(6aR,11aR)-6a and the online spectrum of Peak 2 (see Fig. 2b,
right), thus permitting assignment of the (6aS,11aS)-configur-
ation to the faster eluting enantiomer of 6a and 6c and, conse-
quently, the (6aR,11aR)-configuration to the more slowly eluting
stereoisomer of 6a and 6c.
Recently we have shown that isoflavonoids and pterocarpans
possess osteogenic activity.40 We found that ( )-medicarpin (6a)
inhibits osteoclastogenesis and has non-estrogenic bone-conser-
ving effects in ovary-ectomised mice.41 In order to compare the
osteogenic activity profile of rac-pterocarpan (see Table 2) with
its pure enantiomers, we resolved the enantiomers of the ptero-
carpans 6a and 6c to evaluate the effect of enantiomerically pure
material in two main phases of osteoblast development, viz.
differentiation and mineralisation.
1
negative one in the range of 260–310 nm for the Lb band, in
agreement with the data reported by Szarvas et al.18 Similarly,
the second-eluted compound displayed exactly opposite Cotton
effects as compared to the faster eluting compound. Although
the relative and absolute configuration of the pterocarpans is
known, based on the well established configuration of
(−)-(6aR,11aR)-maackiain and the helicity rule of transition
bands,21 no direct correlation between experimental and compu-
tationally predicted CD spectra has so far been reported as a tool
for the determination of the absolute configurations of natural
and synthetic pterocarpans.
An attribution of the absolute configuration to the respective
enantiomer of 6a and 6c was achieved by the combination of
HPLC-ECD measurements with the quantum-chemical calcu-
lation of the ECD spectra of the respective enantiomers of 6a
and 6c. To investigate the conformational space of (6aR,11aR)-
6a and of (6aR,11aR)-6c, DFT calculations were carried out
using the B3LYP30,31 functional in combination with Pople’s
6-31G* basis set.32
Within the energetically relevant range of 3 kcal mol−1 above
the global minimum the conformational analysis yielded eight
minimum structures for compound 6a while for 6c only four rel-
evant conformers were identified, due to the missing methoxy
group (see the ESI†; Table 1: Cartesian coordinates).
TDB3LYP/SV(P) calculations for these conformers, even in
combination with the COSMO model33,34 to account for solvent
Table 1 B3LYP/6-31G* relative energies of all local minima of
(6aR,11aR)-medicarpin in kcal mol−1 depending on the backbone
structure (Hanti or Oanti; φABCD), OMe (φEFGH) and OH (φIJKL) groups
arrangements
Using our chromatographic system and method (see the ESI;
Fig. S3–S8†) developed for the online-ECD measurements with
a semi-preparative Lux cellulose-1 column, approximately
100 mg of each, (6aS, 11aS)-6a,c (ee > 99.5%) and (6aR,11aR)-
6a,c (ee > 99.5%), were obtained for evaluation of the biological
activity.
b
b
b
Conformer
ΔEa
φABCD
φEFGH
φIJKL
Primary cultures of rat osteoblasts were used for investigating
the effect of enantiomerically pure and racemic pterocarpans on
osteoblast differentiation. Production of alkaline phosphatase
(ALP) served as a differentiation marker of osteoblasts.41
An osteoblast ALP assay was used to screen the activity of
enantiomerically pure and racemic pterocarpans. Briefly, calvar-
ial osteoblasts were cultured to confluence and treated with
various compounds for 48 h in the presence of ascorbate and
glycerophosphate at concentrations ranging from 10−10 M to
10−6 M. At the end of the incubation period, the total ALP
(R,R)-6a1 (Hanti
(R,R)-6a2 (Hanti
(R,R)-6a3 (Hanti
(R,R)-6a4 (Hanti
(R,R)-6a5 (Oanti
(R,R)-6a6 (Oanti
(R,R)-6a7 (Oanti
(R,R)-6a8 (Oanti
)
)
)
)
)
)
)
)
0.00
0.16
0.24
0.37
1.88
2.11
2.16
2.30
−57
−57
−57
−57
53
53
53
−1
−180
−180
1
179
−1
179
0
−179
0
1
179
179
0
53
−179
1
a Relative energies in kcal mol−1 b Dihedral angles in degree [°].
.
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Table 2 Osteoblast differentiation and mechanism studies for
osteogenic activity by pterocarpans (6a–e)
ALP activitya
BMP-2
Runx-2
Compound 10−10
M
10−8
M
10−6
M
expressionb expressionb
( )-6a
(+)-6a
(−)-6a
( )-6b
( )-6c
(+)-6c
(−)-6c
( )-6d
( )-6e
236
95
59
na
47.5
58
45
na
na
263
93
73
331
159
95
na
53.2
51
37
na
na
0.50*
2.33**
1.06
—
3.09**
1.52
1.50*
—
—
0.94
2.28**
2.13**
na
—
47.5
53.2
51
na
na
3.50**
10.54*
1.71**
—
—
a % increase compared with control. b Fold increase compared with
control. ‘na’ means not active. *P value <0.05. **P value <0.01.
Fig. 3 mRNA levels of BMP-2 by qPCR for 6a.
Fig. 4 mRNA levels of Runx-2 by qPCR for 6a.
Fig. 2 Stereochemical assignment of the two enantiomers of medicar-
pin (6a) and its synthetic derivative 9-demethoxy-medicarpin (6c) by (a)
LC-ECD coupling and quantum-chemical ECD calculations, applying
(b) the TDB2GP-PLYP (above) and the combined DFT/MRCI approach
(below).
BMP-2 stimulation, suggesting that Runx-2 is a downstream
transcription factor in BMP-2 signalling. Thus, mRNA levels of
Runx-2 and BMP-2 were monitored by qPCR after treatment
with the compounds. In the case of the medicarpins ( )-6a,
(+)-6a, and (−)-6a, a significant increase in BMP-2 mRNA
levels was found for (+)-6a when compared with the control (in
the absence of a test substance). A decrease in the BMP-2 tran-
scription levels was observed after treatment with (−)-6a, which
reveals the antagonistic effect of the (6aR,11aR)-enantiomer evi-
dencing the importance of the absolute configuration (see
Fig. 3).
Runx-2 transcript levels were significantly increased by (+)-6a
and ( )-6a, while for (−)-6a no change was observed (see
Fig. 4). In the case of 9-demethoxymedicarpin [( )-6c, (+)-6c,
and (−)-6c)], BMP-2 and Runx-2 mRNA levels were increased
for all three test substances (see Fig. 5 and 6). However, the
highest increase in BMP-2 transcript level was obtained by
activity was measured with the method using 4-nitrophenyl-
phosphate (PNPP) as a substrate.41 Treatment with the syn-
thesised pterocarpans 6a–e, only pterocarpans ( )-6a,c, (+)-6a,c,
and (−)-6a,c led to increased osteoblast differentiation as
assessed spectrophotometrically by osteoblast ALP production
(see Table 2; see the ESI; Fig. S9–S14†).
Furthermore the effect of the respective enantiomers on the
expression of osteogenic genes (Runx-2 and BMP-2) in calvarial
osteoblasts by quantitative real-time PCR (qPCR) was studied.
Runx-2, a bone-specific transcription factor, is a key regulator of
osteoblastic differentiation.42 Runx-2 expression is induced by
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Conclusion
We have demonstrated a concise ‘three-step’ synthesis of the
pterocarpan class of compounds. We developed an improved
analytical and semi-preparative HPLC protocol for the resolution
of racemates 6a and 6c using cellulose tris(3,5-dimethyl-phenyl-
carbamate) as a chiral stationary phase. The exemplary optical
resolution of medicarpin (6a) and 9-demethoxymedicarpin (6c)
was achieved by HPLC on a chiral phase and the absolute
configuration of the respective cis-enantiomers was determined
by LC-ECD coupling in combination with quantum-chemical
ECD calculations. The comparison revealed quite good agree-
ment between the simulated ECD spectra (calculated with
TDB2GP-PLYP and MRCI) and the experimental ones, thus per-
mitting assignment of the (6aS,11aS)-configuration to the faster
eluting enantiomers of 6a and 6c, and consequently the
(6aR,11aR)-configuration to the slower ones of 6a and 6c. An
osteogenic study comparing racemic pterocarpans with enantio-
merically pure ones showed that (6aS,11aS)-medicarpin [(+)-6a]
significantly increased both BMP-2 and Runx-2 mRNA levels.
In short, the work described in this paper thus provides a short
and efficient synthesis of racemic cis-pterocarpans and a new
protocol for the resolution of their enantiomers on a chiral phase
through analytical and preparative HPLC, and demonstrates
direct correlation between experimental and computationally pre-
dicted ECD spectra as a powerful tool for the comprehensive
stereochemical characterization of these bioactive substances in
general.
Fig. 5 mRNA levels of BMP-2 by qPCR for 6c.
Fig. 6 mRNA levels of Runx-2 by qPCR for 6c.
Experimental section
Commercial reagents were used without purification. 1H and 13
C
NMR spectra were recorded at 300 MHz, with CDCl3 and
DMSO-d6 as the solvents. Chemical displacements are reported
in parts per million shifts (δ values) from Me4Si (0.00 ppm for
1H) or based on the central peak of the solvent CDCl3
(77.00 ppm for 13C NMR) as the internal standard. Signal pat-
terns are indicated as s, singlet; d, doublet; t, triplet; m, multiplet.
Coupling constants (J values) are given in Hertz (Hz). Infrared
(IR) spectra were recorded on a Perkin-Elmer AX-1 spectropho-
tometer in a KBr disc and are reported in wave number (cm−1).
For mass spectra analysis, an ESI-MS spectrometer was used.
Optical rotations were measured on a Rudolf Autopol III.
Fig. 7 Bone marrow mineralisation alizarin staining for measuring
calcium nodule formation by medicarpins ( )-6a, (+)-6a, and (−)-6a.
Experimental procedures and characterization data
1-(2,4-Dihydroxyphenyl)-2-(2,4-dimethoxyphenyl)ethanone (3a).
A mixture of resorcinol (1a) (4.95 g, 45.0 mmol), 2-(2,4-
dimethoxyphenyl)acetic acid (2a) (7.25 g, 37.0 mmol), and
BF3·OEt2 complex solution (12 mL) was heated at 90–100 °C
for 2 h and quenched with 500 mL of water. The resulting
mixture was extracted with CHCl3 (3 × 75 mL) and the com-
bined organic extracts were washed with brine, dried over anhy-
drous Na2SO4, and concentrated in vacuo. The crude residue
was purified by silica gel column chromatography (CHCl3–
n-hexane, 4 : 1) giving 3a (8.09 g, 76%) as a white solid, mp
150–152 °C (from CHCl3–n-hexane) (lit.,8 mp 154 °C); IR
(KBr): νmax/cm−1 3261br (OH), 1635s (CO); 1H NMR δH
treatment with ( )-6c possibly due to a synergistic effect of the
two enantiomers. Runx-2 transcript level was maximally
increased by (−)-6c followed by ( )-6c. Thus, based on ALP
and qPCR, (+)-6a was found to be the most potent molecule
among racemic and enantiomerically pure medicarpins. In
addition to osteoblast differentiation, all tested compounds,
( )-6a, (+)-6a, and (−)-6a, promoted osteoblast mineralisation
(see Fig. 7). Treatment of bone-marrow derived osteoblast cells
with ( )-6a, (+)-6a or (−)-6a for 21 d in osteoblast differen-
tiation media resulted in enhanced formation of mineralized
nodules compared with control treated cells (see Fig. 7).
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(300 MHz; CDCl3 + DMSO-d6; Me4Si) 3.61 (3 H, s), 3.62 (3 H,
s), 3.98 (2 H, s), 6.11 (1 H, s), 6.23 (1 H, d, J 10.5), 6.28–6.38
(2 H, m), 6.90 (1 H, d, J 9.5), 7.69 (1 H, d, J 8.6), 10.39 (1 H, s,
OH, D2O exchange); 13C NMR δC (75 MHz; CDCl3 + DMSO-
d6) 200.6, 163.2, 163.1, 158.2, 156.2, 131.0, 129.5, 113.9,
110.6, 106.5, 102.9, 101.0, 96.6, 53.7, 53.4, 36.5; MS (ESI) m/z
289 ([M + H]+).
reactants, and purified by silica gel column chromatography
(CHCl3–n-hexane 7 : 3) giving 3e (6.46 g, 60%) as a white solid,
mp 148–150 °C (from n-hexane–CHCl3); IR (KBr): νmax/cm−1
1
1702s (CO); H NMR δH (300 MHz; CDCl3; Me4Si) 3.83 (3 H,
s), 4.19 (2 H, s), 6.24 (1 H, s), 6.49 (1 H, s), 6.81–6.98 (2 H, m),
7.16–7.31 (2 H, m), 7.98 (1 H, s, OH, D2O exchange), 12.45
(1 H, s, OH, D2O exchange); 13C NMR δC (75 MHz; CDCl3)
202.0, 163.9, 157.6, 156.8, 131.1, 130.9, 128.8, 122.5, 120.8,
114.2, 111.0, 110.7, 104.5, 55.4, 39.2; HRMS (ESI) exact mass
calcd for C15H14ClO4: 293.0581 ([M + H]+). Found: 293.0473
([M + H]+).
1-(2,3,4-Trihydroxyphenyl)-2-(2,4-dimethoxyphenyl)ethanone
(3b). The compound was prepared from benzene-1,2,3-triol (1b)
(5.67 g, 45.0 mmol) and 2-(2,4-dimethoxyphenyl)acetic acid
(2a) (7.25 g, 37.0 mmol) as described for 3a, and purified by
silica gel column chromatography (CHCl3–n-hexane, 9 : 1) fur-
nishing 3b (8.10 g, 72%) as a white solid, mp 130–132 °C
(from EtOH) (lit.,43 mp 134–135 °C); IR (KBr): νmax/cm−1
General procedure for the synthesis of compounds (4a–e)
Method A. A mixture of resorcinol (1a–c, 45.0 mmol), 2-
(2,4-dimethoxyphenyl)acetic acid (2a, 37.0 mmol) or 2-(2-meth-
oxyphenyl)acetic acid (2b, 37.0 mmol), and BF3·OEt2 complex
solution (27 mL, 225.0 mmol) was heated at 90–100 °C for 2 h
and mesyl chloride (20.7 mmol) was gradually added to it at
50 °C. The solution was then heated up to 80–90 °C for 3–4 h.
After concentration of the mixture in vacuo the residue was
treated with ice and the solid obtained was purified by silica gel
column chromatography (CHCl3–MeOH, 99 : 1) affording 4a–e.
1
3459br (OH), 1613s (CO); H NMR δH (300 MHz; DMSO-d6;
Me4Si) 3.63 (3 H, s), 3.67 (3 H, s), 4.08 (2 H, s), 6.35 (1 H, d,
J 8.7), 6.40 (1 H, d, J 8.3), 6.47 (1 H, d, J 9.0), 6.98 (1 H, d,
J 9.0), 7.38 (1 H, d, J 9.0), 8.58 (1 H, s, OH, D2O exchange),
10.03 (1 H, s, OH, D2O exchange), 12.45 (1 H, s, OH, D2O
exchange); 13C NMR δC (75 MHz; DMSO-d6) 203.2, 159.8,
158.0, 152.4, 152.4, 132.4, 131.5, 122.5, 115.7, 112.7, 107.8,
104.7, 98.4, 55.5, 55.2, 38.3; HRMS (ESI) exact mass calcd for
C16H16O6: 305.1025 ([M + H]+). Found: 305.1000 ([M + H]+).
Method B. BF3·OEt2 complex (3.5 mL, 27.6 mmol) was
1-(2,4-Dihydroxyphenyl)-2-(2-methoxyphenyl)ethanone (3c).
The compound was prepared as described for 3a starting from
resorcinol (1a) (4.95 g, 45.0 mmol) and 2-(2-methoxyphenyl)
acetic acid (2b) (6.15 g, 37.0 mmol), and purified by silica gel
column chromatography (CHCl3–n-hexane, 9 : 1) furnishing 3c
(7.66 g, 80%) as a white solid, mp 158–160 °C (from CHCl3–
n-hexane) (lit.,44 mp 193 °C); IR (KBr): νmax/cm−1 3284br
(OH), 1631s (CO); 1H NMR δH (300 MHz; CDCl3; Me4Si) 3.80
(3 H, s), 4.23 (2 H, s), 6.33–6.42 (2 H, m), 6.86–6.98 (2 H, m),
7.19 (1 H, d, J 5.9), 7.24–7.32 (1 H, m), 7.83 (1 H, d, J 7.9),
12.68 (1 H, s, OH, D2O exchange); 13C NMR δC (75 MHz;
CDCl3) 202.6, 165.3, 162.5, 132.8, 131.0, 128.6, 123.1, 120.7,
118.9, 118.4, 110.5, 107.7, 103.5, 55.4, 39.1; MS (ESI): m/z 259
([M + 1]+).
gradually added to
a solution of deoxybenzoins (3a–e,
6.9 mmol) in DMF at 0 °C. The resulting solution was stirred for
30 min. After addition of mesyl chloride (1.6 mL, 20.7 mmol) at
50 °C, the solution was heated up to 80–90 °C for 5 h. The
mixture was then concentrated, the residue treated with ice, and
the solid obtained was purified by silica gel column chromato-
graphy (CHCl3–MeOH, 99 : 1) giving 4a–e.
3-(2,4-Dimethoxyphenyl)-7-hydroxy-4H-chromen-4-one (4a). Fol-
lowing the procedure described in Method A, compound 4a
(1.65 g, 80%) was isolated as a white solid, mp 238–240 °C
(from CHCl3–MeOH) (lit.,8 mp 265–267 °C); IR (KBr): νmax
/
cm−1 1621s (conj. CO); 1H NMR δH (300 MHz; CDCl3
+
DMSO-d6; Me4Si) 3.58 (3 H, s), 3.65 (3 H, s), 6.66 (1 H, d, J
2.7), 6.73 (1 H, d, J 2.7, 8.4), 7.00 (1 H, d, J 10.3), 7.71 (1 H,
s), 7.81 (1 H, d, J 10.3); 13C NMR δC (75 MHz; CDCl3
+
2-(2-Methoxyphenyl)-1-(2,3,4-trihydroxyphenyl)ethanone (3d).
The compound was prepared as described for 3a using benzene-
1,2,3-triol (1b) (5.67 g, 45.0 mmol) and 2-(2-methoxyphenyl)
acetic acid (2b) (6.15 g, 37.0 mmol) as the starting materials,
and purified by silica gel column chromatography (CHCl3–
n-hexane, 4 : 1) giving 3d (8.11 g, 80%) as a white solid, mp
132–134 °C (from CHCl3–n-hexane); IR (KBr): νmax/cm−1
DMSO-d6) 174.5, 161.6, 159.9, 157.9, 157.5, 152.7, 131.1,
126.3, 120.6, 116.1, 114.1, 112.6, 103.5, 101.4, 97.7, 54.6, 54.3;
HRMS (ESI) exact mass calcd for C17H15O5: 299.09195
([M + H]+). Found: 299.09241 ([M + H]+).
3-(2,4-Dimethoxyphenyl)-7,8-dihydroxy-4H-chromen-4-one (4b).
Following the procedure described in Method B, compound 4b
(1.52 g, 70%) was obtained as a white solid, mp 212–218 °C
1
3402br (OH), 1629s (CO); H NMR δH (300 MHz; DMSO-d6;
(from CHCl3–MeOH) (lit.,8 mp 238–239 °C); IR (KBr): νmax
/
Me4Si) 3.76 (3 H, s), 4.20 (2 H, s), 6.41 (1 H, d, J 9.0),
6.85–6.90 (2 H, m), 7.12 (1 H, d, J 7.3), 7.19–7.25 (1 H, m),
7.38 (1 H, d, J 9.0), 9.83 (1 H, s, OH, D2O exchange); 13C
NMR δC (75 MHz; DMSO-d6) 202.5, 156.9, 152.2, 152.1,
132.2, 130.7, 128.1, 123.3, 122.1, 120.1, 112.6, 110.4, 107.6,
55.1, 38.6; HRMS (ESI) exact mass calcd for C15H15O5:
275.0919 ([M + H]+). Found: 275.0761 ([M + H]+).
cm−1 3496br (OH), 1652s (conj. CO); H NMR δH (300 MHz;
DMSO-d6; Me4Si) 3.64 (3 H, s), 3.73 (3 H, s), 6.50 (2 H, dd, J
8.4, 2.3), 6.56 (1 H, d, J 2.2), 6.88 (1 H, d, J 8.7), 7.34 (1 H, d,
J 8.7), 8.10 (1 H, s); 13C NMR δC (75 MHz; CDCl3 + DMSO-
d6) 175.4, 161.1, 159.0, 154.2, 150.4, 147.3, 133.4, 132.6,
121.4, 117.9, 116.0, 114.5, 114.1, 105.1, 99.1, 56.0, 55.7;
HRMS (ESI) exact mass calcd for C17H14O6: 315.08686
([M + H]+). Found: 315.0871 ([M + H]+).
1
1-(5-Chloro-2,4-dihydroxyphenyl)-2-(2-methoxyphenyl)ethanone
(3e). The compound was prepared as described for 3a using
4-chlorobenzene-1,3-diol (1c) (6.50 g, 45.0 mmol) and 2-
(2-methoxyphenyl)acetic acid (2b) (6.15 g, 37.0 mmol) as the
7-Hydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one
(4c). Fol-
lowing the procedure described in Method A, compound 4c
(1.39 g, 75%) was delivered as a white solid, mp 222–224 °C
9588 | Org. Biomol. Chem., 2012, 10, 9583–9592
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(from CHCl3–MeOH); IR (KBr): νmax/cm−1 1628s (conj. CO);
1H NMR δH (300 MHz; DMSO-d6; Me4Si) 3.60 (3 H, s), 7.83
(1 H, d, J 8.9), 7.76 (1 H, s), 7.06–7.22 (2 H, m), 6.65–6.85 (4
H, m); 13C NMR δC (75 MHz; DMSO-d6) 173.7, 161.4, 156.6,
156.2, 152.5, 130.4, 128.2, 126.0, 120.6, 119.9, 118.9, 115.7,
113.8, 109.8, 101.1, 54.3; HRMS (ESI) exact mass calcd
for C16H13O4: 269.08138 ([M + H]+). Found: 269.07974
([M + H]+).
7,8-Dihydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one (4d). Fol-
lowing the procedure described in Method B, compound 4d
(1.56 g, 72%) was produced as a white solid, mp 192–194 °C
(from CHCl3–MeOH); IR (KBr): νmax/cm−1 1595s (conj. CO);
1H NMR δH (300 MHz; CDCl3 + DMSO-d6; Me4Si) 3.59 (3 H,
s), 6.83–6.90 (2 H, m), 6.96 (1 H, d, J 9), 7.10–7.13 (1 H, m),
7.23–7.32 (2 H, m), 8.10 (1 H, s), 9.38 (1 H, s, OH), 10.25 (1 H,
s, OH); 13C NMR δC (75 MHz; CDCl3 + DMSO-d6) 172.8,
156.6, 152.7, 148.9, 146.1, 132.0, 130.9, 128.7, 120.6, 120.3,
119.4, 117.1, 115.4, 113.4, 110.3, 54.7; HRMS (ESI) exact mass
calcd for C17H14O6: 315.08686 ([M + H]+). Found: 315.0871
([M + H]+).
6-Chloro-7-hydroxy-3-(2-methoxyphenyl)-4H-chromen-4-one (4e).
Following the procedure described in Method B, compound 4e
(1.29 g, 62%) was obtained as a white solid, mp 228–230 °C
(from CHCl3–MeOH); IR (KBr): νmax/cm−1 1625s (conj. CO);
1H NMR δH (300 MHz; CDCl3 + DMSO-d6; Me4Si) 3.68 (3H,
s), 6.93–6.95 (1 H, m), 6.98 (2 H, s), 7.18–7.20 (1 H, m),
7.32–7.38 (1 H, m), 7.93 (1 H, s), 8.19 (1 H, s), 11.65 (1 H, s,
OH, D2O exchange); 13C NMR δC (75 MHz; DMSO-d6) 158.3,
157.9, 156.1, 154.8, 132.0, 130.2, 126.5, 122.3, 121.3, 120.5,
120.0, 117.4, 111.7, 101.1, 56.0; HRMS (ESI) exact mass
calcd for C16H1335ClO4: 303.0424 ([M + H]+). Found: 303.0421
([M + H]+).
(CHCl3–MeOH 98 : 2) giving 5b (1.17 g, 69%) as a white solid,
mp 170–172 °C (from CHCl3–MeOH) (lit.,43 mp 174–175 °C);
1
IR (KBr): νmax/cm−1 1617s (conj. CO); H NMR δH (300 MHz;
CDCl3 + DMSO-d6; Me4Si) 3.82 (3 H, s), 4.02 (3 H, s), 4.03
(3 H, s), 6.55 (2 H, dd, J 8.3, 2.5), 6.65 (1 H, d, J 2.7), 7.08
(1 H, d, J 8.6), 7.14 (1 H, d, J 9.7), 8.08–8.12 (2 H, m), 9.16
(1 H, s, OH, D2O exchange); 13C NMR δC (75 MHz; CDCl3)
179.0, 162.0, 157.9, 157.0, 154.9, 150.5, 136.4, 130.3, 124.4,
121.9, 117.9, 112.8, 111.0, 107.6, 104.4, 61.7, 56.6, 55.4;
HRMS (ESI) exact mass calcd for C18H16O6: 329.10251
([M + H]+). Found: 329.1029 ([M + H]+).
7-Hydroxy-3-(2-hydroxyphenyl)-4H-chromen-4-one (5c). Follow-
ing the procedure of 5a, compound 5c (1.22 g, 80%) was
obtained as a white solid, mp 196–198 °C (from CHCl3–
1
MeOH); IR (KBr): νmax/cm−1 1621s (conj. CO); H NMR δH
(300 MHz; CDCl3; Me4Si) 6.74–6.90 (4 H, m), 7.05 (1 H, d,
J 7.7), 7.08–7.18 (1 H, m), 7.92 (1 H, s), 7.99 (1 H, d, J 9.7);
13C NMR δC (75 MHz; CDCl3 + DMSO-d6) 178.1, 163.4,
157.8, 156.0, 155.0, 129.9, 129.7, 127.5, 123.9, 120.4, 120.3,
118.7, 116.0, 115.9, 102.1; HRMS (ESI) exact mass calcd for
C15H11O4: 255.06573 ([M
+
H]+). Found: 255.06487
([M + H]+).
7,8-Dimethoxy-3-(2-methoxyphenyl)-4H-chromen-4-one
(5d).
Following the procedure of 5b, compound 5d (1.29 g, 72%)
gave a white solid, mp 168–170 °C (from CHCl3–MeOH); IR
(KBr): νmax/cm−1 1626s (conj. CO); 1H NMR δH (300 MHz;
CDCl3; Me4Si) 4.02 (3 H, s), 4.03 (3 H, s), 6.94–7.02 (1 H, m),
7.07–7.20 (3 H, m), 7.31–7.40 (1 H, m), 8.07–8.12 (1 H, d,
J 9.1), 8.16 (1 H, s), 8.85 (1 H, s, OH, D2O exchange); 13C
NMR δC (75 MHz; CDCl3) 178.8, 157.0, 156.4, 155.6, 150.4,
136.4, 130.5, 129.7, 124.5, 121.9, 120.8, 120.5, 119.5, 117.9,
111.0, 61.7, 56.5; HRMS (ESI) exact mass calcd for C17H14O5:
299.0919 ([M + H]+). Found: 299.0921 ([M + H]+).
7-Hydroxy-3-(2-hydroxy-4-methoxyphenyl)-4H-chromen-4-one
(5a). To a solution of compound 4a (2.00 g, 6.7 mmol) in
CH3CN (25 mL), AlCl3 (2.66 g, 20.0 mmol) was added at 0 °C.
After refluxing for 10–15 h the mixture was cooled and poured
into ice-cooled water. The precipitated solid was filtered, washed
with water and purified by silica gel column chromatography
(CHCl3–MeOH, 98 : 2) furnishing 5a (1.14 g, 60%) as a white
solid, mp 210–212 °C (from CHCl3–MeOH) (lit.,8 mp
6-Chloro-7-hydroxy-3-(2-hydroxyphenyl)-4H-chromen-4-one (5e).
Following the procedure of 5a, compound 5e (1.16 g, 60%) was
synthesized as a white solid, mp > 250 °C (from CHCl3–
1
MeOH); IR (KBr): νmax/cm−1 1636s (conj. CO); H NMR δH
(300 MHz; CDCl3 + DMSO-d6; Me4Si) 7.01–7.06 (1 H, t),
7.09–7.12 (1 H, d), 7.24 (1 H, s), 7.35–7.39 (2 H, t), 8.24 (1 H,
s), 8.27 (1 H, s), 11.30 (1 H, s, OH, D2O exchange); 13C –NMR
δC (75 MHz; DMSO-d6) 174.2, 158.3, 156.1, 155.9, 155.3,
132.1, 129.8, 126.5, 122.2, 120.0, 119.4, 119.1, 117.6, 116.2,
104.1; HRMS (ESI) exact mass calcd for C15H1035ClO4:
289.0268 ([M + H]+). Found: 289.0101 ([M + H]+).
1
212–215 °C); IR (KBr): νmax/cm−1 1619s (conj. CO); H NMR
δH (300 MHz; CDCl3 + DMSO-d6; Me4Si) 3.61 (3 H, s),
6.25–6.39 (2 H, m), 6.68–6.80 (2 H, m), 6.98 (1 H, d, J 9.6),
7.85 (1 H, d, J 8.7), 7.92 (1 H, s), 10.33 (1 H, s, OH, D2O
exchange); 13C NMR δC (75 MHz; DMSO-d6) 175.2, 161.6,
159.4, 156.4, 155.5, 153.1, 130.2, 125.9, 120.9, 115.0, 114.1,
110.9, 104.1, 101.3, 100.8, 53.7; HRMS (ESI) exact mass
calcd for C16H13O5: 285.07630 ([M + H]+). Found: 285.07820
([M + H]+).
3-(2-Hydroxy-4-methoxyphenyl)-7,8-dimethoxy-4H-chromen-4-one (5b).
To a solution of compound 4b (2.10 g, 6.7 mmol) in DMF
(10 mL), K2CO3 (2.78 g, 20.1 mmol) and MeI (1.1 mL,
16.8 mmol) were added and the mixture was stirred for 5–6 h
and then poured into ice-cold water and the solid was filtered.
After dissolving the solid in CH3CN (25 mL), AlCl3 (2.66 g,
20 mmol) was added to the solution at 0 °C. The reaction
mixture was refluxed for 10–15 h and then cooled and poured
into ice-cooled water. The precipitated solid was filtered, washed
with water and purified by silica gel column chromatography
9-Methoxy-6a,11a-dihydro-6H-benzo[4,5]furo[3,2-c] chromen-
3-ol [( )-6a]. NaBH4 (2.4 g, 63.6 mmol) was added to a stirred
solution of chromen-4-one 5a (3.01 g, 10.6 mmol) in absolute
ethanol (30 mL) at 0 °C. The mixture was then stirred for 24 h at
room temperature and the reaction was stopped by addition of
ice-cooled water. Neutralisation of the solution with 10% hydro-
chloric acid delivered a white precipitate, which was washed
with water and purified by silica gel column chromatography
(n-hexane–CHCl3, 1 : 20) giving 6a (1.57 g, 55%) as a white
solid, mp 184–186 °C (from n-hexane–CHCl3) (lit.,8 mp
194–195 °C); IR (KBr): νmax/cm−1 3393br (OH), 1594s, 1451,
1
1381, 1348, 1285, 1155, 1027; H NMR δH (300 MHz; CDCl3;
Me4Si) 3.49–3.70 (2 H, m), 3.79 (3 H, s), 4.26 (1 H, dd, J 4.5,
10.5), 4.97 (1 H, s, OH, D2O exchange), 5.52 (1 H, d, J 6.6),
6.41–6.51 (3 H, m), 6.58 (1 H, dd, J 2.5, 8.4), 7.15 (1 H, d,
This journal is © The Royal Society of Chemistry 2012
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J 8.8), 7.41 (1 H, d, J 8.4); 13C NMR δC (75 MHz; CDCl3 +
CD3OD) 160.8, 160.3, 158.2, 156.3, 131.7, 124.6, 119.1, 111.1,
109.7, 106.1, 103.1, 96.6, 78.6, 66.2, 55.2, 39.2; HRMS (ESI)
exact mass calcd for C16H15O4: 271.09703 ([M + H]+). Found:
271.09729 ([M + H]+). Resolution of the enantiomers of ( )-6a:
HPLC (semi-preparative, see the ESI; Fig. S3–S5†): (6aS,11aS)-
6a: tR = 20.1 min; [α]D25 +78.19 (c 0.1 in CH3CN) (lit.,45 [α]2D5
+113.9 (c 0.23 in CHCl3)). (6aR,11aR)-6a: tR = 22.1 min;
[α]2D5 −79.72 (c 0.1 in CH3CN) (lit.,46a [α]2D5 −87.9° (c 0.5 in
CH3OH); lit.,46b −188 (c 0.1 in CHCl3)).
921, 883, 836, 747; 1H NMR δH (300 MHz; CDCl3; Me4Si)
3.55–3.72 (2 H, m), 4.29 (1 H, dd, J 9.3, 3.3), 5.46 (1 H, d, J
6.4), 5.63 (1 H, s, OH, D2O exchange), 6.62 (1 H, s), 6.81–6.96
(2 H, m), 7.14–7.28 (2 H, m), 7.51 (1 H, s); 13C NMR δC
(75 MHz; CDCl3) 159.1, 155.6, 152.4, 130.8, 129.3, 126.7,
124.7, 121.1, 113.8, 113.4, 110.2, 109.5, 104.6, 66.4, 40.0;
HRMS (ESI) exact mass calcd for C15H1135ClO3: 275.04750
([M + H]+). Found: 275.04340 ([M + H]+).
Material and methods
3,4,9-Trimethoxy-6a,11a-dihydro-6H-benzo[4,5]furo[3,2-c]chromene
[( )-6b]. Application of the protocol described for 6a led to
compound 6b (2.20 g, 66%) as a white solid, mp 110–112 °C
(from n-hexane–CHCl3) (lit.,43 mp 114–115 °C); IR (KBr):
Biological methods: reagents and chemicals
Cell culture media and supplements were purchased from Invi-
trogen (Carlsbad, CA) and all fine chemicals from Sigma-
Aldrich (St. Louis, MO). The BMP-2 ELISA kit was provided
by R&D Systems. The ECL kit was obtained from Amersham
Pharmacia, USA. All antibodies for Western blot analysis were
obtained from Cell Signaling Technologies (Danvers, MA).
HPLC-grade acetonitrile and isopropanol were purchased from
Sisco Research Laboratories (SRL) Pvt. Limited (Mumbai,
India), n-hexane, ammonium acetate and glacial acetic acid
(analytical grade) were provided by E-Merck Limited (Mumbai,
India).
1
νmax/cm−1 2929, 1615, 1497, 1466, 1282, 1106, 1027; H NMR
δH (300 MHz; CDCl3; Me4Si) 3.53–3.64 (2 H, m), 3.78 (3 H, s),
3.86 (3 H, s), 3.88 (3 H, s), 4.36 (1 H, dd, J 4.5, J 10.5), 5.52
(1 H, d, J 6.6), 6.45–6.48 (2 H, m), 6.69 (1 H, d, J 8.8), 7.15
(1 H, d, J 8.8), 7.25 (1 H, d, J 9.3); 13C NMR δC (75 MHz;
CDCl3) 161.2, 160.7, 153.6, 149.5, 137.5, 125.7, 124.8, 119.0,
114.2, 106.4, 106.0, 97.0, 78.6, 66.8, 61.0, 56.2, 55.5, 39.5;
HRMS (ESI) exact mass calcd for C18H18O5: 315.12325
([M + H]+). Found: 315.12290 ([M + H]+).
6a,11a-Dihydro-6H-benzo[4,5]furo[3,2-c]chromen-3-ol [( )-6c].
Following the procedure described for 6a, compound 6c (1.45 g,
57%) was produced as a white solid, mp 148–152 °C (from
CHCl3–MeOH) (lit.,47 mp 147–150 °C); IR (KBr): νmax/cm−1
3396br (OH), 1597, 1516, 1476, 1381, 1351, 1289, 1259, 1222,
1170, 1122, 1082, 1017; 1H NMR δH (300 MHz; CDCl3;
Me4Si) 3.53–3.72 (2 H, m), 4.23–4.32 (1 H, m), 5.21 (1 H, s,
OH, D2O exchange), 5.49 (1 H, d, J 6.4), 6.42 (1 H, d, J 2.3),
6.56 (1 H, dd, J 2.9, 8.7), 6.82–6.95 (2 H, m), 7.14–7.28 (2 H,
m), 7.41 (1 H, d, J 8.1); 13C NMR δC (75 MHz; CDCl3) 159.3,
157.1, 156.6, 132.3, 129.3, 127.1, 124.8, 121.0, 112.5, 110.2,
109.9, 103.7, 77.7, 66.3, 40.1; HRMS (ESI) exact mass calcd
for C15H13O3: 241.08647 ([M + H]+). Found: 241.08666
([M + H]+). Resolution of the enantiomers of ( )-6c: HPLC
(semi-preparative, see the ESI; Fig. S6–S8†): (6aS,11aS)-6c: tR =
Culture of calvarial osteoblasts
Rat calvarial osteoblasts were obtained following our previously
published protocol of sequential digestion.41,48 Briefly, calvaria
from 1- to 2-d-old Sprague–Dawley rats (both sexes) were
pooled. After surgical isolation from the skull and removal of
sutures and adherent mesenchymal tissues, calvaria were sub-
jected to five sequential (10–15 min) digestions at 37 °C in a sol-
ution containing 0.1% dispase and 0.1% collagenase P. Cells
released from the second to fifth digestions were pooled, centri-
fuged, resuspended, and plated in T-25 cm2 flasks in α-MEM
containing 10% FCS and 1% penicillin–streptomycin (complete
growth medium).
17.8 min; [α]2D5 +79.75 (c 0.1 in CH3CN). (6aR,11aR)-6c: tR
=
20.3 min; [α]2D5 −82.06 (c 0.1 in CH3CN). [α]2D5 is not reported
in the literature.20
Osteoblast differentiation
3,4-Dimethoxy-6a,11a-dihydro-6H-benzo[4,5]furo[3,2-c]chromene
[( )-6d]. According to the procedure described for 6a, com-
pound 6d (2.10 g, 70%) was furnished as a white solid, mp
106–108 °C (from n-hexane–CHCl3); IR (KBr): νmax/cm−1
For the measurement of alkaline phosphatase (ALP) activity,41,48
osteoblasts at ∼80% confluence were trypsinised and 2 × 103
cells per well were seeded in 96-well plates. Cells were treated
with different concentrations of the compounds for 48 h in
α-MEM supplemented with 5% charcoal treated FCS, 10 mmol
β-glycerophosphate, 50 μg mL−1 ascorbic acid and 1% penicillin–
streptomycin (osteoblast differentiation medium). At the end of
the incubation period, the total ALP activity was measured
using p-nitrophenylphosphate (PNPP) as a substrate and quan-
tified colourimetrically at 405 nm.
1
2931, 1610, 1471, 1286, 1224, 1108; H NMR δH (300 MHz;
CDCl3; Me4Si) 3.62–3.73 (2 H, m), 3.86 (3 H, s), 3.89 (3 H, s),
4.37–4.41 (1 H, dd, J 4.05, 10.3), 5.51–5.53 (1 H, d, J 6.8),
6.68–6.70 (1 H, d, J 8.6), 6.78–6.87 (2 H, m), 7.09–7.14 (1 H,
m), 7.18–7.20 (2 H, m); 13C NMR δC (75 MHz; CDCl3) 159.5,
153.9, 149.7, 137.7, 129.5, 127.3, 126.0, 125.0, 121.2, 114.3,
110.4, 106.3, 78.0, 66.8, 61.3, 56.4, 40.3; HRMS (ESI) exact
mass calcd for C17H16O4: 285.11260 ([M + H]+). Found:
285.11200 ([M + H]+).
Mineralisation of bone marrow derived osteoblast cells
2-Chloro-6a,11a-dihydro-6H-benzofuro[3,2-c]chromen-3-ol [( )-6e].
Following the procedure described for 6a, compound 6e (1.60 g,
55%) was synthesized as a white solid, mp 138–140 °C (from
n-hexane–CHCl3); IR (KBr): νmax/cm−1 3394br (OH), 1623,
1589, 1490, 1438, 1385, 1316, 1214, 1154, 1076, 1019, 957,
For mineralisation studies, bone marrow cells were cultured in a
medium consisting of α-MEM, supplemented with 10% fetal
bovine serum, 50 μg mL−1 ascorbic acid, and 10 μmol β-glycero-
phosphate. Cells were cultured with and without compounds
9590 | Org. Biomol. Chem., 2012, 10, 9583–9592
This journal is © The Royal Society of Chemistry 2012

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for 21 d at 37 °C in a humidified atmosphere of 5% CO2 and
95% air, and the medium was changed every 48 h. After 7 d, the
attached cells were fixed in 4% formaldehyde for 20 min at
room temperature and rinsed once in PBS. After fixation, the
specimens were processed for staining with 40 mmol alizarin
red-S, which stains areas rich in nascent calcium. BMP-2 was
used as a positive control. For quantification of alizarin red-S
staining, 800 μL of 10% (v/v) acetic acid was added to each
well, and plates were incubated at room temperature for 30 min
with shaking. The monolayer, now loosely attached to the plate,
was then scraped with a cell scraper and transferred with 10%
(v/v) acetic acid to a 1.5 mL tube. After vortexing for 30 s, the
slurry was overlaid with 500 μL mineral oil (Sigma-Aldrich),
heated to exactly 85 °C for 10 min, and transferred to ice for
5 min. The slurry was then centrifuged at 20 000g for 15 min
and 500 μL of the supernatant was removed to a new tube. Then
200 μL of 10% (v/v) ammonium hydroxide was added to neu-
tralize the acid. In some cases, the pH was measured at this point
to ensure that it was between 4.1 and 4.5. OD (405 nm) of
150 μL aliquots of the supernatant were measured in 96-well
format using opaque-walled, transparent-bottomed plates.
denaturation at 94 °C for 2 min, and annealing and extension at
62 °C for 30 s, extension at 72 °C for 30 s. GAPDH was used to
normalize differences in RNA isolation, RNA degradation, and
the efficiencies of the reverse transcription.
Computational details
Conformational analyses for 6a and 6c were done using the
Gaussian03 software package.49 For the geometry optimizations
the B3LYP functional was chosen together with the double-zeta
6-31G* basis set. The TDDFT excited-states calculations of 6a
and 6c were performed using the ORCA software package.50 For
the four conformers of each compound within the energetical
range of 3 kcal mol−1, single UV and ECD spectra were simu-
lated with TDB2GP-PLYP/TZVP) (nstates = 40) with correspond-
ing oscillator and rotational strength values from the length
formalism.51 Furthermore the excited states (nstates = 20) were
calculated with MRCI (CAS 12,12) and the initial orbitals in
these calculations were orbitals from a DFT [B3LYP/G/SV(P)]
calculation, performed also with ORCA. The UV and ECD
curves of 6a and 6c were calculated as sums of Gauss functions,
centered at the wavelength of the corresponding excitations and
multiplied by the respective oscillator and rotational strength
values, with an empirically chosen exponential half width of
0.1 eV. Summation of the single spectra following the Boltzmann
statistics yielded the overall UV and ECD curves. Before com-
parison with the experimental ECD spectra, a UV correction of
42 nm for the curves generated with TDB2GP-PLYP/TZVP and
34 nm for the spectra calculated with the combined approach of
DFT (B3LYP/SVP) and MRCI was accomplished.52 For Gauss
curve generation, UV shifting, comparison with experimental
data, and plotting, SpecDis53 was used.
For measuring BMP-2 production from osteoblasts, 5 × 103
cells per well were seeded in 24-well plates. Cells were exposed
to 10−10 M pterocarpan for 48 h with or without inhibitors in
α-MEM media supplemented with 5% FCS, 10 mmol β-glycero-
phosphate and 50 μg mL−1 ascorbic acid. At the end of incu-
bation, supernatants were collected for determination of BMP-2
by ELISA as per the manufacturer’s instructions. For inhibitor
studies, cells were pre-treated with inhibitors 30 min prior to
compound treatment.
Methodology for Runx-2 and BMP-2 expression by using q-PCR
Total RNA was extracted from the cultured cells using TRIzol®
(Invitrogen, Carlsbad, CA). cDNA was synthesized from 2 μg
total RNA with the Revert Aid™ H Minus first strand cDNA
synthesis kit (Fermentas, Vilnius, Lithuania). SYBR green
chemistry was used for quantitative determination of the mRNAs
for BMP-2 and Runx-2 and a housekeeping gene, GAPDH, fol-
lowing an optimized protocol. The design of sense and antisense
oligonucleotide primers for BMP-2 and Runx-2 was done using
the Universal ProbeLibrary (Roche Diagnostics, Indianapolis,
IN). The primer pair for BMP-2 (Gene Accession Number:
NM_017178.1) was 5′-CGG ACT GCG GTC TCC TAA-3′
(sense) and 5′-GGG GAA GCA GCA ACA CTA GA-3′ (anti-
sense); for Runx-2 (Gene Accession Number: NM_053470.1)
the primer pair was 5′-GCC GGG AAT GAT GAG AAC TAC
T-3′ (sense) and 5′-TCC GGC CTA CAA ATC TCA GAT C-3′
and for GAPDH it was 5′-CAG CAA GGA TAC TGA GAG
CAA GAG-3′ (sense) and 5′-GGA TGG AAT TGT GAG GGA
GAT G-3′ (antisense). For real-time PCR, the cDNA was
amplified with a Light Cycler 480 (Roche Diagnostics Pvt Ltd,
Indianapolis, IN). The double-stranded DNA-specific dye SYBR
Green I was incorporated into the PCR buffer provided in the
Light Cycler 480 SYBR Green I Master (Roche Diagnostics Pvt
Ltd, Indianapolis, IN) to allow for quantitative detection of the
PCR product in a 20 μL reaction volume. The temperature
profile of the reaction was 95 °C for 5 min, 40 cycles of
Acknowledgements
We gratefully acknowledge financial support from the Council of
Scientific and Industrial Research (CSIR), New Delhi, India and
the Alexander-von-Humboldt Foundation, Bonn, Germany for
research support and equipment subsidy (HPLC system). Amit
Kumar and Ashutosh Raghuvanshi are thankful to CSIR, New
Delhi for research fellowships. The spectroscopic data provided
by SAIF, CDRI is gratefully acknowledged. The CDRI com-
munication number is 8295.
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