One step synthesis of 2-hydroxymethylisoflavone and their osteogenic activity

Article abstract of DOI:10.1016/j.bmcl.2011.01.095

An efficient one step synthesis of new 2-hydroxymethylisoflavone is reported. A series of deoxybenzoin was subjected to cyclization with glyoxal in the presence of basic condition (KOH/EtOH) to afford the 2-hydroxymethyl isoflavone. The structures of comp

Full text of DOI:10.1016/j.bmcl.2011.01.095

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1706–1709
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
One step synthesis of 2-hydroxymethylisoflavone and their osteogenic activity q
Manmeet Kumar a, Preeti Rawat a, Jyoti Kureel b, Anuj Kumar Singh b, Divya Singh b, Rakesh Maurya a,
⇑
a Medicinal and Process Chemistry Division, Central Drug Research Institute, CSIR, Lucknow-226 001, UP, India
b Endocrinology Division, Central Drug Research Institute, CSIR, Lucknow-226 001, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient one step synthesis of new 2-hydroxymethylisoflavone is reported. A series of deoxybenzoin
was subjected to cyclization with glyoxal in the presence of basic condition (KOH/EtOH) to afford the 2-
hydroxymethyl isoflavone. The structures of compounds 5a–g were confirmed by NMR experiments
including 1H, 13C, HMBC, HSQC and COSY. These compounds were assessed for stimulation of osteoblast
function using primary culture of rat calvarial osteoblasts in vitro. Compounds 5a, 5d, 5f and 5g were
potent in stimulating differentiation of osteoblasts as assessed by measuring alkaline phosphatase
(ALP) activity. Besides, effect of these analogs was also seen on the transcript levels of osteogenic genes
like Runx-2, osteocalcin and Bone morphogenetic protein-2 (BMP-2), involved in osteoblast differentia-
tion and mineralization. Based on quantitative PCR data, compound 5f was found to be the potent fol-
lowed by 5d. Compound 5f robustly increased the mRNA levels of Runx-2 (8.0 fold), BMP-2 (ꢀ2 fold)
and osteocalcin (ꢀ2.0 fold) in osteoblasts. Collectively, we demonstrate osteogenic activity of the novel
2-hydroxymethyl isoflavones with 5f having the most potent activity.
Received 22 October 2010
Revised 10 January 2011
Accepted 20 January 2011
Available online 26 January 2011
Keywords:
2-Hydroxymethylisoflavone
1,5-Hydride shift
2-Carbaldehydeisoflavone
Osteoblast differentiation
Quantitative PCR
Ó 2011 Elsevier Ltd. All rights reserved.
Isoflavonoids are a group of phenolic compounds produced by
various higher plants to protect themselves from environmental
stress and are present in foods such as beans, cabbage, soya beans,
grains and hops.1 Some of the most popular isoflavones are glyci-
tein, genistein, formononetin, biochanin A and daidzein.
O
C
A
B
O
Figure 1. General structure of isoflavone.
Isoflavones and its derivatives (Fig. 1) possess a diverse range of
biological activities including antimicrobial, antioxidant, stimulat-
ing nerve growth, insecticidal activities,2 anti osteoporotic,3 hypo-
lipidemic activities4 etc. It is also reported that isoflavones have
healthful benefits in human obesity and have a positive influence
on plasma cholestrol.5 They produce weaker estrogenic effects
when endogenous estrogen level is inadequate. In contrast, they
can inhibit the efficacy when estrogens are adequate.6 With the
dual-effects, isoflavones are considered to be effective and safer
estrogen replacements. These observations inspired several groups
to synthesize various derivatives of isoflavone with the intention to
either improve biochemical and pharmacokinetic characteristics of
the parent drug or to obtain compounds containing essential ele-
ments of the parent substance but having novel properties and/
or affecting novel molecular targets.
osteogenic activity and also increase bone mass in growing rats.
In this study we have synthesized analogs of isoformononetin
and validated its osteogenic activity in vitro by assessing effect
on osteoblast differentiation by measuring ALP activity. Our stud-
ies show that these analogs have potent bone forming activity at
concentrations ꢀ100 fold lower than isoformononetin.
The literature synthesis for isoflavones in general involves two
steps wherein a phenol is reacted in the presence of a Lewis acid
with phenylacetic acid to generate an intermediate deoxybenzoin7
which is then cyclized with a one carbon electrophile. A convenient
approach is that of Baker et al.8 who used oxalyl chloride and pyr-
idine as the cyclizing agent, whereas others have used ethyl for-
mate,9 triethylorthoformate10 or carbon disulfide11 and some
other strategy are also reported in the literature.12 To the best of
our knowledge, the synthesis of 2-hydroxymethyl isoflavone using
glyoxal has not been reported. Herein, we report on an efficient,
synthetic pathway to 2-hydroxymethylisoflavone, which was
accomplished through the condensation of deoxybenzoin with gly-
oxal in basic condition. Key step of our synthesis is base catalyzed
enolization of 1,4-diketone intermediate followed by 1,5-hydride
shift to afford 2-hydroxymethylisoflavone.
Recently we have reported the osteogenic activity of Butea
monosperma crude extract. Subsequent studies led to the isolation
of several methoxyisoflavones like isoformononetin and formo-
nonetin.3 These methoxyisoflavones were shown to have in vitro
q
CDRI Communication No. 8019.
⇑
Corresponding author. Tel.: +91 522 2612411 18x4235; fax: +91 522 2623405/
2623938/2629504.
E-mail address: mauryarakesh@rediffmail.com (R. Maurya).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.095

M. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1706–1709
1707
O
R1
OH
R1
O
O
R4
(CHO)2
X
H
R4
R3
R3
R1
OH
R4
R3
.
BF3 OEt2
80 oC
O
KOH, EtOH
RT
O
R2
HO
R2
3a-g
R2
2a-f
1a-c
4a-g
(CHO)2
RT
KOH, EtOH
R1
O
O
OH
R4
R3
R2
5a-g
Scheme 1. Synthesis of compounds 5a–g.
We anticipated that glyoxal could be more effective to synthe-
size 2-carbaldehydeisoflavone (4) using Aldol condensation with
deoxybenzoin (2a–f) and followed by intra molecular Michael
addition (Scheme 1). 2-Carbaldehydeisoflavone (4) could be used
to synthesize various derivative of isoflavone utilising aldol con-
densation reaction with various acetophenones. Deoxybenzoins
(3a–g)13 were prepared by the published procedure from hydro-
xy-phenols (1a–c) and phenylacetic acids (2a–f) by Friedel–Crafts
acylation using boron trifluoride etherate as solvent and also as
the Lewis acid for the acylation. This reaction was carried out at
80 °C and completed in about one and a half hours13 (Scheme 1).
When deoxybenzoin was treated with the glyoxal in ethanol using
potassium hydroxide as a base at RT, afforded 2-hydroxymethyl
isoflavones (5a–g)14 in a respectable yield of 50–80%. Proposed
mechanism of this step is depicted in Figure 2. It is assumed that
condensation of deoxybenzoin with glyoxal undergoes intra
molecular ring cyclization to the formation of 2-carbaldehyde
dihydroisoflavone and dienol intermediates, followed by 1,5-hy-
dride shift to afford 2-hydroxymethylisoflavones (5a–g).
H
H
HO
O
OH
CH3
H
O
H
O
5a
Figure 3. Selected HMBC correlation of compound 5a.
Table 1
Synthesis of 2-isoflavone derivatives 5a–g
Product
R1
R2
R3
R4
Yield (%)
5a
5b
5c
5d
5e
5f
OH
OCH3
OH
OH
OH
H
H
H
H
H
OCH3
OH
H
OCH3
OH
OCH3
H
OCH3
OCH3
H
H
H
OCH3
H
H
78
65
72
75
66
50
80
H
H
5g
H
The structures of compounds 5a–g15–21 were confirmed by 1D
and 2D NMR experiments including 1H, 13C, HMBC, HSQC, and
COSY. Selected HMBC correlations of compound 5a are given in
Figure 3. This newly developed procedure was applied to the syn-
thesis of a number of isoflavone derivatives, afforded in good to
excellent yield depicted in Table 1.
the most active. These compounds increased osteoblast differenti-
ation at ꢀ100 fold lower concentration than isoformononetin
which is known for its osteogenic activity. In order to validate
which amongst the four compounds was the most potent osteo-
genic agent, their activity was further checked by quantitative PCR.
We next studied the effect of compounds on the expression of
osteogenic genes (osteocalcin, Runx-2 and BMP-2)23 in calvarial
osteoblasts by quantitative real time PCR (qPCR).24 Runx-2, a
bone-specific transcription factor, is a key regulator of osteoblast
differentiation (Fig. 5). Runx-2 expression is induced by BMP-2
stimulation. Thus, Runx-2 is a downstream transcription factor in
BMP-2 signaling. Runx-2 transcription is important for expression
of osteocalcin, an important biomarker of osteoblast differentia-
tion. The transcript levels of Runx-2, OCN and BMP-2 were as-
sessed by qPCR after treatment with compounds and results
were expressed as fold change over untreated cells. Runx-2 gene
expression was increased by both 5f (8.0 fold increase) and 5d
(6.5-fold increase) with no effect of 5a and 5g. BMP-2 expression
was increased by 5f (2.0 fold) and 5g (2.5 fold) with no effect of
other two. In case of OCN, mRNA expression was increased by
ꢀ2.0 fold by 5f with no effect of other compounds. Thus based
on real time PCR data 5f was the most active (increased expression
of Runx-2, BMP-2 and OCN) followed by 5d. Taken together,
5f > 5d > 5g > 5a.
Alkaline phosphatase (ALP),22 which is one of the important
markers of osteoblast differentiation, was measured spectrophoto-
metrically and optical density was determined at 405 nm. Com-
pounds 5a–g were evaluated for ALP activity stimulation at
concentrations ranging from 10ꢁ12 to 10ꢁ6 M (Fig. 4). Isoformo-
nonetin (10ꢁ8 M) was used as a positive control. Treatment of cal-
varial rat osteoblast cells led to increased ALP activity by
compounds 5a, 5d, 5f and 5g with compounds 5d and 5f being
R1
OH
O
R1
O
O
R4
R3
(CHO)2, RT
KOH, EtOH
H
R4
R3
O
R2
R2
OH
R1
O
R1
O
OH
R4
R3
R4
R3
In conclusion, we have reported efficient and operationally sim-
ple synthesis of 2-hydroxymethyl isoflavone. This simple one step
process provides the first simple entry into a series of isoflavones
with different substitution in ring A or B at will and should provide
a simple route to substituted analogues for SAR studies. These
O
R2
O
R2
H
Figure 2. A plausible reaction pathway of formation of 2-hydroxymethyl
isoflavone.

1708
M. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1706–1709
Figure 4. Effect of compounds on osteoblast differentiation. 2 ꢂ 103 rat calvarial osteoblasts were seeded in 96 well plates and exposed to various concentrations of
compounds ranging from 10ꢁ12 M to 10ꢁ6 M for 48 h and ALP activity was determined spectrophotometrically at 405 nm. Isoformononetin (Iso) at 10ꢁ8 M was used as a
positive control. Data shown as mean SEM; n = 6; ⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.001 compared with vehicle treated cells.
Figure 5. Effects of compounds on mRNA expression of BMP-2, osteocalcin (OCN) and Runx-2. Osteoblast cells were cultured with or without compounds for 48 h. qPCR for
osteocalcin, Runx-2 and BMP-2 was performed as described in materials and methods. Data shown as mean SEM; n = 3; ⁄P <0.05 compared with vehicle treated cells.
3. Maurya, R.; Yadav, D. K.; Singh, G.; Bhargavan, B.; Murthy, P. S. N.; Sahai, M.;
Singh, M. M. Bioorg. Med. Chem. Lett. 2009, 19, 610.
4. Xiang, H.; Zhao, W.; Xiao, H.; Qian, L.; Yao, Y.; Li, X.-B.; Liao, Q.-J. Bioorg. Med.
2-hydroxymethyl isoflavones are also potential anti-osteoporotic
agents. Further studies regarding the biological activities of such
derivatives are in progress.
Chem. 2010, 18, 3036.
5. Ørgaard, A.; Jensen, L. Exp. Biol. Med. 2008, 233, 1066.
6. Cline, J. M.; Wood, C. E. Am. J. Primatol. 2009, 71, 722.
Acknowledgments
7. Dohme, A. R. L.; Cox, E. H.; Miller, E. J. Am. Chem. Soc. 1926, 48, 1688.
8. Baker, W.; Chadderton, J.; Harborne, J.; Ollis, W. D. J. Chem. Soc. 1953, 1852.
9. Mahal, H. S.; Rai, H. S.; Venkataraman, K. J. Chem. Soc. 1934, 1120.
10. Sathe, V. R.; Venkataraman, K. Curr. Sci. India 1949, 18, 373.
11. Kim, Y. W.; Brueggemeier, R. W. Tetrahedron Lett. 2002, 43, 6113.
12. (a) Ollis, W. D. In The Chemistry of Flavonoid Compounds; Geissman, T. A., Ed.;
Manmeet Kumar and Preeti Rawat are thankful to the CSIR, New
Delhi, India for Senior Research Fellowship and SAIF division, CDRI,
for spectral data.
Pergamon: Oxford, 1962;
p 353; (b) Hepworth, J. D. In Comprehensive
References and notes
Heterocyclic Chemistry; Katritzky, A. R., Rees, C. W., Eds.; Pergamon: New
York, 1984; Vol. 3, p 816; (c) William, T. B.; Gu, Y.-Q. J. Org. Chem. 1988, 53,
1353; (d) Kim, Y.-W.; Mobley, J. A.; Brueggemeier, R. W. Bioorg. Med. Chem. Lett.
2003, 13, 1475; (e) Granados-Covarrubias, E. H.; Maldonado, Luis A.
1. Singh, H.; Pratap, R. Tetrahedron Lett. 2006, 47, 8161.
2. Chen, G. S.; Chang, C.-S.; Kan, W. M.; Chang, C.-L.; Wang, K. C.; Chern, J.-W. J.
Med. Chem. 2001, 44, 3759.

M. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1706–1709
1709
Tetrahedron Lett. 2009, 50, 1542; (f) Biegasiewicz, K. F.; Denis, J. D. St.; Carroll,
V. M.; Priefer, R. Tetrahedron Lett. 2010. doi:10.1016/j.tetlet.2010.06.078; (g) Li,
S.-R.; Chen, P.-Y.; Chen, L.-Y.; Lo, Y.-F.; Tsai, I.-L.; Wang, E.-C. Tetrahedron Lett.
2009, 50, 2121; (h) Skouta, R.; Li, C.-J. Tetrahedron Lett. 2007, 48, 8343.
13. Wähälä, K.; Hase, T. A. J. Chem. Soc., Perkin Trans. 1 1991, 3005.
14. General procedure for synthesis of deoxybenzoin (3a–g): The mixture of p-
methoxy phenyl acetic acid (500 mg, 3.08 mmol) in boron trifluoride etherate
(0.6 ml) and resorcinol (280 mg, 3.00 mmol) were heated at 80 °C for 8 h.
Reaction mixture was poured in water, extracted with ethyl acetate and
dried over anhydrous sodium sulphate. Reaction mixture was concentrated
to give crude product. Purification was done by column chromatography
using silica gel as adsorbent and hexane, ethyl acetate as eluent to yield
compound 3a.
1.2 Hz, H-6), 6.90 (1H, d, J = 1.2 Hz, H-8), 6.97 (1H, d, J = 7.5 Hz, H-60), 6.30 (1H,
d, J = 7.5, 1.1 Hz, H-50), 6.00 (1H, d, J = 1.1 Hz, H-30), 4.17 (2H, s, CH2), 3.82 (3H,
s); 13C NMR (CDCl3, 75 MHz): 175.8 (C-4), 162.7 (C-7), 162.4 (C-2), 157.2 (C-40),
157.6 (C-9), 108.8 (C-10), 132.5 (C-60), 127.2 (C-5), 156.7 (C-20), 123.4 (C-3),
115.1 (C-6), 116.8 (C-10), 103.0 (C-30), 106.6 (C-50), 58.8 (CH2), 57.2 (OCH3);
ESIMS: (m/z) 329 [M+H]+; Elemental Anal. Calcd for C18H16O6: C, 65.85; H, 4.91.
Found: C, 65.78; H, 4.97.
21. Data of compound 5f: Amorphous powder; UV (MeOH): kmax 247, 341 nm; 1H
NMR (CDCl3, 300 MHz): 7.74 (1H, dd, J = 8.5, 1.5 Hz, H-5), 7.40 (1H, m, H-7),
7.34 (1H, m, H-8), 7.21 (1H, m, H-6), 6.96 (1H, d, J = 7.3 Hz, H-60), 6.37(1H, dd,
J = 7.3, 1.2 Hz, H-50), 6.01 (1H, d, J = 1.2 Hz, H-30), 4.16 (2H, s, CH2), 3.82 (3H, s);
13C NMR (CDCl3, 75 MHz): 177.2 (C-4), 133.2 (C-7), 162.5 (C-2), 159.0 (C-40),
156.6 (C-9), 156.7 (C-20), 132.4 (C-60), 128.2 (C-5), 108.5 (C-10), 123.7 (C-
3),125.4 (C-6), 123.4 (C-10), 103.1 (C-30), 106.6 (C-50), 60.5 (CH2), 57.9 (OCH3);
ESIMS: (m/z) 299 [M+H]+; Elemental Anal. Calcd for C17H14O5: C, 68.54; H, 4.73.
Found: C, 68.63; H, 4.66.
15. General procedure for synthesis of 2-hydroxymethyl isoflavone (5a): To
a
magnetically stirred solution of deoxybenzoin 3a (500 mg, 2.06 mmol) and
potassium hydroxide (169 mg, 3,09 mmol) in ethanol (25 ml) was added the
glyoxal (0.15 ml, 3.09 mmol) at room temperature. The reaction mixture was
stirred for 4 h. The reaction mixture was poured into water. The organic layer
was extracted with ethyl acetate, washed with water, dried over Na2SO4 and
the solvent was removed under reduced pressure. The residue was purified by
column chromatography on silica gel using ethyl acetate/hexane (50:50) or
chloroform/methanol (98:2) as the eluting solvents.
22. Data of compound 5g: Amorphous powder; UV (MeOH): kmax 258, 344 nm; 1H
NMR (CDCl3, 300 MHz): 8.21 (1H, dd, J = 8.9, 1.7 Hz, H-5), 7.39 (2H, m, H-20, 60),
7.68 (1H, m, H-7), 7.46 (1H, d, J = 8.0 Hz, H-8), 7.29 (3H, m, H-30, 40, 50), 7.29
(1H, m, H-6), 4.49 (2H, s, CH2); 13C NMR: (CDCl3, 75 MHz): 177.7 (C-4), 162.3
(C-2), 155.9 (C-9), 133.3 (C-7), 131.4 (C-10), 130.4 (C-30, 50), 128.5 (C-20, 60),
128.3 (C-5), 126.3 (C-40), 125.2 (C-6), 123.7 (C-3), 123.5 (C-10), 60.8 (CH2);
ESIMS: (m/z) 253 [M+H]+; Elemental Anal. Calcd for C16H12O3: C, 76.18; H, 4.79.
Found: C, 76.25; H, 4.67.
16. Data of compound 5a: Pale yellow solid. Mp 197–199 °C; UV (MeOH): kmax 248,
330 nm; IR (KBr): 3178, 1645, 1611, 1557, 1306, 1255, 1089 cmꢁ1;1H NMR:
(CD3OD, 300 MHz): 8.18 (1H, d, J = 7.47 Hz, H-5), 7.34 (2H, d, J = 7.2 Hz, H-20,
60), 7.12 (2H, d, J = 7.2 Hz, H-30, 50), 7.03 (1H, d, J = 7.4 Hz, H-6), 7.03 (1H, d,
J = 7.4 Hz, H-8), 4.51 (2H, s, CH2), 3.95 (3H, s), 13C NMR: (CD3OD, 75 MHz):
179.5 (C-4), 164.9 (C-7), 164.8 (C-2), 161.4 (C-40), 159.6 (C-9), 133.0 (C-20, 60),
128.5 (C-5), 125.4 (C-10), 123.9 (C-3), 116.6 (C-6), 117.4 (C-10), 114.9 (C-30&50),
61.0 (CH2), 55.9 (OCH3); ESIMS: (m/z) 299 [M+H]+; Elemental Anal. Calcd for
23. Alkaline phosphatase activity: Calvarial osteoblasts were trypsinized at 70–80%
confluence using 0.02% trypsin-EDTA (Sigma). 2 ꢂ 103 cells were seeded in
a-
MEM containing 10% FBS media in 96-well plate with or without IF-01, IF-04,
IF-07 and IF-08 (Concentration ranging from 10ꢁ12 M to 10ꢁ6 M) for 48 h in
presence of 50 lg/ml ascorbate and 10 mM glycerophosphate. At the end of
incubation period, total ALP activity was measured with a method using p-
nitrophenylphosphate (PNPP) as substrate. Isoformononetin was used as a
positive control. The reaction mixture contained diethanolamine buffer (1 mol/
L, pH 9.8), 0.5 mmol/L MgCl2, and 10 mmol/L PNPP. ALP activity was measured
colorimetrically at 405 nm as described previously.
C
17H14O5: C, 68.54; H, 4.73. Found: C, 68.47; H, 4.75.
17. Data of compound 5b: Amorphous powder, UV (MeOH): kmax 255, 273, 330 nm;
IR (KBr): 3175, 1655, 1600, 1255, 1089 cmꢁ1 1H NMR (DMSO-d6, 300 MHz):
;
7.95 (1H, d, J = 8.3 Hz, H-5), 6.82 (2H, d, J = 7.8 Hz, H-30& 50), 7.10 (2H, d,
J = 7.8 Hz, H-20 & 60), 7.11 (1H, d, J = 8.3 Hz, H-6), 7.11 (1H, s, H-8), 4.28 (2H, s,
CH2), 3.91 (3H, s); 13C NMR (DMSO-d6, 75 MHz): 175.7 (C-4), 163.8 (C-7), 162.9
(C-2), 157.2 (C-40), 157.0 (C-9), 131.7 (C-20&60), 122.2 (C-5), 125.4 (C-10), 122.2
(C-3), 116.6 (C-6), 116.6 (C-10), 114.7 (C-30&50), 59.2 (CH2), 56.1 (OCH3);
ESIMS: (m/z) 299 [M+H]+; Elemental Anal. Calcd for C17H14O5: C, 68.54; H, 4.73.
Found: C, 68.57; H, 4.68.
24. Osteocalcin, Runx-2 and BMP-2 expression by using q-PCR: Total RNA was
extracted from the cultured cells using Trizol (Invitrogen). cDNA was
synthesized from 2 lg total RNA with the Revert Aid™ H Minus first strand
cDNA synthesis kit (Fermentas, USA). SYBR green chemistry was used for
quantitative determination of the mRNAs for Osteocalcin and BMP-2 and a
housekeeping gene, GAPDH, following an optimized protocol. The design of
sense and antisense oligonucleotide primers was based on published cDNA
sequences using the Universal probe library (Roche diagnostics, USA). For real-
time PCR, the cDNA was amplified with Light Cycer 480 (Roche diagnostics pvt
ltd). The double-stranded DNA-specific dye SYBR Green I was incorporated into
the PCR buffer provided in the Light Cycler 480 SYBER green I master (Roche
diagnostics pvt ltd) to allow for quantitative detection of the PCR product in a
18. Data of compound 5c: Yellow solid; Mp 210–211 °C; UV (MeOH): kmax 245,
341 nm; 1H NMR (CDCl3, 300 MHz): 8.03 (1H, d, J = 8.8 Hz, H-5), 7.10 (1H, s, H-
8), 7.09 (1H, d, J = 1.5 Hz, H-20), 7.09 (1H, d, J = 8.8 Hz, H-6), 7.01 (1H, dd,
J = 7.0 Hz,1.5, H-60), 6.97 (1H, d, J = 7.0 Hz, H-50), 4.40 (2H, s, CH2), 3.84 (3H, s),
3.83 (3H, s); 13C NMR (CDCl3, 75 MHz): 174.8 (C-4), 163.8 (C-7), 162.8 (C-2),
157.5 (C-9), 147.6 (C-30), 147.1 (C-40), 127.4 (C-5), 123.2 (C-10), 122.0 (C-3),
119.8 (C-20), 116.5 (C-60), 115.3 (C-6), 117.1 (C-10), 113.5 (C-50), 60.8 (CH2),
56.1(OCH3), 56.2 (OCH3); ESIMS: (m/z) 329 [M+H]+; Elemental Anal. Calcd for
20-ll reaction volume. The temperature profile of the reaction was 95 °C for
5 min, 40 cycles of denaturation at 94 °C for 2 min, and annealing and
extension at 62 °C for 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. Primer pairs used were; for BMP-2, 50-CGG ACT GCG
GTC TCC TAA-30 (sense); 50-GGG GAA GCA GCA ACA CTA GA-30 (antisense); for
osteocalcin, 50-GGA CAT TAC TGA CCG CTC C-30 (sense), 50-TTT TCA GTG TCT
GCC GTG AG-30 (antisense); for Runx-2 were, 50-GCC GGG AAT GAT GAG AAC
TAC T-30 (sense), 5’-TCC GGC CTA CAA ATC TCA GAT C-3’ (antisense); for
GAPDH, 5’-CAG CAA GGA TAC TGA GAG CAA GAG-3’ (sense), 5’-GGA TGG AAT
TGT GAG GGA GAT G-3’ (antisense).
C
18H16O6: C, 65.85; H, 4.91. Found: C, 65.77; H, 4.98.
19. Data of compound 5d: Amorphous powder; UV (MeOH): kmax 238, 350 nm; 1H
NMR (CDCl3, 300 MHz): 7.22 (3H, m, H-30,40 & 50), 7.80 (1H, d, J = 9.0 Hz, H-5),
7.38 (2H, m, H-20 & 60), 7.05 (1H, d, J = 9.0 Hz, H-6), 7.04 (1H, s, H-8), 4.17 (2H, s,
CH2); 13C NMR (CDCl3, 75 MHz): 176.0 (C-4), 163.3 (C-7), 162.3 (C-2), 126.5 (C-
40), 157.8 (C-9), 128.3 (C-20&60), (C-5), 130.9 (C-10), 123.7 (C-3), 114.3 (C-6),
117.9 (C-10), 129.8 (C-30&50), 58.9 (CH2); ESIMS: (m/z) 269 [M+H]+; Elemental
Anal. Calcd for C16H12O4: C, 71.64; H, 4.51. Found: C, 71.59; H, 4.58.
20. Data of compound 5e: Amorphous powder; UV (MeOH): kmax 251, 278, 330 nm;
1H NMR (CDCl3, 300 MHz): 8.07 (1H, d, J = 8.8 Hz, H-5), 6.99 (1H, dd, J = 8.8,