C.-H. Tseng et al. / European Journal of Medicinal Chemistry 44 (2009) 3621–3626
3625
65.37, 66.97 (2C), 70.22, 94.38, 100.07, 114.55 (2C), 114.58, 118.38,
5.2.2. Cell culture and drug treatment
124.62, 124.78, 127.77, 130.15 (2C), 152.10, 157.94, 158.65, 163.97,
175.85. Anal. calcd for C23H25NO6: C 66.40, H 6.19, N 3.37; found: C
66.09, H 6.17, N 3.34.
D1 cell, which is a mesenchymal stem cell line cloned from
bone marrow cells of BALB/c mice, was purchased from American
Type Culture Collection (Rockville, MD). D1 cells can be induced
into osteoblasts, adipocytes and chondrocytes. D1 cells were
maintained in DMEM (Gibco BRL, Gaithersburg, MD) supple-
mented with 10% FBS, 100 U/mL of penicillin and streptomycin.
MC3T3E1 cell, which is a preosteoblast cell line derived from
calvaria of C57BL/6 mice, was obtained from the American Type
Culture Collection (Rockville, MD). MC3T3E1 cells were main-
5.1.5. 3-(4-(3-(Cyclopropylamino)-2-hydroxypropoxy)phenyl)-7-
methoxy-4H-chromen-4-one (5c)
The title compound was obtained by the treatment of 3 with
cyclopropylamine as described for 5a. Yield: 78%. Mp: 105–106 ꢁC.
1H NMR (400 MHz, DMSO-d6): 0.22 (m, 2H, cyclopropyl-H), 0.36
(m, 2H, cyclopropyl-H), 2.11 (m, 1H, cyclopropyl-H), 2.64–2.77 (m,
2H, 30-H), 3.88-4.05 (m, 6H, OMe, 10- and 20-H), 4.97 (1H, NH), 6.99
(m, 2H, Ar–H), 7.08 (dd, 1H, J ¼ 8.8, 2.0 Hz, 6-H), 7.17 (d, 1H,
J ¼ 2.0 Hz, 8-H), 7.51 (m, 2H, Ar–H), 8.03 (d,1H, J ¼ 8.8 Hz, 5-H), 8.43
(s, 1H, 2-H). 13C NMR (100 MHz, DMSO-d6): 6.17 (2C), 30.29, 52.21,
56.13, 67.95, 70.83, 100.07, 114.20 (2C), 114.80, 117.59, 123.38,
124.00, 126.95, 130.05 (2C), 153.48, 157.46, 158.52, 163.71, 174.65.
Anal. calcd for C22H23NO5$0.1H2O: C 68.94, H 6.11, N 3.66; found: C
68.69, H 6.06, N 3.46.
tained in
aMEM (Gibco BRL, Gaithersburg, MD) supplemented
with 10% FBS, 100 U/mL of penicillin and streptomycin. They
exhibit osteogenic properties in Dulbecco modified Eagle medium
(Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum
and 50 mg/mL sodium ascorbate in a humidified atmosphere of 5%
CO2 at 37 ꢁC, and the medium was changed every 2 days. New
synthetic compounds were dissolved in DMSO to a final concen-
tration of 10 mM and stored at ꢂ20 ꢁC. The stock solution was
freshly diluted with medium to a final concentration of 10 mM
containing 0.1% of DMSO. Control cultures were treated with the
same amount of DMSO as used in the corresponding experiments.
5.1.6. 7-(3-(Cyclohexylamino)-2-hydroxypropoxy)-3-(3,4-
dimethoxyphenyl)-4H-chromen-4-one (6a)
The title compound was obtained by the treatment of 4 with
cyclohexylamine as described for 5a. Yield: 54%. Mp: 70–71 ꢁC. 1H
NMR (400 MHz, CDCl3): 1.03–1.32 (m, 5H, cyclohexyl-H), 1.62–1.95
(m, 5H, cyclohexyl-H), 2.45 (m, 1H, cyclohexyl-H), 2.76 (dd, 1H,
J ¼ 12.4, 8.0 Hz, 30-H), 2.96 (d, 1H, J ¼ 12.4, 4.0 Hz, 300-H), 3.92 and
3.93 (two s, 6H, 30- and 40-OMe), 3.99–4.09 (m, 3H, 100- and 200-H),
6.89 (d, 1H, J ¼ 2.4 Hz, 8-H), 6.92 (d, 1H, J ¼ 8.4 Hz, 50-H), 7.01–7.06
(m, 3H, 6- and 60-H), 7.21 (d, 1H, J ¼ 2.4 Hz, 20-H), 7.95 (s, 1H, 2-H),
8.21 (d, 1H, J ¼ 8.8 Hz, 5-H). 13C NMR (100 MHz, CDCl3): 24.96 (2C),
25.98, 33.61, 33.91, 48.52, 55.90, 55.92, 56.75, 68.04, 71.04, 100.82,
111.09, 112.42, 114.82, 118.53, 120.99, 124.55, 124.90, 127.75, 148.71,
149.04, 152.26, 157.77, 163.05, 175.86. Anal. calcd for
C26H31NO6$0.2H2O: C 68.32, H 6.92, N 3.06; found: C 68.51, H 6.99,
N 3.01.
5.2.3. Cell viability by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay
The MTT assay is a colorimetric assay based on the ability of the
viable cells to reduce a soluble yellow tetrazolium salt to blue for-
mazan crystals [33]. After compound treatments, 350
mL of MTT
solution (0.5 g/mL in PBS) was added to each well and incubated
m
for 4 h. DMSO was then added for another 0.5 h to thoroughly
dissolve the dark blue crystals. The absorbance at 570 nm was
measured with an ELISA reader. Inhibition of mitochondrial
metabolism was shown as relative activity (% of control).
5.2.4. Cytotoxicity assay by lactate dehydrogenase (LDH) leakage
Lactate dehydrogenase (LDH) leakage from cells was measured
to quantify the cytotoxicity by using a cytotoxicity detection kit
(Roche, Germany) [34]. Cells were previously seeded into 48-well
plates (4 ꢃ104 cells/well). After drug treatment, the supernatants
and cell layers of the cultures were collected for assay. According
to the manufacturer’s guidelines for the detection kit, cell layers
were lysed with 1% Triton X-100, and cell lysates and supernatants
5.1.7. 3-(3,4-Dimethoxyphenyl)-7-(2-hydroxy-3-
morpholinopropoxy)-4H-chromen-4-one (6b)
The title compound was obtained by the treatment of 4 with
morpholine as described for 5a. Yield: 51%. Mp: 135–136 ꢁC. 1H
NMR (400 MHz, CDCl3): 2.47–2.73 (m, 6H, morpholinyl-H and 300-
H), 3.71–3.80 (m, 4H, morpholinyl-H), 3.92 and 3.93 (two s, 6H, 30-
and 40-OMe), 4.11 (m, 2H, 100-H), 4.17 (m, 1H, 200-H), 6.90 (d, 1H,
J ¼ 2.4 Hz, 8-H), 6.92 (d, 1H, J ¼ 8.4 Hz, 50-H), 7.02–7.06 (m, 2H, 6-
and 60-H), 7.20 (d, 1H, J ¼ 2.0 Hz, 20-H), 7.95 (s, 1H, 2-H), 8.21 (d, 1H,
J ¼ 8.8 Hz, 5-H). 13C NMR (100 MHz, CDCl3): 30.85, 53.69, 55.91,
55.93, 60.78, 65.08, 66.92 (2C), 70.67, 100.89, 111.12, 112.45, 114.77,
118.61, 121.00, 124.54, 124.93, 127.80, 148.73, 149.07, 152.26, 157.75,
162.99, 175.83. Anal. calcd for C24H27NO7: C 65.29, H 6.16, N 3.17;
found: C 65.20, H 6.19, N 3.14.
were assayed in a 96-well plate, respectively. Briefly, 100 mL of
catalyst solution was added in each assay well for 20 min.
Absorbance was measured with an ELISA reader with a 490 nm
filter. LDH leakage was shown as relative activity (% of total cell
toxicity).
5.2.5. Osteogenic differentiation and quantification of
mineralization
Osteogenic differentiation was induced by culturing cells in
osteo-induction medium (OIM, 10% FBS, 0.1
mM dexamethasone,
10 mM -glycerophosphate, and -Ascorbic 2 phosphate 100 m
b
L
M in
low glucose DMEM) for 7–14 days. The extracellular matrix calci-
fication was estimated by using Alizarin red S stain [35]. The Aliz-
arin red S-stained mineral was quantified by the osteogenesis
quantification kit (CHEMICONÒ).
5.2. Pharmacological evaluation
5.2.1. Tartrate resistant acid phosphatase (TRAP) solution assay [28]
103 Raw 264.7 cells were cultured in 96-well plates with 100 ng/
mL RANKL (R&D Systems, Minneapolis, MN). Cultures were incu-
bated at 37 ꢁC in 5% CO2 for 5 days with addition of media con-
taining fresh RANKL on day 3. In the TRAP solution assay, enzyme
Acknowledgment
activity was examined by the conversion of
(4 mmol/L; Sigma Chemical Co.) to -naphthol in the presence of
2 mol/L -tartrate solution (Sigma Chemical Co.) in each well.
Absorbance was measured at 405 nm using a microplate reader
(model 550; Bio-Rad Labs.).
a-naphthyl phosphate
a
Financial support of this work by the Ministry of Economics (94-
96-EC-17-A-17-S1-041, Taiwan, ROC) is gratefully acknowledged.
We also thank the National Center for High-Performance Computing
for providing computer resources and chemical database services.
L