H. Xiang et al. / Bioorg. Med. Chem. 18 (2010) 3036–3042
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(t, 3H –CH3), d 2.61 (t 2H –CO–CH2–), d 3.85 (s 3H –OCH3), d 7.15
(dd 1H C6-H), d 7.29 (d 1H C8-H), d 7.98 (s 1H C2-H), d 8.31 (d
1H C5-H); MS (EI) m/e: 534. Anal. Calcd for C34H46O5ꢁ0.5H2O: C,
75.08; H, 8.78. Found: C, 75.10; H, 8.71.
groups: normal control rats fed with a low fat diet (normal), high-
fat diet-fed control rats treated with vehicle (HFD), high-fat diet-
fed rats treated with sibutramine at a dose of 10 mg/kg (sibutr-
amine), high-fat diet-fed rats treated with the selected compound
at a dose of 50 mg/kg (1a, 1b, 1c, 2a, 2b or 2c). The diet was pur-
chased from Qinglongshan Food Service, Nanjing, China. LFD was
composed of protein 20%, carbohydrate 70%, and fat 10%, whereas
HFD diet was composed of protein 20%, carbohydrate 35%, and fat
45% (of total energy, %kcal). They were allowed free access to tap
water and food. Every group of rats, the normal control rats were
exception, were fed with high-fat diet for two months. Then every
group of rats were given vehicle or compounds by intragastric
administration respectively, once daily for one month. After this
feeding period, the rats were sacrificed, and blood samples were
taken form femoral artery. Plasma was analyzed for triglyceride
(TG), total cholesterol (TC) and free fatty acid (FFA) using diagnos-
tic kits following the manufacturer’s protocol. Epididymal fat de-
pots and retroperitoneal fat depots were excised immediately
following blood collection, washed in cold physiological (isotonic)
saline, gently blotted, and then weighed. Also, the body weight was
determined.
4.1.3.4. 3-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl palmitate
(40-methoxy daidzein-7-palmitate, 1d). 73%; white solid; mp
102–104 °C; IR (KBr) 1763 cmꢀ1, 1638 cmꢀ1 1H NMR (CDCl3) d
;
0.92 (t, 3H –CH3), d 2.65 (t 2H –CO–CH2–), d 3.89 (s 3H –OCH3), d
7.19 (dd 1H C6-H), d 7.32 (d 1H C8-H), d 8.02 (s 1H C2-H), d 8.35
(d 1H C5-H); MS (EI) m/e: 506. Anal. Calcd for C32H42O5: C,
75.90; H, 8.67. Found: C, 75.86; H, 8.35.
4.1.3.5. 3-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl 10-undec-
ylenate (40-methoxy daidzein-7-undecylenate, 1e). 66%; white
solid; mp 79–82 °C; IR (KBr) 1744 cmꢀ1, 1646 cmꢀ1 1H NMR
;
(CDCl3) d 2.60 (t 2H –CO–CH2–), d 3.83 (s 3H –OCH3), d 4.95 (dd
2H CH2@), d 5.82 (m 1H –CH@), d 7.14 (dd 1H C6-H), d 7.27 (d
1H C8-H), d 7.96 (s 1H C2-H), d 8.30 (d 1H C5-H); MS (EI) m/e:
434. Anal. Calcd for C27H30O5: C, 74.53; H, 6.93. Found: C, 74.63;
H, 6.96.
4.1.3.6. 4-(7-Methoxy-4-oxo-4H-chromen-3-yl)phenyl (9Z)-ole-
ate (7-methoxy daidzein-40-oleate, 2a). 81%; white solid; mp
4.2.2. Bioactivities study in male C57BL/6 mice
Male C57BL/6J mice (Experimental Animal Center of Nanjing
University, Nanjing, China) were acclimatized for 3 days, then di-
vided into five groups of 10 mice each, including: normal control
group (fed with low fat diet), high-fat diet-fed group (HFD diet:
10% of lard, 2% of cholesterol and 1% of cholate added to LFD),
‘low-dose’ group (HFD mice treated with 1a at a dose of 50 mg/
kg/d), ‘high-dose’ group (HFD mice treated with 1a at a dose of
100 mg/kg/d) and positive control group (HFD mice treated with
Inositol Nicotinate at a dose of 200 mg/kg/d). The mice were indi-
vidually housed and maintained in a 12-h light/dark cycle at
22 2 °C. Food and water were available ad libitum. The body
weight and food intake amount were recorded every week. Before
feeding the test drug, mice were fed either normal diet or HFD ad
libitum for 4 weeks. Then each of the groups were given normal
diet, HFD or HFD plus drugs for 4 weeks. On completion of the
experiment, after 12 h of fasting, blood samples were taken form
vena orbitalis. Plasma was analyzed for triglyceride (TG), total cho-
lesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-
density lipoprotein cholesterol (LDL-C) and insulin using diagnos-
tic kits following the manufacturer’s protocol.
76–78 °C; IR (KBr) 1746 cmꢀ1, 1639 cmꢀ1 1H NMR (CDCl3) d 0.88
;
(t, 3H –CH3), d 2.56 (t 2H –CO–CH2–), d 3.92 (s 3H –OCH3), d 5.36
(m 2H –CH@CH–), d 6.85 (d 1H C8-H), d 6.99 (dd 1H C6-H), d
7.94 (s 1H C2-H), d 8.21 (d 1H C5-H); MS (EI) m/e: 532. Anal. Calcd
for C34H44O5: C, 76.86; H, 8.48. Found: C, 76.66; H, 8.32.
4.1.3.7. 4-(7-Methoxy-4-oxo-4H-chromen-3-yl)phenyl (9Z,12Z)-
linoleate (7-methoxy daidzein-40-linoleate, 2b). 69%; white so-
lid; mp 58–62 °C; IR (KBr) 1746 cmꢀ1, 1639 cmꢀ1; 1H NMR (CDCl3)
d 0.88 (t, 3H –CH3), d 2.56 (t 2H –CO–CH2–), d 3.90 (s 3H –OCH3), d
6.85 (d 1H C8-H), d 6.99 (dd 1H C6-H), d 7.93 (s 1H C2-H), d 8.21 (d
1H C5-H); MS (EI) m/e: 530. Anal. Calcd for C34H42O5: C, 76.64; H,
8.02. Found: C, 76.95; H, 7.98.
4.1.3.8. 4-(7-Methoxy-4-oxo-4H-chromen-3-yl)phenyl stearate
(7-methoxy daidzein-40-stearate, 2c). 85%; white solid; mp 98–
102 °C; IR (KBr) 1746 cmꢀ1, 1639 cmꢀ1 1H NMR (CDCl3) d 0.88
;
(t, 3H –CH3), d 2.57 (t 2H –CO–CH2–), d 3.92 (s 3H –OCH3), d 6.87
(d 1H C8-H), d 7.00 (dd 1H C6-H), d 7.95 (s 1H C2-H), d 8.21 (d
1H C5-H); MS (EI) m/e: 534.
4.2.3. Acute toxicity test
4.1.3.9. 4-(7-Methoxy-4-oxo-4H-chromen-3-yl)phenyl palmitate
(7-methoxy daidzein-40-palmitate, 2d). 91%; white solid; mp
The toxicity of 1a was tested on KM mice both male and female
in groups of 10 animals per dose. Compound 1a, dissolved in olive
oil, was given orally in doses of 1771, 1968, 2187, 2430 and
2700 mg/kg, respectively. After the administration of the com-
pound, mice were observed for clinical signs of toxicity and num-
ber of deaths. After the test, all mice were sacrificed and checked
macroscopically for possible damage to the heart, liver, and kid-
neys. The LD50 value was calculated using Bliss method.
96–100 °C; IR (KBr) 1745 cmꢀ1, 1638 cmꢀ1 1H NMR (CDCl3) d
;
0.88 (t, 3H –CH3), d 2.56 (t 2H –CO–CH2–), d 3.92 (s 3H –OCH3), d
6.86 (d 1H C8-H), d 7.00 (dd 1H C6-H), d 7.94 (s 1H C2-H), d 8.21
(d 1H C5-H); MS (EI) m/e: 506.
4.1.3.10. 4-(7-Methoxy-4-oxo-4H-chromen-3-yl)phenyl 10-
undecenoate (7-methoxy daidzein-40-undecylenate, 2e). 75%;
white solid; mp 88–90 °C; IR (KBr) 1746 cmꢀ1, 1639 cmꢀ1
;
1H
4.3. 3T3-L1 cell culture experiments
NMR (CDCl3) d 2.56 (t 2H –CO–CH2–), d 3.92 (s 3H –OCH3), d
4.96 (dd 2H CH2@), d 5.81 (m 1H –CH@), d 6.86 (d 1H C8-H), d
7.00 (dd 1H C6-H), d 7.94 (s 1H C2-H), d 8.21 (d 1H C5-H); MS
(EI) m/e: 434. Anal. Calcd for C27H30O5: C, 74.63; H, 7.04. Found:
C, 74.63; H, 6.96.
4.3.1. Cell proliferation assay by MTT
3T3-L1 preadipocytes (5000 cells/well) were seeded in 96-well
plates. After two days’ conventional culture, the cells were incu-
bated with Dulbecco’s Modified Eagle’s medium (DMEM, Gibco)
containing the selected compounds (100 lmol/L) for 48 h. The cul-
4.2. Animal experiment
ture solution containing 0.1% (V/V) ethanol (96%) was given to the
control group. Twenty microliter of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) was
4.2.1. Body weight loss and hypolipidemic activities in SD rats
Male SD rats (Purchased from Experimental Animal Center of
Nanjing Medical University, Nanjing, China) were divided into nine
placed in each well for 4 h at 37 °C. Consecutively, 100
lL of di-
methyl sulfoxide (DMSO) was added to extract the MTT formazan,