Synthesis and cytotoxic activity of genistein derivatives

Article abstract of DOI:10.1007/s00044-009-9271-z

A series of genistein derivatives was prepared and tested in vitro against leukocythemia (HL-60), colorectal adenocarcinoma (HT-29), and human gastric adenocarcinoma (SGC-7901) cell lines. Among these derivatives, 4′,5-di-n-octoxy-7-gem-difluoromethylenegenistein, 9f, had the strongest activity against HL-60, HT-29, and SGC-7901 cells.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-009-9271-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:1296–1306
DOI 10.1007/s00044-009-9271-z
ORIGINAL RESEARCH
Synthesis and cytotoxic activity of genistein derivatives
•
Xing Zheng Xu Yao Yunmei Liu Zitong Zheng
•
•
•
•
Jianguo Cao Duanfang Liao
Received: 4 May 2009 / Accepted: 17 September 2009 / Published online: 14 October 2009
¨
Ó Birkhauser Boston 2009
Abstract A series of genistein derivatives was prepared and tested in vitro against
leukocythemia (HL-60), colorectal adenocarcinoma (HT-29), and human gastric
adenocarcinoma (SGC-7901) cell lines. Among these derivatives, 40,5-di-n-octoxy-
7-gem-difluoromethylenegenistein, 9f, had the strongest activity against HL-60,
HT-29, and SGC-7901 cells.
HF CO
O
2
OR
O
OR
9f R = n-C8H17
Keywords Synthesis ꢀ Genistein ꢀ Cytotoxicity ꢀ MTT assay
X. Zheng (&) ꢀ X. Yao ꢀ Y. Liu ꢀ Z. Zheng ꢀ D. Liao
Institute of Pharmacy and Pharmacology, Nanhua University, Hengyang 421001, People’s Republic
of China
e-mail: zhengxing5018@yahoo.com
J. Cao
Medical College, Hunan Normal University, Hunan Changsha 410006, People’s Republic of China

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Introduction
Cancer represents one of the most serious health problems in the world. Major
advances have been made in early detection, prevention, and treatment. However,
anticancer agents generally act on metabolically active or rapidly proliferating cells,
and cannot distinguish between cancer and normal cells (Kumar et al., 2007; Zhang
et al., 2005). Thus, toxicity to normal cells limits the amount of drugs that can be
given to patients (Kumar et al., 2003).Therefore, in recent years, there has been
growing interest in the search for anticancer substances with high efficacy, low
toxicity, and minimum side effects. Since many clinically successful anticancer
drugs either are natural products or have been developed from naturally occurring
lead compounds, one approach to finding new anticancer drugs is to search
chemicals of plant origin. Although isoflavones enjoy a limited distribution in the
plant kingdom, these compounds have received much attention recently due to their
interesting biological activities (Gao et al., 2003; Peterson and Barnes, 1991). Apart
from their important biological property of estrogenic activity, isoflavones possess a
broad range of pharmacological properties including antioxidant or possible
therapeutic applications in the prevention of coronary heart diseases and cancers
(Uckun et al., 1995; MerzDemlow et al., 2000). It is well known that Asian
populations, who have low rates of breast and prostate cancers, consume 20–80 mg
of genistein/day, whereas the dietary intake of genistein in the United States is only
1–3 mg/day. A typical isoflavone is the commonly occurring genistein. Its many
biological activities have made it the subject of more than 3,600 published studies
(listed in Biological Abstracts) in the past 10 years. Most of these studies have
focused on the pharmacological activities of genistein as a tyrosine kinase inhibitor,
its chemoprotectant activities against cancers and cardiovascular diseases, and its
phytoestrogen activities (Dixon and Ferreira, 2002). In the anticancer area, genistein
can reduce the risk of breast (Peterson and Barnes, 1996; Kousidou et al., 2006),
uterus, ovary, stomach, and lung cancers and leukemia and mortality of prostate
cancer in humans because it not only inhibits angiogenesis in vitro and in vivo but
also inhibits tumor cell growth and tumor cell proliferation (Mitchell et al., 2000;
Zhou et al., 1998). However, its anticancer activities are low. Numerous studies in
vitro have shown a close relationship between the chemical structure and the
biological activity of genistein derivatives, whereby their basic structure can be
modified to increase or decrease their biological activity (Kim et al., 2003; Gao
et al., 2001). As part of our current work (Zheng et al., 2003), searching for
anticancer substances, we describe herein the synthesis of genistein derivatives and
their anticancer activities against leukocythemia (HL-60), colorectal adenocarci-
noma (HT-29), and human gastric adenocarcinoma (SGC-7901) cell lines.
Chemistry and biological activity
Melting points were measured on a Kofler apparatus, uncorrected. 1H NMR and 13
C
NMR spectra were recorded on a Bruker AM 300(300-MHz) spectrometer with
Me4Si as internal standard. 19F NMR spectra were obtained on a Bruker AM-300

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spectrometer in CDCl3 with CFCl3 as external standard, downfield shifts being
designated as negative. All chemical shifts (d) are expressed as parts per million,
and coupling constants (J) are given as hertz. Mass spectra were recorded on
a Finnigan-MAT-8430 instrument using EI ionization at 70 eV. IR spectra were
recorded on a Shimadzu IR-440 spectrometer. Elemental analyses (C and H)
were performed on an Italian Carlo-Erba 1106 elemental analyzer. All reagents
were commercial materials used without further purification.
40,5-Dihydroxy-7-methoxygenistein (2)
A mixture of 1 mmol (270 mg) 1, 980 mg K2CO3, 6 mmol MeI, and 10 ml acetone
was stirred at room temperature for 24 h, and then the mixture was filtered to
remove inorganic salts. After distillation of the acetone, 7-methoxygenistein, 2
(4.1%), was obtained by column chromatography on silica gel and elution with 98:8
petroleum ether:ethyl acetate. MS (EI, 70 eV) m/z: 284 (M?, 100.00), 166 (23.73),
1
167 (20.38), 285 (20.07), 138 (17.21), 95 (12.47), 118 (12.18), 255 (11.72). H
NMR (300 MHz, DMSO-d6): 3.830 (3H, s, OCH3), 6.366 (1H, d, J = 2.4 Hz, H-6),
6.604 (1H, d, J = 2.4 Hz, H-8), 6.798 (2H, d, J = 8.7 Hz, H-30 and H-50), 7.361
(2H, d, J = 8.7 Hz, H-20 and H-60), 8.358 (1H, s, H-2), 9.561 (1H, s, OH-40), 12.890
(1H, s, OH-5).
General procedures for preparation of 3a–3d, 4a, and 4b
A mixture of 1 mmol (270 mg) 1, 980 mg K2CO3, 6 mmol RX, and 10 ml acetone
was stirred at reflux for 6–24 h, and then the mixture was filtered to remove
inorganic salts. After distillation of the acetone, dialkylated products 3a–3d and
trialkylated 4a and 4b were isolated by column chromatography on silica gel and
elution with 98:2 or 90:10 petroleum ether:ethyl acetate.
40,7-Dimethoxy-5-hydroxygenistein (3a)
Yield, 83.5%. MS (EI, 70 eV) m/z: 298 (M?, 100.00), 271 (34.39), 132 (21.39), 299
1
(19.86), 166 (19.24), 283 (12.00), 138 (11.48), 89 (10.61). H NMR (300 MHz,
CDCl3): 3.381 (3H, s, OCH3), 3.900 (3H, s, OCH3), 6.407 (1H, d, J = 2.4 Hz, H-6),
6.422 (1H, d, J = 2.4 Hz, H-8), 7.003 (2H, d, J = 9.9 Hz, H-30 and H-50), 7.484
(2H, d, J = 9.9 Hz, H-20 and H-60), 7.893 (1H, s, H-2), 12.890 (1H, s, OH).
40,7-Diallyloxy-5-hydroxygenistein (3b)
Yield, 77.5%; m.p., 117–119.5°C. MS (EI, 70 eV) m/z: 349 (100.00), 308 (37.62),
350 (M?, 23.86), 275 (17.25), 291 (16.94), 41 (14.77), 293 (14.48). IR tmax (cm-1
,
1
KBr): 1649 (C=O), 2954 (OH). H NMR (300 MHz, CDCl3): 4.556–5.087 (4H, m,
2 9 CH2O), 5.283–5.485 (4H, m, 2 9 CH2), 5.928–6.114 (2H, m, 2 9 CH), 6.372
(2H, d, J = 8.7 Hz, H-30 and H-50), 6.982 (1H, d, J = 2.1 Hz, H-6), 7.428 (1H, d,
J = 2.1 Hz, H-8), 7.833 (2H, d, J = 8.7 Hz, H-20 and H-60), 7.862 (1H, s, H-2),
12.981 (1H, s, OH). 13C NMR(CDCl3): 180.824 (C=O, C-4), 164.461 (C-7),

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162.722 (C-5), 158.816 (C-40), 157.877 (C-9), 152.675 (C-2), 133.077 (allyl-40,
CH), 132.047 (allyl-7, CH), 130.056 (2C, C-20 and C-60), 123.633 (C-3), 123.076
(C-10), 118.507 (allyl-40, CH2), 117.797 (allyl-7, CH2), 114.906 (2C, C-30 and C-50),
114.654 (C-10), 98.772 (C-6), 93.134 (C-8), 69.120 (allyl-40, OCH2), 68.868 (allyl-
7, OCH2). Anal. Calcd. for C21H18O5: C, 71.99, H, 5.18. Found: C, 71.86, H, 5.16.
40,7-Dioctyloxy-5-hydroxygenistein (3c)
Yield, 75.6%; m.p., 85.5–87°C. MS (EI, 70 eV) m/z: 494 (M?, 100.00), 270 (73.92),
495 (34.42), 382 (28.34), 242 (18.08), 271 (18.07), 43 (16.34), 269 (15.52). IR tmax
1
(cm-1, KBr): 1666 (C=O), 3128 (OH). H NMR (300 MHz, CDCl3): 0.867–2.171
(30H, m, 2 9 n-C7H15), 3.957-4.028 (4H, m, 2 9 CH2O), 6.358 (1H, d, J =
2.1 Hz, H-6), 6.380 (1H, d, J = 2.1 Hz, H-8), 6.960 (2H, d, J = 8.4 Hz, H-30 and
H-50), 7.437 (2H, d, J = 8.4 Hz, H-20 and H-60), 7.845 (1H, s, H-2), 12.857 (1H, s,
OH). 13C NMR(CDCl3): 180.839 (C=O, C-4), 165.125 (C-7), 162.283 (C-5),
159.373 (C-40), 157.946 (C-9), 152.576 (C-2), 130.041 (2C, C-20 and C-60), 123.671
(C-3), 122.733 (C-10), 114.670 (2C, C-30 and C-50), 106.126 (C-10), 98.566 (C-6),
92.844 (C-8), 68.693 (C-1000), 68.113 (C-100), 31.809 (C-6000), 31.786 (C-600), 29.345-
28.933 (6C, C-200 and C-2000, C-400 and C-4000, C-500 and C-5000), 26.026 (C-3000),
25.927 (C-300), 22.647 (2C, C-700 and C-7000), 14.088 (2C, C-800 and C-8000). Anal.
Calcd. for C31H42O5: C, 75.27, H, 8.56. Found: C, 75.12, H, 8.47.
40,7-Didecyloxy-5-hydroxygenistein (3d)
Yield, 73.3%; m.p., 89–90°C. MS (EI, 70 eV) m/z: 550 (M?, 100.00), 270 (60.92),
551 (38.64), 410 (26.75), 271 (17.81), 43 (16.01), 242 (15.18), 269 (14.23). IR tmax
1
(cm-1, KBr): 1665 (C=O), 3123 (OH). H NMR (300 MHz, CDCl3): 0.863–1.827
(38H, m, 2 9 n-C9H19), 3.982–4.031 (4H, m, 2 9 CH2O), 6.364 (1H, d,
J = 2.4 Hz, H-6), 6.381 (1H, d, J = 2.4 Hz, H-8), 6.963 (2H, d, J = 8.4 Hz, H-30
and H-50), 7.439 (2H, d, J = 8.4 Hz, H-20 and H-60), 7.847 (1H, s, H-2), 12.859 (1H,
s, OH). 13C NMR(CDCl3): 180.832 (C=O, C-4), 165.125 (C-7), 162.283 (C-5),
159.373 (C-40), 157.938 (C-9), 152.560 (C-2), 130.041 (2C, C-20 and C-60), 123.664
(C-3), 122.733 (C-10), 114.662 (2C, C-30 and C-50), 106.126 (C-10), 98.566 (C-6),
92.837 (C-8), 68.693 (C-1000), 68.113 (C-100), 31.885 (2C, C-800 and C-8000), 29.558–
28.933 (10C, C-200 and C-2000, C-400 and C-4000,C-500 and C-5000, C-600 and C-6000, C-700
and C-7000), 26.026 (C-3000), 25.920 (C-300), 22.670 (2C, C-900 and C-9000), 14.111 (2C,
C-1000 and C-10000). Anal. Calcd. for C35H50O5: C, 76.33, H, 9.15. Found: C, 76.12,
H, 9.15.
40,5,7-Trimethoxygenistein (4a)
Yield, 10.1%. MS (EI, 70 eV) m/z: 312 (100.00), 313 (75.82), 266 (29.62), 267
1
(25.53), 132 (22.94), 283 (20.88), 281 (19.92), 282 (17.92). H NMR (300 MHz,
DMSO-d6): 3.759 (3H, s, OCH3), 3.806 (3H, s, OCH3), 3.857 (3H, s, OCH3), 6.484
(1H, d, J = 2.4 Hz, H-6), 6.635 (1H, d, J = 2.4 Hz, H-8), 6.940 (2H, d, J = 8.7 Hz,
H-30 and H-50), 7.416 (2H, d, J = 8.7 Hz, H-20 and H-60), 8.167 (1H, s, H-2).

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40,5,7-Triallyloxygenistein (4b)
Yield, 11.6%; m.p., 101–104°C. MS (EI, 70 eV) m/z: 390 (M?, 100.00), 361
(95.87), 293 (88.82), 41 (45.92), 321 (44.10), 349 (37.56), 252 (37.22), 362 (34.97).
IR tmax (cm-1, KBr): 1644 (C=O). 1H NMR (300 MHz, CDCl3): 4.546-4.637(6H,
m, 3 9 CH2O), 5.262–5.688(6H, m, 3 9 CH2), 6.002–6.110 (3H, m, 3 9 CH),
6.383(2H, d, J = 2.1 Hz, H-6), 6.425(1H, d, J = 2.1 Hz, H-8), 6.939(2H, d,
J = 8.7 Hz, H-30 and H-50), 7.440(2H, d, J = 8.7 Hz, H-20 and H-60), 7.719 (1H, s,
H-2). 13C NMR(CDCl3): 175.179 (C=O, C-4), 162.546 (C-7), 160.303 (C-5),
159.762 (C-40), 158.419 (C-9), 149.970 (C-2), 133.237 (allyl-5, CH), 132.108 (2C,
allyl-7, allyl-40, CH), 130.361 (2C, C-20 and C-60), 126.028 (C-3), 124.548 (C-10),
118.545 (allyl-5, CH2), 118.003 (allyl-40, CH2), 117.614 (allyl-7, CH2), 114.555
(2C, C-30 and C-50), 110.268 (C-10), 97.803 (C-6), 93.592 (C-8), 69.837 (allyl-5,
OCH2), 69.158 (allyl-40, OCH2), 68.799 (allyl-7, OCH2). Anal. Calcd. for
C24H22O5: C, 73.83, H, 5.68. Found: C, 73.87, H, 5.72.
40,7-Diacetoxy-5-hydroxygenistein (5) and 7-acetoxy-40,5-dihydroxy-genistein
(6)
A mixture of 1 mmol (270 mg) 1, 10 ml acetic anhydride and a drop of pyridine
was heated at reflux temperature for 7 h and poured into ice-cold water. The yellow
solid precipitate was filtered, and 5 and 6 were isolated by column chromatography
on silica gel and elution with CH2Cl2 or 98:2 CH2Cl2:MeOH.
40,7-Diacetoxy-5-hydroxygenistein (5)
Yield, 87.2%; m.p., 200–202°C. MS (EI, 70 eV) m/z: 270 (100.00), 312 (38.98), 43
(18.93), 269 (17.18), 354 (16.99), 271 (16.51), 153 (9.27), 246 (7.99). IR tmax
1
(cm-1, KBr): 1656 (C=O), 3076 (OH). H NMR (300 MHz, CDCl3): 2.338 (3H, s,
CH3COO), 2.346 (3H, s, CH3COO), 6.610 (1H, d, J = 1.8 Hz, H-6), 6.783 (1H, d,
J = 1.8 Hz, H-8), 7.199 (2H, d, J = 8.7 Hz, H-30 and H-50), 7.563 (2H, d,
J = 8.7 Hz, H-20 and H-60), 7.982 (1H, s, H-2), 12.748 (1H, s, OH).
7-Acetoxy-40,5-dihydroxygenistein (6)
Yield, 5.3%; m.p., 265°C. MS (EI, 70 eV) m/z: 284 (100.00), 166 (22.33), 167
(16.82), 138 (14.94), 255 (11.75), 312 (M?, 10.48), 118 (9.26), 95 (9.23). IR tmax
(cm-1, KBr): 1670 (C=O), 3013 (OH), 3384 (OH). 1H NMR (300 MHz, DMSO-d6):
2.863 (3H, s, CH3COO), 6.404 (1H, d, J = 2.1 Hz, H-6), 6.649 (1H, d, J = 2.1 Hz,
H-8), 6.827 (2H, d, J = 8.4 Hz, H-30 and H-50), 6.391 (2H, d, J = 8.4 Hz, H-20 and
H-60), 7.400 (1H, s, H-2), 8.597 (1H, s, OH-40), 11.951 (1H, s, OH-5). Anal. Calcd.
for C17H12O6: C, 65.39, H, 3.87. Found: C, 65.46, H, 3.93.

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1301
7-gem-Difluoromethylenated genistein (7) and 40,7-di-gem-difluoromethylenated
genistein (8)
A mixture of 51 mmol genistein, 20 g NaOH, 12.5 ml H2O, and 15 ml dioxane was
prepared in a four-necked flask equipped with thermometer, stirrer, gas-inlet tube
extending below the liquid surface, and reflux condenser. The temperature was
adjusted to 70–75°C and HCF2Cl was introduced for 8 h. The mixture was allowed
to cool to room temperature, then diluted with 50 ml H2O, 10 ml ether was added,
and the mixture was filtered to remove precipitated inorganic salts. The water layer
was extracted with three 10-ml portions of ether and dried over anhydrous Na2SO4.
After distillation of the ether, purification of the residue by column chromatography
on silica gel and elution with 95:5 petroleum ether:ethyl acetate gave compound 7 in
a 42.0% yield and 8 in a 15.8% yield.
7-gem-Difluoromethylenated genistein (7)
M.p., 194–195°C. MS (EI, 70 eV) m/z: 320 (M?, 100.00), 319 (27.42), 203 (22.57),
118 (18.14), 321 (17.72), 202 (7.10), 89 (5.25), 51 (4.90). IR tmax (cm-1, KBr):
1
1662 (C=O), 3319 (OH), 3528 (OH). H NMR (300 MHz, CDCl3): 6.599 (1H, d,
J = 2.4 Hz, H-6), 6.820 (2H, d, J = 12.0 Hz, H-30 and H-50), 6.867 (1H, d,
J = 2.4 Hz, H-8), 7.371 (2H, d, J = 12.0 Hz, H-20 and H-60), 7.427 (1H, t,
J = 72.9 Hz, HF2CO), 8.244 (1H, s, H-2), 9.550 (1H, s, OH-40), 12.994 (1H, s, OH-
5). 19F NMR (300 MHz): -84.372 (d, J = 72.9 Hz). Anal. Calcd. for C16H10F2O5:
C, 60.08, H, 3.15. Found: C, 60.07, H, 2.94.
40,7-Di-gem-difluoromethylenated genistein (8)
m.p., 103–106°C. MS (EI, 70 eV) m/z: 370 (M?, 100.00), 253 (50.40), 369 (26.68),
202 (22.67), 371 (18.52), 118 (11.40), 155 (10.85), 174 (10.81). IR tmax (cm-1
,
KBr): 1653 (C=O), 3074 (OH). 1H NMR (300 MHz, CDCl3): 6.561 (1H, t,
J = 73.8 Hz, HF2CO), 6.590 (1H, d, J = 2.4 Hz, H-6), 6.638 (1H, t, J = 72.3 Hz,
HF2CO), 6.684 (1H, d, J = 2.4 Hz, H-8), 7.229 (2H, d, J = 9.0 Hz, H-30 and H-50),
7.551 (2H, d, J = 9.0 Hz, H-20 and H-60), 7.975 (1H, s, H-2), 12.774 (1H, s, OH).
19F NMR (300 MHz): -80.953 (d, J = 73.8 Hz), -2.414 (d, J = 72.3 Hz). Anal.
Calcd. for C17H10F2O5: C, 55.31, H, 2.74. Found: C, 55.40, H, 2.53.
General procedures for preparation of 9a–9g
A mixture of 1 mmol (320 mg) 7, 980 mg K2CO3, 6 mmol RX, and 10 ml acetone
was stirred at reflux for 6–2 4 h, then the mixture was filtered to remove inorganic
salts. After distillation of the acetone, purification of the residue by column
chromatography on silica gel and elution with 95:5 or 90:10 petroleum ether:ethyl
acetate gave compounds 9a–9g in a 81–95% yield, respectively.

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40,5-Dimethoxy-7-gem-difluoromethylenated genistein (9a)
Yield, 92.1%; m.p., 136–138°C. MS (EI, 70 eV) m/z: 348 (M?, 100.00), 347
(41.07), 302 (25.34), 349 (19.45), 132 (18.11), 319 (15.70), 317 (13.69), 331
1
(11.19). IR tmax (cm-1, KBr): 1637 (C=O). H NMR (300 MHz, CDCl3): 3.834
(3H, s, OCH3), 3.970 (3H, s, OCH3), 6.546 (1H, d, J = 2.1 Hz, H-6), 6.642 (1H, t,
J = 72.3 Hz, HF2CO), 6.724 (1H, d, J = 2.1 Hz, H-8), 6.940 (2H, d, J = 8.7 Hz,
H-30 and H-50), 7.472 (2H, d, J = 8.7 Hz, H-20 and H-60), 7.811 (1H, s, H-2). 19F
NMR (300 MHz): -82.150 (d, J = 72.3 Hz). Anal. Calcd. for C18H14F2O5: C,
62.07, H, 4.05. Found: C, 62.26, H, 4.15.
40,5-Diethoxy-7-gem-difluoromethylenated genistein (9b)
Yield, 89.7%; m.p., 96–97°C. MS (EI, 70 eV) m/z: 376 (M?, 100.00), 361 (97.06),
303 (42.10), 358 (32.81), 304 (32.33), 118 (27.28), 375 (26.81), 377 (21.98). IR
tmax (cm-1, KBr): 1613 (C=O). 1H NMR (300 MHz, CDCl3): 1.386–1.572 (6H, m,
2 9 CH3), 4.023–4.181 (4H, m, 2 9 OCH2), 6.525 (1H, d, J = 2.4 Hz, H-6), 6.628
(1H, t, J = 72.6 Hz, HF2CO), 6.699 (1H, d, J = 2.4 Hz, H-8), 6.931 (2H, d,
J = 8.4 Hz, H-30 and H-50), 7.436 (2H, d, J = 8.4 Hz, H-20 and H-60), 7.780 (1H, s,
H-2). 19F NMR (300 MHz): -82.085 (d, J = 72.6 Hz). Anal. Calcd. for
C20H18F2O5: C, 63.82, H, 4.82. Found: C, 63.69, H, 4.77.
40,5-Di-n-propoxy-7-gem-difluoromethylenated genistein (9c)
Yield, 86.3%. MS (EI, 70 eV) m/z: 375 (100.00), 43 (39.12), 404 (M?, 26.74), 41
(26.73), 118(25.13), 304(23.05), 376(22.01), 303(19.05). IRtmax(cm-1, KBr):1651
(C=O). 1H NMR (300 MHz, CDCl3): 0.972–1.120 (4H, m, 2 9 CH2), 1.157 (3H, t,
J = 7.5 Hz, CH3), 1.223 (3H, t, J = 7.5 Hz, CH3), 3.987–4.164 (4H, m, 2 9 CH2O),
6.643 (1H, d, J = 2.4 Hz, H-6), 6.761 (1H, t, J = 72.6 Hz, HF2CO), 6.807 (1H, d,
J = 2.4 Hz, H-8), 7.053 (2H, d, J = 9.0 Hz, H-30 and H-50), 7.547 (2H, d, J = 9.0 Hz,
H-20 and H-60), 7.886 (1H, s, H-2). 19F NMR (300 MHz): –82.142 (d, J = 72.6 Hz).
Anal. Calcd. for C22H22F2O5: C, 65.34, H, 5.48. Found: C, 65.56, H, 5.66.
40,5-Dibenzyloxy-7-gem-difluoromethylenated genistein (9d)
Yield, 94.7%. MS (EI, 70 eV) m/z: 91 (100.00), 500 (M?, 9.06), 92 (8.20), 65
(8.09), 57 (6.32), 43 (4.67), 89 (4.12), 71 (3.91). IR tmax (cm-1, KBr): 1647 (C=O).
1H NMR (300 MHz, CDCl3): 4.636 (4H, s, 2 9 CH2O), 7.314–7.342 (18H, m,
2 9 C6H5, HF2CO, H-6, H-8, H-30, H-50, H-20 H-60, H-2). 19F NMR (300 MHz): –
82.161 (d, J = 72.2 Hz). Anal. Calcd. for C30H22F2O5: C, 71.99, H, 4.34. Found: C,
72.03, H, 4.52.
40,5-Di-n-heptoxy-7-gem-difluoromethylenated genistein (9e)
Yield, 80.9%. MS (EI, 70 eV) m/z: 431 (100.00), 516 (M?, 60.10), 517 (56.18), 41
(33.58), 57 (32.98), 43 (29.15), 432 (29.02), 320 (23.46). IR tmax (cm-1, KBr):

Med Chem Res (2010) 19:1296–1306
1
1303
1652 (C=O). H NMR (300 MHz, CDCl3): 0.906–2.016 (26H, m, 2 9 n-C6H13),
3.950 (2H, t, J = 6.6 Hz, CH2O), 4.047 (2H, t, J = 6.6 Hz, CH2O), 6.512 (1H, d,
J = 2.1 Hz, H-6), 6.648 (1H, d, J = 2.1 Hz, H-8), 6.764 (1H, t, J = 72.9 Hz,
HF2CO), 6.893 (2H, d, J = 9.0 Hz, H-30 and H-50), 7.412 (2H, d, J = 9.0 Hz, H-20
and H-60), 7.759 (1H, s, H-2). 19F NMR (300 MHz): -82.897 (d, J = 72.9 Hz).
Anal. Calcd. for C30H38F2O5: C, 69.75, H, 7.41. Found: C, 69.68, H, 7.36.
40,5-Di-n-octoxy-7-gem-difluoromethylenated genistein (9f)
Yield, 82.0%. MS (EI, 70 eV) m/z: 445 (100.00), 446 (28.67), 320 (19.66), 544
(M?, 15.24), 459 (12.97), 321 (12.45), 43 (11.29), 333 (10.55). IR tmax (cm-1
,
1
KBr): 1651 (C=O). H NMR (300 MHz, CDCl3): 0.860–2.181 (30H, m, 2 9 n-
C7H15), 3.985–4.064 (4H, m, 2 9 CH2O), 6.378 (1H, d, J = 2.1 Hz, H-6), 6.423
(1H, d, J = 2.1 Hz, H-8), 6.531 (1H, t, J = 74.1 Hz, HF2CO), 7.155 (2H, d,
J = 8.7 Hz, H-30 and H-50), 7.543 (2H, d, J = 8.7 Hz, H-20 and H-60), 7.748 (1H, s,
H-2). 19F NMR (300 MHz): -80.555 (d, J = 74.1 Hz). Anal. Calcd. for
C32H42F2O5: C, 70.56, H, 7.77. Found: C, 70.67, H, 7.85.
40,5-Di-n-decoxy-7-gem-difluoromethylenated genistein (9g)
Yield, 81.2%; m.p., 60–62°C. MS (EI, 70 eV) m/z: 473 (100.00), 43 (73.36), 57
(44.90), 320 (37.26), 55 (34.87), 41 (33.91), 474 (31.13), 600 (M?, 24.62). IR tmax
1
(cm-1, KBr): 1658 (C=O). H NMR (300 MHz, CDCl3): 0.848–1.783 (38H, m,
2 9 n-C9H19), 3.966 (2H, t, J = 6.6 Hz, CH2O), 4.040 (2H, t, J = 6.6 Hz, CH2O),
6.513 (1H, d, J = 2.1 Hz, H-6), 6.627 (1H, t, J = 72.3 Hz, HF2CO), 6.674 (1H, d,
J = 2.1 Hz, H-8), 6.925 (2H, d, J = 8.4 Hz, H-30 and H-50), 7.423 (2H, d, J = 8.4 Hz,
H-20 and H-60), 7.753 (1H, s, H-2). 19F NMR (300 MHz): -82.052 (d, J = 72.3 Hz).
Anal. Calcd. for C36H50F2O5: C, 71.97, H, 8.39. Found: C, 72.13, H, 8.57.
Biological activity
HL-60, HT-29, and SGC-7901 cell lines were cultured and passaged in RPMI-1640
medium, supplemented with 10% (v/v) inactive calf bovine serum, 100 U/ml
penicillin, and 100 U/ml streptomycin in a humidified incubator at 37°C under an
atmosphere of 5% CO2. Stock solutions of tested compounds were prepared in
DMSO and were used for serial dilutions in culture medium. The cytotoxicity assay
was performed by a modification of the MTT method (Mosmann, 1983).
Results and discussion
The readily available genistein (1) was used as the starting material for preparation
of derivatives of genistein. Upon treatment of genistein (1) with CH3I in the
presence of K2CO3 in acetone at room temperature, 7-methoxygenistein (2) was
obtained as the single product in a very low yield, together with the recovery of
genistein. The reason might be that the acidity of the 7- and 40-hydroxy groups of

1304
Med Chem Res (2010) 19:1296–1306
genistein are different enough to allow selective ionization of the 7-OH group at
relatively low temperatures. However, when the reaction mixture was stirred at
reflux, dialkylated products (3a–3d) were isolated, as well as trialkylated products
(4a and 4b) in the case of CH3I and allyl bromide. The ratios of 3a/4a and 3b/4b
were about 8/1 and 9/1, respectively. Tri-n-octyloxygenistein and tri-n-decyloxy-
genistein were not obtained, probably due to the steric hindrance of octyl and decyl
groups. A mixture of 1, acetic anhydride (used as both reactant and solvent), and a
drop of pyridine was heated at reflux temperature for 7 h to provide 5 and 6
(Scheme 1).
The insertion reaction of difluoromethylene carbene was carried out for the
introduction of a gem-difluoromethylene group to genistein. Treatment of 1 with
HCF2Cl gas in the presence of NaOH in dioxane/H2O (Miller and Thanassi, 1960)
gave 7 and 8. The reaction of 7 with RX in the presence of K2CO3 at reflux
temperature gave 9a–9g (Scheme 2).
Assays were performed in 96-well plates essentially as described by Mosmann
(1983). Pharmacological results are summarized in Table 1. It appears that these
closely related molecules display remarkable differences in cytotoxicity. As
reported in Table 1, we discovered that compounds 2, 3b, 3c, 3d, 7, and 9a–9g
show stronger cytotoxicity toward HL-60 cells, compounds 9c, 9d, and 9f have
stronger cytotoxicity against HT-29 cells, and compounds 2, 3c, 3d, 7, 8, and 9b–9g
possess stronger cytotoxicity toward SGC-7901 cells than genistein 1. 5,40-Di-n-
octoxyl-7-gem-difluoromethylene-genistein (9f) has the strongest activity against
HL-60, HT-29, and SGC-7901 tumor cells.
Although the general structure–activity relationship of these genistein derivatives
was not elucidated from these data, the following points are noteworthy. (1)
Compounds 2, 6, and 7 have stronger activities than 3a, 5, and 8 against HL-60 and
SGC-7901 tumor cells, respectively. These results show that a 40-hydroxy moiety in
isoflavonoids is necessary. (2) Comparing 9a–9g with 1, the results show that, apart
from 9a, the 5,40-dialkylated compounds with a 7-gem-difluoromethyleneoxy
moiety are more active against HL-60 and SGC-7901 tumor cells than 1. Moreover,
MeO
O
O
CH3I / K2CO3
HO
O
O
rt
OH
OH
OH
OH
Ac2O
2
1
reflux
RX / K2CO3
AcO
O
AcO
O
O
+
RO
O
RO
O
+
OH
O
OH
OH
OAc
6
5
OR
O
OH
O
OR
OR
4
3
3a R = CH3, 3b R= allyl;
3cR = n-C8H17, 3d R = n-C10H21
4a R = CH3,4b R= ally
Scheme 1 General synthesis of 2, 3a–3d, 4a, 4b, and 5

Med Chem Res (2010) 19:1296–1306
1305
HF2CO
O
O
HO
O
O
HF2CO
O
O
+
NaOH / HCF2Cl
OH
OH
OH
OCF2H
OH
OH
7
8
1
reflux
RX / K2CO3
HF2CO
O
9a R = CH3, 9b R= CH2CH3;
9c R = n-C3H7, 9d R = C6H5CH2;
9e R = n- C7H15, 9f R = n-C8H17
9g R = n-C10H21
;
OR
O
OR
9
Scheme 2 General synthesis of 7, 8, and 9a–9g
Table 1 Human cancer cell line growth inhibition values (lM) for the synthesized compounds
Compound
Cancer cell linea
HL-60
HT-29
SGC-7901
1b
2
4.82
4.35
5.14
3.44
1.62
3.09
5.86
4.95
3.53
5.50
4.61
1.75
0.54
1.29
1.41
0.47
1.63
4.11
5.11
5.78
3.94
7.78
6.51
4.82
4.27
11.24
8.11
4.59
5.54
6.41
5.51
2.15
4.24
3.39
1.64
3.30
3a
3b
3c
3d
5
4.18
14.20
10.80
11.60
19.40
4.21
6
7
4.56
8
5.35
9a
9b
9c
9d
9e
9f
9g
a
9.83
11.20
2.50
2.82
5.25
1.86
14.26
Cancer type: SGC-7901(gastric carcinoma), HT-29 (colon), and HL-60 (promyelocytic leukemia)
b
Genistein (1; 99 ? %) from Arcos Organics (USA) as positive control
apart from 9c and 9g, the activity against SGC-7901 was enhanced with a longer
alkyl chain. In addition, the different alkylated substituents had little effect against
HL-60 cells.

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Med Chem Res (2010) 19:1296–1306
Acknowledgments This research was supported by the Key Project of the Chinese Ministry of
Education (No. 20809), Scientific Research Fund of Hunan Provincial Education Department (No.
07B065), Science and Technology Bureau of Hunan Province (No. 2007FJ4155), and Hunan Provincial
Natural Science Foundation (No. 07JJ6157).
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