Synthesis and antitumoractivity of liquiritigenin thiosemicarbazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.04.036

In an attempt to develop potent and selective antitumor agents,a series of liquiritigenin thio-semicarbazone derivatives were designed and synthesized. The cytotoxicities of these compounds were evaluated in vitro against K562,DU-145,SGC-7901,HCT-116 and Hela cell lines. The pharmacological results showed that most of the prepared compounds displayed excellent selective cytotoxicity toward K562 and DU-145 cells. From the structureeactivity relationships we may conclude that the introduction of a thiosemicarbazone functional group at the 4-position in the skeleton of liquiritigenin is associated with an increase in cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2010.04.036

European Journal of Medicinal Chemistry 45 (2010) 3453e3458
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activity of liquiritigenin thiosemicarbazone derivatives
*
Kun Hu, Ze-hua Yang, Sha-Sha Pan, Hua-jin Xu, Jie Ren
Faculty of Chemistry & Chemical Engineering, Jiangsu Polytechnic University, 1 Gehu Road, Changzhou, Jiangsu 213164, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to develop potent and selective antitumor agents, a series of liquiritigenin thio-
semicarbazone derivatives were designed and synthesized. The cytotoxicities of these compounds were
evaluated in vitro against K562, DU-145, SGC-7901, HCT-116 and Hela cell lines. The pharmacological
results showed that most of the prepared compounds displayed excellent selective cytotoxicity toward
K562 and DU-145 cells. From the structureeactivity relationships we may conclude that the introduction
of a thiosemicarbazone functional group at the 4-position in the skeleton of liquiritigenin is associated
with an increase in cytotoxicity.
Received 20 February 2010
Received in revised form
29 April 2010
Accepted 30 April 2010
Available online 6 May 2010
Keywords:
Ó 2010 Elsevier Masson SAS. All rights reserved.
Liquiritigenin
Thiosemicarbazone
Synthesis
Antitumor activity
1. Introduction
antitumor activity of these compounds seems to be due to an
inhibition of DNA synthesis produced by the modification in the
Many clinically successful anticancer drugs are either natural
products or have been developed from naturally occurring lead
compounds, such as taxol, topotecan, irinotecan and vinblastine.
Flavonoids are very well known family possessing a broad range of
pharmacological properties. Liquiritigenin is a kind of flavonoids
existed in Radix Glycyrrhizae. It has been reported that liquiritigenin
could possess cytotoxic activity against five human cancer cell lines
in vitro [1]. Shi-ping Zhang et al. [2] also found liquiritigenin could
inhibit the growth of SGC-7901, SMMC-7721 and Lovo cells. And
several mechanisms of antitumor action of liquiritigenin have been
proposed. For example, it could inhibit the activity of aromatase [3].
Besides, liquiritigenin and its derivatives are selective estrogen
reductive conversion of ribonucleotides to deoxyribonucleotides
[19].
Recently, several kinds of thiosemicarbazone derivatives have
been synthesized and their antitumor activities were also reported
[6]. But there were few literatures reported for flavonoids thio-
semicarbazone derivatives. In our study, we designed and synthe-
sized a series of liquiritigenin thiosemicarbazone derivatives to
screen their antitumor activity against different cancer cell lines in
vitro in order to get more potent and selective anticancer agents,
especially for estrogen-dependent cancer.
2. Chemistry
receptor
b
agonists [4] just like other phytoestrogens which have
preventive effect against various
been suggested to have
a
The preparation of target compounds is illustrated in Scheme 1.
The resorcinol 1 and 4-hydroxybenzaldehyde were commercially
available. A solution of resorcinol 1 and zinc chloride in acetic acid
was refluxed for 1 h. After cooling to room temperature, ice water
was added to the solution to form 1-(2,4-dihydroxyphenyl) etha-
none 2. Thereafter, the 4-hydroxyl of compound 2 was protected
with chloromethyl methyl ether (MOMCl) in acetone in the pres-
ence of K2CO3 to give compound 3. Meanwhile, the protection of
hydroxyl in 4-hydroxybenzaldehyde was accomplished using the
same procedure and reagents as synthesizing compound 2. Next,
compound 3 and compound 4 were treated with potassium
hydroxide in ethanol at room temperature for 24 h, followed by
neutralizing with hydrochloric acid, compound 5 was obtained. The
obtained compound 5 was refluxed in ethanol in the presence of
estrogen-dependent cancer such as breast cancer and prostate
cancer [5].
Thiosemicarbazone,
a large group of thiourea derivatives,
exhibits various biological activities and have therefore attracted
considerable pharmaceutical interest [6]. They have been evaluated
as antiviral [7], antibacterial [8e10] and anticancer [11e16] thera-
peutics over the last 50 years, whose biological activities are
a function of parent aldehyde or ketone moiety [17,18]. Conjugated
NeNeS tridentate ligand system of thiosemicarbazide
(NH2eCSeNHeNH2) seems essential for anticancer activity. The
* Corresponding author. Tel./fax: þ86 519 89819963.
E-mail address: hukun1979@163.com (J. Ren).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.036

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K. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 3453e3458
Scheme 1. Synthesis of liquiritigenin thiosemicarbazone derivatives.
sodium acetate for 12 h to give compound 6. Finally, liquiritigenin
was obtained by refluxing compound 6 in ethanol under acid
conditions. From resorcinol to liquiritigenin, the over all yield was
36.4%.
potencies of some compounds were better or comparative to the
lead compound liquiritigenin, some compounds showed moderate
cytotoxicity against HCT-116 cancer cell line, although they were
less potent than liquiritigenin. For SGC-7901 and Hela cell lines,
most of the compounds showed low activity, but they were more
potent than liquiritigenin. In conclusion, the cytotoxicities of the
tested compounds against K562 and DU-145 were higher than SGC-
7901, HCT-116 and Hela cell lines, reflecting the excellent selectivity
for a particular human leukemia and human prostate carcinoma
cancer cell types.
Next, we turned our attention to the introduction of thio-
semicarbazone to liquiritigenin. Compound 8 was easily prepared
by following the previously reported method in high yield [18]. The
condensation of compound 8 and liquiritigenin 7 was first carried
out in ethanol under refluxing condition, however, invariably low
yield of target compound was obtained after refluxing for long
period. Nevertheless, the reaction proceeded smoothly until
consumption of the starting material when using ethanol as solvent
in the presence of concentrated hydrochloric acid. The alkyldi-
thioate in compound 8 was hydrolyzed to thionocarboxylic acid at
the same time. Finally, compound 9 was treated with various
amines and refluxed in ethanol for 3e4 h in the presence of dicy-
clohexylcarbodiimide (DCC). All the new compounds synthesized
were characterized by 1H NMR, 13C NMR, MS, and IR spectroscopy.
The cytotoxicities of the resulting liquiritigenin thio-
semicarbazone derivatives appeared to be related to the nature of
the substituent group at thiocarbonyl. For K562 cell line, most of
the prepared compounds showed potent inhibitory activities with
the IC50 values lower than 30
mM, which were superior to
Table 1
The antitumor activities of liquiritigenin thiosemicarbazone derivatives.
a
3. Results and discussion
Compounds
IC50/(
K562
m )
mol Lꢀ1
SGC-7901
DU-145
HCT-116
Hela
The in vitro cytotoxicities of the liquiritigenin thio-
semicarbazone derivatives against K562 (human leukemia cell
line), DU-145 (human prostate carcinoma cell line), SGC-7901
(human gastric cancer cell line), HCT-116 (human colon cancer cell
line) and Hela (human cervical carcinoma cell line) cells were
evaluated by the standard MTT Assay using 5-FU(5-Fluorouracil) as
a positive control. Antitumor potency of the compounds was
indicated by IC50 values that were calculated by linear regression
analysis of the concentrationeresponse curves obtained for each
compound. Data were reported in Table 1.
Liquiritigenin
10a
10b
10c
10d
10e
10f
10g
10h
>100
31.0
21.3
17.0
8.9
12.4
17.3
13.7
26.2
23.1
29.4
45.4
>100
74.7
60.4
57.7
42.9
62.1
51.7
55.3
>100
98.8
84.5
2.19
92.4
31.2
35.2
50.8
31.0
32.2
37.0
40.0
22.5
10.1
52.2
2.95
37.2
54.0
38.2
49.3
25.9
41.9
52.3
50.9
79.4
60.4
63.5
1.93
>100
40.2
68.1
>100
50.0
75.3
65.2
74.4
>100
65.8
76.3
9.7
10i
10j
5-FU
As shown in Table 1, most of prepared compounds showed
potent inhibitory effects on K562 and DU-145 cell lines, and the
a
Date present the mean values of three independent determinations.

K. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 3453e3458
3455
liquiritigenin (IC50 > 100
m
M) and 5-FU(IC50 ¼ 45.4
m
M). It has been
room temperature, the reaction mixture was added with ice water
(100 mL), and separated by suction and washed with water to give
1-(2,4-dihydroxyphenyl)ethanone 2 (1.1 g, 69.6%) as light yellow
observed from Table 1 that most of the compounds with aliphatic
amine moieties have higher cytotoxicity than those with hetero-
cyclic amine groups. For example, among 10be10e bearing
acicular crystal. 1H NMR (DMSO-d6)
d: 2.51(3H, s, CH3), 6.25(1H, d,
aliphatic amine moieties, the IC50 values of them are 21.3
17.0 M, 8.9 M and 12.4 M respectively, which were superior to
10he10j bearing heterocyclic amine moieties with the IC50 values
of 26.2 M, 23.1 M, 29.4 M. However, there were some excep-
m
M,
J ¼ 2.3 Hz, 3-H), 6.38(1H, dd, J ¼ 8.8 Hz, 2.3 Hz, 5-H), 7.76(1H, d,
m
m
m
J ¼ 8.8 Hz, 6-H), 10.64(1H, s, 4-OH), 12.63(1H, s, 2-OH). 13C NMR
(DMSO-d6) d: 26.3(CH3), 102.3(1-C), 108.1(5-C), 112.8(6-C), 133.6(3-
C), 164.2(4-C), 164.8(2-C), 202.6(C]O).
m
m
m
tions, such as 10a has the lowest cytotoxicity and 10g has relatively
higher cytotoxicity in K562 cell line. For all derivatives with
aliphatic amine moieties, the activities appeared to be related to the
size of substituent. For example, the derivatives with the substit-
uents as tert-butyl (10d), dipropyl (10e), butyl (10c), isopropyl (10b)
5.1.2. 1-(2-Hydroxy-4-(methoxymethoxy)phenyl)ethanone(3)
To a solution of 1-(2,4-dihydroxyphenyl)ethanone 2 (152 mg,
1 mmol) in ethanol (10 mL) were added K2CO3. After stirring for
1 h, MOMCl (0.114 mL, 1.5 mmol) was added. After stirring for
another 4 h, distilled water (10 mL) was poured into the reaction
mixture and then extracted with ethyl acetate (3 ꢁ 10 mL). The
combined organic layer was dried over anhydrous Na2CO3 and
concentrated in vacuo. The residual oil was purified by flash
chromatography on silica gel (ethyl acetate:petroleum
ether ¼ 1:15) to give 1-(2-hydroxy-4-(methoxymethoxy)phenyl)
ethanone 3 (194.8 mg, 91.4% yield) as achromatous oil. 1H NMR
and propyl (10a), with the IC50 values of 8.9
mM, 12.4 mM, 17.0 mM,
21.3 M and 31.0 M respectively. A similar tendency was observed
m
m
against SGC-7901 cell line. The results indicated that the intro-
duction of aliphatic amine functional group at the thiocarbonyl
position facilitated the increase of their cytotoxic activities, while
heterocyclic amine functional group has
promoting.
a smaller role in
For DU-145, the introduction of thiosemicarbazone has greatly
enhanced the cytotoxicity of prepared compounds compared to
liquiritigenin, which was in agreement with the aim for inhibiting
the estrogen-dependent cancer. As demonstrated in Table 1, the
best active compound was 10i with a methyl piperazine moiety
(CDCl3)
6.53(1H, dd, J ¼ 8.8 Hz, 2.4 Hz, 5-H), 6.57(1H, d, J ¼ 2.4 Hz, 6-H),
12.62(1H, s, 2-OH). 13C NMR (CDCl3)
: 26.1(CH3), 56.2(OCH3), 93.9
(-CH2), 103.6 (1-C), 108.1(5-C), 114.6(6-C), 132.3(3-C), 163.6(2-C),
164.7(4-C), 202.7(C]O).
d: 2.54(3H, s, COCH3), 3.47(3H, s, OCH3), 5.19(2H, s, CH2),
d
with the IC50 value of 10.1
mM, followed by 10h with a piperazine
moiety with the IC50 value of 22.5
mM, the derivative 10j with
5.1.3. 4-(Methoxymethoxy)benzaldehyde(4)
a Morpholine moiety and 10c with a n-butylamine moiety exhibi-
ted the worst activity. The data in Table 1 for DU-145 cell line also
showed that the compounds with aliphatic amine moieties had
stronger cytotoxicity than those with heterocyclic amine groups.
However, compounds 10h and 10i, which contained a piperazine
moiety and methyl piperazine moiety, were the exceptions. They
exhibited extraordinary cytotoxicity in DU-145cell line.
4-(methoxymethoxy)benzaldehyde 4 was prepared using the
same procedure and reagents as synthesizing 1-(2-hydroxy-4-
(methoxymethoxy)phenyl)ethanone 3. Yield 95.4%. 1H NMR
(CDCl3)
d
: 3.49(3H, s, CH3), 5.25(2H, s, CH2), 7.14(2H, d, J ¼ 8.7 Hz,
3,5-2H), 7.83(2H, d, J ¼ 8.7 Hz, 2,6-2H), 9.89 (1H, s, CHO). 13C NMR
(CDCl3) d: 56.3(CH3), 94.1(CH2), 116.3(3,5-2C), 131.8 (2,6-2C), 162.2
(4-C), 190.8 (CHO).
4. Conclusion
5.1.4. 1-(2-Hydroxy-4-(methoxymethoxy)phenyl)-3-(4-
(methoxymethoxy)phenyl)prop-2-en-1-one (5)
In summary, we have designed and synthesized ten liquir-
itigenin thiosemicarbazone derivatives, and evaluated their anti-
tumor activities against five cell lines (K562, DU-145, SGC-7901,
HCT-116, Hela). Most of the prepared compounds displayed the
selective cytotoxicity toward K562 and DU-145 cell lines. From the
structureeactivity relationships we may conclude that the intro-
duction of thiosemicarbazone at 4-position in liquiritigenin is
associated with enhanced cytotoxic activity. When the thio-
semicarbazone contained aliphatic amine moieties, the effect was
more obvious. This study may provide valuable information for
further designing and developing more potent anticancer agents.
1-(2-hydroxy-4-(methoxymethoxy)phenyl)ethanone 3(196 mg,
1 mmol), 4-(methoxymethoxy)benzaldehyde 4 (166 mg, 1 mmol),
KOH (1.12 g, 20 mmol), ethanol (10 mL) and distilled water (1 mL)
were stirred for 48 h. Crushed ice (5 g) was added into the
reaction mixture and neutralized with 3 M HCl and then extrac-
ted with ethyl acetate (3 ꢁ 10 mL). The combined organic layer
was dried over anhydrous Na2CO3 and concentrated in vacuo. The
residual oil was purified by flash chromatography on silica gel
(ethyl acetate:petroleum ether ¼ 1:10) to give 1-(2-hydroxy-4-
(methoxymethoxy)phenyl)-3-(4-(methoxymethoxy)phenyl)prop-
2-en-1-one 5 (258.1 mg, 75.1% yield) as yellow acicular crystal. 1H
NMR (CDCl3) d: 3.49(6H, s, 2CH3), 5.22(4H, s, 2CH2), 6.59(1H, dd,
5. Experimental protocols
J ¼ 9.1 Hz, 2.3 Hz, 60-H), 6.6(1H, d, J ¼ 2.3 Hz, 30-H), 7.1(2H, d,
J ¼ 8.7 Hz, 3,5-2H), 7.47(1H, d, J ¼ 15.5 Hz, 50-H), 7.60(2H, d,
5.1. Synthesis
J ¼ 8.6 Hz, 2,6-2H), 7.84(1H, d, J ¼ 6.5 Hz,
b
-H), 7.86(1H, d,
: 56.2
(CH3), 56.4(CH3), 94.1(CH2), 94.3(CH2), 104.0(10-C), 108.2 (
-C),
115.1(50-C), 116.6(3,5-2C), 118.4(60-C), 128.6 (1-C), 130.3(2,6-2C),
131.3(30-C), 144.3( -C), 159.5 (4-C), 163.6(20-C), 166.2(40-C), 192.1
(C]O). IR (KBr disc)
J ¼ 13.0 Hz,
a d
-H), 13.35(1H, s, 20-OH). 13C NMR (CDCl3)
Reagents and solvents were obtained from commercial
suppliers. Solvents were dried and purified using standard tech-
niques. All reactions involving air or moisture sensitive reagents or
intermediates were performed under nitrogen. NMR spectra were
recorded on a Bruker AVANCE III 500 Hz spectrometer. IR spectra
were recorded on a Bruker Optics TENSOR 27 infrared spectrom-
eter. Mass spectra were recorded on a Shimadzu VG-Autospec-
3000 mass spectrometer.
b
a
n
: 3440, 2597, 2900, 2826, 1644, 1569, 1510,
1233, 1150, 1078, 1004, 835, 652 cmꢀ1. FAB-MS: m/z ¼ 345
(M þ 1)
5.1.5. 7,40-Dimethoxymethoxy flavanone (6)
To a solution of 1-(2-hydroxy-4-(methoxymethoxy)phenyl)-3-
(4-(methoxymethoxy)phenyl)prop-2-en-1-one 5 (344 mg,1 mmol)
in ethanol (10 mL) were added anhydrous NaOAc (820 mg,
10 mmol) and distilled water (3 mL). The reaction mixture was
5.1.1. 1-(2,4-Dihydroxyphenyl)ethanone (2)
Resorcinol 1 (1.1 g, 0.01 mol), zinc chloride (1.36 g, 0.01 mol) and
glacial acetic acid (30 mL) were refluxed for 1 h. After cooling to

3456
K. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 3453e3458
refluxed for 24 h, cooled to room temperature, added distilled
water (15 mL) and extracted with ethyl acetate (3 ꢁ 10 mL). The
combined organic layer was dried over anhydrous Na2CO3 and
concentrated in vacuo. The residual oil was purified by flash chro-
matography on silica gel (ethyl acetate:petroleum ether ¼ 1:10) to
give 7,40-dimethoxymethoxy flavanone 6 (283.2 mg, 82.3% yield) as
5.1.9. General procedure for preparation of liquiritigenin
thiosemicarbazone derivatives (10ae10j)
To a solution of 2-(7-hydroxy-2-(4-hydroxyphenyl)chroman-4-
ylidene)hydrazinecarbothioic O-acid 9 (200 mg, 0.61 mmol) in
ethanol (12 mL) were added amine (0.61 mmol) and DCC (125.9 mg,
0.61 mmol). The reaction mixture was refluxed for 4 h, and then
distilled water (20 mL) was added, extracted with acetic ether
(3 ꢁ 15 mL), dried over anhydrous Mg2SO4 and concentrated in
vacuo. The residue was purified by flash chromatography on silica
gel to afford 10a-10j as light yellow oil.
light yellow solid. 1H NMR (CDCl3)
d: 2.80(1H, dd, J ¼ 17.0 Hz, 3.2 Hz,
3 cis-H), 3.04(1H, dd, J ¼ 17.2 Hz, 13.6 Hz, 3 syn-H), 3.48(6H, s,
2CH3), 5.20(4H, s, 2CH2), 5.42(1H, dd, J ¼ 13.4 Hz, 2.5 Hz, 2-H), 6.67
(1H, d, J ¼ 2.2 Hz, 8-H), 6.71(1H, dd, J ¼ 8.7 Hz, 2.2 Hz, 5-H), 7.09(2H,
d, J ¼ 8.7 Hz, 30,50-2H), 7.40(2H, d, J ¼ 8.7 Hz, 20,60-2H), 7.87(1H, d,
J ¼ 8.7 Hz, 6-H). 13C NMR (CDCl3)
d
: 44.2 (3-C), 56.0(CH3), 56.4(CH3),
5.1.9.1. 2-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)-N-
79.7(2-C), 94.2(CH2), 94.5(CH2), 103.7(10-C), 111.2(6-C), 115.7(5-C),
propylhydrazinecarbothioamide (10a). Yield 86.2%. 1H NMR (DMSO-
116.5(30,50-2C), 127.7(20,40-2C), 128.8(10-C), 132.2(8-C), 157.6(40-C),
d6)
d
: 0.88(3H, t, J ¼ 7.38 Hz, CH3), 1.24(2H, br, CH2), 1.59(2H, q,
163.3(9-C), 163.7(7-C), 190.8(4-C). IR (KBr disc)
n
: 3439, 2963, 2928,
J ¼ 7.38, CH2), 2.75(1H, dd, J ¼ 18.3 Hz,12.2 Hz, 3 cis-H), 3.30(1H, dd,
J ¼ 18.1 Hz, 2.7 Hz, 3 syn-H), 5.03(1H, dd, J ¼ 12.3 Hz, 2.60 Hz, 2-H),
6.28(1H, d, J ¼ 2.3 Hz, 8-H), 6.45(1H, dd, J ¼ 8.9 Hz, 2.3 Hz, 6-H), 6.79
(2H, d, J ¼ 8.6 Hz, 30,50-2H), 7.31(2H, d, J ¼ 8.6 Hz, 20,60-2H), 8.09(1H,
d, J ¼ 8.7 Hz, 5-H), 8.50(1H, t, J ¼ 5.9 Hz, NH). 9.52(1H, s, 40-OH), 9.92
2832, 1682, 1606, 1511, 1444, 1245, 1154, 1079, 997, 829, 653,
522 cmꢀ1. FAB-MS: m/z ¼ 345 (M þ 1)
5.1.6. Liquiritigenin (7)
To a solution of 7,40-dimethoxymethoxy flavanone 6 (344 mg,
1 mmol) in ethanol (10 mL) were added 3 M HCl (1 mL). The
reaction mixture was refluxed for 1 h, cooled to room temperature,
added distilled water (15 mL) and extracted with ethyl acetate
(3 ꢁ 10 mL). The combined organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo. The residual oil was purified by
flash chromatography on silica gel (ethyl acetate:petroleum
ether ¼ 1:2) to give liquiritigenin 7 (37.4 mg, 92.7% yield) as light
(1H, s, 7-OH), 10.20(1H, s, NNH). 13C NMR (DMSO-d6)
d: 11.2(CH3),
22.1(CH2), 31.8(CH2NH), 45.1(3-C), 76.3(2-C), 102.8(10-C), 110.0(6-
C),111.5(5-C),115.0(30,50-2C),126.6(10-C),127.8(20,60-2C),130.0(8-C),
143.1(C]S), 157.3(40-C), 158.5(9-C), 160.3(7-C), 172.3(C]N). IR (KBr
disc) n: 3356, 2929,1625,1514,1442,1346,1219,1167,1125, 972, 834,
639, 550, 515 cmꢀ1. FAB-MS: m/z ¼ 372 (M þ 1)
5.1.9.2. 2-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)-N-
yellow solid. 1H NMR (DMSO-d6)
d
: 2.63(1H, dd, J ¼ 16.5 Hz, 2.8 Hz,
isopropylhydrazinecarbothioamide (10b). Yield 88.5%. 1H NMR
3 syn-H), 3.11(1H, dd, J ¼ 16.8 Hz, 12.9 Hz, 3 cis-H), 5.43(1H, dd,
J ¼ 13.7 Hz, 2.2 Hz, 2-H), 6.33(1H, d, J ¼ 2.1 Hz, 8-H), 6.50(1H, dd,
J ¼ 8.8 Hz, 2.1 Hz, 5-H), 6.78(1H, d, J ¼ 8.5 Hz, 30,50-2H), 7.32(1H, d,
J ¼ 8.5 Hz, 20,40-2H), 7.64(1H, d, J ¼ 8.7 Hz, 6-H), 9.56(1H, s, eOH),
(DMSO-d6)
d
: 1.17(6H, t, J ¼ 6.5 Hz, 2CH3), 3.10(1H, dd, J ¼ 18.0 Hz,
3.0 Hz, 3 syn-H), 3.81(1H, dd, J ¼ 18.2 Hz, 11.3 Hz, 3 cis-H), 4.44(1H,
q, J ¼ 6.9 Hz, CH), 5.79(1H, dd, J ¼ 11.2 Hz, 2.9 Hz, 2-H), 6.34(2H, m,
5,8-2H), 6.66(2H, d, J ¼ 8.6 Hz, 30,50-2H), 6.91(2H, d, J ¼ 8.5 Hz, 20,60-
2H), 8.0(1H, d, J ¼ 8.4 Hz, NHCH), 9.31(1H, s, 40-OH), 9.76(1H, s, 7-
10.56(1H, s, eOH). 13C NMR (DMSO-d6)
d: 43.1(3-C), 78.9(2-C),
102.5(10-C), 110.4(6-C), 113.5(5-C), 115.0(30,50-2C), 128.1(20,60 -2C),
OH),10.01(1H, s, NNH). 13C NMR (DMSO-d6)
d: 21.9(CH3), 22.0(CH3),
128.3(10-C), 129.2(8-C), 157.5(40-C), 163.1(9-C), 164.5(7-C), 190.0(4-
43.5(NCH), 45.9(3-C), 61.4(2-C), 102.8(10-C), 107.9(6-C), 108.1(5-C),
C). IR (KBr disc) n: 3257, 3030, 2918, 2826, 2709, 1652, 1602, 1519,
115.0(30,50-2C), 126.6(20,60-2C), 130.8(10-C), 133.5(8-C), 156.0(40-C),
1471, 1329, 1257, 1164, 1117, 832, 534 cmꢀ1. FAB-MS: m/z ¼ 257
(M þ 1)
156.2(9-C), 158.2(7-C), 161.0(C]S), 173.5(C]N). IR(KBr disc) n:
3307, 2967, 2925, 1619, 1526, 1499, 1456, 1367, 1335, 1306, 1228,
1159, 1118, 1067, 1041, 984, 872, 834, 795, 657, 572 cmꢀ1. FAB-MS:
m/z ¼ 372 (M þ 1)
5.1.7. Methyl thiocarbamate (8)
Methyl thiocarbamate 8 was obtained as recently described
procedure [20]. m.p. 81e82 ꢂC. IR (KBr disc)
n
: 3455, 3260, 3155,
2974, 1600, 1507, 1423, 1373, 1290, 1152, 1005, 943, 707, 665,
466 cmꢀ1
5.1.9.3. N-Butyl-2-(7-hydroxy-2-(4-hydroxyphenyl)chroman-4-yli-
dene)hydrazinecarbothioamide(10c). Yield 83.5%. 1H NMR (DMSO-
.
d6)
d
: 0.92(3H, t, J ¼ 7.36 Hz, CH3), 1.31(2H, m, J ¼ 7.5 Hz, CH2), 1.57
(2H, m, J ¼ 7.5 Hz, CH2), 2.75(1H, dd, J ¼ 17.5 Hz, 12.2 Hz, 3 cis-H),
3.31(1H, dd, J ¼ 17.2 Hz, 2.7 Hz, 3 syn-H), 3.57(2H, m, J ¼ 6.8 Hz,
CH2), 5.03(1H, dd, J ¼ 12.2 Hz, 2.5 Hz, 2-H), 6.28(1H, d, J ¼ 2.2 Hz, 8-
H), 6.45(1H, dd, J ¼ 8.7 Hz, 2.2 Hz, 6-H), 6.79(2H, d, J ¼ 8.5 Hz, 30,50-
2H), 7.31(2H, d, J ¼ 8.5 Hz, 20,60-2H), 8.09(1H, d, J ¼ 8.7 Hz, 5-H),
8.49(1H, t, J ¼ 5.9 Hz, NH), 9.54(1H, s, 40-OH), 10.03(1H, s, 7-OH),
5.1.8. 2-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)
hydrazinecarbothioic O-acid (9)
To a solution of liquiritigenin 7 (256.3 mg, 1 mmol) and methyl
thiocarbamate 8 (366.6 mg, 3 mmol) in ethanol (10 mL) were added
concentrated hydrochloric acid (0.2 mL). After refluxing for 3 h,
distilled water was added, the product that precipitated was
separated by suction and washed with water. The solid was
recrystallized by ethanol to give 2-(7-hydroxy-2-(4-hydrox-
10.20(1H, s, NNH). 13C NMR (DMSO-d6)
d: 13.9(CH3), 19.6(CH2), 31.1
(CH2), 31.9(CH2), 43.2(3-C), 76.3(2-C), 102.9(10-C), 110.0(6-C), 111.6
(5-C), 115.0(3050-2C), 126.7(10-C), 127.9(20,60-2C), 130.1(8-C), 143.1
(C]S), 157.3(40-C), 158.5(9-C), 160.4(7-C), 177.3(C]N). IR(KBr disc)
yphenyl)chroman-4-ylidene)hydrazinecarbothioic
O-acid
9
(281.8 mg, 85.3% yield) as a light yellow solid. 1H NMR (DMSO-d6)
d:
n: 3356, 2929,1625, 1514, 1442, 1346, 1219, 1167, 1125, 1021, 972,
2.85(1H, dd, J ¼ 19.7 Hz, 12.7 Hz, 3 cis-H), 3.41(1H, d, J ¼ 15.9 Hz, 3
syn-H), 5.12(1H, d, J ¼ 12.8 Hz, 2-H), 6.31(1H, s, 8-H), 6.50(1H, d,
J ¼ 8.1 Hz, 6-H), 6.79(2H, d, J ¼ 8.1 Hz, 30,50-2H), 7.31(2H, d,
J ¼ 8.1 Hz, 20,60-2H), 7.80(1H, d, J ¼ 8.5 Hz, 5-H), 9.54(1H, s, 40-OH),
834, 639, 550, 515 cmꢀ1. FAB-MS: m/z ¼ 387 (M þ 1)
5.1.9.4. N-tert-Butyl-2-(7-hydroxy-2-(4-hydroxyphenyl)chroman-4-
ylidene)hydrazinecarbothioamide (10d). Yield 86.7%. 1H NMR
10.10(1H, s, 7-OH),12.39(1H, s, eCSOH). 13C NMR (DMSO-d6)
d: 32.3
(DMSO-d6)
d
:1.54(9H, s, 3CH3), 2.77(1H, dd, J ¼ 18.22 Hz, 12.2 Hz, 3
(3-C), 76.4(2-C), 103.0(10-C), 110.5(6-C), 110.9(5-C), 115.0(30,50-2C),
cis-H), 3.30(1H, dd, J ¼ 17.3 Hz, 2.3 Hz, 3 syn-H), 5.04(1H, dd,
J ¼ 12.2 Hz, 2-H), 6.30(1H, d, J ¼ 2.2 Hz, 8-H), 6.48(1H, dd, J ¼ 8.8 Hz,
2.2 Hz, 6-H), 6.79(2H, d, J ¼ 8.5 Hz, 30,50-2H), 7.31(2H, d, J ¼ 8.5 Hz,
206’-2H), 7.73(1H, s, NH), 7.76(1H, d, J ¼ 8.7 Hz, 5-H), 9.54(1H, s, 40-
126.0(10-C), 127.9(20,60-2C), 129.8(8-C), 147.1(C]S), 157.4(40-C),
159.2(9-C), 161.2(7-C), 198.4(eC]N). IR (KBr disc) n: 3313, 3137,
2926,1892,1603,1512,1478,1403,1351,1309,1214,1159,1129,1106,
1058, 989, 837, 750, 633, 527, 507 cm-1. FAB-MS: m/z ¼ 331 (M þ 1)
OH), 10.01(1H, s, 7-OH), 10.16(1H, s, NNH). 13C NMR (DMSO-d6)
d:

K. Hu et al. / European Journal of Medicinal Chemistry 45 (2010) 3453e3458
3457
28.6(3CH3), 32.0(-C(CH3)3), 52.5(3-C), 76.3(2-C), 103.1(10-C), 110.4
(6-C), 111.2(5-C), 115.0(30,50-2C), 125.6(10-C), 127.9(20,60-2C), 129.9
(8-C), 143.2(C]S), 157.3(40-C), 158.6(9-C), 160.5(7-C), 175.9(C]N).
5.1.9.9. N0-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)-4-
methylpiperazine-1-carbothiohydrazide (10i). Yield 74.7%. 1H NMR
(DMSO-d6)
d: 2.21(3H, s, CH3), 2.33(2H, t, J ¼ 7.2 Hz, CH2), 2.43(2H, t,
IR (KBr disc)
n: 3326, 2967, 2927, 1702, 1616, 1536, 1497, 1451, 1404,
J ¼ 6.7 Hz, CH2), 3.09(1H, dd, J ¼ 18.5 Hz, 7.4 Hz, 3 syn-H), 3.62(2H, t,
J ¼ 6.4 Hz, CH2), 3.81(2H, t, J ¼ 6.4 Hz, CH2), 3.87(1H, dd, J ¼ 18.5 Hz,
11.5 Hz, 3 cis-H), 5.83(1H, dd, J ¼ 11.8 Hz, 7.7 Hz, 6-H), 6.40(1H, d,
J ¼ 2.0 Hz, 8-H), 6.72(2H, d, J ¼ 8.4 Hz, 30,50-2H), 7.08(2H, d,
J ¼ 8.4 Hz, 20,60-2H), 7.30(1H, d, J ¼ 9.1 Hz, 5-H), 10.30(1H, s, 7-OH).
1364, 1323, 1268, 1214, 1161, 1108, 1070, 1024, 994, 875, 835, 756,
698, 659, 574, 501, 439 cmꢀ1. FAB-MS: m/z ¼ 387 (M þ 1)
5.1.9.5. 2-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)-N,N-
dipropylhydrazinecarbothioamide (10e). Yield 83.2%. 1H NMR
13C NMR (DMSO-d6)
d: 42.5(3-C), 45.4(CH3), 50.3(2CH2), 54.4
(DMSO-d6)
d
: 0.79(6H, t, J ¼ 7.3 Hz, 2CH3), 1.59(4H, m, 2CH2), 3.09
(2CH2), 62.9(2-C), 102.6(10-C), 107.5(6-C), 108.1(5-C), 115.2(30,50-
2C), 127.7(20,60-2C), 130.4(2-C), 132.6(8-C), 155.7(40-C), 156.5(9-C),
(1H, dd, J ¼ 18.2 Hz, 8.4 Hz, 3 cis-H), 3.37(1H, dd, J ¼ 17.2 Hz, 2.3 Hz,
3 syn-H), 3.85(4H, m, 2CH2), 5.80(1H, dd, J ¼ 11.6 Hz, 8.8 Hz, 2-H),
6.36(1H, s, 8-H), 6.37(1H, d, J ¼ 9.0 Hz, 6-H), 6.69(2H, d, J ¼ 8.6 Hz,
30,50-2H), 7.09(2H, d, J ¼ 8.4 Hz, 20,60-2H), 7.32(1H, d, J ¼ 9.1 Hz, 5-
H), 9.36(1H, s, 40-OH), 10.02(1H, s, 7-OH), 10.27(1H, s, NNH). 13C
158.7(7-C), 161.1(C]S), 182.0(C]N). IR (KBr disc) n: 3416, 2925,
2855, 1628, 1514, 1467, 1432, 1389, 1333, 1249, 1167, 1103, 1019, 973,
832, 804, 652, 595, 555, 520 cmꢀ1. FAB-MS: m/z ¼ 413 (M þ 1)
NMR (DMSO-d6)
d
: 11.1(CH3), 20.5(CH2), 33.4(CH2), 53.5(3-C), 63.2
5.1.9.10. N0-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)
(2-C), 102.6(10-C), 107.8(6-C), 107.9(5-C), 115.0(30,50-2C), 128.2
morpholine-4-carbothiohydrazide (10j). Yield 78.5%. 1H NMR
(20,60-2C), 130.2(10-C), 132.6(8-C), 154.8(C]S), 156.4(40-C), 158.6(9-
(DMSO-d6)
d
: 3.10(1H, dd, J ¼ 17.8 Hz, 7.0 Hz, 3 syn-H), 3.60(4H, br,
C), 160.8(2-C), 182.8(C]N). IR (KBr disc)
n
: 3325, 2962, 2930, 1628,
2CH2), 3.67(2H, br, CH2), 3.85(3H, br, CH2, 3cis-H), 5.83(1H, dd,
J ¼ 12.2 Hz, 7.4 Hz, 2-H), 6.38(2H, m, 6,8-2H), 6.71(2H, d, J ¼ 8.3 Hz,
30,50-2H), 7.07(2H, d, J ¼ 8.3 Hz, 20,60-2H), 7.32(1H, d, J ¼ 9.0 Hz, 5-
H), 9.35(1H, br, 40-OH), 10.03(1H, br, 7-OH), 10.24(1H, br, NH). 13C
1515, 1431, 1390, 1307, 1236, 1169, 1138, 1099, 1023, 971, 833, 809,
743, 644, 596, 557, 518 cmꢀ1. FAB-MS: m/z ¼ 415 (M þ 1)
5.1.9.6. N0-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)pyr-
NMR (DMSO-d6) d: 42.6(3-C), 51.1(2CH2), 59.7(2-C), 66.0(2CH2),
rolidine-1-carbothiohydrazide(10f). Yield 83.2%. 1H NMR (DMSO-d6)
102.6(10-C), 107.6(6-C), 108.0(5-C), 115.2(30,50-2C), 127.7(20,60-2C),
d
: 1.77(2H, br, CH2), 1.93(2H, br, CH2), 3.05(1H, dd, J ¼ 18.3 Hz,
130.4(11-C), 132.6(8-C), 155.7(C]S), 156.4(40-C), 158.7(9-C), 161.1
7.0 Hz, 3 syn-H), 3.66(4H, br, 2CH2), 3.83(1H, dd, J ¼ 18.2, 11.4 Hz, 3
cis-H), 5.85(1H, dd, J ¼ 11.8 Hz, 7.2 Hz, 2-H), 6.35(1H, s, 8-H), 6.36
(1H, d, J ¼ 2.1 Hz, 6-H), 8.69(2H, d, J ¼ 8.7 Hz, 30,50-2H), 7.06(2H, d,
J ¼ 8.7 Hz, 20,60-2H), 7.33(1H, d, J ¼ 9.0 Hz, 5-H), 9.35(1H, s, 40-OH),
(7-C), 182.0(C]N). IR (KBr disc) n: 3251, 2962, 2922, 1702, 1628,
1603, 1515, 1472,1434, 1389, 1306, 1270, 1234, 1168, 1110, 1028, 970,
893, 834, 642, 596, 556 cmꢀ1. FAB-MS: m/z ¼ 401 (M þ 1)
10.01(1H, s, 7-OH), 10.20(1H, s, NH). 13C NMR (DMSO-d6)
d: 25.0
5.2. Biology
(2CH2), 42.6(3-C), 53.3(2CH2), 63.0(2-C), 102.5(10-C), 107.9(5,6-2C),
115.1(30,50-2C), 127.7(20,60-2C), 130.2(10-C), 133.1(8-C), 154.2(C]S),
The tumor cell lines (K562, DU-145, SGC-7901, HCT-116, Hela)
were obtained from Shanghai Institute of Pharmaceutical Industry.
The cytotoxic activity in vitro was measured using the MTT assay.
156.3(40-C),158.4(9-C),160.7(7-C),177.5(C]N). IR (KBr disc)
n: 3237,
2969, 2877, 1702, 1628, 1602, 1516, 1471, 1448, 1384, 1338, 1302,
1238, 1171, 1131, 1101, 1038, 970, 833, 803, 643, 595, 553, 519, 493,
459 cmꢀ1. FAB-MS: m/z ¼ 385 (M þ 1)
The MTT solution (10.0 mL/well) was added in RPMI-1640 media
(Sigma, St. Louis, MO) after cells were treated with the drug for
72 h, and cells were incubated for further 4 h at 37 ꢂC. The purple
5.1.9.7. N0-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)
formazan crystals were dissolved in 100.0 mL DMSO. After 10 min,
piperidine-1-carbothiohydrazide(10g). Yield 88.5%. 1H NMR (DMSO-
the plates were read on an automated microplate spectropho-
tometer (Bio-Tek Instruments, Winooski, VT) at 570 nm and
630 nm. Assays were performed in triplicate on three independent
experiments. The concentration required for 50% inhibition of cell
viability (IC50) was calculated using the software “DoseeEffect
Analysis with Microcomputers”. The tumor cell lines panel con-
sisted of K562. DU-145, SGC-7901, HCT-116, Hela. In all of these
experiments, three replicate wells were used to determine each
point.
d6)
d
: 1.48(2H, br, CH2), 1.62(4H, br, 2CH2), 3.08(1H, dd, J ¼ 17.9 Hz,
7.6 Hz, 3 syn-H), 3.66(2H, t, J ¼ 6.1 Hz, CH2), 3.74(2H, t, J ¼ 6.1 Hz,
CH2), 3.85(1H, dd, J ¼ 18.1 Hz, 11.6 Hz, 3 cis-H), 5.84(1H, dd,
J ¼ 12.8 Hz, 7.9 Hz, 2-H), 6.37(1H, s, 8-H), 6.40(1H, d, J ¼ 10.8 Hz, 5-
H), 6.70(2H, d, J ¼ 8.3 Hz, 30,50-2H), 7.08(2H, d, J ¼ 8.3 Hz, 20,60-2H),
7.29(1H, d, J ¼ 8.9 Hz, 6-H), 9.33(1H, s, 40-OH), 10.02(1H, s, 7-OH),
10.32(1H, s, NH). 13C NMR (DMSO-d6)
d: 24.0(CH2), 25.6(2CH2), 42.4
(3-C), 51.5(2CH2), 62.8(2-C), 102.5(10-C), 107.0(6-C), 108.5(5-C),
115.1(30,40-2C), 130.3(10-C), 132.7(8-C), 155.2(C]S), 156.4(40-C),
158.6(9-C), 160.9(7-C), 182.0(C]N). IR (KBr disc) n: 3372, 2926,
2853, 1660, 1626, 1515, 1390, 1248, 1166, 1130, 1017, 969, 891, 830,
Acknowledgements
641, 594, 552, 517 cmꢀ1. FAB-MS: m/z ¼ 398 (M þ 1)
We thank the Jiangsu Provincial Natural Science Foundation (No.
BK2009549) for financial support.
5.1.9.8. N0-(7-Hydroxy-2-(4-hydroxyphenyl)chroman-4-ylidene)
piperazine-1-carbothiohydrazid(10h). Yield 84.9%. 1H NMR (DMSO-
d6)
d
: 2.73(4H, s, 2CH2), 2.89(4H, s, 2CH2), 3.34(1H, dd, J ¼ 18.6 Hz,
References
2.9 Hz, 3 syn-H), 3.98(1H, dd, J ¼ 18.6 Hz, 10.8 Hz, 3 cis-H), 5.89(1H,
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5-H), 6.69(2H, d, J ¼ 8.5 Hz, 30,50-2H), 6.95(2H, d, J ¼ 8.5 Hz, 20,60-
2H), 7.48(1H, d, J ¼ 8.3 Hz, 6-H), 9.42(1H, s, 40-OH), 10.25(2H, s, 70-
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