Synthesis, crystal structure and anticancer activities of tetrahydropyrido[4,3-d]dihydropyrimidine-2-thiones

Article abstract of DOI:10.1002/cjoc.201200568

A new series of tetrahydropyrido[4,3-d]dihydropyrimidine-2-thiones (3a-3x) were designed and synthesized. Their structures were confirmed by¹H NMR, IR, MS and elemental analysis, and the conformation of compound 3j was confirmed by X-ray diffraction. Preliminary bioassays indicated that most of the target compounds presented good antiproliferative activities against leukemic K562 cells, ovarian cancer HO-8910 cells and liver cancer SMMC-7721 cells in vitro. Among them the compounds 3i and 3m afford the best activity, the IC ₅₀ of them were 3.22 and 3.65 iμg/mL against leukemic K562 cells, respectively, which were lower than the anticancer drug of clinical practice 5-FU (IC₅₀=8.56 iμg/mL). Preliminary mechanism of action studies revealed that compound 3i caused DNA fragmentation and activated caspase-3/7 in leukemic K562 cells. A new series of tetrahydropyrido[4,3-d] dihydropyrimidine-2-thiones (3a-3x) were synthesized and the conformation of compound 3j was confirmed by X-ray diffraction. Preliminary bioassays indicated that most of the target compounds presented good antiproliferative activities against leukemic K562 cells, ovarian cancer HO-8910 cells and liver cancer SMMC-7721 cells in vitro. Preliminary mechanism of action studies revealed that compound 3i caused DNA fragmentation and activated caspase-3/7 in leukemic K562 cells. Copyright

Full text of DOI:10.1002/cjoc.201200568

FULL PAPER
DOI: 10.1002/cjoc. 201200568
Synthesis, Crystal Structure and Anticancer Activities of
Tetrahydropyrido[4,3-d]dihydropyrimidine-2-thiones
Ding, Lia(丁丽)
Wang, Jinga(王晶)
Xue, Sijia*,a(薛思佳)
Li, Jinga(李静)
Xiao, Dia(肖笛)
Hao, Zhibinga(郝志兵)
Pang, Chunchenga(庞春成)
Department of Chemistry, College of Life and Environmental Science, Shanghai Normal University, Shanghai
200234, China
A new series of tetrahydropyrido[4,3-d]dihydropyrimidine-2-thiones (3a—3x) were designed and synthesized.
Their structures were confirmed by 1H NMR, IR, MS and elemental analysis, and the conformation of compound 3j
was confirmed by X-ray diffraction. Preliminary bioassays indicated that most of the target compounds presented
good antiproliferative activities against leukemic K562 cells, ovarian cancer HO-8910 cells and liver cancer
SMMC-7721 cells in vitro. Among them the compounds 3i and 3m afford the best activity, the IC50 of them were
3.22 and 3.65 μg/mL against leukemic K562 cells, respectively, which were lower than the anticancer drug of clini-
cal practice 5-FU (IC50＝8.56 μg/mL). Preliminary mechanism of action studies revealed that compound 3i caused
DNA fragmentation and activated caspase-3/7 in leukemic K562 cells.
Keywords tetrahydropyrido[4,3-d]dihydropyrimidine, thione, synthesis, crystal structure, anticancer activities
Introduction
ments commonly present in natural products, they were
found applications as antihypertensive (SQ32926),[8]
ala-adrenergic receptor antagonists,[9] antibacterial and
antitumour agents.[10] On the other hand, pyridopyrimi-
dine skeleton exists in many natural or synthetic bio-
logically active materials, and its derivatives are applied
in various pharmaceutical and biochemical fields.[11,12]
Structure-activity studies disclose that anticancer activ-
ity of pyrimidinones and pyrimidinethiones is ascribable
to the presence of (i) nitrogen heterocyclic ring and (ii)
thione functionality, which generally enhance the activ-
ity.[13,14] This may provide some useful information for
future design of new pyrimidinethiones.
Pursuing our searches on design, synthesis, and bio-
logical validation of small molecule modulators of
pharmacologically targets, in 2011 we had reported the
synthesis and antiproliferative activities against leuke-
mic K562 cells of N-(substitutedbenzyl)-3,5-bis(benz-
ylidene)-4-piperidone (2).[15] The data indicated that
most of the compounds (2) have good antiproliferative
activities against leukemic K562 cells, and some of
them showed excllent antiproliferative activities, the
IC50 values were lower than that of the anticancer drug
of clinical practice 5-FU. While compared with the
5-FU, these results still could not do better, as well as
their narrow anticancer spectrum. So many efforts
should be made to develop 2 with the goal of increasing
antiproliferative activities against cancer cells and ex-
Cancer, the uncontrolled, rapid and pathological pro-
liferation of abnormal cells, is the second leading cause
of human death after cardiovascular diseases in devel-
oping as well as advanced countries.[1] Among them,
coronic myelogenous leukemia (CML), ovarian cancer
and liver cancer are main diseases to death, which are
caused by clonal expansion of pluripotent stem cells
retaining their differentiation potential.[2] At present,
most of cancer are lack of special treatment methods,
and cytotoxic drugs are still used in combination che-
motherapy to treat these malignant diseases. Chemo-
therapy can only through a lot of damage to the prolif-
eration of cancer cells and high patients get short-term
relief, but can not cure this malignant disease.[3] And
because the chemotherapy drugs currently used lack
specificity, toxic effect is very large. The most difficulty
is that most patients with recurrent use of the chemo-
therapy drug appear resistance to it.[4,5] Hence, the dis-
covery of fast-acting new drugs to effectively cure can-
cer is imperative.
Pyrimidinone sub-structure is prevalent in many
natural/synthetic biologically active compounds, which
were found applications in pharmaceutical and bio-
chemical areas.[6] Because dihydropyrimidinethiones
(DHPMs)[7] can be converted into either guanidines or
1,3-diamines, which constitute the core structural ele-
*
E-mail: sjxue@shnu.edu.cn
Received June 12, 2012; accepted September 4, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200568 or from the author.
Chin. J. Chem. 2012, 30, 2509—2516 © 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Ding et al.
FULL PAPER
panding the anticancer spectra.
4-piperidones (1) were prepared based on the proce-
dures in the literatures.[15] In a microwave reactor, the
title compounds (3a —3x) were synthesized by a
multicomponent reaction of N-(4-substituted benzyl)-4-
piperidones (1), aromatic aldehydes, thiourea and solid
sodium hydroxide in ethanol at 65 ℃ for 15—25 min.
Compared with the conventional synthetic methods,
controlled microwave heating had been shown to dra-
matically reduce reaction times, increase product yields,
and enhance product purities by reducing side reac-
tions.[18]
On the basis of above observations and obeying the
principle of active-factor-addition,[16] in order to find
some potent antiproliferative activity lead compounds,
we have focused our attentions on derivatization of N-
(substituted benzyl)-3,5-bis(benzylidene)-4-piperidones to
get tetrahydropyrido[4,3-d]dihydropyrimidine-2-thiones
(3a—3x), which contain not only DHPMs but also pyri-
dopyrimidine skeleton.
Multi-component reactions (MCRs) constitute a
highly valuable synthetic tool for the construction of
polyfunctionalized heterocyclic compounds required for
drug discovery programmes.[17] In the present investiga-
tion, a series of new tetrahydropyrido[4,3-d]dihydro-
pyrimidine-2-thiones (3a—3x) were synthesized by a
multicomponent reaction of N-(4-substituted benzyl)-
4-piperidones (1), substituted aromatic aldehydes, thio-
urea and solid sodium hydroxide in ethanol. The struc-
Scheme 1 Synthesis of the title compounds (3a—3x)
N
a
d
R1
CH2NH2
R1
O
1
1
CHO
tures of the title compounds were confirmed by H
S
e
1
NMR, IR, MS and elemental analysis, and the confor-
mation of the compound 3j was confirmed by X-ray
diffraction. Preliminary bioassays indicated that most of
the target compounds have good antiproliferative activi-
ties against leukemic K562 cells, ovarian cancer
HO-8910 cells and liver cancer SMMC-7721 cells in
vitro. The IC50 (μg/mL) of 3i and 3m against leukemic
K562 cells were 3.22 and 3.65 respectively, which were
lower than the standard 5-fluorouracil (IC50 ＝8.56
μg/mL). Preliminary mechanism studies revealed that
compound 3i caused DNA fragmentation and activated
caspase-3/7 in leukemic K562 cells.
R2
H2N
NH2
S
HN
N
NH
R2
R2
R1
3
R1 = Me, F, Br
R2 = 2-F, 4-OMe, 4-CH(CH3)2, 4-Me, 4-F, 2-Cl, H, 4-Cl
Experimental
Reagents and conditions: (a) methyl acrylate, absolute methanol,
refluxing; (b) sodium/absolute methanol, toluene, refluxing; (c) 25%
HCl, refluxing; (d) 35% sodium hydroxide; (e) solid sodium hydrox-
ide, absolute ethanol, 65 ℃, microwave reaction, 15—25 min.
Materials and apparatus
All the chemical reagents purchased were of ana-
lytical grade and used without further purification, ex-
cept for the toluene, which was dried by refluxing in the
presence of sodium and distilled prior to use. RPMI
1640 was obtained from Gibco BRL (Grand Island, NY,
USA) and bovine calf serum was supplied by Beijing
Dingguo Biotechnology Co, China. Thin-layer chroma-
tography (TLC) was carried out on silica gel 60 F254
Possible mechanism of the multicomponent reaction
for the synthesis of compounds 3 is described in
Scheme 2. The intermediates 2 were formed by the aldol
condensation of aromatic aldehyde with N-(4-substi-
tuted benzyl)-4-piperidone (1) , then an aza-Michael
addition of thiourea to intermediate 2 leads to the for-
mation of Michael adduct. Finally the tetrahydropyrido-
[4,3-d]-dihydropyrimidine-2-thiones ring can be closed
via nucleophilic attack of an NH2 group on a carbonyl
carbon, and the desired product 3 were obtained by
eliminating a hydroxyl group. It is an efficient and
promising method to construct the tetrahydropyrido-
[4,3-d]dihydropyrimidine-2-thione skeleton.
1
plates (Merck KGaA). H NMR spectra were recorded
on a Bruker Avance 400 (400 MHz) spectrometer, using
DMSO as solvents and tetramethylsilane (TMS) as in-
ternal standard. Melting points were determined by an
RK1 micro scopic melting apparatus and uncorrected.
Elemental analysis was performed with a Perkin-Elmer
2400 instrument. IR spectra were obtained on a Nicolet
5DX FT-IR spectrophotometer in the region 4000—400
cm－ using KBr discs. MS spectra were recorded on a
1
General synthetic procedures for the target com-
pounds (3a—3x)
Trace DSQ mass spectrograph. X-ray diffraction data
were recorded on a Bruker Smart CCD diffractometer.
N-(4-Substituted benzyl)-4-piperidones (1) were
synthesized according to the literature.[15]
Chemistry
In a microwave reactor, a mixture of N-(4-substi-
tuted benzyl))-4-piperidones 1a—1x (1 mmol), substi-
tuted aromatic aldehydes (2 mmol), thiourea (1 mmol),
Synthesis procedures for the title compounds are
summarized in Scheme 1, N-(4-substituted benzyl)-
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 2509—2516

Synthesis, and Anticancer Activities of Tetrahydropyrido[4,3-d]dihydropyrimidine-2-thiones
Scheme 2 Proposed reaction mechanism for the synthesis of the
compounds 3
2.22 (s, 3H) ; IR (KBr) ν: 3429, 1605, 1506, 1440, 1077,
－ ＋
1
988 cm ; ESI-MS ([M＋H] ) m/z: 498.21. Anal. calcd
for C30H31N3O2S: C 72.40, H 6.28, N 8.44; found C
72.41, H 6.27, N 8.46.
O
CHO
8-(4-Isopropylbenzylidene)-4-(4-isopropylphenyl)-
6-(4-methylbenzyl)-1,2,5,6-tetrahydropyrido[4,3-d]-
3,4-dihydropyrimidine-2-thione (3c) Yield: 83%;
R2
-H2O
N
1
OH-
yellow solid, m.p.: 179—181 ℃; H NMR (400 MHz,
DMSO) δ: 9.48 (s, 1H), 9.07 (s, 1H), 7.25—7.07 (m,
13H), 4.77 (s, 1H), 3.59—3.37 (m, 4H), 3.08 (d, J＝
16.6 Hz, 1H), 2.87 (tt, J＝13.5, 6.8 Hz, 2H), 2.75 (d,
J＝16.4 Hz, 1H), 1.19—1.17 (m, 12H); IR (KBr) ν:
3434, 2390, 2340, 1718, 1637, 1421, 1363, 1120, 861
cm ; ESI-MS ([M＋H] ) m/z: 521.29. Anal. calcd for
C34H39N3S: C 78.27, H 7.53, N 8.05; found C 78.25, H
7.52, N 8.06.
R1
1
O
N
S
R2
－
＋
R2
1
H2N
OH-
NH2
4-(4-Methylphenyl)-6-(4-methylbenzyl)-8-(4-meth-
ylbenzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
R1
2
dihydropyrimidine-2-thione (3d)
Yield: 87%;
NH2
NH
1
yellow solid, m.p.: 178—181 ℃; H NMR (400 MHz,
DMSO) δ: 9.48 (s, 1H), 9.04 (s, 1H), 7.17—6.85 (m,
13H), 4.77 (s, 1H), 3.59—3.28 (m, 4H), 3.03 (d, J＝
16.45 Hz, 1H), 2.70 (d, J＝16.4 Hz, 1H), 2.21 (s, 3H),
1.94 (s, 6H); IR (KBr) ν: 3764, 2396, 1637, 1577, 1428,
S
O
N
OH-
-H2O
R2
R2
－
＋
1
1363, 1064, 861 cm ; ESI-MS ([M＋H] ) m/z: 465.65.
Anal. calcd for C30H31N3S C: 77.38, H 6.71, N 9.02;
found C 77.37, H 6.72, N 9.02.
R1
S
4-(4-Fluorophenyl)-6-(4-methylbenzyl)-8-(4-fluo-
robenzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3e) Yield: 78%; yellow
solid, m.p.: 175—177 ℃; 1H NMR (400 MHz, DMSO)
δ: 9.52 (s,1H) , 9.14 (s, 1H), 7.29—6.97 (m, 13H), 4.87
(s, 1H), 3.92—3.52 (m, 4H), 3.07 (d, J＝16.7 Hz, 1H),
2.70 (d, J＝16.5 Hz, 1H), 2.21 (s, 3H); IR (KBr) ν: 3422,
2851, 2026, 1633, 1600, 1564, 1506, 1155, 1077, 854
HN
NH
N
R2
R2
3
R1
and NaOH (2 mmol) in ethanol (50 mL), was heated to
65 ℃ for 5 min, and stirred at the same temperature for
10—20 min. After cooling to room temperature, 20 mL
of water was added and the mixture was neutralized to
pH 6 using 10% HCl solution. The separated solid was
filtered, washed with water, dried, and recrystallized
(ethanol) to afford the target compounds.
8-(2-Fluorobenzylidene)-4-(2-fluorophenyl)-6-(4-
methylbezyl)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3a) Yield: 92%; white
solid, m.p.: 132—134 ℃; 1H NMR (400 MHz, DMSO)
δ: 9.75 (s, 1 H), 9.07 (s, 1H), 6.91—7.40 (m, 13H), 5.13
(s, 1H), 3.31—3.42 (m, 4H), 3.11 (d, J＝18.0 Hz, 1H),
2.73 (d, J＝16.6 Hz, 1H), 2.21 (s, 3H); IR (KBr) ν: 3424,
1567, 1485, 1454, 1099, 988, 857 cm ; ESI-MS ([M＋
H]＋) m/z: 474.17. Anal. calcd for C28H25F2N3S: C 71.17,
H 5.32, N 8.87; found C 71.20, H 5.30, N 8.88.
4-(4-Methoxylphenyl)-6-(4-methylbenzyl)-8-(4-
methoxylbezylidene)-1,2,5,6-tetrahydropyrido[4,3-
d]-3,4-dihydropyrimidine-2-thione (3b) Yield: 78%;
－
＋
1
cm ; ESI-MS ([M＋H] ) m/z: 473.71. Anal. calcd for
C28H28F2N3S: C 71.01, H 5.32, N 8.87; found C 71.00,
H 5.30, N 8.86.
8-(2-Chlorobenzylidene)-4-(2-chlorophenyl)-6-(4-
methylbenzyl)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3f) Yield: 86%; white
solid, m.p.: 102—104 ℃; 1H NMR (400 MHz, DMSO)
δ: 9.59 (s, 1H), 9.20 (s, 1H), 7.47—6.96 (m, 13H), 4.92
(s, 1H), 3.53—3.34 (m, 4H), 3.12 (d, J＝16.7 Hz, 1H),
2.75 (d, J＝16.6 Hz, 1H), 2.22 (s, 3H); IR (KBr) ν: 3422,
－
1
2342, 1560, 1506, 1451, 1075, 990, 856 cm ; ESI-MS
([M＋H]＋) m/z: 507.11. Anal. calcd for C28H25Cl2N3S:
C 66.40, H 4.98, N 8.30; found C 66.41, H 4.98, N 8.31.
8-Benzylidene-4-phenyl-6-(4-methylbenzyl)-1,2,5,
6-tetrahydropyrido[4,3-d]-3,4-dihydropyrimidine-2-
thione (3g) Yield: 91%; white solid, m.p.: 174—176
℃; 1H NMR (400 MHz, DMSO) δ: 9.53 (s, 1H), 9.08 (s,
1H), 7.53—6.96 (m, 15H), 4.85 (s, 1H), 3.61—3.36 (m,
4H), 3.12 (d, J＝16.7 Hz, 1H), 2.74 (d, J＝16.5 Hz, 1H),
2.22 (s, 3H); IR (KBr) ν: 3425, 2347, 1561, 1546, 1456,
－
1
1
white solid, m.p.: 110—112 ℃; H NMR (400 MHz,
－
＋
1
DMSO) δ: 9.40 (s, 1H), 9.04 (s, 1H), 7.17—6.85 (m,
13H), 4.74 (s, 1H), 3.74 (s, 6H), 3.28—3.55 (m, 4H),
3.03 (d, J＝16.45 Hz, 1H), 2.69 (d, J＝16.3 Hz, 1H),
1175, 990, 856 cm ; ESI-MS ([M＋H] ) m/z: 437.20.
Anal. calcd for C28H27N3S: C 76.85, H 6.22, N 9.60;
found C 76.85, H 6.20, N 9.61.
Chin. J. Chem. 2012, 30, 2509—2516
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Ding et al.
FULL PAPER
8-(4-Chlorobenzylidene)-4-(4-chlorophenyl)-6-(4-
methylbenzyl)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3h) Yield: 76%; white
(s, 1H), 3.61—3.36 (m, 4H), 3.08 (d, J＝16.75 Hz, 1H),
2.70 (d, J＝16.61 Hz, 1H); IR (KBr) ν: 3416, 1570,
1500, 1473, 1077, 989, 879, 547 cm ; ESI-MS ([M＋
H]＋) m/z: 478.15. Anal. calcd for C27H22F3N3S: C 67.91,
H 4.64, N 8.80; found C 67.90, H 4.65, N 8.81.
－
1
1
solid, m.p.: 108—109 ℃; H NMR (400 MHz, DMSO)
δ: 9.59 (s, 1H), 9.20 (s, 1H), 7.46—6.65 (m, 13H), 4.92
(s, 1H), 3.60—3.34 (m, 4H), 3.12 (d, J＝16.7 Hz, 1H),
2.75 (d, J＝16.7 Hz, 1H), 2.20 (s, 3H); IR (KBr) ν: 3426,
4-(2-Chlorophenyl)-8-(2-chlorobenzylidene)-6-(4-
fluorobenzyl)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
－
1
＋
1605, 1506, 1440, 1077, 988 cm ; ESI-MS ([M＋H] )
m/z: 486.17. Anal. calcd for C28H25Cl2N3S: C 66.40, H
6.98, N 8.30; found C 66.43, H 6.97, N 8.28.
dihydropyrimidine-2-thione (3n)
Yield: 77%;
1
yellow solid, m.p.: 110—112 ℃; H NMR (400 MHz,
DMSO) δ: 9.81 (s, 1H) , 9.08 (s, 1H), 7.56—6.92 (m,
13H), 5.32 (s, 1H), 3.47—3.12 (m, 4H), 3.14 (d, J＝
16.7 Hz, 1H), 2.71 (d, J＝16.8 Hz, 1H); IR (KBr) ν:
3426, 1601, 1569, 1457, 1157, 1077, 858, 544 cm ;
MS (ESI) ([M＋H]＋) m/z: 509.21. Anal. calcd for
C27H22FCl2N3S: C 63.53, H 4.34, N 8.23; found C 63.52,
H 4.31, N 8.24.
4-(2-Fluorophenyl)-6-(4-fluorobenzyl)-8-(2-fluoro-
benzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3i) Yield: 94%; white
－
1
1
solid, m.p.: 106—108 ℃; H NMR (400 MHz, DMSO)
δ: 9.53 (s, 1H), 9.14 (s, 1H), 7.40—6.90 (m, 13H), 5.14
(m, 4H), 3.13 (d, J＝17.0 Hz, 1H), 2.74 (d, J＝16.7 Hz,
1H); IR (KBr) ν: 3426, 1567, 1465, 1453, 1077, 858;
ESI-MS ([M ＋H] ＋ ) m/z: 478.15. Anal. calcd for
C27H22F3N3S: C 67.91, H 4.64, N 8.80; found C 67.90,
H 4.65, N 8.81.
6-(4-Fluorobenzyl)-8-benzylidene-4-phenyl-1,2,5,
6-tetrahydropyrido[4,3-d]-3,4-dihydropyrimidine-2-
thione (3o) Yield: 91%; white solid, m.p.: 174—176
℃; 1H NMR (400 MHz, DMSO) δ: 9.53 (s, 1H), 9.13 (s,
1H), 7.41—6.97 (m, 15H), 4.85 (s, 1H), 3.61—3.20 (m,
4H), 3.12 (d, J＝16.5 Hz, 1H), 2.74 (d, J＝16.4 Hz, 1H);
IR (KBr) ν: 3425, 1568, 1510, 1452, 1076, 985, 879,
6-(4-Fluorobenzyl)-4-(4-methoxylphenyl)-8-(4-
methoxylbenzylidene)-1,2,5,6-tetrahydropyrido[4,3-
d]-3,4-dihydropyrimidine-2-thione (3j) Yield: 75%;
－
＋
1
1
yellow solid, m.p.: 117—119 ℃; H NMR (400 MHz,
858 cm ; ESI-MS ([M＋H] ) m/z: 441.19. Anal. calcd
for C27H24FN3S: C 73.44, H 5.48, N 9.52; found C
73.45, H 5.50, N 9.54.
DMSO) δ: 9.42 (s, 1H), 9.05 (s, 1H), 7.17—6.95 (m,
13H), 4.77 (s, 1H), 3.74 (s, 6H), 3.61—3.34 (m, 4H),
3.03 (d, J＝15.7 Hz, 1H), 2.69 (d, J＝17.6 Hz, 1H); IR
(KBr) ν: 3414, 1605, 1568, 1509, 1441, 1174, 1078, 858
4-(4-Chlorophenyl)-8-(4-chlorobenzylidene)-6-(4-
fluorobenzyl)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
－
＋
1
cm ; ESI-MS ([M＋H] ) m/z: 501.19. Anal. calcd for
C29H28FN3O2S: C 69.44, H 5.63, N 8.38; found C 59.45,
H 5.61, N 8.40.
dihydropyrimidine-2-thione (3p)
Yield: 75%;
1
yellow solid, m.p.: 116—118 ℃; H NMR (400 MHz,
DMSO) δ: 9.81 (s, 1H) , 9.05 (s, 1H), 7.55—6.92 (m,
13H), 5.32 (s, 1H), 3.42—3.12 (m, 4H), 3.14 (d, J＝
17.3 Hz, 1H), 2.71 (d, J＝16.8 Hz, 1H); IR (KBr) ν:
3427, 1601, 1569, 1457, 1157, 1077, 858 cm ; MS
(ESI) ([M ＋H] ＋ ) m/z: 510.09. Anal. calcd for
C27H22Cl2N3S: C 63.53, H 4.34, N 8.23; found C 63.52,
H 4.31, N 8.24.
6-(4-Fluorobenzyl)-4-(4-isopropylphenyl)-8-(4-
isopropylbenzylidene)-1,2,5,6-tetrahydropyrido[4,3-
d]-3,4-dihydropyrimidine-2-thione (3k) Yield: 85%;
－
1
1
white solid, m.p.: 117—119 ℃; H NMR (400 MHz,
DMSO) δ: 9.48 (s, 1H), 9.06 (s, 1H), 7.34—6.92 (m,
13H), 4.78 (s, 1H), 3.59—3.31 (m, 4H), 3.07 (d, J＝
16.7 Hz, 1H), 2.87 (d, J＝12.1 Hz, 2H), 2.74 (d, J＝
16.4 Hz, 1H), 1.19—1.16 (m, 12H); IR (KBr) ν: 3414,
6-(4-Bromobenzyl)-4-(2-fluorophenyl)-8-(2-fluoro-
benzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
－
1
1604, 1568, 1509, 1441, 1174, 988, 858 cm ; ESI-MS
([M＋H]＋) m/z: 526.26. Anal. calcd for C33H36FN3S: C
75.39, H 6.90, N 7.99; found C 75.41, H 6.88, N 8.00.
6-(4-Fluorobenzyl)-4-(4-methylphenyl)-8-(4-meth-
ylbenzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3l) Yield: 80%; yellow
dihydropyrimidine-2-thione (3q)
Yield: 80%;
1
yellow solid, m.p.: 119—121 ℃; H NMR (400 MHz,
DMSO) δ: 9.75 (s, 1H), 9.07 (s, 1H), 7.41—7.00 (m,
13H), 5.14 (s, 1H), 3.43—3.15 (m, 4H), 3.14 (d, J＝
16.7 Hz, 1H), 2.73 (d, J＝16.8 Hz, 1H); IR (KBr) ν:
3417, 1556, 1487, 1454, 1152, 1099, 1071, 856, 758 cm
－
solid, m.p.: 132—134 ℃; H NMR (400 MHz, DMSO)
1; MS (ESI) ([M＋H]＋) m/z: 537.06. Anal. calcd for
1
δ: 9.46 (s, 1H), 9.08 (s, 1H), 7.20—6.99 (m, 13H), 4.78
(s, 1H), 3.56—3.26 (m, 4H), 3.06 (d, J＝16.6 Hz, 1H),
2.71 (d, J＝16.3 Hz, 1H), 2.30 (s, 3H), 2.28 (s, 3H); IR
(KBr) ν: 3426, 1567, 1500, 1453, 1077, 989, 879, 858
cm ; ESI-MS ([M＋H] ) m/z: 469.70. Anal. calcd for
C29H28FN3S: C 74.17, H 6.01, N 8.95; found C 74.14, H
6.00, N 8.97.
C28H25F2BrN3S: C 60.23, H 4.12, N 7.80; found C 60.25,
H 4.12, N 7.83.
6-(4-Bromobenzyl)-4-(4-methoxylphenyl)-8-(4-
methoxylbenzylidene)-1,2,5,6-tetrahydropyrido[4,3-
d]-3,4-dihydropyrimidine-2-thione (3r) Yield: 86%;
－
＋
1
1
white solid, m.p.: 175—177 ℃; H NMR (400 MHz,
DMSO) δ: 9.46 (s, 1H), 9.08 (s, 1H), 7.45—6.89 (m,
13H), 4.78 (s, 1H), 3.29—3.59 (m, 4H), 3.07 (d, J＝
16.6 Hz, 1H), 2.70 (d, J＝16.4 Hz, 1H), 2.29 (s, 6H); IR
(KBr) ν: 3427, 1566, 1530, 1403, 1078, 988, 877, 856
4-(4-Fluorophenyl)-6-(4-fluorobenzyl)-8-(4-fluoro-
benzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3m) Yield: 94%; white
－
＋
1
1
solid, m.p.: 106—108 ℃; H NMR (400 MHz, DMSO)
cm ; ESI-MS ([M＋H] ) m/z: 562.12. Anal. calcd for
δ: 9.53 (s, 1H), 9.14 (s, 1H), 7.30—6.98 (m, 13H), 4.88
C29H28BrN3O2S: C 65.65, H 5.32, N 7.92; found C
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 2509—2516

Synthesis, and Anticancer Activities of Tetrahydropyrido[4,3-d]dihydropyrimidine-2-thiones
65.64, H 5.33, N 7.93.
δ: 9.58 (s, 1H), 9.16 (s, 1H), 7.47—7.04 (m, 13H), 4.89
6-(4-Bromobenzyl)-4-(4-isopropylphenyl)-8-(4-
isopropylbenzylidene)-1,2,5,6-tetrahydropyrido[4,3-
d]-3,4-dihydropyrimidine-2-thione (3s) Yield: 89%;
(s, 1H), 3.53—3.07 (m, 4H), 3.10 (d, J＝16.8 Hz, 1H),
2.71 (d, J＝16.6 Hz, 1H); IR (KBr) ν: 3426, 1567, 1500,
1453, 1077, 989, 879, 858 cm ; ESI-MS ([M＋H] )
m/z: 570.01. Anal. calcd for C27H22BrCl2N3S: C 56.76,
H 3.88, N 7.35; found C 56.75, H 3.88, N 7.36.
－
＋
1
1
white solid, m.p.: 121—123 ℃; H NMR (400 MHz,
DMSO) δ: 9.48 (s, 1H), 9.05 (s, 1H), 7.34—6.82 (m,
13H), 4.78 (s, 1H), 3.55—3.31 (m, 4H), 3.07 (d, J＝
16.7 Hz, 1H), 2.87 (d, J＝12.1 Hz, 2H), 2.74 (d, J＝
16.4 Hz, 1H), 1.19—1.16 (m, 12H); IR (KBr) ν: 3426,
X-ray crystallography
A chartreuse crystal of compound 3j recrystallized
by slow evaporation from ethyl acetate at room tem-
perature, with dimensions of 0.12 mm×0.10 mm×0.10
mm was mounted on a glass fiber for data collection
which was made on α BRUKER SMART APEX 1000
CCD diffractometer equipped with a graphite-mono-
chromatic MoKα radiation (λ＝0.71073 Å) by using a
φ-ω scan mode in the range of 0.85°≤θ≤22.87° (－36
≤h≤36, 0≤k≤52, 0≤l≤28) at 173 K. A total of
21124 reflections were collected with 6366 unique ones
(Rint＝0.0545), of which 3804 with I＞2σ(I) were con-
sidered as observed and used in the succeeding refine-
ments. The intensity data were corrected for Lp factors
and empirical absorption. All of the non-hydrogen at-
oms were refined anisotropically, and hydrogen atoms
were located at their idealized positions. The structure
was solved by direct methods and refined by full-matrix
least-squares techniques on F2 using the SHELXTL
program package. The final R＝0.0601, wR＝0.1505
－
1
1567, 1500, 1453, 1077, 989, 879, 858 cm ; ESI-MS
([M＋H]＋) m/z: 587.26. Anal. calcd for C33H36BrN3S: C
75.39, H 6.90, N 7.99; found C 75.41, H 6.88, N 8.00.
6-(4-Bromobenzyl)-4-(4-methylphenyl)-8-(4-meth-
ylbenzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3t) Yield: 86%; white
solid, m.p.: 175—177 ℃; 1H NMR (400 MHz, DMSO)
δ: 9.48 (s, 1H), 9.07 (s, 1H), 7.37—7.04 (m, 13H), 4.78
(s, 1H), 3.59—3.29 (m, 4H), 3.07 (d, J＝16.6 Hz, 1H),
2.70 (d, J＝16.4 Hz, 1H), 2.31—2.29 (m, 6H); IR (KBr)
－
1
ν: 3427, 1567, 1500, 1453, 1077, 989, 879, 858 cm ;
ESI-MS ([M ＋H] ＋ ) m/z: 530.12. Anal. calcd for
C29H28BrN3S: C 65.65, H 5.32, N 7.92; found C 65.64,
H 5.33, N 7.93.
6-(4-Bromobenzyl)-4-(4-fluorophenyl)-8-(4-fluoro-
benzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-di-
hydropyrimidine-2-thione (3u) Yield: 81%; orange
1
solid, m.p.: 134—136 ℃; H NMR (400 MHz, DMSO)
δ: 9.53 (s, 1H), 9.15 (s, 1H), 7.47—7.02 (m, 13H), 4.88
(s, 1H), 3.53—3.29 (m, 4H), 3.09 (d, J＝16.7 Hz, 1H),
2.70 (d, J＝16.5 Hz, 1H); IR (KBr) ν: 3416, 1601, 1569,
(w＝1/[σ2( F2 )＋(0.0568P)2 ＋0.2960P], where P＝
o
( F2 ＋2 F2 )/3), S＝1.025, (Δ/σ)max＝0.143, (Δρ)max＝
o
c
0.519 and (Δρ)min＝－0.315 e/Å3. The structural graph-
ics were drawn with SHELXTL-97 software package.
Other details of the structure have been deposited with
the Cambridge Crystallographic Data Centre, 120302F.
－
1
1506, 1484, 1429, 1157, 1073, 841, 647 cm ; MS (ESI)
([M＋H]＋) m/z: 537.89. Anal. calcd for C27H22BrF2N3S:
C 60.23, H 4.12, N 7.80; found C 60.20, H 4.10, N 7.81.
6-(4-Bromobenzyl)-4-(2-chlorophenyl)-8-(2-chlo-
robenzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3v) Yield: 77%; yel-
Treatment of tumor cell lines
The antiproliferative activities of the title com-
pounds and 5-FU against leukemia K562 cells,
HO-8910 cells and liver cancer SMMC-7721 cells were
determined by the MTT assay according to the standard
bioactivity test procedures.[19] Leukemic K562 cells,
ovarian cancer HO-8910 cells and liver cancer
SMMC-7721 cells were cultured in RPMI 1640 medium
at 37 ℃ with 5% CO2, and 95% air, supplemented
with 10% (V/V) bovine calf serum and 80 U/mL gen-
tamicin. The cells were seeded onto 96-well plates or
other appropriate dishes containing the medium at the
density of 6250/cm2. About 10 mg of the title com-
pounds were dissolved in 100 μL of dimethylsulfoxide
(DMSO), then concentrations of 1.0, 10.0, 100.0 μg/mL
were adjusted stepwise in each 96-well plates, and the
concentration of DMSO was 1‰. One hundred thou-
sand cells, 20 μL of FBS, RPMI-1640 and diversified
concentration title compounds were seeded in each 100
μL well of a 96-well plate. Each group had eight wells.
Set up the control well of remedy colour (5-FU) and
incubate the well (containing cells). After 44 h in a hu-
midified atmosphere with 5% CO2 at 37 ℃, add 10 μL
of MTT (5 mg/mL) into every well, then rejoin 100 μL
1
low solid, m.p.: 110—112 ℃; H NMR (400 MHz,
DMSO) δ: 9.58 (s, 1H) , 9.16 (s, 1H), 7.56—7.14 (m,
13H), 5.35 (s, 1H), 3.53—3.32 (m, 4H), 3.10 (d, J＝
16.7 Hz, 1H), 2.71 (d, J＝16.8 Hz, 1H); IR (KBr) ν:
－
1
3427, 1601, 1569, 1457, 1157, 1077, 858 cm ; MS
(ESI) ([M ＋H] ＋ ) m/z: 571.31. Anal. calcd for
C27H22BrCl2N3S: C 56.76, H 3.88, N 7.35; found C
56.77, H 3.89, N 7.34.
6-(4-Bromobenzyl)-8-benzylidene-4-phenyl-1,2,5,
6-tetrahydropyrido[4,3-d]-3,4-dihydropyrimidine-2-
thione (3w) Yield: 91%; white solid, m.p.: 174—176
℃; 1H NMR (400 MHz, DMSO) δ: 9.55 (s, 1H), 9.13 (s,
1H), 7.53—7.04 (m, 15H), 4.85 (s, 1H), 3.61—3.36 (m,
4H), 3.12 (d, J＝16.7 Hz, 1H), 2.74 (d, J＝16.5 Hz, 1H);
IR (KBr) ν: 3428, 1565, 1506, 1455, 1077, 989, 879,
858; ESI-MS([M＋H]＋) m/z: 502.09. Anal. calcd for
C27H24BrN3S: C 64.54, H 4.81, N 8.36; found C 64.55,
H 4.80, N 8.36.
6-(4-Bromobenzyl)-4-(4-chlorophenyl)-8-(4-chlo-
robenzylidene)-1,2,5,6-tetrahydropyrido[4,3-d]-3,4-
dihydropyrimidine-2-thione (3x) Yield: 89%; white
1
solid, m.p.: 179—181 ℃; H NMR (400 MHz, DMSO)
Chin. J. Chem. 2012, 30, 2509—2516
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2513

Ding et al.
FULL PAPER
of 10% SDS to stop the reaction after 4 h. One night
later at 37 ℃, the number of absorbency (A) of every
well was measured by enzyme immunity at 570 nm.
Equation of growth inhibition of cells was shown as
follows.
was separated on 1.5% agarose gels containing 0.5
mg/mL ethidium bromide and photographed by Bio-Rad
GD2000 (Bio-Rad, Hercules, CA, USA).
Statistical analysis
Caspase-3/7 data are presented as means ±SE of at
least three independent experiments. In addition, each
caspase-3/7 experiment was performed using triplicate
and duplicate wells, respectively. All fluorescence cas-
pase-3/7 data were first blank corrected and then con-
verted into rates of AFC produced per hour. These val-
ues were statistically compared to the controls using
one-way ANOVA with a Dunnett's post test using InStat
version 3.0a (GraphPad) where p＜0.05 was considered
significant.
Inhibition [1 A(experiment)/A(control)] 100%
＝ －
×
Apoptosis detection
Fluorescein isothiocyanate (FITC)-conjugated an-
nexin V has been utilized to detect the externalization of
phosphatidylserine that occurs at an early stage of
apoptosis. Propidium iodide (PI) is used as a marker of
necrosis due to cell membrane destruction.[21] To eluci-
date whether compound-induced cell death involved
apoptosis or necrosis, we performed a biparametric cy-
tofluorimetric analysis using annexin V and PI dou-
ble-staining as shown in Figure 2. Treatment of leuke-
mic K562 cells with 1, 10 and 100 μg/mL 3i respec-
tively, for 24 h induced apoptosis effects (annexin V＋/
PI－) in 2.70% (Control), 6.05% (1 μg/mL), 33.00% (10
μg/mL) and 81.26% (100 μg/mL) of annexin V-FITC
cells.
Result and Discussion
Evaluation of anticancer activities
The antiproliferative activities (IC50) of compounds
3a—3x and 5-FU were presented in Table 1, and the
effects of 3i on the morphology of leukemic K562 cells
at 1, 10, 100 μg/mL cultured after 44 h are shown in
Figre 1.
Western blotting
After treatment, cell pellets were collected and lysed
in a lysis buffer [150 mmol/L NaCl, 50 mmol/L Tris-
HCl pH 8.0, 2 mmol/L ethylene glycol-bis(β-aminoethyl
ether), 2 mmol/L EDTA, 25 mmol/L NaF, 25 mmol/L
β-glycerophosphate, 0.2% Triton X-100, 0.3% Nonidet
P-40, and 0.1 mmol/L phenylmethylsulfonyl fluoride].
Total protein concentrations of whole cell lysis were
determined using BioRad BCA method (PIERCE,
Rockford, IL). Equal amounts of protein sampled from
whole cell lysis were subjected to electrophoresis on
10%—12% Tris-Glycine pre-cast gels (Novex, San
Diego, CA) and electroblotted onto Immobilon-P
Transfer Membrane (Millipore Corporation, Billerica,
Massachusetts), and probed with primary antibodies and
then incubated with a horseradish peroxidase (HRP)
conjugated secondary antibodies. Proteins were visual-
ized using enhanced chemiluminescence (ECL) Western
Blotting detection reagents (Amersham Biosciences,
Piscataway, NJ).
Control
1 µg/mL
10 µg/mL
100 µg/mL
Figure 1 Leukemic K562 cells act on 3i at 1, 10, 100 μg/mL
cultured after 44 h.
Electrophoretic analysis of DNA fragmentation
According to the Table 1, most of the title com-
pounds exhibited good antiproliferative activities
against leukemic K562 cells, ovarian cancer HO-8910
cells and liver cancer SMMC-7721 cells in vitro.
Among them 3i and 3m afforded the best activities in
vitro. The IC50 of them were 3.22 and 3.65 μg/mL
against leukemic K562 cells respectively, which were
lower than the anticancer drug of clinical practice 5-FU
(IC50＝8.56 μg/mL).
As shown in Table 1, when the different substituents
R1 and R2 were introduced to the title compounds, the
antiproliferative activities varied greatly. Considering
the influence of R1 group, when the R2 group is same,
the compounds bearing F (3i—3p) had shown relatively
Leukemia K562 cells were lysed in 200.0 mL lysis
buffer (10.0 mmol/L EDTA; 50.0 mmol/L Tris-HCl, pH
8.0; 0.5% sodium lauryl sulfate; 100.0 mg/mL pro-
teinase K) at 37 ℃ for 12 h, then incubated with RNase
(50.0 mg/mL) at 37 ℃ for an additional 1 h. After
incubation, DNA in the lysate was extracted with equal
volume of phenol/chloroform/isoamyl alcohol (25∶
24∶1), then with chloroform. DNA was precipitated
with two volumes of ethanol in the presence of 0.3
mol/L sodium acetate. After centrifugation at 12000 g
for 15 min, the DNA pellets were washed with 70%
ethanol, air-dried, and resuspended in 20.0 mL TE (10.0
mmol/L Tris-HCl and 1.0 mmol/L EDTA, pH 8.0). DNA
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Chin. J. Chem. 2012, 30, 2509—2516

Synthesis, and Anticancer Activities of Tetrahydropyrido[4,3-d]dihydropyrimidine-2-thiones
better antiproliferative activities than those bearing Br
and Me, the activities increased in the order: 3e (R1＝
Me, R2＝4-F)＜3u (R1＝Br, R2＝4-F)＜3m (R1＝F,
R2＝4-F). When the titie compounds bearing different
R2, such as 3i—3p, their activities decreased in the orde:
3i (R2＝2-F)＞3m (R2＝4-F)＞3p (R2＝4-Cl)＞3n
(R2＝2-Cl)＞3o (R2＝H)＞3j (R2＝4-OMe)＞3l (R2＝
4Me) ＞3k [R2 ＝4-CH(Me)2]. These results clearly
suggest that, when R were electron-withdrawing sub-
stituents, such as single F or Cl atom, the activities of
the title compounds were higher than those bearing
electron-donating substituents, such as OMe, Me,
CH(Me)2. Considering the discussion above, we found
that the antiproliferative activities of our designed
compounds could be increased by the introduction of
fluoro, chloro on benzene rings, further study is under-
way. The results further suggested that small differences
between the structures could lead to large differences in
the overall activities, which implies further possibilities
for lead compound development.
Figure 3s. Figures 1s—3s are in the Supporting Infor-
mation.
As shown in Figure 1s, treatment of leukemic K562
cells with 1, 10 and 100 μg/mL 3i respectively, for 24 h,
induced apoptosis effects (annexin V＋/PI－) in 2.70%
(Control), 6.05% (1 μg/mL), 33.00% (10 μg/mL) and
81.26% (100 μg/mL) of annexin V-FITC cells. The re-
sults showed that 3i induced more apoptotic cells than
that of the untreated control. In addition, the apoptosis
of leukemic K562 cells was determined by caspase-3/7
Kit and Electrophoretic Analysis of DNA Fragmenta-
tion (Figure 2s and Figure 3s). We found that compared
with control cells, cells exposed to 3i slightly increased
caspase-3/7 activity, and resulted in a characteristic
fragmentation of DNA, a common feature of apoptotic
cell death. DNA fragmentation was later conducted in
apoptotic cells, compared with increasing caspase-3/7
activity. These theoretical events may explain that
treating with 10 µg/mL 3i, a severely high dose, could
induce leukemic K562 cells DNA fragmentation. Taken
together, all results suggested that apoptosis pathway
was involved in 3i induced antiproliferative activity in
Leukemic K562 cells.
Table 1 Antiproliferative activities (IC50) of the compounds 3a
—3x and 5-FU at 48 h
IC50/(μg•mL－
)
1
To confirm the three-dimensional information of the
title compounds, the single-crystal structure of com-
pound 3j was investigated and determined by X-ray
diffraction analysis. Molecular structure of compound 3j
with atom-labeling is shown in Figure 2 and the packing
diagram in Figure 4s. The crystallographic information
files of 3j and Figure 4s are in the Supporting Informa-
tion.
Compd. R1
R2
Leukemic Ovarian can- Liver cancer
K562 cer HO-8910 SMMC-7721
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
17.17
38.43
55.67
45.42
16.21
17.45
31.06
19.67
3.22
14.32
24.56
21.65
3.65
33.21
62.54
87.65
63.17
39.46
40.21
43.55
41.36
20.17
32.65
78.32
34.65
19.65
24.33
34.72
22.56
24.36
43.54
78.45
35.32
32.17
33.46
39.43
35.71
14.46
47.17
88.43
145.67
105.42
46.21
48.45
51.06
46.67
21.22
64.32
124.5
71.65
23.65
26.74
31.21
27.11
29.32
83.24
127.82
91.42
31.27
46.56
40.06
47.32
13.7
Me 2-F
Me 4-OMe
Me 4-CH(CH3)2
Me 4-Me
Me 4-F
Me 2-Cl
Me 4-H
Me 4-Cl
F
F
F
2-F
4-OMe
4-CH(CH3)2
4-Me
4-F
F
F
F
F
9.74
11.21
9.11
2-Cl
4-H
4-Cl
2-F
F
7.32
Br
Br
Br
Br
Br
Br
Br
Br
11.24
27.82
21.42
6.27
13.56
15.06
17.32
8.56
4-OMe
4-CH(CH3)2
4-Me
4-F
3s
3t
Figure 2
Molecular structure of compound 3j (Number
3u
3v
3w
3x
5-FU
120302F) with atom-labeling.
2-Cl
4-H
4-Cl
Conclusions
A new series of tetrahydropyrido[4,3-d]dihydropyri-
midine-2-thiones (3a—3x) have been designed and
synthesized. The conformation of the compound 3j was
confirmed by X-ray diffraction. Most of the title com-
pounds showed good antiproliferative activity against
human cancer cell lines, including leukemic K562,
ovarian cancer HO-8910 and liver cancer SMMC-7721
in vitro. Among them, the 3i and 3m were the most
The results of preliminary mechanism studaies are
shown in Figure 1s—3s. The Figure 1s is the flow
analysis figure of 3i induced apoptosis of K562 cells,
the effect of 3i on the activation of caspase-3/7 is shown
in Figure 2s and the DNA electrophoresis is shown in
Chin. J. Chem. 2012, 30, 2509—2516
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2515

Ding et al.
FULL PAPER
[3] Petzer, A. L.; Gunsiliusb, E. Arch. Med. Res. 2003, 34, 496.
[4] El-Subbagh, H. I.; Abuzaid, S. M.; Mahran, M. A.; Badria, F. A.;
Al-Obaid, A. M. J. Med. Chem. 2000, 43, 2915.
[5] Dimmock, J. R.; Pati, H. N.; Das, U.; Das, S.; Bandy, B.; DeClercq,
E.; Balzarini, J.; Kawase, M.; Sakagami, H.; Quail, J. W.; Stables, J.
P. Eur. J. Med. Chem. 2010, 45, 4838.
[6] (a) Atwal, K. S.; Rovnyak, G. C.; O'Reilly, B. C.; Schwartz, J. J.
Org. Chem. 1989, 54, 5898; (b) Kappe, C. O.; Fabian, W. M. F.;
Semones, M. A. Tetrahedron 1997, 53, 2803.
[7] Stephen, M. R.; Raju, S. K.; Lawzer, A. L. Bioorg. Med. Chem. Lett.
2011, 21, 3012.
[8] (a) Rovnyak, G. C.; Kimball, S. D.; Moreland, S.; Gougoutas, G. Z.;
O'Reilly, B. C.; Schwartz, J.; Malley, M. F. J. Med. Chem. 1992, 35,
3254; (b) Alajarin, R. Bioorg. Med. Chem. 1994, 2, 323; (c)
Schleifer, K.-J. J. Med. Chem. 1999, 42, 2204.
promising compounds against the leukemic K562 cells,
the IC50 of them were 3.22 and 3.65 μg/mL, respectively,
which were lower than the anticancer drug of clinical
practice 5-FU (IC50＝8.56 μg/mL). In addition, pre-
liminary mechanism of action studies revealed that
compound 3i caused DNA fragmentation, and activated
caspase-3/7 in leukemia K562 cells. The results pro-
vided valuable information for the design of anticancer
agents. Future progress on related series will be reported
in due course.
Acknowledgment
The bioassay was supported by Zooblast-molecular
Biology Laboratory of Shanghai Normal University.
This work was supported by the National Natural Sci-
ence Foundation of China (Nos. 21042010, 21102092
and 30870560), the Key Scientific “Twelfth Five-Year”
[9] Pati, H. N.; Das, U.; Quail, J. W.; Kawase, M.; Sakagami, H.;
Dimmock, J. R. Eur. J. Med. Chem. 2008, 43, 1.
[10] Kappe, C. O.; Fabian, W. M. F.; Semones, M. A. Tetrahedron 1997,
53, 2803.
[11] Cotta, C. V.; Bueso-Ramos, C. E. Ann. Diagn. Pathol. 2007, 11, 68.
[12] Guo, H.; Tian, J. Chin. J. Org. Chem. 2011, 31, 1752.
[13] Stephen, M. R.; Raju, S. K.; Lawzer, A. L. Bioorg. Med. Chem. Lett.
2011, 21, 3012.
[14] Ashraf, M. M.; Abdel-Galil, E. A.; Musaed, A. A. Am. J. Biochem.
Biotech. 2011, 7, 43.
National
Technology
Support
Program
(No.
2011BAE06B01-17), the Innovation Project of Shang-
hai Education Commission (No. 12YZ078), the Key
Project of Science and Technology Commission of
Shanghai 105405503400), the Leading Academic Dis-
cipline Project of Shanghai Normal University (No.
DZL808), Shanghai Key Laboratory of Rare Earth
Functional Materials, Shanghai Normal University (No.
07dz22303). We are also grateful for the support from
Branch of National Pesticide R&D South Center.
[15] Wang, J.; Meng, W.; Ni, Z. J.; Xue, S. J. Chin. J. Chem. 2011, 29,
2421.
[16] Perez-Rebolledo, A.; Ayala, J. D.; de Lima, G. M.; Marchini, N.;
Atwal, K. S.; Hedberg, A.; Bombieri, G.; Zani, C. L.; Souza-
Fagundes, E. M.; Beraldo, H. Eur. J. Med. Chem. 2005, 40, 467.
[17] Rostom, S. A. F.; Hassan, G. S.; El-Subbagh, H. I. Arch. Der.
Pharm. (Weinheim) 2005, 338, 175.
[18] Huang, S.; Lin, R.; Yu, Y.; Lu, Y.; Lu, Y.; Li, S.; Emanuel, S. L.;
Middleton, S. A. Bioorg. Med. Chem. Lett. 2007, 17, 1243.
[19] Fang, Z. K.; Xue, S. J.; Chen, L.; Xu, Y.; Yin, A. Q.; Li, C. Y. Chin.
J. Chem. 2009, 27, 397.
[20] Tian, J. J.; Guo, H. Y. Chin. J. Org. Chem. 2011, 31, 2009.
[21] Wang, H. F.; Xue, S. J.; Zhu, J.; Yang, D. R.; Jin, J.; Fang, Z. K.
Chin. J. Struct. Chem. 2009, 28, 742.
References
[1] O’Connor, R. Curr. Cancer Drug Targets 2009, 9, 273.
[2] Simona, F.; Riccardo, A.; Marilisa, B.; Margherita, G.; Chiara, C.;
Isabelle, Z. C.; Michel, B.; Elise, C. K.; Giacomo, D. L. J. Proteome
Res. 2007, 6, 4330.
(Pan, B.; Qin, X.)
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