Synthesis of 9-(heteroarylmethylidene)amino derivatives of homocamptothecin with biological activities

Article abstract of DOI:10.1002/cbdv.201000271

Six 9-(heteroarylmethylidene)amino derivatives, 2a-2f, of homocamptothecin were synthesized for the first time by total synthesis in 22 steps and biologically evaluated as inhibitors of topoisomerase I. Moreover, the antitumor activities of 2a-2f against three human tumor cell lines, i.e., A-549, MDA-MB-435, and HCT-116, were determined and the results showed that compound 2c was the most active homocamptothecin derivative against the A-549 (IC ₅₀=0.046 μM) and HTC-116 tumor cells (IC₅₀=3.67 μM), with a ca. 50 times higher activity than the reference drug topotecan (TPT) against the lung cancer cell line A-549. Copyright

Full text of DOI:10.1002/cbdv.201000271

1266
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Synthesis of 9-(Heteroarylmethylidene)amino Derivatives of
Homocamptothecin with Biological Activities
by Wei Guo, Zhenyuan Miao*, Chunquan Sheng*, Jianzhong Yao, Wenfeng Liu, Lingjian Zhu,
Yongqiang Zhang, Pengfei Cheng, Guoqiang Dong, Chunlin Zhuang, and Wannian Zhang*
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, P. R. China
(W. Zhang, phone/fax: þ86-21-81871243; e-mail: zhangwnk@hotmail.com; Z. Miao,
phone/fax: þ86-21-81871233; e-mail: miaozhenyuan@hotmail.com; C. Sheng,
phone/fax: þ86-21-81871233; e-mail: shengcq@hotmail.com)
Six 9-(heteroarylmethylidene)amino derivatives, 2a–2f, of homocamptothecin were synthesized for
the first time by total synthesis in 22 steps and biologically evaluated as inhibitors of topoisomerase I.
Moreover, the antitumor activities of 2a–2f against three human tumor cell lines, i.e., A-549, MDA-MB-
435, and HCT-116, were determined and the results showed that compound 2c was the most active
homocamptothecin derivative against the A-549 (IC50 ¼0.046 mm) and HTC-116 tumor cells (IC50
¼
3.67 mm), with a ca. 50 times higher activity than the reference drug topotecan (TPT) against the lung
cancer cell line A-549.
Introduction. – Camptothecin (CPT), a pentacyclic alkaloid isolated in 1966 by
Wall et al. from Camptotheca acuminata, a tree native to China, showed broad-
spectrum antitumor activity [1]. Although CPT is a potent cytotoxic agent, early studies
in the 1970s revealed its severe and unpredictable toxicities [2]. Interest in CPT
derivatives was revitalized in 1985 by the discovery that CPT exhibits a unique
mechanism of action, because its target is the nuclear enzyme topoisomerase I (Topo I)
[3]. Numerous CPT derivatives were synthesized over the last 20 years and much of the
efforts were focusing on improving the H2O solubility and the activity of CPT
derivatives. Now, topotecan (TPT) [4], belotecan [5], and irinotecan [6] (Fig. 1) are
successfully used in clinical practice. Irinotecan, a prodrug marketed by Pfizer as
Camptosarꢀ, has been approved for metastatic colorectal carcinoma, while TPT, sold by
GlaxoSmithKline under the name Hycamtinꢀ, has been used to treat ovarian and small-
cell lung cancer [7].
In 1997, Lavergne et al. [8] described homocamptothecin (hCPT), a semi-synthetic
CPT analog with a modified E ring, in which the natural a-hydroxy-d-lactone E ring
was replaced by a b-hydroxy-e-lactone ring [8]. hCPT was found to inhibit Topo I-
mediated DNA relaxation with a potency comparable to that of CPT [9]. Furthermore,
hCPT also inhibited the growth of L1210 cells and it proved to be considerably more
potent than the reference compounds CPTand TPT. hCPT conserved Topo I-mediated
activity in vitro and in vivo, while showing increased plasma stability. And in contrast to
the lactone ring of CPT, which opened rapidly and reversibly, the lactone ring of hCPT
opened slowly and irreversibly [10][11]. Therefore, enlarging the E ring in hCPT
proved to be a real breakthrough in the competitive field of research of new CPT
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢂrich

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1267
Fig. 1. Chemical structures of camptothecin analogues
analogues, and this discovery has stimulated the design and synthesis of other CPT
derivatives.
In our previous study, a series of novel 9-benzylidenamino derivatives of hCPTwere
designed and synthesized via Friedlꢀnder cyclization [12]. Various substitutions,
including Me-, Me2N-, and MeO-group substitution, of the benzylidenamino residue
allowed obtaining derivatives with excellent cytotoxic activity in vitro and a Topo I-
inhibitory activity comparable or superior to that of TPT. Indeed, some compounds
showed higher inhibitory activities with IC50 values of 2.3 to 9.8 nm against breast
cancer cells than TPT. However, structureꢀactivity relationship (SAR) studies
indicated that hetaryl imine substitution could also improve the activities of CPT. In
this study, we present the synthesis of 9-(heteroarylmethylidene)amino derivatives of
hCPT, their spectroscopic characterization, and the results of biological activity assays,
in an attempt to find compounds with improved biological activity.
Results and Discussion. – Chemistry. The title compounds were prepared via
Friedlꢀnder cyclization of N-(2-acetyl-3-aminophenyl)acetamide (ring A) and a
building block that introduces rings C, D, and E into the final hCPT derivatives,
according to a previously reported synthetic method [12–19]. Various substituted
heteroaromatic aldehydes were reacted with 9-amino-7-methylhomocamptothecin (1)
to give the 9-(heteroarylmethylidene)amino derivatives 2a–2f (Scheme) [12].
Cytotoxicity. The six target compounds, 2a–2f, along with the reference compound
TPTwere tested, for their cytotoxicity against three cancer cell lines, i.e., A-549 (human

1268
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Scheme
a) Aromatic aldehydes, Yb(OTf)3, pyridine, r.t., 12–48 h; yield 20–33%.
lung adenocarcinoma epithelial cell line), MDA-MB-435 (human breast carcinoma cell
line), and HCT-116 (human colon carcinoma cell line), using the MTT method in vitro.
The IC50 values of the tested compounds, listed in the Table, showed that all the six
synthesized compounds exhibited obvious in vitro antitumor activity, especially against
the A-549 cell line. Among them, the 2’-thiophene substituted derivative 2c tended to
be the most active compound with a cytotoxic activity that was ca. 50 times higher than
that of the reference compound TPT against the A-549 cells. Moreover, it can be seen
that the compounds with a heteroatom at position 2’, i.e., 2b, 2c, and 2d, revealed better
antitumor activities against the lung adenocarcinoma epithelial cells than the other
hCPT derivatives.
Table. Cytotoxic Activity of the 9-(Heteroarylmethylidene)aminohomocamptothecin Derivatives against
Three Human Tumor Cell Lines
Compounds
IC50 [mm]a)
A-549
MDA-MB-435
HCT-116
2a
2b
2c
2d
2e
2f
0.31
0.36
0.046
0.97
1.24
3.52
2.44
24.80
>100
55.05
>100
81.46
6.45
10.85
3.67
21.67
28.79
27.06
4.97
87.46
21.79
Topotecan
a) The IC50 values were determined by using the MTT method.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1269
Topoisomerase I-Mediated DNACleavage. Negatively supercoiled plasmid pBR322
was incubated with human Topo I and concentrations of CPTand hCPTs at 10 mm. The
gel shown in Fig. 2 indicates that the level of negatively supercoiled DNA molecules
was considerably increased in the presence of the hCPT derivatives, and the Topo I
inhibitory activity was more potent than that observed for CPT under identical
conditions. It can be hypothesized that the presence of a heterocycle or a phenylimine
substituent in hCPT favors the Topo I–DNA–drug tertiary complex stability and
improves the inhibition of Topo I-mediated DNA cleavage compared to CPT.
Furthermore, compounds 2d, 2e, and 2f, substituted with a pyridine ring having the N-
atom at different positions, showed no distinguished difference at the same concen-
tration in this assay.
Fig. 2. Effect of compounds 2a–2f on Topoisomerase I-mediated DNA relaxation. Lane 1: supercoiled
DNA pBR322; Lane 2: supercoiled DNA and Topo I. Lanes 3–9: supercoiled DNA, Topo I, and 10 mm
of CPT, 2a, 2b, 2c, 2d, 2e, or 2f, respectively.
Molecular Docking. To improve our understanding of the interaction between the
hCPT derivatives and Topo I, the binding mode of 2d within the active site of Topo I in
comparison to that of hCPT was investigated using AutoDock 4.0. The results showed
that the binding mode of the hCPT derivatives within the active site of Topo I was
similar to that of hCPT. Their orientation in the active site cavity is perpendicular to the
main axis of the DNA and parallel to the bases. Both hCPT and compound 2d formed
two H-bonding interactions with Arg364 and Asp533 (Figs. 3 and 4, resp.). For
example, rings A and B of compound 2d are on the minor groove side of the
intercalation binding pocket and its N-atom formed a H-bond with Arg364 at a distance
of 1.91 ꢃ. The OH group at C(20) formed a H-bond with Asp533, a residue known to be
required for the enzyme sensitivity to CPT (Fig. 4). The molecular-docking results
suggest that compound 2d actually interacted with the active site, which provided a
good structural basis to explain the efficient inhibitory action.
Conclusions. – In summary, a series of new 9-(heteroarylmethylidene)amino
derivatives of hCPT have been synthesized and biologically evaluated as inhibitors of
Topo I. Within these series of compounds, 2c was found to have the best antitumor
activity with the broadest spectrum. The Topo I-mediated DNA cleavage assay showed
that all six hCPT derivatives were more potent Topo I inhibitors than CPT at a
concentration of 10 mm. Further in vivo evaluation and mechanistic studies on this novel
class of anticancer agents are currently in progress and will be reported in due course.

1270
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 3. Predicted binding mode of homocamptothecin to the topoisomerase I active site. The figure was
generated using PyMOL [20].
Fig. 4. Predicted binding mode of compound 2d to the topoisomerase I active site. The figure was
generated using PyMOL [20].
This research was supported in part by the Key Project of Science and Technology of Shanghai (No.
09431901700), the Major Special Project for the Creation of New Drugs (No. 2009ZX09301-011), and the
Shanghai Leading Academic Discipline Project (Project No. B906).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1271
Experimental Part
General. Commercial solvents were used without any further purification or pretreatment. Column
chromatography (CC): silica gel 60 G (SiO2; Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R.
China). TLC: precoated GF254 silica-gel plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R.
China). 1H- and 13C-NMR Spectra: Bruker 500 NMR spectrometer, at 500 and 125 MHz, resp., in
(D6)DMSO; d in ppm rel. to Me4Si, J in Hz. ESI-MS: API-3000 mass spectrometer; in m/z. Elemental
analysis: MOD-1106 instrument; analses were consistent with the theoretical values within ꢁ0.4%.
General Procedure for the Synthesis of the 9-(Heteroarylmethylidene)amino Derivatives, 2a–2f, of
Homocamptothecin. 9-Amino-7-methylhomocamptothecin (1; 100 mg, 0.26 mmol) and a substituted
aromatic aldehyde (0.78 mmol) were dissolved in pyridine (10 ml) with Yb(OTf)3 (16 mg, 0.03 mmol) as
the catalyst [12]. The resulting mixture was stirred at r.t. for 12 to 48 h. After filtration, the solvent was
removed, and the crude product was purified by flash chromatography (SiO2, CH2Cl2/MeOH 100 :2).
9-(Phenylmethylidene)aminohomocamptothecin (¼11-[(E)-Benzylideneamino]-5-ethyl-1,4,5,13-
tetrahydro-5-hydroxy-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 2a).
The general synthetic method described above afforded 2a (23%). Yellow solid. M.p. >3008.
1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.87 (q, J¼7.6, CH2(19)); 2.92 (s, Me(7)); 3.32 (q, J¼13.9,
CH2(21)); 5.25 (s, CH2(5)); 5.51 (q, J¼15.1, CH2(17)); 6.03 (s, HOꢀC(20)); 7.19 (d, J¼7.5, HꢀC(10));
7.39 (s, HꢀC(14)); 7.50–7.51 (m, HꢀC(3’), HꢀC(5’)); 7.80 (t, J¼8.0, HꢀC(11)); 7.93–8.04 (m, HꢀC(2’),
HꢀC(4’), HꢀC(6’), HꢀC(12)); 8.58 (s, HꢀC¼N). 13C-NMR: 8.6; 21.2; 36.7; 42.8; 50.9; 61.7; 73.5; 99.9;
116.5; 122.8; 123.0; 127.7; 129.4; 129.5; 130.4; 130.6; 131.2; 132.2; 133.2; 136.3; 141.7; 145.4; 150.3; 152.0;
156.2; 159.4; 160.8; 167.7; 172.2. ESI-MS: 478.45 ([MꢀH]ꢀ ), 981.27 ([2 MþNa]þ ). Anal. calc. for
C29H25N3O4 (479.18): C 66.99, H 5.03, N 10.66; found: C 66.90, H 5.02, N 10.67.
9-(Furylmethylidene)aminohomocamptothecin (¼ 5-Ethyl-11-[(E)-furan-2-ylmethylidene)amino]-
1,4,5,13-tetrahydro-5-hydroxy-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-di-
one; 2b). The general synthetic method described above afforded 2b (33%). Yellow solid. M.p. 228–
2308. 1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.87 (q, J¼7.6, CH2(19)); 2.93 (s, Me(7)); 3.32 (q, J¼13.8,
CH2(21)); 5.22 (s, CH2(5)); 5.54 (q, J¼15.3, CH2(17)); 6.03 (s, HOꢀC(20)); 6.79 (s, HꢀC(4’)); 7.13 (d, J¼
7.5, HꢀC(10)); 7.28 (d, J ¼ 3.3, HꢀC(5’)); 7.37 (s, HꢀC(14)); 7.76 (t, J¼8.0, HꢀC(11)); 7.94 (d, J¼8.4,
HꢀC(12)); 8.05 (s, HꢀC(3’)); 8.37 (s, HꢀC¼N). 13C-NMR: 8.6; 21.1; 36.6; 42.8; 50.9; 61.7; 73.5; 100.0;
116.5; 122.1; 122.8; 126.3; 128.3; 130.3; 130.5; 137.7; 141.5; 145.3; 150.2; 150.3; 150.9; 151.9; 154.1; 156.2;
159.4; 161.0; 172.2. ESI-MS: 468.31 ([MꢀH]ꢀ ), 961.21 ([2 MþNa]þ ). Anal. calc. for C27H23N3O5
(469.16): C 69.07, H 4.94, N 8.95; found: C 66.10, H 4.99, N 8.93.
9-(Thienylmethylidene)aminohomocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-meth-
yl-11-[(E)-(thiophen-2-ylmethylidene)amino]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-
3,15-dione; 2c). The general synthetic method described above afforded 2c (20%). Yellow solid. M.p.
1
>3008. H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.87 (q, J¼7.6, CH2(19)); 2.91 (s, Me(7)); 3.32 (q, J¼13.9,
CH2(21)); 5.25 (s, CH2(5)); 5.54 (q, J¼15.1, CH2(17)); 6.03 (s, HOꢀC(20)); 7.20 (d, J ¼ 7.3, HꢀC(10));
7.22–7.31 (t, J¼8.0, HꢀC(11)); 7.38 (s, HꢀC(14)); 7.79–7.99 (m, HꢀC(2’), HꢀC(4’), HꢀC(5’), HꢀC(12));
8.73 (s, HꢀC¼N). 13C-NMR: 8.6; 21.1; 36.6; 42.8; 50.9; 61.7; 73.5; 99.9; 116.5; 122.8; 123.1; 127.7; 128.9;
130.3; 130.6; 132.5; 134.1; 141.7; 142.7; 145.4; 150.3; 151.1; 151.9; 153.8; 156.2; 159.4; 172.2. ESI-MS:
994.69 ([2 MþNa]þ ). Anal. calc. for C27H23N3O4S (485.14): C 66.79, H 4.77, N 8.65; found: C 66.83, H
4.78, N 8.63.
9-(2-Pyridylmethylidene)aminohomocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-
methyl-11-[(E)-(pyridin-2-ylmethylidene)amino]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-
3,15-dione; 2d). The general synthetic method described above afforded 2d (24%). Yellow solid. M.p.
1
>3008. H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.86 (q, J¼7.9, CH2(19)); 2.92 (s, Me(7)); 3.38 (q, J¼13.7,
CH2(21)); 5.26 (s, CH2(5)); 5.42 (q, J¼15.1, CH2(17)); 6.04 (s, HOꢀC(20)); 7.25(d, J¼7.3, HꢀC(10)); 7.40
(s, HꢀC(14)); 7.62–7.63 (m, HꢀC(4’)); 7.79 (t, J¼7.9, HꢀC(11)); 8.03 (d, 1 H, 12-H, J¼8.3, HꢀC(12));
8.04–8.06 (m, HꢀC(5’)); 8.26 (d, J¼7.8, HꢀC(6’)); 8.54 (s, HꢀC¼N); 8.80 (d, J ¼ 4.2, HꢀC(3’)).
13C-NMR: 8.7; 21.1; 36.7; 39.0; 42.9; 51.0; 61.8; 73.6; 100.0; 113.2; 116.3; 117.7; 122.9; 123.2; 127.7; 130.5;
130.6; 142.0; 145.5; 147.5; 148.4; 150.4; 151.9; 152.0; 152.5; 156.3; 159.5; 172.3. ESI-MS: 479.98 ([MꢀH]ꢀ ).
Anal. calc. for C28H24N4O4 (480.18): C 69.99, H 5.03, N 11.66; found: C 66.89, H 5.02, N 11.68.

1272
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
9-(3-Pyridylmethylidene)aminohomocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-
methyl-11-[(E)-(pyridin-3-ylmethylidene)amino]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-
3,15-dione; 2e). The general synthetic method described above afforded 2e (21%). Yellow solid. M.p.
>3008 .1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.87 (q, J¼7.5, CH2(19)); 2.92 (s, Me(7)); 3.38 (q, J ¼ 13.7,
CH2(21)); 5.27 (s, CH2(5)); 5.54 (q, J¼15.1, CH2(17)); 6.03 (s, HOꢀC(20)); 7.23 (d, J¼7.3, HꢀC(10));
7.40 (s, HꢀC(14)); 7.60–7.80 (m, HꢀC(5’)); 7.82 (s, HꢀC(11)); 8.03 (d, J ¼ 8.3, HꢀC(12)); 8.66–8.67 (m,
HꢀC(6’)); 8.70 (s, HꢀC¼N); 8.78–8.79 (m, HꢀC(4’)); 9.17 (s, HꢀC(2’)). 13C-NMR: 8.6; 21.2; 29.4; 36.7;
42.9; 50.9; 61.8; 73.6; 100.1; 116.5; 122.8; 123.1; 127.7; 129.0; 130.4; 130.5; 132.5; 134.2; 141.7; 142.8; 145.4;
150.3; 151.1; 151.9; 153.8; 156.3; 159.5; 172.3. ESI-MS: 479.63 ([MꢀH]ꢀ ). Anal. calc. for C28H24N4O4
(480.18): C 69.98, H 5.02, N 11.67; found: C 69.90, H 5.01, N 11.69.
9-(4-Pyridylmethylidene)aminohomocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-
methyl-11-[(E)-(pyridin-4-ylmethylidene)amino]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-
3,15-dione; 2f). The general synthetic method described above afforded 2f (20%). Yellow solid. M.p.
>3008 .1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.87 (q, J¼7.5, CH2(19)); 2.92 (s, Me(7)); 3.38 (q, 2 H, J¼13.7,
CH2(21)); 5.27 (s, CH2(5)); 5.54 (q, J¼15.1, CH2(17)); 6.03 (s, HOꢀC(20)); 7.23 (d, J¼7.3, HꢀC(10));
7.40 (s, HꢀC(14)); 7.80–7.81 (m, HꢀC(11)); 7.82–7.84 (m, HꢀC(2’), HꢀC(5’)); 8.03 (d, J¼8.3, HꢀC(12));
8.70 (s, HꢀC¼N); 8.83–8.85 (m, HꢀC(4’)). 13C-NMR: 8.6; 21.1; 29.4; 36.6; 41.0; 42.8; 50.9; 61.7; 73.5;
100.0; 116.7; 122.8; 123.1; 128.5; 130.3; 130.8; 141.5; 142.6; 145.3; 149.5; 150.2; 151.1; 151.5; 152.1; 156.2;
159.4; 159.6; 172.2. ESI-MS: 479.36 ([MꢀH]ꢀ ). Anal. calc. for C28H24N4O4 (480.18): C 69.97, H 5.02, N
11.68; found: C 69.91, H 5.01, N 11.70.
Cytotoxicity Assay. The prepared compounds were tested for their in vitro cytotoxic activities against
three cell lines, i.e., A-549 (human lung adenocarcinoma epithelial cell line), MDA-MB-435 (human
breast carcinoma cell line), and HCT-116 (human colon carcinoma cell line), using a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)-based assay. The cells were plated
in 96-well plates at 1200 cells/well and incubated at 378 for 24 h. Then, the test compounds were added at
different concentrations into triplicate wells, and 0.1% DMSO was used for the control. After 3 d of
incubation at 378, 20 ml of MTT soln. (5 mg/ml) was added to each well, and after shaking for 1 min the
plate was incubated for further 4 h. The formazan crystals were dissolved with 100 ml of DMSO. The
absorbance (OD) at 570 nm was quantified with a microplate reader (WellscanMK-2, Labsystems Co.,
Ltd., Finnland). Wells containing no test compounds were used as blanks. The survival of the cells was
expressed as percentage relative to untreated control cells.
Inhibition of Topoisomerase I-Mediated DNA Relaxation. Camptothecin (CPT) was obtained from
Tianzunzezhong, P. R. China, and calf thymus topoisomerase I (Topo I), reaction buffer, bovine serum
albumin (BSA), loading buffer, and supercoilded DNA pBR322 were purchased from TaKaRa
Biotechnology Co., Ltd., P. R. China.
To 16 ml dist. H2O, 2 ml reaction buffer containing 0.25 mg supercoiled DNA and 0.5 U Topo I, 2 ml
0.1% BSA, and 0.02 ml test compound soln. in 1.5 ml sample tube (final concentration 100 mm) were
added. The mixture was incubated at 378 for 15 min. The reactions were stopped by adding SDS (0.5%
final concentration), and 3.5 ml of 6ꢂloading buffer (0.1 mm EDTA, 7% glycerol, 0.01% xylene cyanol
FF, and 0.01% bromopenol blue) was added to the mixtures, which were electrophoresed in 0.8% agarose
gel in TAE buffer (0.4m TRIS, 0.01m EDTA, and 0.2m AcOH, pH 8.5) for 40 min at 120 V. The gels were
stained with ethidium bromide at r.t. and photographed with an UV transilluminator (Tannon 2500,
Tianneng Co., Ltd., P. R. China).
Binding Mode Prediction with AutoDock 4.0. The AutoDock 4.0 tools were used to check and
prepare the ligand and receptors files. A grid of 44, 44, and 42 points in the x, y, and z directions, resp., was
constructed centered on the center of the mass of the inhibitors. A grid spacing of 0.375 ꢃ, a distance-
dependent function of the dielectric constant for the energetic map calculations, and a Lamarckian
genetic algorithm were used. The docked compounds were subjected to 50 runs of the AutoDock search,
using 500 000 steps of energy evaluation and the default values for the other parameters. Cluster analysis
was performed on the results using an rms tolerance of 1.0 ꢃ. The docking pose analysis was carried out
for the first pose of the first cluster and the first pose of the most populated cluster.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
REFERENCES
1273
[1] M. E. Wall, M. C. Wani, C. E. Cook, K. H. Palmer, A. T. McPhail, G. A. Sim, J. Am. Chem. Soc.
1966, 88, 3888.
[2] C. G. Moertel, A. J. Schutt, R. J. Reitemeier, R. G. Hahn, Cancer Chemother. Rep. 1972, 56, 649.
[3] Y. H. Hsiang, R. Hertzberg, S. Hecht, L. F. Liu, J. Biol. Chem. 1985, 260, 14873.
[4] W. D. Kingsbury, J. C. Boehm, D. R. Jakas, K. G. Holden, S. M. Hecht, G. Gallagher, M. J. Caranfa,
F. L. McCabe, L. F. Faucette, R. K. Johnson, R. P. Hertzberg, J. Med. Chem. 1991, 34, 98.
[5] A. I. Graul, J. R. Prous, Drug News Perspect. 2005, 18, 21.
[6] L. P. Rivory, J. Robert, Phamacol. Ther. 1995, 68, 269.
[7] S. T. Liew, L.-X. Yang, Curr. Pharm. Des. 2008, 14, 1078.
[8] O. Lavergne, L. Lesueur-Ginot, F. P. Rodas, D. C. H. Bigg, Bioorg. Med. Chem. Lett. 1997, 7, 2235.
[9] Y. Urasaki, Y. Takebayashi, Y. Pommier, Cancer Res. 2000, 60, 6557.
[10] W. Du, D. P. Curran, R. L. Bevins, S. G. Zimmer, J. Zhang, T. G. Burke, Bioorg. Med. Chem. 2002,
10, 103.
[11] L. Lesueur-Ginot, D. Demarquay, R. Kiss, P. G. Kasprazyk, L. Dassonneville, C. Bailly, J. Camara,
O. Lavergne, D. C. H. Bigg, Cancer Res. 1999, 59, 2939.
[12] W. Guo, Z. Miao, C. Sheng, J. Yao, H. Feng, W. Zhang, L. Zhu, W. Liu, P. Cheng, J. Zhang, X. Che,
W. Wang, C. Luo, Y. Xu, Eur. J. Med. Chem. 2010, 45, 2223.
[13] Z.-Y. Miao, C.-Q. Sheng, W.-N. Zhang, H.-T. Ji, J. Zhang, L.-C. Shao, L. You, M. Zhang, J. -Z. Yao,
X.-Y. Che, Bioorg. Med. Chem. 2008, 16, 1493.
[14] S. Yang, W. N. Zhang, Y. J. Zhou, J. Z. Yao, H. T. Ji, Chin. J. Med. Chem. 2004, 14, 106.
[15] S. Yang, W. N. Zhang, Y. J. Zhou, J. Z. Yao, J. X. Yu, C. Q. Sheng, Chin. J. Med. Chem. 2004, 14, 242.
[16] N. Moskalev, M. Barbasiewicz, M. Makosza, Tetrahedron 2004, 60, 347.
[17] M. Mentel, R. Breinbauer, Curr. Org. Chem. 2007, 11, 159.
[18] T. Yaegashi, S. Sawada, H. Hagata, T. Furuta, T. Yokokura, T. Miyasaka, Chem. Pharm. Bull. 1994,
42, 2518.
[19] S. Dallavalle, A. Ferrari, L. Merlini, S. Penco, N. Carenini, M. De Cesare, P. Perego, G. Pratesi, F.
Zunino, Bioorg. Med. Chem. Lett. 2001, 11, 291.
[20] PyMOL, Molecular visualization system, http://www.pymol.org.
Received September 21, 2010