Synthesis and biological evaluation of 7-alkenyl homocamptothecins as potent topoisomerase I inhibitors

Article abstract of DOI:10.1002/cbdv.201100195

Homocamptothecin (hCPT) is a camptothecin (CPT) derivative with a seven-membered β-hydroxylactone E ring, which shows higher lactone stability and improves topoisomerase I (Topo I) inhibition activity. In an attempt to improve the antitumor activity of homocamptothecins, a series of 7- alkenyl-homocamptothecin derivatives was designed and synthesized based on a semisynthetic route starting from CPT. Most of the synthesized compounds exhibit higher cytotoxic activities on the A-549 tumor cell line than topotecan (TPT). Some compounds such as 2a and 2o show a broad in vitro antitumor spectrum and exhibit superior Topo I-inhibition activity.

Full text of DOI:10.1002/cbdv.201100195

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Synthesis and Biological Evaluation of 7-Alkenyl Homocamptothecins as
Potent Topoisomerase I Inhibitors
by Lingjian Zhua)1), Xianghua Zhangb)1), Ning Leic), Wenfeng Liua), Zhenyuan Miao*a), Chunlin
Zhuanga), Chunquan Shenga), Wei Guoa), Guoqiang Donga), Jianzhong Yaoa), Pengfei Chenga), and
Wannian Zhang*a)
a) School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
P. R. China (W. Z.: phone/fax: þ86-21-81871243; e-mail: zhangwnk@hotmail.com; Z. M.: phone:
þ86-21-81871233; e-mail: miaozhenyuan@hotmail.com)
b) First Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military
Medical University, 225 Changhai Road, Shanghai 200438, P. R. China
c) Department of Quality Assurance, Lian Yun Gang JARI Pharmaceutical Co., Ltd., 18 Zhenhua Road,
Lianyungang 222100, P. R. China
Homocamptothecin (hCPT) is a camptothecin (CPT) derivative with a seven-membered b-
hydroxylactone E ring, which shows higher lactone stability and improves topoisomerase I (Topo I)
inhibition activity. In an attempt to improve the antitumor activity of homocamptothecins, a series of 7-
alkenyl-homocamptothecin derivatives was designed and synthesized based on a semisynthetic route
starting from CPT. Most of the synthesized compounds exhibit higher cytotoxic activities on the A-549
tumor cell line than topotecan (TPT). Some compounds such as 2a and 2o show a broad in vitro
antitumor spectrum and exhibit superior Topo I-inhibition activity.
Introduction. – Isolated from the Chinese tree Camptotheca acumunata by Wall,
Wani, and co-workers in 1966 [1], camptothecin (CPT) is the parent of a large and
important family of antitumor agents. Topotecan (TPT) and irinotecan are analogs of
CPT that are now being marketed in several countries, and a number of other analogs
are the subject of ongoing clinical or preclinical evaluation [2].
However, the severe toxicity, the instability of the lactone, and other unpredictable
and adverse effects of CPTs [3][4] have promoted intensive efforts to reduce their
toxicity in patients and overcome the tumor drug resistance for this class of compounds.
A novel and interesting option would be to modify the metabolically liable CPT lactone
ring from a six-membered a-hydroxy lactone to a seven-membered b-hydroxy lactone
ring, which leads to the development of the homocamptothecin (hCPT) family of
compounds [5]. The expanded seven-membered lactone E ring that characterizes
hCPT enhances the plasma stability of the drug and reinforces the inhibition of
topoisomerase I (Topo I) compared with the conventional CPTs [6]. On the basis of
these encouraging results, many hCPT analogs have been synthesized and evaluated in
vitro and in vivo as antitumor agents [7–11]. Among them, BN80915 (diflomotecan)
1
)
These two authors contributed equally to this work.
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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and BN80927 (Fig. 1) are representatives and have been in clinical trial, and
encouraging results have been obtained [12][13].
Fig. 1. Chemical structures of CPT, hCPT, and representative analogues
Several reports indicated that the CPT derivatives with substituents at quinoline A/
B ring [14], especially at C(7), C(9), and C(10), are of great interest, and several good
drug candidates (such as exatecan, lurtotecan, and silatecan) are under clinical trials.
The importance of the lipophilic groups at C(7) of camptothecin has been
demonstrated by the increase of inhibitor intracellular uptake and cytotoxic activities
[15]. Previous studies have also revealed that substitution at C(7) did not decrease
camptothecinꢂs antitumor activity [16].
In our laboratory, a series of analogs with substituents at C(7) of homocampto-
thecin derivatives has been synthesized, and many compounds showed superior
activities comparable to the marketed CPTs [17][18]. Among these compounds, a
substituted C¼N group has shown to be useful to increase antitumor activity for
antitumor agents such as (arylimino)methyl and (oxyimino)methyl hCPTs (Fig. 2)
[19]. Here, we describe the adoption of a C¼C bond as a linker according to the
principle of biological isostere. Furthermore, the C¼C bond has been successfully
employed in the structural modification of CPTs [20]. Based on the ternary complex of
DNAꢀTopo IꢀCPT [21], we present here a presumed binding mode of this class of
compounds with a DNAꢀTopo I complex. In this model, we found a large space around
the C(7)-atom of homocamptothecin that allows the introduction of a hydrophobic side
chain (Fig. 3). Therefore, we designed and synthesized a series of 7-alkenyl [22] and 7-
(alkoxycarbonyl)ethenyl hCPT derivatives and explored whether this modification
could enhance the antitumor activity of hCPTs.

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Fig. 2. Chemical structures of 7-iminomethyl-substituted homocamptothecins
Fig. 3. Hypothetical model for the binding of 7-N¼C and 7-C¼C linked homocamptothecins with the
DNAꢀTopo I complex. a) Superimposition of the docked conformations of 7-[(phenylimino)methyl]-
homocamptothecin with 7-(phenylethenyl)homocamptothecin. b) Binding mode of 7-[(phenylimino)-
methyl]homocamptothecin in the active site of the Topo IꢀDNA complex. c) Binding mode of
compound 2b in the active site of the Topo IꢀDNA complex. The figures were prepared from PDB entry
1T8I, using PyMol (http://pymol.souceforge.net/).
Results and Discussion. – Chemistry. The chemical synthesis of the targeted
compounds was outlined in the Scheme. hCPT was synthesized by a semisynthetic
approach. Starting from CPT, a four-step route, including reduction, oxidative cleavage,
Reformatsky reaction, and ring closure, was applied to synthesize the racemic hCPT
[23]. 7-Formylhomocamptothecin (1) was prepared according to Sawada et al. [24].
Treatment of hCPT with H2O2 and FeSO4 in an aqueous MeOH/H2SO4 solution
furnished 7-(hydroxymethyl)homocamptothecin in 79% yield. The HOꢀCH2 group

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was oxidized to form 7-formylhomocamptothecin (1). In this process, we used SeO2 as
the oxidant instead of the previously reported AcOH in order to avoid the formation of
the by-product acetoxymethyl-hCPT. For the preparation of C(7)-vinyl derivatives
starting from aldehyde 1, the Wittig reaction was an obvious choice. However, attempts
to react compound 1 with unstabilized Wittig ylides in the presence of strong bases
failed. Due to the sensitivity of 1 to strongly basic conditions, the Wittig reactions were
then conducted in the presence of weak bases and stabilized ylides, which allowed us to
prepare derivatives 2a–2l as mixtures of (E) and (Z) isomers, which were not
separated. Compounds 2m–2p were obtained as pure (E)-isomers.
Scheme
a) Wittig reagents, DMAP, CHCl3, 658.
Biological Activity. The in vitro antitumor activities of these 7-vinylhomocampto-
thecin derivatives against solid tumor cell lines, A-549 (human lung adenocarcinoma
epithelial cell line), HCT-116 (human colon carcinoma cell line), and MDA-MB-435
(human breast carcinoma cell line), were evaluated. Topotecan (TPT) was used as a
reference compound, since it is a clinically effective CPT and does not require
metabolic activation.
As expected, the results indicated that the alkenyl and (alkoxycarbonyl)ethenyl
groups at C(7) could promote the antitumor activity of the hCPTs (Table). The results
also revealed that these compounds were more sensitive against the A-549 cell line than
against the HCT-116 and MDA-MB-435 cell lines, which was consistent with the clinical
behavior of CPT derivatives [25]. For the A-549 cell line, many compounds, such as 2a,
2b, 2e, and 2m, showed higher in vitro inhibitory activities than TPT. With few
exceptions (i.e., 2f and 2i), the tested compounds showed increased cytotoxic potency
against the HCT-116 cell line compared to TPT. With regard to the MDA-MB-435 cell
line, only compounds 2a and 2o exhibited antitumor activities comparable to TPT. In
particular, the activity of the most promising compounds, 2a and 2o, showed a broad in
vitro antitumor spectrum. Further pharmacological and toxicological evaluation of
these promising compounds is in progress. Comparison of the inhibition ratio revealed

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Table. In vitro Antitumor Activity of 7-Alkenyl and 7-[(Alkoxycarbonyl)ethenyl] hCPTs 2a–2p against
Three Human Tumor Cell Linesa)
Compounds
IC50 [mm]b)
A-549
HCT-116
MDA-MB-435
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
TPT
0.259ꢁ0.012
0.493ꢁ0.014
3.25ꢁ0.17
2.62ꢁ0.36
0.431ꢁ0.018
4.38ꢁ0.92
4.68ꢁ0.04
3.35ꢁ0.56
2.65ꢁ0.15
3.15ꢁ0.69
0.387ꢁ0.033
20.6ꢁ0.9
1.72ꢁ0.05
1.32ꢁ0.18
4.67ꢁ0.47
4.93ꢁ0.89
4.63ꢁ0.70
36.6ꢁ1.6
4.35ꢁ0.37
23.4ꢁ1.2
71.1ꢁ2.6
3.53ꢁ0.70
1.86ꢁ0.27
27.6ꢁ1.4
4.49ꢁ0.74
3.59ꢁ0.59
2.44ꢁ0.02
10.5ꢁ0.9
27.5ꢁ0.6
13.6ꢁ0.3
34.9ꢁ0.01
182ꢁ1
204ꢁ1
207ꢁ2
207ꢁ1
180ꢁ1
136ꢁ2
200ꢁ3
32.2ꢁ0.9
40.6ꢁ1.0
34.2ꢁ0.5
204ꢁ1
0.811ꢁ0.014
2.54ꢁ0.88
0.486ꢁ0.017
5.29ꢁ0.15
2.64ꢁ0.12
36.2ꢁ0.3
19.2ꢁ0.3
35.2ꢁ0.1
18.5ꢁ1.0
a) Cancer cells lines: A-549, Human lung adenocarcinoma epithelial cell line; HCT-116, human colon
carcinoma cell line; MDA-MB-435, human breast carcinoma cell line. b) The IC50 values were
determined by using the MTT method.
that compounds 2a, 2b, 2j, and 2k possessed a high growth-inhibitory effect at the
concentrations of 0.1, 1, 10, and 100 mg/ml, while a lower activity was observed at the
same concentrations for TPT (Fig. 4).
Fig. 4. Comparison of the inhibition ratio of compounds 2a, 2b, 2j, 2k, and TPTagainst the cancer cell line
A-549
Topoisomerase I-Mediated DNA Cleavage. Generally, CPT and its clinically used
derivatives act by stabilizing a covalent Topo IꢀDNA complex called the cleavable
complex. Therefore, Topo I-mediated DNA cleavage assays were used to investigate
the ability of the new compounds 2a–2p to inhibit Topo I. Cleavable complexes were

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revealed by the appearance of short DNA fragments, when the samples were analyzed
by gel electrophoresis under denaturing conditions. The results (Fig. 5) indicated that
the cytotoxic agents 2a, 2b, 2j, 2k, 2l, 2m, and 2o were also very potent as Topo I
inhibitors. Furthermore, the addition of increasing concentrations of CPT, 2k, or 2m is
accompanied by a dose-dependent increase in the level of cleavable complexes.
Importantly, 2k and 2m showed lower intensity of the migrating band (corresponding to
nicked DNA) than CPT regardless of low (1 mm) or high concentration (100 mm). The
relative potency between the three compounds, as indicated by the remaining amount
of intact DNA, is CPT, 2m, and 2k in increasing order (Fig. 6).
Fig. 5. Effect of the selected compounds on Topo I-mediated DNA relaxation. Lane 1: supercoiled DNA
pBR322. Lane 2: supercoiled DNA and Topo I. Lanes 3–10: supercoiled DNA, Topo I, and 100 mm of
CPT, 2m, 2o, 2a, 2b, 2j, 2l, and 2k, resp.
Fig. 6. Effect of compounds CPT, 2m, and 2k on Topo I-mediated DNA relaxation in a concentration
gradient. Lane 1: supercoiled DNA pBR322. Lane 2: supercoiled DNA and Topo I. Lanes 3–5:
supercoiled DNA, Topo I, and CPTat 1, 50, and 100 mm, resp. Lanes 6–8: supercoiled DNA, Topo I, and
2m at 1, 50, and 100 mm, resp. Lanes 9–11: supercoiled DNA, Topo I, and 2k at 1, 50, and 100 mm, resp.
Conclusions. – Taken together, our results clearly indicate that C(7) of hCPT is a
favorable position for the introduction of an alkenyl or an (alkoxycarbonyl)ethenyl
group. It is likely that the improvement of cytotoxic activity is the result of multiple
favorable events, including increased drug uptake (as expected on the basis of increased
lipophilicity) and increased stability (as documented also for 7-silyl-modified CPTs
[26]). A better understanding of this feature could provide further insight for the
development of more effective Topo I inhibitors.
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).

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Experimental Part
General Methods. All reagents and solvents were reagent grade or were purified by standard
methods before use. Anh. solvents and reagents were all anal. pure and dried through routine protocols.
TLC: silica gel plates GF254 (Qindao Haiyang Chemical, P. R. China). Flash column chromatography
(CC): silica gel (SiO2; 200–300 mesh; Qingdao Haiyang Chemical). M.p.: electrothermal apparatus;
uncorrected. 1H- and 13C-NMR spectra: Bruker 500 MHz spectrometer; with TMS as internal standard
and CDCl3 or (D6)DMSO as solvent; chemical shifts (d values) and coupling constants (J values) are
given in ppm and Hz, resp.
General Procedure for the Synthesis of the 7-Alkenyl and 7-[(Alkoxycarbonyl)ethenyl] Derivatives
2a–2p of Homocamptothecin. (E/Z)-7-[(4-Nitrophenyl)ethenyl]homocamptothecin (¼ 5-Ethyl-1,4,5,13-
tetrahydro-5-hydroxy-12-[(E/Z)-2-(4-nitrophenyl)ethenyl]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-
b]quinoline-3,15-dione; 2a). A soln. of 7-formylhomocamptothecin (1; 100 mg, 0.25 mmol), DMAP
(2.4 mg, 0.02 mmol), and [(4-nitrophenyl)methylene](triphenyl)phosphorane (120 mg, 0.30 mmol) in
30 ml of CHCl3 was heated to reflux for 5 h. The solvent was evaporated in vacuo, and the residual slurry
was purified by flash chromatography using CH2Cl2/MeOH 100 :1 as eluent. Extensive CC was required
to separate most of the Ph3PO from the desired product 2a. Yellow powder (a mixture of (Z) and (E)
isomers, (Z)/(E) 1:1). Yield 77%. M.p. >308. IR (KBr): 3341, 3087, 3078, 2974, 2931, 1748, 1717, 1687,
1655, 1650, 1618, 1593, 1580, 1535, 1497, 1438, 1372, 1343, 1311, 1268, 1210, 1079, 973, 858, 829, 761, 699,
632. 1H-NMR (500 MHz, (D6)DMSO): 0.89 (t, J¼7.4, MeCH2, (Z)); 0.94 (t, J¼7.4, MeCH2, (E)); 1.85
(q, J¼7.3, MeCH2, (Z)); 1.91 (q, J¼7.3, MeCH2, (E)); 3.35 (q, J¼2.3, CH2CO, (Z)); 3.37 (q, J¼2.3,
CH2CO, (E)); 4.68 (s, CH2N, (Z)); 5.19 (s, CH2N, (E)); 5.43 (q, J¼15.0, CH2O, (E)); 5.45 (q, J¼15.0,
CH2O, (Z)); 6.07 (s, OH); 7.25 (d, J¼12.8, CH¼, (Z)); 7.33 (d, J¼12.8, CH¼, (Z)); 7.33 (s, HꢀC(14),
(E)); 7.37 (s, HꢀC(14), (Z)); 7.42 (d, J¼16.6, CH¼, (E)); 7.56–7.65 (m, HꢀC(10), HꢀC(11), (Z)); 7.72 (t,
J¼8.0, HꢀC(10), (E)); 7.81 (t, J¼8.1, HꢀC(11), (E)); 7.94 (d, J¼8.8, HꢀC(12), (Z)); 7.98 (d, J¼8.3,
HꢀC(12), (E)); 8.05 (d, J ¼ 16.6, CH¼, (E)); 8.15 (dd, J¼8.6, 8.6, 1 arom. H); 8.12 (d, J¼8.4, HꢀC(9),
(Z)); 8.32 (d, J¼8.4, HꢀC(9), (E)). 13C-NMR: 8.6; 14.6; 29.7; 35.2; 42.7; 61.8; 73.6; 100.1; 122.6; 123.5;
124.0; 125.7; 127.6; 127.7; 128.5; 129.0; 130.2; 131.5; 131.9; 132.0; 135.97; 135.99; 137.56; 137.58; 144.7;
148.7; 148.9; 152.95; 152.96; 156.3; 159.3; 165.5; 172.4. HR-EI-MS: 509.1620 (Mþ ; calc. 509.1587).
(E/Z)-7-(Phenylethenyl)homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-[(E/Z)-2-
phenylethenyl]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 2b) [22]. With (phenyl-
methylene)(triphenyl)phosphorane. Yellow powder (a mixture of (Z) and (E) isomers, (Z)/(E) 1:1).
Yield 34%. M.p. >3008. 1H-NMR (500 MHz, (D6)DMSO): 0.89 (t, J¼7.4, MeCH2, (Z)); 0.94 (t, J¼7.4,
MeCH2, (E)); 1.85 (q, J¼7.3, MeCH2, (Z)); 1.91 (q, J¼7.3, MeCH2, (E)); 3.24 (q, J¼2.3, CH2CO, (Z));
3.26 (q, J¼2.3, CH2CO, (E)); 4.62 (s, CH2N, (Z)); 5.44 (s, CH2N, (E)); 5.44 (q, J¼15.0, CH2O, (E)); 5.45
(q, J¼15.0, CH2O, (Z)); 6.06 (s, OH); 7.02–7.04 (m, 2 arom. H); 7.09 (d, J¼12.5, CH¼, (Z)); 7.12–7.14
(m, 3 arom. H); 7.23 (d, J¼12.3, CH¼, (Z)); 7.38 (s, HꢀC(14), (E)); 7.42 (s, HꢀC(14), (Z)); 7.43 (d, J¼
16.6, CH¼, (E)); 7.50 (t, J¼7.9, 2 arom. H); 7.62–7.71 (m, HꢀC(10), HꢀC(11), (Z)); 7.83–7.88 (m,
HꢀC(10), HꢀC(11), (E)); 8.00 (d, J ¼ 16.6, CH¼, (E)); 8.09 (d, J¼8.3, HꢀC(12), (Z)); 8.13 (d, J¼8.3,
HꢀC(12), (E)); 8.17 (d, J¼8.4, HꢀC(9), (Z)); 8.45 (d, J¼8.4, HꢀC(9), (E)). HR-EI-MS: 464.1708
(Mþ ; calc. 464.1736).
(E/Z)-7-[(2-Bromophenyl)ethenyl]homocamptothecin (¼12-[(E/Z)-2-(2-Bromophenyl)ethenyl]-5-
ethyl-1,4,5,13-tetrahydro-5-hydroxy-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione;
2c). With [(2-bromophenyl)methylene](triphenyl)phosphorane. Yellow powder (a mixture of (Z) and
(E) isomers, (Z)/(E) ¼ 2 :1). Yield 24%. M.p. >308. 1H-NMR (500 MHz, CDCl3): 0.86–0.93 (m,
MeCH2, (Z/E); 1.75–2.04 (m, MeCH2, (Z/E)); 3.30 (dq, J¼2.3, 2.4, CH2CO, (Z/E)); 4.65 (dd, J¼14.3,
CH2N, (Z)); 5.44 (s, CH2N, (E)); 5.49 (dq, J¼14.9, 14.9, CH2O, (Z/E)); 6.62 (d, J¼7.5, 1 arom. H); 6.77
(t, J¼7.4, 1 arom. H); 7.00 (t, J¼6.7, 1 arom. H); 7.06 (d, J¼12.1, CH¼, (Z)); 7.32 (d, J¼12.1, CH¼, (Z));
7.47 (t, J¼8.0, 1 arom. H); 7.48 (s, HꢀC(14), (Z)); 7.57 (s, HꢀC(14), (E)); 7.53–7.62 (m, HꢀC(10),
HꢀC(11), (Z)); 7.67 (d, J¼16.0, CH¼, (E)); 7.72–7.77 (m, HꢀC(10), HꢀC(11), (E)); 7.84 (d, J¼7.5,
HꢀC(12)); 8.04 (d, J¼8.3, HꢀC(9)); 8.08 (d, J ¼ 16.0, CH¼, (E)). HR-EI-MS: 542.0810 (Mþ ; calc.
542.0841).

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(E/Z)-7-[(4-Cyanophenyl)ethenyl]homocamptothecin (¼4-[(E/Z)-2-(5-Ethyl-4,5,13,15-tetrahydro-
5-hydroxy-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-12-yl)ethenyl]benzonitrile;
2d). With [(4-cyanophenyl)methylene](triphenyl)phosphorane. Yellow powder (a mixture of (Z) and
(E) isomers, (Z/E) ¼ 1:1). Yield 77%. M.p. >3008. 1H-NMR (500 MHz, CDCl3): 0.86–1.04 (m, MeCH2,
(Z/E)); 1.98–2.06 (m, MeCH2, (Z/E)); 3.30 (dq, CH2CO, (Z/E)); 5.30 (s, CH2N, (Z)); 5.42 (q, J¼14.9,
CH2N, (E)); 5.47 (dq, J¼14.9, J¼15.0, CH2O, (Z/E)); 7.13 (d, J¼12.3, CH¼, (Z)); 7.17 (s, HꢀC(14));
7.23 (d, J¼12.3, CH¼, (E)); 7.34 (d, J¼16.8, CH¼, (E)); 7.57 (dd, J¼8.5, 4 arom. H); 7.56–7.65 (m,
HꢀC(10), HꢀC(11), (Z)); 7.72–7.89 (m, HꢀC(11), HꢀC(10), (E)); 7.99 (d, J ¼ 16.8, CH¼, (E)); 8.05 (d,
J¼8.3, HꢀC(12), (E)); 8.09–8.18 (m, HꢀC(12)/HꢀC(9), (Z)); 8.18 (d, J¼8.3, HꢀC(9), (E)). HR-EI-
MS: 489.1702 (Mþ ; calc. 489.1689).
(E/Z)-7-[(3-Fluorophenyl)ethenyl]homocamptothecin (¼ 5-Ethyl-12-[(E/Z)-2-(3-fluorophenyl)-
ethenyl]-1,4,5,13-tetrahydro-5-hydroxy-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione;
2e). With [(3-fluorophenyl)methylene]triphenylphosphorane. Yellow powder (a mixture of (Z) and (E)
isomers, (Z)/(E) 1:1). Yield 34%. M.p. >3008. 1H-NMR (500 MHz, CDCl3): 0.89–1.14 (m, MeCH2, (Z/
E); 1.97–2.05 (m, MeCH2, (Z/E); 3.30 (dq, J¼13.5, 13.6, CH2CO, (Z/E); 4.73 (q, J¼18.6, CH2O, (Z);
5.30 (s, CH2N, (Z); 5.34 (s, CH2N, (E)); 5.49 (q, J¼15.0, CH2O, (E)); 6.66 (d, J¼9.4, 1 arom. H); 6.78 (d,
J¼7.5, 1 arom. H); 6.84 (t, J¼7.4, 1 arom. H); 6.97 (d, J¼12.4, CH¼, (Z)); 7.06–7.09 (m, 1 arom. H); 7.12
(d, J¼12.4, CH¼, (E)); 7.40–7.47 (m, HꢀC(10), HꢀC(11), (Z)); 7.44 (s, HꢀC(14)); 7.59 (d, J¼17.0, CH¼,
(E)); 7.57–7.68 (m, HꢀC(11), HꢀC(10), (E)); 8.05 (d, J¼8.3, HꢀC(12), (E)); 8.17 (d, J ¼ 8.4, HꢀC(9),
(E)); 8.18 (d, J ¼ 17.0, CH¼, (E)). HR-EI-MS: 482.1611 (Mþ ; calc. 482.1642).
(E/Z)-7-[(2-Fluorophenyl)ethenyl]homocamptothecin (¼ 5-Ethyl-12-[(E/Z)-2-(2-fluorophenyl)-
ethenyl]-1,4,5,13-tetrahydro-5-hydroxy-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione;
2f). With [(2-fluorophenyl)methylene](triphenyl)phosphorane. Yellow powder (a mixture of (Z)- and
(E)-isomers, (Z)/(E) 1:1). Yield 20%. M.p. >3008. 1H-NMR (500 MHz, CDCl3): 0.91–1.05 (m, MeCH2,
(Z/E)); 1.95–2.09 (m, MeCH2, (Z/E)); 3.30 (dq, J¼13.6, 13.6, CH2CO, (Z/E)); 4.70 (dd, J¼19.1, CH2N,
(Z)); 5.27 (s, CH2N, (E)); 5.45 (dq, J¼14.9, 15.0, CH2O, (Z/E)); 6.67–6.70 (m, 2 arom.); 6.77 (t, J¼7.4, 1
arom. H); 7.01 (t, J¼6.7, 1 arom. H); 7.03 (d, J¼11.8, CH¼, (Z)); 7.09–7.14 (m, 1 arom. H); 7.21 (t, J¼7.4,
1 arom. H); 7.28 (d, J¼12.1, CH¼, (Z)); 7.34 (d, J¼16.2, CH¼, (E)); 7.49 (s, HꢀC(14)); 7.53–7.62 (m,
HꢀC(10), HꢀC(11), (Z)); 7.69 (d, J ¼ 16.2, CH¼, (E)); 7.71–7.74 (m, HꢀC(10), HꢀC(11), (E)); 7.98 (d,
J¼7.5, HꢀC(12), (E)); 8.07 (d, J¼8.3, HꢀC(9), (Z/E)). HR-EI-MS: 482.1610 (Mþ ; calc. 482.1642).
(E)-7-[(2,3,4,5,6-Pentafluorophenyl)ethenyl]homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-
hydroxy-12-[(E)-2-(pentafluorophenyl)ethenyl]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-
3,15-dione; 2g). With [(2,3,4,5,6-pentafluorophenyl)methylene](triphenyl)phosphorane. Yellow powder.
Yield 10%. M.p. >3008. 1H-NMR (500 MHz, CDCl3): 0.88 (t, J¼7.4, MeCH2); 1.88 (q, J¼7.3, MeCH2);
3.34 (q, J¼13.6, CH2CO); 5.33 (dd, J¼18.9, 18.9, CH2N); 5.51 (dd, J¼15.4, 15.4, CH2O); 6.67–6.70 (m, 2
arom. H); 6.77 (t, J¼7.4, 1 arom. H); 7.01 (t, J¼6.7, 1 arom. H); 7.03 (d, J¼11.8, CH¼, (Z)); 7.09–7.14
(m, 1 arom. H); 7.21 (t, J¼7.4, 1 arom. H); 7.28 (d, J¼12.1, CH¼, (Z)); 7.13 (d, J¼17.0, CH¼ ); 7.45 (s,
HꢀC(14)); 7.67 (t, J¼7.4, HꢀC(10)); 7.93 (d, J ¼ 17.0, CH¼ ); 7.78 (t, J¼7.3, HꢀC(11)); 8.00 (d, J¼7.5,
HꢀC(12)); 8.13 (d, J¼8.3, HꢀC(9)). HR-EI-MS: 554.1302 (Mþ ; calc. 554.1265).
(E/Z)-7-[(4-Methylphenyl)ethenyl]homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-
[(E)-2-(4-methylphenyl)ethenyl]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 2h).
With [(4-methylphenyl)methylene](triphenyl)phosphorane. Yellow powder (a mixture of (Z)- and
(E)-isomers, (Z)/(E) 1:1). Yield 44%. M.p. >3008. 1H-NMR (500 MHz, CDCl3): 0.89–1.04 (m, MeCH2,
(Z/E)); 1.97–2.04 (m, MeCH2, (Z/E)); 2.18 (s, MeꢀAr); 3.31 (dq, J¼13.5, 13.6, CH2CO, (Z/E)); 5.32
(dd, J¼18.9, CH2N); 5.44 (dq, J¼15.0, 15.1, CH2O, (Z/E)); 6.81 (d, J¼12.3, CH¼, (Z)); 7.09 (d, J¼12.3,
CH¼, (Z)); 7.19 (d, J¼16.5, CH¼, (E)); 7.44 (dd, J¼7.8, 4 arom. H); 7.48 (s, HꢀC(14)); 7.50–7.58 (m,
HꢀC(10), (Z/E)); 7.53 (d, J ¼ 16.5, CH¼, (E)); 7.70 (t, J¼7.9, HꢀC(11), (Z/E)); 7.97 (d, J¼8.2,
HꢀC(12), (E)); 8.00–8.03 (m, HꢀC(12)/ HꢀC(9), (Z)); 8.05 (d, J¼8.4, HꢀC(9), (E)). HR-EI-MS:
478.1904 (Mþ ; calc. 478.1893).
(E)-7-[(2-Chlorophenyl)ethenyl]homocamptothecin (¼12-[(E)-2-(2-Chlorophenyl)ethenyl]-5-eth-
yl-1,4,5,13-tetrahydro-5-hydroxy-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 2i).
With [(2-chlorophenyl)methylene](triphenyl)phosphorane. Yellow powder. Yield 35%. M.p. >3008.
1H-NMR (500 MHz, CDCl3): 0.97 (t, J¼7.4, MeCH2); 1.99 (q, J¼7.3, MeCH2); 3.29 (q, J¼13.6,

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
CH2CO); 4.65 (dd, J¼19.2, CH2N); 5.46 (dd, J¼15.3, CH2O); 6.63 (d, J¼7.3, 1 arom. H); 6.73 (t, J¼7.4,
1 arom. H); 7.08 (t, J¼6.7, 1 arom. H); 7.09 (d, J¼17.0, CH¼); 7.38 (t, 1 arom. H); 7.40 (s, HꢀC(14)); 7.45
(d, J ¼ 17.0, CH¼); 7.56 (t, J¼7.4, HꢀC(10)); 7.72 (t, J¼7.4, HꢀC(11)); 8.01 (d, J¼8.2, HꢀC(12)); 8.07 (d,
J¼8.3, HꢀC(9)). HR-EI-MS: 498.1307 (Mþ ; calc. 498.1346).
(E/Z)-7-[(3-Chlorophenyl)ethenyl]homocamptothecin (¼12-[(E)-2-(3-Chlorophenyl)ethenyl]-5-
ethyl-1,4,5,13-tetrahydro-5-hydroxy-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione;
2j). With [(3-chlorophenyl)methylene](triphenyl)phosphorane. Yellow powder (a mixture of (Z) and
1
(E) isomers, (Z)/(E) 1:1). Yield 34%. M.p. >3008. H-NMR (500 MHz, (D6)DMSO): 0.81–0.93 (m,
MeCH2, (Z/E)); 1.81–1.94 (m, MeCH2, (Z/E)); 3.04–3.49 (m, CH2CO, (Z/E)); 4.77 (s, CH2N, (Z)); 5.48
(s, CH2N, (E)); 5.44 (dq, J¼15.2, 15.1, CH2O, (Z/E)); 6.02 (s, OH, (Z)); 6.05 (s, OH, (E)); 6.88 (d, J¼7.8,
1 arom. H); 7.08 (t, J¼7.8, 1 arom. H); 7.19 (d, J¼12.4, CH¼, (Z)); 7.18–7.21 (m, 2 arom. H); 7.40 (s,
HꢀC(14), (Z)); 7.42 (s, HꢀC(14), (E)); 7.46 (d, J¼12.4, CH¼, (Z)); 7.68 (t, J¼7.6, HꢀC(10), (Z)); 7.73 (t,
J¼7.4, HꢀC(10), (E)); 7.85 (d, J¼17.0, CH¼, (E)); 7.84–7.89 (m, HꢀC(11), (Z/E)); 8.12–8.20 (m,
HꢀC(12), (Z/E)); HꢀC(9), (Z)); 8.06 (d, J ¼ 17.0, CH¼, (E)); 8.53 (d, J¼8.4, HꢀC(9), (E)). HR-EI-
MS: 498.1306 (Mþ ; calc. 498.1346).
(E/Z)-7-{[(2-Trifluoromethyl)phenyl]ethenyl}homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-
hydroxy-12-{(E/Z)-2-[2-(trifluoromethyl)phenyl]ethenyl}-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-
b]quinoline-3,15-dione; 2k). With {[2-(trifluoromethyl)phenyl]methylene}(triphenyl)phosphorane. Yel-
low powder (a mixture of (Z) and (E) isomers, (Z)/(E) 2 :1). Yield 24%. M.p. >3008. 1H-NMR
(500 MHz, (D6)DMSO): 0.80–0.92 (m, MeCH2, (Z/E)); 1.81–1.85 (m, MeCH2, (Z/E)); 3.28 (dq, J¼
13.6, 13.5, CH2CO, (Z/E)); 4.60 (s, CH2N, (Z)); 5.27 (s, CH2N, (E)); 5.45 (dq, J¼15.0, 15.1, CH2O, (Z/
E)); 6.02 (s, OH); 6.81 (d, J¼7.7, 1 arom. H); 7.11 (t, J¼7.5, 1 arom. H); 7.16 (d, J¼12.4, CH¼, (Z)); 7.36
(s, HꢀC(14), (Z)); 7.40 (s, HꢀC(14), (E)); 7.32–7.44 (m, 2 arom. H); 7.48 (d, J¼12.4, CH¼, (Z)); 7.63 (t,
J¼7.7, HꢀC(10), (Z)); 7.70 (t, J¼7.8, HꢀC(10), (E)); 7.56 (d, J¼16.2, CH¼, (E)); 7.75–7.87 (m,
HꢀC(11), (Z/E)); 8.00–8.05 (m, HꢀC(12), (Z/E)); 7.86 (d, J ¼ 16.2, CH¼, (E)); 8.35 (d, J¼7.8, HꢀC(9),
(Z)); 8.43 (d, J¼8.2, HꢀC(9), (E)). HR-EI-MS: 532.1602 (Mþ ; calc. 532.1610).
(E/Z)-7-{[4-(Trifluoromethyl)phenyl]ethenyl}homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-
hydroxy-12-{(E/Z)-2-[4-(trifluoromethyl)phenyl]ethenyl}-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-
b]quinoline-3,15-dione; 2l). With {[4-(trifluoromethyl)phenyl]methylene}(triphenyl)phosphorane. Yel-
low powder (a mixture of (Z) and (E) isomers, (Z)/(E) 1:1). Yield 35%. M.p. >3008. 1H-NMR
(500 MHz, CDCl3): 0.91–1.03 (m, MeCH2, (Z/E)); 1.98–2.04 (m, MeCH2, (Z/E)); 3.31 (dq, J¼13.7, 13.6,
CH2CO, (Z/E)); 4.06 (br. s, OH); 5.32 (dd, J¼18.5, CH2N); 5.40 (dq, J¼15.1, 15.2, CH2O, (Z/E)); 7.00
(d, J¼12.4, CH¼, (Z)); 7.16 (d, J¼12.3, CH¼, (Z)); 7.37 (d, J¼17.5, CH¼, (E)); 7.47 (s, HꢀC(14)); 7.46–
7.54 (m, HꢀC(10), (Z/E)); 7.69 (d, J ¼ 17.5, CH¼, (E)); 7.62–7.68 (m, HꢀC(11), (Z/E)); 7.77 (dd, J¼8.2,
4 arom. H); 7.88 (d, J¼8.3, HꢀC(12), (Z/E)); 7.95 (d, J¼8.4, HꢀC(9), (Z)); 8.00 (d, J¼8.4, HꢀC(9),
(E)). HR-EI-MS: 532.1601 (Mþ ; calc. 532.1610).
(E)-7-[(tert-Butyloxycarbonyl)ethenyl]homocamptothecin (¼tert-Butyl (2E)-3-(5-Ethyl-5-hy-
droxy-4,5,13,15-tetrahydro-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-12-yl)prop-2-
enoate; 2m). With [(tert-butyloxycarbonyl)methylene](triphenyl)phosphorane. A yellow powder of 2m
was obtained. Yield 72%. M.p. >3008. IR (KBr): 3339, 3084, 2974, 2938, 1735, 1717, 1678, 1654, 1629,
1593, 1580, 1497, 1438, 1372, 1343, 1311, 1268, 1079, 973, 858, 761, 632. 1H-NMR (500 MHz, (D6)DMSO):
0.87 (t, J¼7.4, MeCH2); 1.57 (s, Me3CO); 1.86 (q, J¼7.3, MeCH2); 3.27 (q, J¼13.8, CH2CO); 5.42 (q, J¼
15.0, CH2O); 5.46 (d, J¼8.4, CH2N); 6.05 (s, OH); 6.75 (d, J¼16.2, COCHCH¼); 7.43 (s, HꢀC(14)); 7.79
(t, J¼7.4, HꢀC(10)); 7.92 (t, J¼7.5, HꢀC(11)); 8.21 (d, J¼8.4, HꢀC(12)); 8.27 (d, J¼8.3, HꢀC(9)); 8.28
(d, J¼16.0, COCHCH¼). 13C-NMR: 8.6; 14.6; 29.7; 36.7; 42.9; 51.2; 61.3; 61.7; 73.6; 100.8; 123.2; 124.7;
125.7; 128.4; 128.6; 130.1; 131.0; 136.0; 137.8; 144.7; 148.7; 153.0; 156.3; 159.3; 165.7; 172.2. HR-EI-MS:
488.1903 (Mþ ; calc. 488.1947).
(E)-7-[(Ethoxycarbonyl)ethenyl]homocamptothecin (¼ Ethyl (2E)-3-(5-Ethyl-4,5,13,15-tetrahydro-
5-hydroxy-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-12-yl)prop-2-enoate; 2n). With
[(ethoxycarbonyl)methylene](triphenyl)phosphorane. Yellow powder. Yield 66%. M.p. >3008.
1H-NMR (500 MHz, (D6)DMSO): 0.87 (t, J¼7.4, MeCH2); 1.35 (t, J¼7.1, MeCH2O); 1.87 (q, J¼7.3,
MeCH2); 3.27 (q, J¼13.8, CH2CO); 4.31 (q, J¼7.3, MeCH2O); 5.42 (q, J¼15.0, CH2O); 5.46 (d, J¼6.3,
CH2N); 6.05 (s, OH); 6.85 (d, J¼16.2, COCHCH¼); 7.43 (s, HꢀC(14)); 7.79 (t, J¼7.4, HꢀC(10)); 7.92 (t,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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J¼7.5, HꢀC(11)); 8.21 (d, J¼8.4, HꢀC(12)); 8.26 (d, J¼8.3, HꢀC(9)); 8.34 (d, J¼16.2, COCHCH¼).
13C-NMR: 8.6; 14.6; 29.7; 36.7; 42.8; 51.2; 61.3; 61.7; 73.6; 100.1; 123.2; 124.7; 125.7; 128.4; 128.9; 130.1;
131.0; 136.0; 137.6; 144.7; 148.7; 153.0; 156.3; 159.3; 165.7; 172.2. HR-EI-MS: 460.1605 (Mþ ; calc.
460.1634).
(E)-7-[(Methoxycarbonyl)ethenyl]homocamptothecin (¼ Methyl (2E)-3-(5-Ethyl-4,5,13,15-tetrahy-
dro-5-hydroxy-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-12-yl)prop-2-enoate; 2o).
With [(methoxycarbonyl)methylene](triphenyl)phosphorane. Yellow powder. Yield 67%. M.p. >3008.
1H-NMR (500 MHz, (D6)DMSO): 0.87 (t, J¼7.4, MeCH2); 1.87 (q, J¼7.3, MeCH2); 3.28 (q, J¼13.8,
CH2CO); 3.85 (s, MeO); 5.42 (q, J¼15.0, CH2O); 5.46 (d, J¼5.2, CH2N); 6.06 (s, OH); 6.86 (d, J¼16.2,
COCHCH¼); 7.43 (s, HꢀC(14)); 7.78 (t, J¼7.4, HꢀC(10)); 7.92 (t, J¼7.5, HꢀC(11)); 8.21 (d, J¼8.4,
HꢀC(12)); 8.26 (d, J¼8.4, HꢀC(9)); 8.34 (d, J¼16.2, COCHCH¼). HR-EI-MS: 446.1521 (Mþ ; calc.
446.1476).
(E)-7-[(Ethoxycarbonyl)(methyl)ethenyl]homocamptothecin (¼ Ethyl (2E)-3-(5-Ethyl-4,5,13,15-
tetrahydro-5-hydroxy-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-12-yl)-2-methyl-
prop-2-enoate; 2p). With [(ethoxycarbonyl)(methyl)methylene](triphenyl)phosphorane. Yellow pow-
der. Yield 45%. M.p. >3008. 1H-NMR (500 MHz, (D6)DMSO): 0.86 (t, J¼7.4, MeCH2); 1.35 (t, J¼7.0,
MeCH2O); 1.73 (s, CHCH¼Me); 1.87 (q, J¼7.3, MeCH2); 3.27 (q, J¼13.8, CH2CO); 4.31 (q, J¼7.3,
MeCH2O); 5.11 (d, J¼4.4, CH2N); 5.46 (q, J¼15.0, CH2O); 6.04 (s, OH); 7.44 (s, HꢀC(14)); 7.74 (t, J¼
7.4, HꢀC(10)); 7.88–7.94 (m, HꢀC(11)/HꢀC(12)); 7.92 (s, CH¼); 8.21 (d, J¼8.4, HꢀC(9)). HR-EI-MS:
474.1811 (Mþ ; calc. 474.1791).
Cytotoxicity 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 (¼ 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-2H-tetrazolium bromide) soln. (5 mg/ml) was added to each well, and, after shaking for
1 min, the plate was incubated further for 4 h. Formazan crystals were dissolved with 100 ml 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 of 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 supercoiled DNA pBR322 were purchased from TaKaRa
Biotechnology Co., Ltd., P. R. China.
To 16 ml of dist. H2O, 2 ml of reaction buffer containing 0.25 mg of supercoiled DNA and 0.5 U Topo I,
2 ml of 0.1% BSA, and 0.02 ml of 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 quenched 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% bromophenol blue) was added to the mixtures, which were subjected to
electrophoresis 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).
Computational Protocols. The crystallographic coordinates of the ternary complex of
CPTꢀDNAꢀTopo I (3.0-ꢃ resolution, Rcryst ¼0.244) were obtained from the Brookhaven Protein
Databank entry 1T8I. All crystallographic H2O molecules were removed from the coordinate set. The
binding region was defined as all the atoms that are 15 ꢃ around the centroid of CPT in the crystal
structure. The ligands were extracted from the X-ray complexes, which were subsequently docked into
their corresponding proteins by means of Gold 3.0.1.
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Received June 3, 2011