Trifluoromethyl-promoted homocamptothecins: Synthesis and biological activity

Article abstract of DOI:10.1016/j.ejmech.2010.02.051

The homocamptothecin (hCPT) represents a new class of topoisomerase inhibitor which combines enhanced plasma stability and strong antitumor activity. Fluorine imparts desirable characteristics to drugs by modulating both the pharmacokinetics and pharmacodynamic properties of a drug. Therefore, in an attempt to improve the antitumor activity of homocamptothecins, seven new 7-trifluoromethylated homocamptothecin derivatives were prepared by proline-catalyzed Friedlander annulation. The antitumor activity in vitro and in vivo on cancer cell lines, and inhibitory properties of topoisomerase I-mediated DNA cleavage of compounds 6c and 8b were evaluated. Several of these trifluoromethylated hCPT derivatives (such as 6a, 6b and 6c) possessed higher in vitro antitumor activity than topotecan (TPT). Especially, the compound 6c showed effective in vivo antitumor activity comparable to that of TPT.

Full text of DOI:10.1016/j.ejmech.2010.02.051

European Journal of Medicinal Chemistry 45 (2010) 2726e2732
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Trifluoromethyl-promoted homocamptothecins: Synthesis and biological activity
*
Lingjian Zhu, Zhenyuan Miao , Chunquan Sheng, Wei Guo, Jianzhong Yao, Wenfeng Liu,
**
Xiaoying Che, Wenya Wang, Pengfei Cheng, Wannian Zhang
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
The homocamptothecin (hCPT) represents a new class of topoisomerase inhibitor which combines
enhanced plasma stability and strong antitumor activity. Fluorine imparts desirable characteristics to
drugs by modulating both the pharmacokinetics and pharmacodynamic properties of a drug. Therefore, in
an attempt to improve the antitumor activity of homocamptothecins, seven new 7-trifluoromethylated
homocamptothecin derivatives were prepared by proline-catalyzed Friedlander annulation. The anti-
tumor activity in vitro and in vivo on cancer cell lines, and inhibitory properties of topoisomerase
I-mediated DNA cleavage of compounds 6c and 8b were evaluated. Several of these trifluoromethylated
hCPT derivatives (such as 6a, 6b and 6c) possessed higher in vitro antitumor activity than topotecan (TPT).
Especially, the compound 6c showed effective in vivo antitumor activity comparable to that of TPT.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 19 January 2010
Received in revised form
21 February 2010
Accepted 22 February 2010
Available online 1 March 2010
Keywords:
Homocamptothecin
Camptothecin
Synthesis
Trifluoromethyl group
Biological activity
1. Introduction
reinforces the inhibition of topoisomerase I compared with conven-
tional six-membered CPTs.
Camptothecin (CPT) [1], a pentacyclic alkaloid isolated from
Camptothecin acuminata (Xi Su) by Wall and Wani in 1966, has
shown excellent anticancer activity, and thus became a prominent
lead for anticancer drug development. It has already been discov-
ered that the cytotoxicity of camptothecin is due to a novel
mechanism of action involving selective inhibition of DNA top-
oisomerase I (topo I) [2,3].
Three of its analogs, topotecan, irinotecan and belotecan (Fig. 1),
are being used in clinic for the treatment of cancers and several other
analogs are currently under clinical development at various stages
[4]. However, the severe toxicity, the instability of the lactone and
other defects of CPTs [5] have promoted intensive efforts to increase
the efficacy of these agents, 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 camptothecin lactone ring from a six-membered a-hydroxy-
The high stability of hCPT is with no doubt directly attributed to
its expanded lactone E-ring but the stability and anti-
topoisomerase I activity can be enhanced further by adding
a substituent to the native scaffold. Numerous strategies have been
utilized in efforts to improve hCPT's pharmacological profile in
order to obtain more effective antitumor candidates. Among them,
fluorine substitution imparts unique and advantageous physi-
ochemical properties that can be shrewdly employed to construc-
tively alter pharmacological agents. Fluorine compounds have
specificity as compared with hydrocarbons. It is considered that this
specificity is based on higher electronegativity of the fluorine atom
than the other atoms, sterically small molecular volume next to the
hydrogen, and large carbon-fluorine bond energy [6]. Therefore,
when a compound is modified with a fluorine atom, especially
a trifluoromethyl group, chemical properties and reactivity of the
compound could undergo conspicuous change. The adornment of
natural products with fluorine has proven beneficial in many
examples [7]. With regard to homocamptothecin, some fluorinated
derivatives have also been studied [8e11]. A systematic analysis of
the effect of a mono or bis-fluorination of the quinoline A-ring of
hCPT indicated very clearly that the fluorine substitution of the
hCPT skeleton is a key feature for activity enhancement [12]. This
research has led to two drug candidates (Fig. 2). Exatecan, fluori-
nated on the A aromatic ring, is under phase III development,
despite the lack of confirmation of initial hopes during the phase II
lactone to a seven-membered b-hydroxylactone ring, which leads to
the development of the homocamptothecin (hCPT) family of
compounds. The expanded seven-membered lactone E-ring that
characterizes hCPTs enhances the plasma stability of the drug and
* Corresponding author. Tel.: þ86 21 81871233.
** Corresponding author. Tel./fax: þ86 21 81871243.
E-mail addresses: miaozhenyuan@hotmail.com (Z. Miao), zhangwnk@hotmail.
com (W. Zhang).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.051

L. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 2726e2732
2727
9
7
O
O
10
N
N
A
B
N
C
N
D
O
O
E
N
O
OH
hCPT
OH O
CPT
N
N
O
N
H
HO
O
HO
O
O
O
O
N
N
N
N
N
N
O
O
belotecan
OH O
topotecan
irinotecan
OH O
OH O
Fig. 1. Camptothecin, homocamptothecin and representative analogs.
trials [13]. Diflomotecan (BN80915), a homocamptothecin difluori-
nated on the A aromatic ring, is currently in phase II [12]. Diflo-
motecan is remarkably efficient at inhibiting the growth of human
colon cancer cells in vivo and, administered orally, it also shows
superior activities against human prostate cancers compared with
the benchmark products topotecan (TPT) and irinotecan (IRT). This
9, 10-difluoro-hCPT derivative is one of the most promising new
members of the ‘tecan’ family.
From the structureeactivity relationship (SAR) of CPT [14], it
appears that substituents on position 7 are very important to the
activity of the CPT analogues. The previous studies have demon-
strated that substitution on position 7 is no steric compromise [15].
A number of analogues with substituents on position 7 show
enhanced biological profiles [16]. Therefore, in an effort to find new
fluorinated hCPTs with promising biological properties, our present
study introduced the trifluoromethyl group to the position 7 of the
homocamptothecin skeleton in order to explore whether this
modification improves the biological properties of hCPTs.
species with ethyl trifluoroacetate resulted in the formation of
ketoamides 3 which can be isolated with moderate yields by
crystallization. A 50% excess of n-butyllithium was required to
make up for its consumption by the solvent (tetrahydrofuran) [19].
Hydrolysis of the amide in situ (conc. HCl) provided the hydro-
chloride of 4. Treatment of the salt with aqueous NaHCO3 provided
the corresponding free base 4.
The key intermediate CDE ring 5 was synthesized according to the
previously reported method [20], and the synthetic process was
optimized in our present studies [21]. Friedlander condensation of the
tricyclic 5 with o-trifluoroacetylanilines 4 in the presence of an acid
catalyst gave the novel 7- trifluoromethylated homocamptothecins 6.
Previously, the synthesis of CPTs or hCPTs by Friedlander condensa-
tion was usually catalyzed by toluene-4-sulfonic acid (p-TSA)
[22]. Herein, we firstly utilized proline-catalyzed Friedlander annu-
lation [23] toward the synthesis of 7-trifluoromethylated homo-
camptothecin derivatives successfully.
In order to demonstrate the superiority of trifluoromethyl
group compared to other alkyl groups, we synthesized six
7-methylated homocamptothecin derivatives corresponding to the
7-trifluoromethylated ones (Scheme 3).
2. Chemistry
In order to test the hypothesis concerning the effect of tri-
fluoromethyl group on position 7 of hCPT, we prepared seven
derivatives of 7-trifluoromethylated hCPT in racemeric mixtures by
the Friedlander route. Scheme 1 shows the synthetic strategy for
7-CF3-hCPTs based on the coupling of o-trifluoroacetylaniline ring
A with tricyclic keto pyridone ring CDE.
As key reaction intermediates, the o-trifluoroacetylanilines 4
were prepared from anilines 1 as shown in Scheme 2 [17,18].
Reaction of anilines 1 with pivaloyl chloride in a two-phase mixture
of tert-butyl methyl ether (MTBE) and aqueous sodium hydroxide
afforded pivaloylamides 2 in high yield. Directed orthometalation
of 2 (1.5 equiv of n-BuLi or n-hexylLi, THF, TMEDA) generated
the corresponding dilithio intermediates. Reaction of the dilithio
3. Pharmacology
The in vitro antitumor activities of new 7-trifluoromethylated
homocamptothecin derivatives against the solid tumor cell lines,
A549 (non-small cell lung cancer), LOVO (colon cancer), and MDA-
MB-435 (breast cancer), were evaluated, and the comparative assay
of antitumor activity between 7-CH3-hCPTs and 7-CF3-hCPTs was
also investigated. Topotecan was used as a reference compound
since it is a clinically effective CPT and does not require metabolic
activation. In addition, the in vivo antitumor activity of compounds
6c, 6d, 8b, and 8c against mice Lewis lung cancer was estimated.
4. Results and discussion
NH2
F
4.1. In vitro antitumor activity of 7-CF3-hCPTs
O
N
O
The results of in vitro antitumor studies are summarized in Table
1. The inhibitory activities suggested that the trifluoromethyl group
on the position 7 can promote antitumor activity to some extent.
The results revealed that these compounds were more sensitive
against A549 and LOVO cell lines than against MDA-MB-435 cell
line, which was consistent with the clinical behavior of CPT deriv-
atives [24]. For A549 cell line, compounds 6a, 6b, and 6c showed
F
N
N
O
F
N
O
O
OH
OH O
diflomotecan
exatecan
Fig. 2. Structures of representative fluorinated CPT and hCPT.

2728
L. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 2726e2732
R2
CF3
O
6a
6b
R2=H
R2=H
R2=H
R2=CH3O
R2=H
R2=H
R2=H
R2
O
R1 = Cl
R1 = CH3O
R1
N
O
R1
CF3
N
6c R1 = H
6d R1 = H
6e R1 = Br
6f
6g
+
O
O
N
NH2
O
O
R1 = CF3
R1 = OH
OH
5
O
4a-g
6a-g
OH
Scheme 1. Reagents and conditions: proline, DMSO, rt, 24 h.
higher in vitro inhibitory activities than topotecan. Several
compounds, such as 6c, 6d and 6g, also showed better inhibitory
activity against LOVO cell line than topotecan. With regard to MDA-
MB-435 cell line, compounds 6c and 6d exhibited antitumor
activities comparable to TPT. In particular, the activity of the most
promising compound 6c showed broad in vitro antitumor spec-
trum, which is a good candidate for further evaluation.
4.4. In vivo activity
Among the prepared compounds, two pairs of ones, 6c and 8b,
6d and 8c, were selected for further detail in vivo antitumor
activity studies on mice Lewis lung cancer model which exhibited
appreciable responsiveness to topotecan. As a consequence of low
water solubility, these compounds and topotecan (used for posi-
tive control drug) were administered only by intraperitoneal
injection. The results (Table 2) revealed that these compounds
were very potent. At low dose level (0.5 mg/kg), they were
effective in tumor growth inhibition, especially for 6c and 8b
(with inhibition of 45% and 41%, respectively). Although these
compounds didn't exhibit superior efficacy compared with
topotecan (inhibition of around 48%), the trifluoromethylated
compounds 6c and 6d showed more effective tumor growth
inhibition than that of the corresponding 8b and 8c, which was
consistent with the in vitro results (Table 1). Consequently, the
obtained results demonstrated that the trifluoromethyl group on
position 7 of hCPT indeed played a positive role in improving the
antiproliferative activity of hCPT derivatives.
4.2. Comparative evaluation of in vitro activity between
7-CF3-hCPTs and 7-CH3-hCPTs
According to the comparison of antitumor activities between
7-CH3 and 7-CF3-hCPTs (Table 1), it is clearly discovered that most
7-CF3-hCPTs showed higher in vitro antiproliferative activity than
the corresponding 7-CH3-hCPTs, which coincides with our initial
design idea. 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),
changed electronic distributions and increased stability, which can
be accounted for by the unique nature of the trifluoromethyl group.
Thus, it can be speculated that introducing a trifluoromethyl group
to the position 7 of the hCPT skeleton has a positive effect on
cytotoxicity of hCPTs.
5. Conclusion
In summary, homocamptothecin and its analogs are proving to
be an especially effective class of camptothecins that merits
considerable attention. In response to the emergent need for novel
antitumor agents with improved activity, we utilized the unique
properties of trifluoromethyl group to improve the antiproliferative
activity of hCPTs. In our present study, seven novel 7-tri-
fluoromethylated homocamptothecins were designed and synthe-
sized. The proline-catalyzed Friedlander annulation was firstly used
to prepare 7-CF3-hCPTs. Taken together, the results in this study
provide, for the first time, convincing evidence that the position 7
of hCPT is a favorable position for introduction of a trifluoromethyl
group, while the mechanism of these trifluoromethylated homo-
camptothecins is still unknown. Further studies will clearly be
required to understand the role of trifluoromethyl group of position
7 in enhancing the antitumor activity of hCPTs. A better under-
standing of this feature could provide meaningful insights for
further optimization of drug design. We hope that 7-CF3-hCPT
would represent not only an useful design idea but also a promising
template for the preparation of improved anticancer agents in the
future.
4.3. Inhibition of topo I-induced DNA relaxation
Generally, camptothecin and its clinically used derivatives act by
stabilizing a covalent topoisomerase I-DNA complex called the
cleavable complex. Therefore, topoisomerase I-mediated DNA
cleavage assays with topoisomerase I were used to investigate the
ability of 7-CF3-hCPTs to inhibit Topo I. The ability of 7-CF3-hCPT 6c
and 7-CH3-hCPT 8b to stabilize the cleavable complex was evaluated
by incubating Topo I and supercoilded DNA pBR322 in the presence
of drugs. Cleavable complexes were revealed by the appearance of
short DNA fragments when the samples were analyzed by gel
electrophoresis under denaturing conditions. The results (Fig. 3)
indicated that the increasing concentration of CPT, 8b, or 6c was
accompanied by a dose-dependent increase in the level of cleavable
complex. Most importantly, 6c showed lower intensity of the
migrating band (corresponding to nicked DNA) than 8b regardless of
low concentration (1 mM) or high concentration (50 mM). Accord-
ingly, it can be concluded that 7-CF3-hCPT is a more potent inducer
of topoisomerase I-mediated DNA cleavage than 7-CH3-hCPT.
R1
R1
R2
O
R1
R2=H
4a R1 = Cl
R2=H
R2=H
R2
4b R1 = CH3O
R2
R1
R2
a
c
b
CF3
4c
R1 = H
4d R1 = H
R2=CH3O
R2=H
R2=H
F3COC
NH2
4e R1 = Br
4f R1 = CF3
4g R1 = OH
NHCO(CH3)3
3a-f
NHCO(CH3)3
2a-f
NH2
R2=H
4a-f
1a-f
d
4b
4g
Scheme 2. Reagents and conditions: (a) (CH3)3COCl, MTBE, 40% NaOH aq; (b) n-BuLi, CF3CO2C2H5, THF, TMEDA, ꢀ40 ꢁC; (c) conc. HCl, ref., 6 h; (d) 48% HBreAcOH, 80 ꢁC, 2 h, 89.2%.

L. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 2726e2732
2729
R2
CH3
O
R2
O
R2=H
R2=H
R2=CH3O
R2=H
R2=H
8a R1 = CH3O
8b
8c R1 = H
8d R1 = Br
8e R1 = CF3
8f R1 = OH
N
R1
R1
R1 = H
O
CH3
+
N
O
O
N
NH2
O
O
R2=H
OH
5
O
8a-f
7a-f
OH
Scheme 3. Reagents and conditions: toluene, p-TSA, 110ꢁC, 2.5 h.
6. Experimental protocols
tetrahydrofuran (THF) (500 mL), and the mixture was cooled
to ꢀ40 ꢁC. The n-butyllithium solution (600 mL of a 2.5 M solution
in hexane, 1.5 mol) was added dropwise to the cold amide solution
slowly over 2 h, while the temperature was kept below 15ꢁC. The
mixture was aged at 15ꢀ20v for 4 h and cooled below ꢀ25 ꢁC again.
To this mixture was added ethyl trifluoroacetate or ethyl acetate
(2.5 mol) rapidly. After 5 h, the resulting clear solution was
quenched with 15% aqueous HCl, keeping the temperature below
10 ꢁC. The mixture was diluted with 200 mL ethyl ether and the
layers were separated. The organic phase was washed with brine,
dried, filed and concentrated in vacuo, to give a yellow oil. This
material was cooled to 25 ꢁC,12 M HCl (100 mL,1.2 mol) was added,
and the mixture was heated to 75ꢁC and held for 6 h. The cooled
solution was diluted with 100 mL of EtOAc and the mixture was
made basic with a solution of sodium bicarbonate (84.3 g, 1.0 mol)
in water (150 mL). Then the mixture was extracted with ethyl
acetate (50 mL ꢃ 3). The combined organic phase was washed with
brine and dried over anhydrous Na2SO4. After the removal of
solvent, the residue was purified by flash chromatography on silica
gel (petroleum ether/ethyl acetate ¼ 20/1) to afford 4aef or 7aee.
To a solution of acetic acid (50 mL) and aqueous hydrobromic
acid (48%, 50 mL) heated at 80ꢁC was added 4b or 7a (10 mmol).
The mixture was heated at 80 ꢁC for 2 h. After cooling, the mixture
was diluted with cold water (200 mL) and extracted with ethyl
acetate. The organic layer was washed with water and brine, dried
over MgSO4. On evaporation of the solvent, the product 4g or 7f was
obtained.
6.1. General
All reagents and solvents were reagent grade or were purified by
standard methods before use. The melting points were determined
using an electrothermal apparatus and are uncorrected. 1H and 13
C
NMR spectra were recorded at either 300 MHz or 500 MHz with
a Bruker instrument, and reported with TMS as internal standard
and CDCl3 or DMSO-d6 as solvent. Chemical shifts (d values) and
coupling constants (J values) are given in ppm and Hz, respectively.
Elemental analyses were performed with a MOD-1106 instrument
and were consistent with theoretical values within ꢂ0.4%. TLC
analysis was carried out on silica gel plates GF254 (Qindao Haiyang
Chemical, China). Flash column chromatography was carried out on
silica gel 200e300 mesh. Anhydrous solvent and reagents were all
analytically pure and dried through routine protocols. All reactions
requiring anhydrous conditions were performed under a positive
nitrogen flow, and all glassware was oven-dried and/or flame-
dried.
6.2. Typical experimental procedure for the synthesis of 2aef
Anilines (1, 50 mmol) was charged into a mixture of tert-butyl
methyl ether (60 mL) and 40% aqueous sodiumhydroxide (5.5 g,
55 mmol), and the mixture was cooled to 0 ꢁC. To the resulting
slurry was added trimethylacetyl chloride (PVCL) (6.6 g, 55 mmol)
over 0.5 h, keeping the temperature below 15 ꢁC. After being stirred
for 2.5 h at 25 ꢁC, the slurry was cooled to 0 ꢁC and held for 1.5 h.
The product was collected by filtration, washed with a solution of
9/1 water/methanol (150 mL) and water (100 mL), and dried in
vacuo to give pivaloylamide 2 as white solid.
6.3.1. 1-(2-amino-5-chlorophenyl)-2, 2, 2-trifluoroethanone (4a)
A bright yellow solid; yield (76%); 1H NMR (300 MHz, CDCl3)
d
: 6.46 (br s, 2H), 6.70 (d, 1H, J ¼ 9.0 Hz), 7.32 (dd, 1H, J ¼ 2.1 Hz,
9.0 Hz), 7.71 (d, 1H, J ¼ 2.1 Hz); MS (ESI) m/z: 222 (M ꢀ H); Anal.
calcd. for C8H5ClF3NO: C, 42.98; H, 2.25; N, 6.26. Found: C, 42.92;
H, 2.25; N, 6.24.
6.3. General procedure for synthesis of 4aef, 7aeg
6.3.2. 1-(2-amino-5-methoxyphenyl)-2, 2, 2-trifluoroethanone (4b)
An orange solid; yield (80%); 1H NMR (300 MHz, CDCl3)
d: 3.78
Under an atmosphere of nitrogen, compound 2 (1.0 mol) was
charged to a solution of TMEDA (11.6 g, 1.0 mol) in anhydrous
(s, 3H), 6.25 (br s, 2H), 6.69 (d,1H, J ¼ 9.0 Hz), 7.11 (dd,1H, J ¼ 2.1 Hz,
9.0 Hz), 7.15 (d, 1H, J ¼ 2.1 Hz); MS (ESI): 218 (M ꢀ H); Anal. calcd.
for C9H8F3NO2: C, 49.32; H, 3.68; N, 6.39. Found: C, 49.28; H, 3.69;
N, 6.38.
Table 1
The in vitro antitumor activity of 7-CF3-hCPTs and 7-CH3-hCPTsa.
R1
R2
Compounds
IC50
(
m
M)
6.3.3. 1-(2-aminophenyl)-2, 2, 2-trifluoroethanone (4c)
A bright yellow needles; yield (85%); 1H NMR (300 MHz, CDCl3)
A549
LOVO
1.317
14.608
1.162
MDA-MB-435
CH3O
H
H
8a
6b
8b
6c
8c
6d
8d
6e
8e
6f
3.522
0.832
0.468
2.610
21.413
3.882
2.325
7.733
d
: 6.24 (br s, 2H), 6.67 (d, 1H, J ¼ 1.2 Hz), 6.73 (dd, 1H, J ¼ 1.8 Hz,
3.9 Hz), 7.38 (dd, 1H, J ¼ 1.2 Hz, J ¼ 3.9 Hz), 6.67 (d, 1H, J ¼ 1.8 Hz);
MS (ESI): 188 (M ꢀ H); Anal. calcd. for C8H6F3NO: C, 50.80; H, 3.20;
N, 7.41. Found: C, 50.87; H, 3.21; N, 7.39.
H
0.313
1.290
5.467
3.434
2.312
H
CH3O
H
10.911
5.130
2.869
Br
29.052
27.913
>225.22
20.301
9.890
30.859
11.001
>225.22
5.321
3.393
11.213
6.3.4. 6.3.4.1-(2-amino-6-methoxyphenyl)-2, 2,
2-trifluoroethanone (4d)
13.327
>225.22
22.931
7.091
CF3
OH
H
A lemon-yellow solid; yield (83%); 1H NMR (500 MHz, CDCl3)
d
: 3.84 (s, 3H), 6.05 (br s, 2H), 6.20 (d, 1H, J ¼ 8.1 Hz), 6.31 (d, 1H,
H
8f
6g
16.210
4.013
J ¼ 8.3 Hz), 7.25 (t, 1H, J ¼ 8.2 Hz); MS (ESI): 220 (M þ H); Anal.
calcd. for C9H8F3NO2: C, 49.32; H, 3.68; N, 6.39. Found: C, 49.29;
H, 3.67; N, 6.37.
a
Cancer cells: A549: Lung cancer cell; LOVO: Colon cancer cell; MDA-MB-435:
Breast cancer cell.

2730
L. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 2726e2732
Fig. 3. Gel electrophoresis of Topo1-induced DNA cleavage assay for 7-CF3-hCPT 6c and 7-CH3-hCPT 8b. Topo I was topoisomerase I and DNA without drugs. The samples were
reacted with 1, 10, and 50
M drug at 37 ꢁC for 15 min. Reactions were then stopped by adding 0.5% SDS. Gels were photographed under UV transilluminator.
m
6.3.5. 1-(2-amino-5-bromophenyl)-2, 2, 2-trifluoroethanone (4e)
An orange solid; yield (70%); 1H NMR (500 MHz, CDCl3)
: 6.42
6.3.11. 1-(2-amino-5-bromophenyl)ethanone (7d)
d
A brown solid; yield (75%); 1H NMR (500 MHz, CDCl3)
d: 2.55
(br s, 2H), 6.64(d, 1H, J ¼ 8.9 Hz), 7.44 (dd, 1H, J ¼ 2.0 Hz, 8.9 Hz),
7.84 (d, 1H, J ¼ 2.0 Hz); MS (ESI): 266 (M ꢀ H); Anal. calcd. for
C8H5BrF3NO: C, 35.85; H, 1.88; N, 5.23. Found: C, 35.90; H, 1.88; N,
5.24.
(s, 1H), 6.29 (br s, 2H), 6.55 (d, 1H, J ¼ 8.8 Hz), 7.32 (dd, 1H,
J ¼ 2.3 Hz, 8.8 Hz), 7.84 (d, 1H, J ¼ 2.3 Hz); MS (ESI): 214 (M þ H);
Anal. calcd. for C8H8BrNO: C, 44.89; H, 3.77; N, 6.54. Found: C,
44.86; H, 3.78; N, 6.55.
6.3.6. 1-(2-amino-5-trifluoromethylphenyl)-2, 2,
2-trifluoroethanone (4f)
6.3.12. 1-(2-amino-5-(trifluoromethyl)phenyl)ethanone (7e)
A light green solid; yield (80%); 1H NMR (500 MHz, CDCl3)
d:
A bright yellow needles; yield (90%); 1H NMR (500 MHz, CDCl3)
2.61 (s, 1H), 6.67 (br s, 2H), 6.81 (d, 1H, J ¼ 8.7 Hz), 7.45 (dd, 1H,
J ¼ 1.8 Hz, 8.7 Hz), 7.95 (d, 1H, J ¼ 1.8 Hz); MS (ESI): 202 (M ꢀ H);
Anal. calcd. for C9H8F3NO: C, 53.21; H, 3.97; N, 6.89. Found: C,
53.24; H, 3.96; N, 6.87.
d: 6.66 (br s, 2H), 6.81 (d, 1H, J ¼ 8.9 Hz), 7.57 (dd, 1H, J ¼ 1.2 Hz,
8.9 Hz), 7.15 (d, 1H, J ¼ 1.2 Hz); MS (ESI): 256 (M ꢀ H); Anal. calcd.
for C9H5F6NO: C, 42.04; H, 1.96; N, 5.45. Found: C, 42.09; H, 1.96;
N, 5.46.
6.3.13. 1-(2-amino-5-hydroxyphenyl)ethanone (7f)
6.3.7. 1-(2-amino-5-hydroxyphenyl)-2, 2, 2-trifluoroethanone (4g)
A brown solid; yield (66%); 1H NMR (500 MHz, CDCl3)
d: 4.44
A bright yellow solid; yield (76%); 1H NMR (500 MHz, CDCl3)
d
:
(s, 1H), 6.05 (br s, 2H), 6.75 (d, 1H, J ¼ 8.8 Hz), 7.01 (dd, 1H,
J ¼ 3.0 Hz, 8.8 Hz), 7.12 (d, 1H, J ¼ 3.0 Hz); MS (ESI): 150 (M ꢀ H);
Anal. calcd. for C8H9NO: C, 63.56; H, 6.00; N, 9.27. Found: C, 63.51;
H, 6.01; N, 9.24.
4.46 (s, 1H), 6.19 (br s, 2H), 6.67 (d, 1H, J ¼ 9.0 Hz), 7.05 (dd, 1H,
J ¼ 2.8 Hz, 9.0 Hz), 7.17 (d, 1H, J ¼ 2.8 Hz); MS (ESI): 204 (M ꢀ H);
Anal. calcd. for C8H6F3NO2: C, 46.84; H, 2.95; N, 6.83. Found:
C, 46.79; H, 2.96; N, 6.82.
6.4. Typical procedure for preparation of 6aeg
6.3.8. 1-(2-amino-5-methoxyphenyl)ethanone (7a)
A gray solid; yield (50%); 1H NMR (500 MHz, CDCl3) dd: 2.57 (s,
3H), 3.78 (s, 3H), 6.01 (br s, 2H), 6.63 (d, 1H, J ¼ 8.9 Hz), 6.97 (dd, 1H,
J ¼ 2.9 Hz, 8.9 Hz), 7.18 (d, 1H, J ¼ 2.9 Hz); MS (ESI): 165 (M þ H);
Anal. calcd. for C9H11NO2: C, 65.44; H, 6.71; N, 8.48. Found: C, 65.40;
H, 6.72; N, 8.50.
A solution of compound 4 (2.4 mmol) and tricyclic ketone 5
(0.8 g, 3.6 mmol) in DMSO (10 mL) was charged in a three-necked
flask for 20 min. The proline (0.1 g) was then added and the reac-
tion was continued for an additional 24 h at room temperature.
After the reaction was complete, H2O (6 mL) was added .The yellow
solid was filtered off and washed by methanol (15 mL) and acetone
(15 mL) to give 6 as yellow solid.
6.3.9. 1-(2-aminophenyl)ethanone (7b)
A brown oil; yield (70%); 1H NMR (500 MHz, CDCl3)
d: 2.57 (s,
3H), 6.25 (br s, 2H), 6.63e6.66 (m, 2H), 7.26 (dd, 1H, J ¼ 1.3 Hz,
5.8 Hz), 7.71 (dd, 1H, J ¼ 1.3 Hz, J ¼ 5.9 Hz); MS (ESI): 134 (M ꢀ H);
Anal. calcd. for C8H9NO: C, 71.09; H, 6.71; N, 10.36. Found: C, 71.04;
H, 6.72; N, 10.33.
6.4.1. 10-Chloro-7-trifluoromethylhomocamptothecin (6a)
A yellow solid; yield (40%); mp > 300 ꢁC; 1H NMR (500 MHz,
DMSO-d6)
d
: 0.87 (t, 3H, J ¼ 7.3 Hz, CH3), 1.86 (q, 2H, J ¼ 7.4 Hz,
CH3CH2), 3.07e3.49 (q, 2H, J ¼ 13.9Hz, CH2CO), 5.39e5.55 (q,
2H, J ¼ 15.3 Hz, NCH2), 5.46 (s, 2H, OCH2), 6.08 (s, 1H, OH), 7.42 (s,
1H, C14eH), 8.05 (dd, 1H, J ¼ 2.4 Hz, 9.1 Hz, C11eH), 8.26 (d, 1H,
6.3.10. 6.3.10.1-(2-amino-6-methoxyphenyl)ethanone (7c)
A brown solid; yield (55%); 1H NMR (300 MHz, CDCl3)
d: 2.58 (s,
J ¼ 9.1 Hz, C12eH), 8.39 (d, 1H, J ¼ 2.4 Hz, C9eH); 13C NMR
d (ppm):
1H), 3.85 (s, 3H), 5.93 (br s, 2H), 6.19 (dd,1H, J ¼ 0.6 Hz, 8.7 Hz), 6.26
(dd, 1H, J ¼ 0.9 Hz, 7.8 Hz), 7.13 (t, 1H, J ¼ 8.1 Hz); MS (ESI): 164 (M-
H); Anal. calcd. for C9H11NO2: C, 65.44; H, 6.71; N, 8.48. Found: C,
65.41; H, 6.73; N, 8.50.
8.57, 36.64, 42.73, 51.51, 61.57, 73.49, 100.70, 122.77, 123.18, 124.14,
124.95, 127.87, 130.28, 132.25, 132.73, 134.96, 143.16, 147.49, 154.10,
156.21, 159.06, 172.10; MS (ESI): 465 (M þ H); Anal. calcd. for
C22H16ClF3N2O4: C, 56.85; H, 3.47; N, 6.03. Found: C, 56.89; H, 3.48;
N, 6.02.
Table 2
The in vivo antitumor activity of compounds 6c, 6d, 8b and 8c against mice Lewis
lung cancer model.
6.4.2. 10-Methoxy-7-trifluoromethylhomocamptothecin (6b)
A yellow solid; yield (45%); mp > 300ꢁC; 1H NMR (500 MHz,
Sample Dose
(mg/kg)
Mice number
Body weight Tumor
(g, tumor
removed)
Tumor
weight (g) growth
inhibition %
Begin
End
DMSO-d6)
d
: 0.87 (t, 3H, J ¼ 7.4 Hz, CH3), 1.85 (q, 2H, J ¼ 7.5 Hz,
CH3CH2), 3.05e3.49 (q, 2H, J ¼ 13.9 Hz, CH2CO), 4.01 (s, 3H, OCH3),
5.38e5.54 (q, 2H, J ¼ 15.0 Hz, NCH2), 5.45 (s, 2H, OCH2), 6.05 (s, 1H,
OH), 7.39 (d, 1H, J ¼ 2.7 Hz, C9eH), 7.40 (s, 1H, C14eH), 7.71 (dd, 1H,
J ¼ 2.7 Hz, 9.3 Hz, C11eH), 8.26 (d, 1H, J ¼ 9.3 Hz, C12eH); MS (ESI):
459 (M ꢀ H); Anal. calcd. for C23H19F3N2O5: C, 60.00; H, 4.16; N,
6.08. Found: C, 60.06; H, 4.15; N, 6.06.
Control 25
10
6
10
6
19.06 ꢂ 1.35 1.80 ꢂ 0.47
6c
0.5
19.16 ꢂ 0.44 0.98 ꢂ 0.14 45.59
18.80 ꢂ 0.98 1.06 ꢂ 0.34 41.23
19.41 ꢂ 1.17 1.26 ꢂ 0.38 30.08
19.77 ꢂ 1.27 1.38 ꢂ 0.34 23.21
19.24 ꢂ 0.85 0.94 ꢂ 0.26 47.45
8b
6d
8c
0.5
0.5
0.5
0.5
6
6
6
6
6
6
TPT
6
6

L. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 2726e2732
2731
6.4.3. 7-Trifluoromethylhomocamptothecin (6c)
6.5.1. 10-Methoxy-7-methylhomocamptothecin (8a)
A yellow solid; yield (50%); mp > 260 ꢁC; 1H NMR (500 MHz,
DMSO-d6)
CH3CH2), 2.74 (s, 3H, CH3), 3.03e3.47 (q, 2H, J ¼ 13.8 Hz, CH2CO),
3.98 (s, 3H, OCH3), 5.25 (s, 2H, OCH2), 5.39e5.52 (q, 2H, J ¼ 15.0 Hz,
NCH2), 6.00 (s, 1H, OH), 7.32 (d, 1H, J ¼ 2.5 Hz, C9eH), 7.46
(s, 1H, C14eH), 7.50 (dd, 1H, J ¼ 2.5 Hz, 9.2 Hz, C11eH), 8.05 (d, 1H,
J ¼ 9.2 Hz, C12eH); MS (ESI): 405 (M-H); Anal. calcd. for C23H22N2O5:
C, 67.97; H, 5.46; N, 6.89. Found: C, 67.91; H, 5.45; N, 6.90.
A yellow solid; yield (50%); mp > 300 ꢁC; 1H NMR (500 MHz,
DMSO-d6)
d
: 0.87 (t, 3H, J ¼ 7.4 Hz, CH3), 1.86 (q, 2H, J ¼ 7.5 Hz,
d
: 0.86 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 1.85 (q, 2H, J ¼ 7.0 Hz,
CH3CH2), 3.07e3.48 (q, 2H, J ¼ 13.9 Hz, CH2CO), 5.40e5.55
(q, 2H, J ¼ 15.2 Hz, NCH2), 5.47 (s, 2H, OCH2), 6.07 (s, 1H, OH), 7.48
(s, 1H, C14eH), 7.93 (t, 1H, J ¼ 7.2 Hz, C10eH), 8.03 (t, 1H, J ¼ 7.4 Hz,
C
11eH), 8.22 (d,1H, J ¼ 8.4 Hz, C9eH), 8.34 (d,1H, J ¼ 8.4 Hz, C12eH);
13C NMR
d (ppm): 8.57, 36.65, 42.73, 51.50, 61.57, 73.47, 100.58,
122.77, 123.18, 124.12, 124.94, 127.85, 130.28, 132.25, 132.72, 134.96,
143.16, 147.49, 154.40, 156.21, 159.06, 172.14; MS (ESI): 429 (M ꢀ H);
Anal. calcd. for C22H17F3N2O4: C, 61.40; H, 3.98; N, 6.51. Found:
C, 61.35; H, 3.99; N, 6.49.
6.5.2. 7-Methylhomocamptothecin (8b)
A yellow power; yield (52%); mp > 260 ꢁC; 1H NMR (500 MHz,
DMSO-d6)
d
: 0.87 (t, 3H, J ¼ 7.4 Hz, CH3CH2), 1.86 (q, 2H, J ¼ 7.5 Hz,
6.4.4. 9-Methoxy-7-trifluoromethylhomocamptothecin (6d)
A yellow power; yield (25%); mp > 300 ꢁC; 1H NMR (500 MHz,
CH3CH2), 2.78 (s, 3H, CH3), 3.05e3.47 (q, 2H, J ¼ 13.8 Hz, CH2CO),
5.26 (s, 2H, OCH2), 5.41e5.53 (q, 2H, J ¼ 15.1 Hz, NCH2), 6.03 (s, 1H,
OH), 7.39 (s, 1H, C14eH), 7.72 (t, 1H, J ¼ 7.1 Hz, C10-H), 7.86
(t, 1H, J ¼ 7.2 Hz, C11eH), 8.14 (d, 1H, J ¼ 8.2 Hz, C9eH), 8.25 (d, 1H,
DMSO-d6)
d
: 0.87 (t, 3H, J ¼ 7.4 Hz, CH3), 1.85 (q, 2H, J ¼ 7.5 Hz,
CH3CH2), 3.05e3.45 (q, 2H, J ¼ 13.6 Hz, CH2CO), 4.05 (s, 3H, OCH3),
5.39e5.58 (q, 2H, J ¼ 15.3 Hz, NCH2), 5.49 (s, 2H, OCH2), 6.07 (s, 1H,
OH), 7.25 (s, 1H, C14eH),7.29 (d, 1H, J ¼ 2.5 Hz, C10eH), 7.49 (d, 1H,
J ¼ 9.1 Hz, C12eH), 7.71 (dd, 1H, J ¼ 2.5 Hz, 9.1 Hz, C11eH); 13C NMR
J ¼ 8.2 Hz, C12eH); 13C NMR
d (ppm): 8.62, 15.35, 36.68, 42.79,
50.52, 61.70, 73.54, 99.88, 122.78, 124.72, 127.77, 128.07, 129.22,
130.01, 130.36, 140.50, 145.47, 147.25, 152.16, 156.81, 159.49, 172.22;
MS (ESI): 375 (M-H); Anal. calcd. for C22H20N2O4: C, 70.20; H, 5.36;
N, 7.44. Found: C, 70.25; H, 5.37; N, 7.46.
d
(ppm): 8.56, 36.63, 42.73, 51.51, 57.08, 61.55, 73.49, 100.71, 122.75,
123.01, 124.10, 124.89, 127.88, 130.14, 132.21, 132.69, 134.96, 143.16,
147.49, 154.35, 156.02, 159.01, 172.09; MS (ESI): 459 (M-H); Anal.
calcd. for C23H19F3N2O5: C, 60.00; H, 4.16; N, 6.08. Found: C, 60.05;
H, 4.17; N, 6.07.
6.5.3. 9-Methoxy-7-methylhomocamptothecin (8c)
A yellow power; yield (25%); mp > 260 ꢁC; 1H NMR (500 MHz,
DMSO-d6)
d
: 0.86 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 1.85 (q, 2H, J ¼ 7.1 Hz,
CH3CH2), 2.79 (s, 3H, CH3), 3.03e3.44 (q, 2H, J ¼ 13.5 Hz, CH2CO),
4.03 (s, 3H, OCH3), 5.33e5.56 (q, 2H, J ¼ 15.3 Hz, NCH2), 5.45 (s, 2H,
OCH2), 6.05 (s, 1H, OH), 7.20 (s, 1H, C14eH),7.25 (d, 1H, J ¼ 2.2 Hz,
6.4.5. 10-Bromo-7-trifluoromethylhomocamptothecin (6e)
A yellow power; yield (40%); mp > 300 ꢁC; 1H NMR (500 MHz,
DMSO-d6)
d
: 0.87 (t, 3H, J ¼ 7.3 Hz, CH3), 1.86 (q, 2H, J ¼ 7.4 Hz,
C
10eH), 7.40 (d, 1H, J ¼ 8.5 Hz, C12eH), 7.67 (dd, 1H, J ¼ 2.2 Hz,
CH3CH2), 3.07e3.48 (q, 2H, J ¼ 13.9 Hz, CH2CO), 5.40e5.56
(q, 2H, J ¼ 15.4 Hz, NCH2), 5.45 (s, 2H, OCH2), 6.07 (s, 1H, OH), 7.47
(s, 1H, C14eH), 8.09 (dd, 1H, J ¼ 2.5 Hz, 9.5 Hz, C11eH), 8.31 (d, 1H,
J ¼ 9.5 Hz, C12eH), 8.43 (d, 1H, J ¼ 2.5 Hz, C9-H). MS (ESI): 509
(M þ H); Anal. calcd. for C22H16F3N2O4: C, 51.88; H, 3.17; N, 5.50.
Found: C, 51.92; H, 3.18; N, 5.52.
8.5 Hz, C11-H); 13C NMR
d (ppm): 8.58, 15.41, 36.64, 42.74, 51.55,
57.09, 61.65, 73.51, 100.73, 122.77, 123.05, 124.13, 124.91, 130.17,
132.24, 132.74, 138.47, 145.04, 147.01, 152.85, 155.88, 159.55, 172.14;
MS (ESI): 405 (M-H); Anal. calcd. for C23H22N2O5: C, 67.97; H, 5.46;
N, 6.89. Found: C, 67.92; H, 5.47; N, 6.87.
6.5.4. 10-Bromo-7-methylhomocamptothecin (8d)
A yellow power; yield (40%); mp > 260 ꢁC; 1H NMR (500 MHz,
6.4.6. 10- Trifluoromethyl-7-trifluoromethylhomocamptothecin (6f)
A pale solid; yield (20%); mp > 300 ꢁC; 1H NMR (500 MHz,
DMSO-d6)
d
: 0.85 (t, 3H, J ¼ 7.2 Hz, CH3CH2), 1.85 (q, 2H, J ¼ 7.3 Hz,
DMSO-d6)
d: 0.88 (t, 3H, J ¼ 7.3 Hz, CH3), 1.87(q, 2H, J ¼ 7.4 Hz,
CH3CH2), 2.80 (s, 3H, CH3), 3.06e3.47 (q, 2H, J ¼ 13.5 Hz, CH2CO),
5.36e5.52 (q, 2H, J ¼ 15.0 Hz, NCH2), 5.40 (s, 2H, OCH2), 6.01 (s, 1H,
OH), 7.42 (s,1H, C14eH), 8.01 (dd,1H, J ¼ 2.2 Hz, 8.9 Hz, C11eH), 8.25
(d, 1H, J ¼ 8.9 Hz, C12eH), 8.40 (d, 1H, J ¼ 2.3 Hz, C9eH). MS (ESI):
455 (M þ H); Anal. calcd. for C22H19BrN2O4: C, 58.04; H, 4.21;
N, 6.15. Found: C, 58.07; H, 4.22; N, 6.13.
CH3CH2), 3.09e3.49 (q, 2H, J ¼ 13.9 Hz, CH2CO), 5.44e5.59 (q, 2H,
J ¼ 15.4 Hz, NCH2), 5.48 (s, 2H, OCH2), 6.12 (s, 1H, OH), 7.50
(s, 1H, C14eH), 8.21 (dd, 1H, J ¼ 2.6 Hz, 9.7 Hz, C11eH), 8.35 (d, 1H,
J ¼ 9.7 Hz, C12eH), 8.48 (d, 1H, J ¼ 2.6 Hz, C9eH); MS (ESI): 499
(M þ H); Anal. calcd. for C23H16F6N2O4: C, 55.43; H, 3.24; N, 5.62.
Found: C, 55.47; H, 3.23; N, 5.61.
6.5.5. 10-Trifluoromethyl-7-methylhomocamptothecin (8e)
6.4.7. 10-Hydroxy-7-trifluoromethylhomocamptothecin (6g)
A pale yellow power; yield (22%); mp > 300 ꢁC; 1H NMR
(500 MHz, DMSO-d6) d: 0.87 (t, 3H, CH3), 1.86 (q, 2H, CH3CH2),
3.08e3.50 (q, 1H, CH2CO), 5.39e5.55 (q, 2H, J ¼ 15.4 Hz, NCH2), 5.30
(s, 2H, OCH2), 6.04 (s, 1H, C20eOH), 7.40 (d, 1H, J ¼ 2.6 Hz, C9-H),7.41
(s, 1H, C14eH), 7.87 (dd, 1H, J ¼ 2.6 Hz, J ¼ 9.2 Hz, C11eH), 8.29 (d,
1H, J ¼ 9.2 Hz, C12eH), 10.35 (s, 1H, C10-OH), MS (ESI): 445 (M ꢀ H);
Anal. calcd. for C22H17F3N2O5: C, 59.20; H, 3.84; N, 6.28. Found: C,
59.25; H, 3.85; N, 6.30.
A pale solid; yield (20%); mp > 260 ꢁC; 1H NMR (500 MHz,
DMSO-d6)
d
: 0.87 (t, 3H, J ¼ 7.3 Hz, CH3CH2), 1.85 (q, 2H, J ¼ 7.2 Hz,
CH3CH2), 2.81 (s, 3H, CH3), 3.07e3.47 (q, 2H, J ¼ 13.5 Hz, CH2CO),
5.41e5.58 (q, 2H, J ¼ 15.0 Hz, NCH2), 5.46 (s, 2H, OCH2), 6.07 (s, 1H,
OH), 7.46 (s, 1H, C14eH), 8.19 (dd, 1H, J ¼ 2.4 Hz, 9.1 Hz, C11eH), 8.32
(d, 1H, J ¼ 9.1 Hz, C12eH), 8.45 (d, 1H, J ¼ 2.4 Hz, C9eH); MS (ESI):
443 (MeH); Anal. calcd. for C23H19F3N2O4: C, 62.16; H, 4.31; N, 6.30.
Found: C, 62.11; H, 4.32; N, 6.32.
6.5.6. 10-Hydroxy-7-methylhomocamptothecin (8f)
6.5. General Procedure for preparation of 8aef
A pale yellow power; yield (22%); mp > 260 ꢁC; 1H NMR
(500 MHz, DMSO-d6) d: 0.87 (t, 3H, CH3CH2), 1.86 (q, 2H, CH3CH2),
A solution of compound 4 (2.4 mmol) and tricyclic ketone 5
(0.8 g, 3.6 mmol) in toluene (400 mL) was refluxed in a three-
necked flask for 30 min. p-TSA (0.1 g) was then added and refluxing
was continued for an additional 2.5 h. The solution was cooled to
room temperature and the solid was filtered off and washed by
methanol (15 mL) and acetone (15 mL) to give 8 as yellow solid.
2.78 (s, 3H, CH3), 3.06e3.49 (q, 2H, CH2CO), 5.28 (s, 2H, OCH2),
5.39e5.53 (q, 2H, J ¼ 15.4 Hz, NCH2), 6.03 (s, 1H, C20eOH), 7.42 (s,
1H, C14eH), 7.72 (d, 1H, J ¼ 2.6 Hz, C9-H), 8.15 (dd, 1H, J ¼ 2.6 Hz,
J ¼ 9.2 Hz, C11eH), 8.69 (d, 1H, J ¼ 9.2 Hz, C12-H), 10.32 (s, 1H,
C
10eOH), MS (ESI): 391 (MeH); Anal. calcd. for C22H20N2O5: C,
67.34; H, 5.14; N, 7.14. Found: C, 67.38; H, 5.13; N, 7.16.

2732
L. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 2726e2732
6.6. Cytotoxicity
Growth inhibition ¼ ðATWCG ꢀ ATWTGÞ=ATWTG ꢃ 100%
One thousand two hundred cells per well were plated in 96-well
plates. After culturing for 24 h, test compounds were added onto
triplicate wells with different concentration, and 0.1% DMSO for
Acknowledgments
control. After three days of incubation, 20 mL MTT (3-[4,5-dime-
This research was supported in part by the National Natural
Science Foundation of China (No. 30371689), the Key Project of
Science and Technology of Shanghai (No. 09431901700), Major
Special Project for the Creation of New Drugs (Grant Nos.
2009ZX09301-011) and Shanghai Leading Academic Discipline
Project (Project Nos. B906).
thylthiazol-2-yl]-2,5-diphenyl- tetrazolium bromide) solution
(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 dis-
solved with 100 mL DMSO. The absorbance (OD) was quantitated
with microplate spectrophotometer at 570 nm. Wells containing no
drugs were used as blanks for the spectrophotometer. The survival
of the cells was expressed as percentage of untreated control wells.
References
6.7. Topoisomerase I inhibition activity assays
[1] M.E. Wall, M.C. Wani, C.E. Cook, K.H. Palmer, A.T. McPhail, G.A. Sim, J. Am.
Chem. Soc. 88 (1966) 3888e3890.
Camptothecin was obtained from the company of Tianzunzez-
hong in China. Topo I (calf thymus), buffer, BSA, loading buffer
and supercoilded DNA pBR322 were all from TaKaRa Biotechnology
Co., Ltd.
[2] Y.H. Hsiang, R. Hertzberg, S. Hecht, L.F. Liu, J. Biol. Chem. 260 (1985) 14873e14878.
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All reactions were carried out in 20
distilled water, 2 L DNA TopoI buffer, 2
0.25
m
L volumes (16
mL double
m
mL 0.1% BSA) including
m
g supercoiled DNA, 0.5 U Topo I with or without drug. The
reaction was incubated at 37 ꢁC for 15 min. Reactions were stopped
by adding SDS (0.5% final concentration). To the reaction mixtures,
3.5
m
L 6ꢃ loading buffer (0.1 mM EDTA, 7% Glycerol, 0.01% Xylene
[10] C. Bailly, Crit. Rev. Oncol/Hematol. 45 (2003) 91e108.
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