Synthesis and anticancer evaluation of novel 10-methoxycamptothecin derivatives

Article abstract of DOI:10.14233/ajchem.2015.17599

As part of our continuing search for potential anticancer drug candidates, we have synthesized four 10-methoxycamptothecin derivatives. The compounds were synthesized by facile procedures and characterized by ¹H NMR, ¹³C NMR and mass spectra study. The synthesized compounds were examined for their cytotoxic effect on a panel of five human cancer cell lines. Three out of five compounds were found to exhibit moderate anticarcinogenic activities in all cell lines. The result of the present investigation encourage us to develop more other related compounds and to screen them for a wide range of biological activity.

Full text of DOI:10.14233/ajchem.2015.17599

Asian Journal of Chemistry; Vol. 27, No. 3 (2015), 932-934
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2015.17599
Synthesis and Anticancer Evaluation of Novel 10-Methoxycamptothecin Derivatives
*
MEIXUAN ZHU, LIJIA JING, SHAOMING WANG, SHENG ZHANG and YANG WANG
Alkali Soil Nature Environmental Science Center, Northeast Forestry University, Harbin 150040, P.R. China
*Corresponding author: Tel/Fax: +86 451 82192185, E-mail: meixuanzhu@sohu.com, ywang@nefu.edu.cn
Received: 14 March 2014;
Accepted: 19 May 2014;
Published online: 19 January 2015;
AJC-16698
As part of our continuing search for potential anticancer drug candidates, we have synthesized four 10-methoxycamptothecin derivatives.
The compounds were synthesized by facile procedures and characterized by 1H NMR, 13C NMR and mass spectra study. The synthesized
compounds were examined for their cytotoxic effect on a panel of five human cancer cell lines. Three out of five compounds were found
to exhibit moderate anticarcinogenic activities in all cell lines. The result of the present investigation encourage us to develop more other
related compounds and to screen them for a wide range of biological activity.
Keywords: 10-Methoxycamptothecin derivative, Anticancer, Cytotoxic.
anticancer activity would decrease when the open-ring
carboxylate formed. In this article, esterified prodrugs of
10-methoxycamptothecin were synthesized with different
amino acid derivatives at C-20 position and their cytotoxic
effect were examined on a panel of five human cancer cell
lines. The result showed that three of the derivatives exhibit
moderate anticarcinogenic activities against all cell lines. It
brings new intrest to the potential use of 10-methoxycamp-
tothecin in the development of novel anticancer drugs.
INTRODUCTION
Camptothecin (CPT), a naturally occurring pentacyclic
indole alkaloid, was isolated from the oriental tree Camptotheca
acuminata1, a native tree of China. It appeared antitumor activity
and inhibiting effect against solid tumors and leukemia2,3.
Camptothecin can make cells die by inhibiting the replication
of DNA4, which makes it become one of unique anticancer
drugs. The E-ring lactone structure of camptothecin undergo
a rapid, reversible and non-enzymatic hydrolysis to the water-
soluble carboxylate form at basic pH. The water-soluble
carboxylate form of camptothecin, which was used in early
clinical trials due to the poor water-solublity of camptothecin,
is easy to conjugate with serum protein and makes it lose
anticancer activity5. Even worse, it showed severe side effect
on the urinary and digestive systems6 implying that the intact
E-ring lactone structure is the critical structural feature with
respect to anticancer activity7. In recent years, a number of
scientists have engaged in modifing the structure of campto-
thecin to enhance its activity and reduce its toxicity8-13. In order
to stablize the lactone ring, a esterification with the hydroxyl
group at C-20 position can derive prodrugs which can be
converted back to camptothecin by carboxylesterase in vivo14.
A series of novel CPT-bile acids and some simple amino acid
prodrugs were synthesized as well15,16. 10-Methoxycam-
ptothecin (MCPT), a native derivative of camptothecin, few
exist in C. acuminata, possessing better activities against
several cell lines in vitro and showed higher cytotoxicity than
10-hydroxycamptothecin17. However 10-methoxycam-
ptothecin shares the same E-ring lactone structure, its in vivo
EXPERIMENTAL
Melting points of the synthesized compounds are uncor-
rected and performed by melting point apparatus. Mass spectra
were taken on Micromass Quattro-Micro TM. The NMR
spectra were recorded using the Varian Unity Inova 500 NB.
The purity of each compound was detected by a Waters
HPLC system equipped with a 1525 binary pump, a 717 plus
autosampler and a 2996 PDA detector (Milford, MA, USA). A
Thermo ODS C18 column (4.6 × 250 mm i.d., 5 µm) was used.
Preparation of 10-methoxy-camptothecin: A mixture
of different anhydrous potassium carbonate (1 g, 0.007 mol)
and methyl iodide (1 mL) were added into acetone (15 mL)
with 10-hydroxycamptothecin (0.5 g, 0.0014 mol). To this
solution methyl iodide (1 mL) was added in at room tempe-
rature when it was stirred for 0.5 h. Stirring was continued for
an additional 12 h. The solution was dissolved in dichloro-
methane (200 mL), filtered, washed with water and dried with
anhydrous sodium sulfate, recrystallized with anhydrous ethanol
after removing the dichloromethane to obtain 10-methoxycam-
ptothecine (MCPT).

Vol. 27, No. 3 (2015)
Synthesis and Anticancer Evaluation of Novel 10-Methoxycamptothecin Derivatives 933
General procedure for preparation of 10-methoxy-
camptothecin N-t-boc-amino acid derivatives (C1-C4):
N-t-boc-amino acid (0.003 mol) was dissolved in 20 mL of
anhydrous dimethyl formamide in a round-bottom flask at
room temperature. To this solution a mixture of 10-methoxy-
camptothecin (0.2 g, 0.0006 mol), dicyclohexylcarbodiimide
(DCC, 0.52 g, 0.0025 mol) and 4-dimethylaminopyridine
(DMAP, 0.06 g, 0.0005 mol) was added drop wise at 0 °C.
The solution was allowed back to room temperature after
stirring at 0 °C for 1 h. Filtered the resulted dicyclohexylurea
followed by a dilution with 100 mL distilled water to form
precipitate. The precipitate was filtered, washed with water
and dried, purified by column chromatography (dichloromethane/
ethyl alcohol =1:100) to obtain 10-methoxycamptothecin
N-t-boc-amino acid derivatives (C1- C4).
Cytotoxicity assay: The cell lines MIA PaCa-2, HT-29,
DU-145, NCI-H520 and 2774 were included in this study. Each
of the cell lines was grown in RPMI 1640 medium containing
100 IU/mL G-penicillin, 100 µg/mL streptomycin and supple-
mented with 10 % fetal calf serum. Cells were maintained at
37 °C in a humidified atmosphere with 5 % of CO2. Cells
were subcultured every 4-5 days by total replacement using
0.25 % (w/v) trypsin.
Cells were seeded in 96-well culture plates in 0.2 mL of
growth medium per well and allowed to attach for 24 h. Then
the culture medium was replaced with each compound at
different concentrations in four replicates followed by 72 h of
incubation. Each concentration Blank wells only filled with
fresh medium but not cells. camptothecin was used as positive
control. After incubation, 30 µL of MTT solution at a concen-
tration of 3 mg/mL was added to each well followed by 4 h of
incubation. MTT solution was then aspirated and 150 µL of
DMSO was added to each well to dissolve the dark blue crystals
thoroughly. The absorbance was measured at 490 nm using a
microplate reader. The relative growth rate (%) was calculated
as (mean absorbance of the sample/mean absorbance of
the control) × 100 %, considering the optical density of the
control18,19 as 100 %.
powder; m.p. 129-130 °C, MS: m/z 548.4 (M + H)+; 1H NMR
(400 MHz, DMSO-d6) δ 0.96 (3H, t, J = 7.2 Hz, H-18), 1.33
(3H, t, J = 7.2 Hz, CH3), 1.44 (9H, s, t-boc), 2.09 (2H, m, H-
19), 3.90 (3H, s, OCH3), 4.09 (1H, t, J = 7.2 Hz, CH), 5.17
(2H, q, J = 7.2 Hz, H-5), 5.48 (2H, s, H-17), 7.18 (1H, s, H-
14), 7.39 (1H, d, J = 2.8 Hz, H-11), 7.46 (1H, d, J = 6.4 Hz,
NH), 7.61 (1H, d, J = 6.4 Hz, H-9), 7.90 (1H, d, J = 5.2 Hz, H-
12), 8.42 (1H, s, H-7); 13C NMR (400 MHz, DMSO-d6): δ
8.02, 16.93, 19, 28.85, 30.56, 50.55, 56.08, 56.50, 66.52, 76.50,
79.10, 95.66, 106.54, 118.28, 123.37, 129.72, 130.18, 130.40,
130.63, 144.40, 146.11, 146.48, 150.31, 156.10, 157.01,
158.51, 167.63, 172.46.
10-Methoxylcamptothecin-20-O-2-(tert-butoxycar-
bonylamino)-3-henylpropanoic acid ester (C3): Pale
yellowish powder; m.p. 134-136 °C, MS: m/z 624.4 (M + H)+;
1H NMR (400 MHz, DMSO-d6): δ 0.89 (3H, t, J = 7.2 Hz, H-
18), 1.89 (3H, t, J = 7.2 Hz, CH3), 1.87 (2H, m, H-19), 2.68
(2H, q, H-CH2), 5.25 (2H, s, H-5), 5.42 (2H, s, H-17), 6.54
(1H, s, 20-OH), 7.32 (1H, s, H-14), 7.64 (1H, d, H-11), 7.87
(1H, d, J = 9.2 Hz, H-9), 8.16 (1H, d, H-12), 8.63 (1H, s, H-
7); 13C NMR (300 MHz, DMSO-d6): δ 8.24, 9.28, 27.43, 30.75,
50.69, 55.38, 65.71, 72.84, 97.16, 119.59, 119.68, 126.55,
128.79, 130.86, 131.64, 145.81, 146.33, 149.53, 150.47,
152.99, 157.26, 172.91, 173.10.
10-Methoxylcamptothecin-20-O-2-(tert-butoxycar-
bonylamino)-3-methyl-butanoic acid ester (C4): Pale
yellowish powder; m.p. 141-143 °C, MS: m/z 576.4 (M + H)+;
1H NMR (400 MHz, DMSO-d6): δ 0.96 (3H, t, J = 7.2 Hz, H-
18), 1.33 (3H, t, J = 7.2 Hz, CH3), 1.44 (9H, s, t-boc), 2.09
(2H, m, H-19), 3.90 (3H, s, OCH3), 4.09 (1H, t, J = 7.2 Hz,
CH), 5.17 (2H, q, J = 7.2 Hz, H-5), 5.48 (2H, s, H-17), 7.18
(1H, s, H-14), 7.39 (1H, d, J = 2.8 Hz, H-11), 7.46 (1H, d, J =
6.4 Hz, NH), 7.61 (1H, d, J = 6.4 Hz, H-9), 7.90 (1H, d, J =
5.2 Hz, H-12), 8.42 (1H, s, H-7); 13C NMR (400 MHz, DMSO-
d6): δ 8.02, 16.93, 19, 28.85, 30.56, 50.55, 56.08, 56.50, 66.52,
76.50, 79.10, 95.66, 106.54, 118.28, 123.37, 129.72, 130.18,
130.40, 130.63, 144.40, 146.11, 146.48, 150.31, 156.10,
157.01, 158.51, 167.63, 172.46.
Statistical analysis: All the measurements were done in
triplicate and statistical analysis was performed by Microsoft
Excel 2010. Results are presented as mean sem.
RESULTS AND DISCUSSION
The synthesis of 10-methoxycamptothecin bearing a
methoxy side chain at C-10 was realized starting from 10-
hydroxycamptothecin. The reaction with iodomethane in the
presence of acetone proceeded smoothly. N-t-boc-amino acids
were dissolved in DMF and an acyl born at C-terminal. DMAP
was added as a catalyzer for acylation. Dicyclohexyl-
carbodiimide was used to connect the acyl to the C-20 of
10-methoxycamptothecin (Fig. 1).
The compouds were assayed for their cytotoxic activity
against five cancer cell lines consisting of MIA PaCa-2 (human
pancreatic cancer cell line), HT-29 (human colon cancer cell),
DU-145 (human prostate cancer cell), NCI-H520 (human lung
cancer cell), 2774 (human ovarian cancer cell) using the thia-
zolyl blue tetrazolium bromide (MTT) method. Medium was
used as the solvent and blank.As shown in Table-1, compound
C2 was more effective against all the cell lines. Compound
C4 has poor cytotoxicity against all 5 cell lines. Other examined
compounds exhibited a moderate inhibitory effect, depending
Characterization of the synthesized compounds
10-Methoxylcamptothecin-20-O-2-(tert-butoxycar-
bonylamino) acetic acid ester (C1): Pale yellowish powder;
1
m.p. 139-141 °C, MS: m/z 533.3 (M + H)+; H NMR (400
MHz, DMSO-d6): δ 0.94 (3H, t, J = 7.2 Hz, H-18), 1.32 (1H,
s, t-boc), 1.39 ( 8H, s, t-boc), 2.13 (2H, m, H-19), 3.81 (1H,
dd, J = 24 Hz, CH2), 3.90 (3H, s , OCH3), 3.98 (1H, dd, J = 24
Hz, CH2), 5.17 (2H, s, H-5), 5.48 (2H, s, H-17), 7.13 (1H, s,
H-14), 7.43 (1H, d, J = 2.8 Hz, H-11), 7.44 (1H, t, J = 2.8 Hz,
NH), 7.47 (1H, d, J = 2.8Hz, H-9), 7.97 (1H, d, J = 9.2 Hz, H-
12), 8.44 (1H, s, H-7); 13C NMR (400 MHz, DMSO-d6): δ
7.99, 19, 28.27, 28.62, 30.77, 42.55, 50.56, 56.11, 56.50, 66.66,
76.69, 78.95, 95.29, 106.56, 118.50, 123.41, 129.77, 130.28,
130.42, 130.71, 144.38, 145.82, 146.60, 150.26, 156.34,
156.97, 158.55, 167.58, 169.99.
10-Methoxylcamptothecin-20-O-2-(tert-butoxycar-
bonylamino) propanoic acid ester (C2): Pale yellowish

934 Zhu et al.
Asian J. Chem.
TABLE-1
in vitro ANTITUMOR ACTIVITY OF THE DERIVATES OF 10-METHOXYCAMPTOTHECIN
IC50(nM)
Sample
HT29
MPC2
DU145
33.45 1.54
16.22 0.59
306.59 27.48
76.06 3.64
165.80 9.63
> 1000
NCI-H520
25.01 1.24
13.05 1.26
151.47 1.17
79.62 5.60
104.37 9.16
> 1000
2774
Camptothecin
10-Methoxycamptothecin
83.67 3.05
76.80 6.41
884.00 94.11
408.67 16.04
541.33 39.58
> 1000
90.64 4.93
35.34 1.37
540.42 69.95
229.90 14.81
371.59 11.08
> 1000
12.09 1.20
8.72 0.73
105.66 2.12
39.02 0.40
53.34 2.81
> 1000
C1
C2
C3
C4
MPC-2: human pancreatic cancer cell line; HT-29: human colon cancer cell; DU-145: human prostate cancer cell; NCI-H520: human lung cancer
cell; 2774: human ovarian cancer cell.
HO
In conclusion, CPT > MCPT> C2 > C3 > C1> C4. In our
study, all of the 10-methoxycamptothecin derivatives were
formed prodrugs by esterification at C-20 hydroxyl group. The
IC50 values of compounds C1 to C3, range of 79.62-884 nM,
are higher than that of 10-methoxycamptothecin, which implies
that the prodrugs could cleave by carboxylesterase and released
the active 10-methoxycamptothecin and they have great poten-
tial for further study.
O
N
N
O
OH
O
a
H3CO
ACKNOWLEDGEMENTS
O
C1: R=
C2: R=
C3: R=
C4: R=
H
N
This research is financially supported by the "948" Project
of the State Forestry Administration, China (2011-4-16).
N
CH3
O
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C
H2
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O
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a - K2CO3, CH3I, acetone
b - N-t-boc-amino acids,
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O
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Fig. 1. Synthesis of novel camptothecin derivatives
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The anticancer screening of synthesized compounds (C1
to C4) were evaluated against cancer cell lines and the synthe-
sized compounds were found to exhibit mild to moderate
anticancer in all cell lines.