Synthesis and antitumor activity evaluation of a novel series of camptothecin analogs

Article abstract of DOI:10.1080/10286020.2013.804068

A series of novel 10-substituted camptothecin analogs (3-10) with a carbamate linker were synthesized, and their biological activities were evaluated. The amino acid-linked carbamate derivatives (8-10) of the camptothecin-type natural product not only pos

Full text of DOI:10.1080/10286020.2013.804068

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Synthesis and antitumor activity
evaluation of a novel series of
camptothecin analogs
Jian Lv a b , Na Guo a b , Shao-Peng Wen a b , Yu-Ou Teng a b
Meng-Xin Ma c & Peng Yu a b
,
a Key Laboratory of Industrial Microbiology, Ministry of Education,
College of Biotechnology, Tianjin University of Science and
Technology , Tianjin , 300457 , China
b Tianjin Key Laboratory of Industry Microbiology, College of
Biotechnology, Tianjin University of Science and Technology ,
Tianjin , 300457 , China
c College of Chemistry and Chemical Engineering, Southeast
Unversity , Nanjing , 211189 , China
Published online: 25 Jun 2013.
To cite this article: Jian Lv , Na Guo , Shao-Peng Wen , Yu-Ou Teng , Meng-Xin Ma & Peng Yu (2013)
Synthesis and antitumor activity evaluation of a novel series of camptothecin analogs, Journal of
Asian Natural Products Research, 15:8, 867-874, DOI: 10.1080/10286020.2013.804068
To link to this article: http://dx.doi.org/10.1080/10286020.2013.804068
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Journal of Asian Natural Products Research, 2013
Vol. 15, No. 8, 867–874, http://dx.doi.org/10.1080/10286020.2013.804068
Synthesis and antitumor activity evaluation of a novel series
of camptothecin analogs
Jian Lvab, Na Guoab*, Shao-Peng Wenab, Yu-Ou Tengab, Meng-Xin Mac and Peng Yuab
*
aKey Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology,
b
Tianjin University of Science and Technology, Tianjin 300457, China; Tianjin Key Laboratory of
Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology,
Tianjin 300457, China; College of Chemistry and Chemical Engineering,
c
Southeast Unversity, Nanjing 211189, China
(Received 9 April 2013; final version received 7 May 2013)
A series of novel 10-substituted camptothecin analogs (3–10) with a carbamate linker
were synthesized, and their biological activities were evaluated. The amino acid-linked
carbamate derivatives (8–10) of the camptothecin-type natural product not only
possessed good to excellent inhibitory activity against three human tumor cell lines
K562, HepG2, and HT-29, but also showed significantly less cytotoxicity against
normal human cell HEK293 (half maximal inhibiting concentration .10 mM). The
selectivity of compound 9 toward tumor cells relative to normal cells is at least 250
times better than that of camptothecin. The preliminary testing result indicated that the
solubility of these compounds was also improved compared to that of 10-hydroxy
camptothecin.
Keywords: anticancer; solubility; alkaloid
1. Introduction
their antitumor activities and increased
toxicities [9–11]. It was later reported that
the closed lactone form is a prerequisite for
antitumor activity of CPT-type drug
[12,13]. Subsequent effort to develop
water-soluble camptothecin derivatives
was focused on the quinoline portion of
the molecule, which led to the discovery
of topotecan and irinotecan (Figure 1).
Topotecan introduces a nitrogenous group
at the 7-position, and irinotecan is
considered as a prodrug formed between
7-ethyl HCPT and [1,40-bipiperidine]-
10-carboxylic acid. Both topotecan and
irinotecan possess a much improved
solubility, and have been approved for
the treatment of several types of cancer
patients [14,15]. However, they still have
some drawbacks, such as severe neutro-
philic granulocytopenia and diarrhea [16].
Therefore, it is very important to further
Camptothecin (CPT) and 10-hydroxy-
camptothecin (HCPT) are two pentacyclic
alkaloids isolated from a natural plant [1],
and both of them have been found to
possess good inhibitory activities against a
broad spectrum of tumors. Interest in CPT
and HCPT derivatives was revitalized in
1985 by the discovery that the camptothe-
cins exhibited a unique mechanism of
action, characterized by their ability to
bind to the transient topoisomerase I-DNA
complex during DNA replication and
induce double-strand breaks and cell
death [2–5].
The clinical applications of CPT and
HCPT were limited by their side effects
[6,7] and poor solubility [8]. Early
attempts to form a water-soluble sodium
salt by opening the lactone ring with
sodium hydroxide resulted in the loss of
*Corresponding authors. Email: guona@tust.edu.cn; yupeng@tust.edu.cn
q 2013 Taylor & Francis

868
J. Lv et al.
Figure 1. Structures of HCPT, SN38, irinotecan, and topotecan.
study the origin of the side effects and
develop new generation of less toxic
CPT/HCPT derivatives that could extend
the application of these very potent natural
product anticancer drugs.
SN-38 derivatives containing nitrogenous
groups at the 10-position, and the water
solubility of these compounds was indeed
enhanced [21], with some compounds
showing cytotoxic activity similar to that
of CPT in vitro. However, their toxicities to
normal cells were not evaluated. Appro-
priate modification at the 10-position of
CPT could not only be beneficial for its
antitumor activity and physicochemical
property, but may also reduce its toxic
effect. Carbamate was shown to be a
promising linker to form CPT prodrugs
modified at the 20-position [22–25], but
10-amino acid-linked carbamate CPTs
have not previously been reported.
Previous studies indicated that the
substitution at 7 and 9 positions of CPT
derivatives could enhance their antitumor
activities and improve their physicochem-
ical properties [17]. In this study, we aim to
address the possibility that 10-hydroxy
group of HCPT could be a key position
responsible for the excellent potency as
well as a potential cause of its toxicity.
Irinotecan is a 10-bipiperidine-substituted
carbamate of 7-ethyl HCPT, but the side
chain causes a 1000 times decrease in
activity when compared to its parent
molecule SN38 [18]. Li and coworkers
reported the conversion of HCPT into CPT
quaternary ammonium salts bearing sev-
eral water-solubilizing groups with an
ether linker at the 10-position of CPT, and
these salts showed good water solubility
and different cytotoxicities in vitro [19,20].
They also reported a series of HCPT and
In order to clarify whether the 10-
hydroxy group is the major cause of the
toxic effect of HCPT and to find novel
CPT anticancer drugs with improved
solubility and low toxicity, we report in
this study the design, synthesis, and
antitumor activities of a series of novel
10-position nitrogenous group-linked car-
bamate CPTs as well as a preliminary
in vitro toxic evaluation by comparing

Journal of Asian Natural Products Research
869
their cell toxicity difference between
normal human cell HEK293 and cancer
cells K562, HepG2, and HT29.
against three human tumor cells, K562,
HepG2, and HT-29, were evaluated by 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) assay with CPT
as the positive control. As can be seen from
Table 1, all the compounds exhibited
inhibitory activities against tumor cells,
among which five compounds (2 and 6–9)
have very comparable half maximal
inhibiting concentration (IC50) values
with CPT, especially against K562 cell line.
In order to evaluate their toxicities, the
inhibition of compounds 2 and 6–10
against normal human cell HEK293 was
also tested (Table 2). The result showed
that compounds 2, 6, and 8–10 exhibited
much less toxicities against HEK293 than
CPT. Especially, compounds 8–10 were
2. Results and discussion
A series of novel 10-substituted camp-
tothecin analogs (3–10) were synthesized
by a convenient two-step approach. As
illustrated in Scheme 1, HCPT was first
reacted with 4-nitrophenyl chloroformate
in the presence of a base to form the key
intermediate 4-nitrophenyl camptothecin-
10-yl carbonate (2), which was then
treated with different nitrogenous reagents
to afford the target compounds 3–10
(Scheme 1).
The in vitro antitumor activities of the
target compounds 3–10 and intermediate 2
Scheme 1. Synthesis of compounds 3–10. (a) 4-Nitrophenyl chloroformate (4.0 equiv.), CH2Cl2,
triethylamine (10.0 equiv.), 08C to rt, 9 h, 64%. (b) Nitrogenous reagent (2.0 equiv.), DMF, 08C to rt,
2 h, 40–60%.

870
J. Lv et al.
structural modifications made on these
compounds have, therefore, resulted in
significantly higher selectivity toward
tumor cells than normal cells.
Table 1. Antitumor activities of compounds
2–10 in vitro.
In vitro cytotoxicity
(IC50, mM)
To further evaluate the drug-like proper-
ties of these potent compounds, the
predicted theoretical log P and c log P
values of 7–10 were analyzed (Table 3)
and their solubilities in DMSO, acetone, and
deionized water were also briefly tested
(Table4). The data inTables3 and 4 showed
that compounds 9 and 10 have better
properties and compounds 7–10 have
much better solubility in organic solvents
(DMSO and acetone) than HCPT. The water
solubility of only 7 and 8 has been slightly
improved. The compound 9, which is 11 and
25timesmoresolubleinDMSOandacetone
than that of HCPT, possessed excellent
antitumor activities, low toxicity, and a
relatively ideal c log P value and should
deserve further investigation.
Compound
K562
HepG2
HT-29
CPT
2
3
4
5
6
7
8
9
10
0.07
0.07
0.89
0.41
1.72
0.06
0.04
0.06
0.04
0.11
0.06
0.33
0.48
0.69
0.59
0.17
0.28
0.31
0.27
0.30
0.06
0.22
1.11
0.56
1.06
0.15
0.25
0.26
0.19
0.30
found to be at least 250 times less toxic
against HEK293 (IC50 . 10 mM). Con-
sidering the good to excellent antitumor
activities of 8–10, it could be expected
that they might have a much larger safety
window. Interestingly, the cytotoxicity of
compound 7 against HEK293 was iden-
tical to that of CPT. One possible reason is
that compound 7 with a sterically less
hindered glycine-linked carbamate could
be very quickly hydrolyzed back into the
active natural drug HCPT, which itself
also has the similar toxic effect to that of
CPT. These results also indicated that the
10-hydroxy group may be responsible for
the toxicity of HCPT to some extent.
Compounds 8–10 all contain sterically
hindered groups next to carbamates so that
they are difficult to turn back into HCPT,
and therefore could avoid the toxic effect
caused by the free 10-hydroxy group of
HCPT. Taken together, compounds 8–10
possess both good to excellent antitumor
activities and much less toxic effect
against human normal cell HEK293. The
3. Experimental
3.1 General experimental procedures
1H NMR spectra were recorded on a
Bruker AM-400 NMR spectrometer (Bill-
erica, Middlesex, MA, USA) in CDCl3 or
DMSO-d6. The chemical shifts are
reported in d (ppm) relative to tetra-
methylsilane as an internal standard. Mass
spectra were obtained on a Q-TOF mass
spectrometer (Agilent, Santa Clara, CA,
USA). All reagents and solvents used in
this study were of reagent grade. Thin
layer chromatography was carried out
using E. Merck silica gel 60 GF254
precoated plates (Darmstadt, Germany)
and visualized using a combination of
UV254 and UV365. Silica gel (particle size
200–400 mesh, Marine Chemical Group
Table 2. Toxicities against HEK293 of compounds CPT, 2, and 6–10.
Compound
CPT
0.01
2
6
7
8
9
10
Toxicity (IC50, mM)
1.71
0.25
0.009
.10
.10
.10

Journal of Asian Natural Products Research
871
controls were calculated as a cell viability
value of 100%. The IC50 values were
obtained by nonlinear regression using
GraphPad Prism 4.0. IC50 measurements
for each compound were done three times.
Table 3. log P and c log P of compounds CPT,
HCPT, 2, and 6–10.
Compound
log P
c log P
CPT
HCPT
7
8
9
10
1.33
0.94
0.35
0.84
1.73
2.07
0.897
0.9458
0.21
0.5217
1.4497
1.9787
3.3 Synthesis of compounds 2–10
3.3.1 4-Nitrophenyl camptothecin-10-yl
carbonate (2)
Note: Predicted via Chemdraw Ultra 8.0.
To a stirred solution of 10-hydroxy
camptothecin (5.00 g, 13.7 mmol) in dry
CH2Cl2 (500 ml) were added triethylamine
(19.2 ml, 137.2 mmol) and then 4-nitro-
phenyl chloroformate (11.06 g, 54.9 mmol)
at 08C. The reaction mixture was stirred at
08C for 15 min, before warming to room
temperature. After stirring at room tem-
perature for another 9 h, the mixture was
washed with brine (3 £ 500 ml) and the
organic layer was dried with anhydrous
sodium sulfate. Removal of solvent under
vacuum gave a brownish residue that was
purified by column chromatography (200
mesh silica gel, CH2Cl2/CH3OH, 200:1) to
afford compound 2 (4.65 g, 64% yield) as a
pale solid.
1H NMR (DMSO-d6, 400 MHz) d
(ppm): 0.89–0.92 (m, 3H, H-18), 1.85–
1.92 (m, 2H, H-19), 5.32 (s, 2H, H-5), 5.44
(s, 2H, H-17), 6.54 (s, 1H, 20-OH), 7.37 (s,
1H, H-14), 7.78 (d, 2H, J ¼ 9.2 Hz, H-
nitrophenyl), 7.97–7.80 (m, 1H, H-11),
8.20 (d, J ¼ 2.4 Hz, 1H, H-9), 8.30 (d,
J ¼ 9.2 Hz, 1H, H-12), 8.40 (d, J ¼ 9.2 Hz,
2H, H-nitrophenyl), 8.74 (s, 1H, H-7).
Co., Qingdao, China) was used for flash
chromatography.
3.2 Cytotoxicity assay
The cytotoxicities of the target com-
pounds were evaluated by MTT assay.
Cells were seeded at a density of 5 £
104 cells/ml in 96-well microplate
(100 ml/well). After 2 h for K562 cells
and 24 h for HEK293, HepG2, and HT-29
cells, media containing tested compounds
were added in triplicate. After 48-h
incubation, the media were replaced by
phosphate-buffered saline medium con-
taining 0.5 mg/ml MTT and incubated for
another 4 h. Then, the medium was
removed and 100 ml DMSO was added
in each well to dissolve formazan.
The absorbances at 570/630 nm were
measured for K562 cells, and the absor-
bances at 490/630 nm were measured for
HEK293, HepG2, and HT-29 cells using
Thermo microplate reader. The untreated
Table 4. Solubilities of compounds 7–10.
Solubility (mg/ml)
Compound
DMSO
Acetone
Deionized water
HCPTa
7
8
9
10
11.1a
24
27.14
135
45
0.23a
2.4
0.56
6
,0.1a
0.14
0.12
,0.1
,0.1
4.17
Note: 2.0 mg of each compound was weighed, and the solvent was then carefully added until compound was
dissolved.
a The test result of HCPT is consistent with the literature report [23].

872
J. Lv et al.
3.3.2 Camptothecin-10-yl pyrrolidine-1-
carboxylate (3)
3.3.3.2 Camptothecin-10-yl diethylcar-
bamate (5). 1H NMR (DMSO-d6,
400 MHz) d (ppm): 0.89 (t, J ¼ 7.2 Hz,
3H, H-18), 1.14–1.17 (m, 3H, CH3-
diethylamine), 1.25–1.28 (m, 3H, CH3-
diethylamine), 1.84–1.91 (m, 2H, H-19),
3.32–3.49 (m, 2 £ CH2-diethylamine),
5.30 (s, 2H, H-5), 5.43 (s, 2H, H-17),
6.52 (s, 1H, 20-OH), 7.34 (s, 1H, H-14),
7.65–7.68 (dd, J ¼ 9.2, 2.4 Hz, 1H, H-11),
7.91 (d, J ¼ 2.4 Hz, 1H, H-9), 8.17 (d,
J ¼ 9.2 Hz, 1H, H-12), 8.66 (s, 1H, H-7).
ESI-MS (m/z): 464.3 [M þ H]þ.
Compound 2 (0.5 g, 0.9 mmol) was
dissolved in dry N,N-Dimethylformamide
(DMF) (3 ml), and pyrrolidine (0.16 ml,
1.9 mmol) was then added at 08C. The
reaction mixture was stirred for 2 h at
room temperature before quenched with
15 ml icy water. The mixture was
extracted with CH2Cl2 (3 £ 25 ml). The
organic layer was combined and dried
with anhydrous sodium sulfate and con-
centrated under reduced pressure to give a
crude product. Compound 3 (0.19 g, 45%
yield) was then obtained by column
chromatography (200 mesh silica gel,
CH2Cl2/CH3OH, 175:1) as a pale solid.
1H NMR (DMSO-d6, 400 MHz) d
(ppm): 0.89 (t, J ¼ 7.2 Hz, 3H, H-18),
1.86–1.97 (m, 2H, H-19), 3.33 (over-
lapped with H2O in d6-DMSO, 4H, H-
pyrrolidine), 3.34–3.45 (m, 2H, H-pyrro-
lidine), 3.57–3.60 (m, 2H, H-pyrrolidine),
5.30 (s, 2H, H-5), 5.43 (s, 2H, H-17),
6.52 (s, 1H, 20-OH), 7.35 (s, 1H, H-14),
7.67–7.70 (dd, J ¼ 9.2 and 2.4 Hz, 1H,
H-11), 7.90 (d, J ¼ 2.4 Hz, 1H, H-9),
8.18 (d, J ¼ 9.2 Hz, 1H, H-12), 8.66
(s, 1H, H-7). ESI-MS (m/z): 462.2
[M þ H]þ.
3.3.3.3 Camptothecin-10-yl butylcarba-
mate (6). 1H NMR (DMSO-d6, 400 MHz) d
(ppm): 0.87–0.94 (m, 6H, H-18 and CH3-
butylamine), 1.33–1.54 (m, 4H, CH3CH2-
CH2CH2NHZ), 1.84–1.93 (m, 2H, H-19),
3.10–3.15 (m, 2H, CH3CH2CH2CH2-
NHZ), 5.28 (s, 2H, H-5), 5.43 (s, 2H, H-
17), 6.52 (s, 1H, 20-OH), 7.33 (s, 1H, H-14),
7.62–7.65 (dd, J ¼ 9.2 and 2.4 Hz, 1H, H-
11), 7.87 (d, J ¼ 2.4 Hz, 1H, H-9), 7.95 (t,
J ¼ 5.6 Hz, 1H, CH3CH2CH2CH2NHZ),
8.15 (d, J ¼ 9.2 Hz, 1H, H-12), 8.65 (s,
1H, H-7). ESI-MS (m/z): 464.3 [M þ H]þ.
3.3.3.4 N-[(10-Camptothecinyloxy) car-
1
bonyl]-glycine methyl ester (7). H NMR
(DMSO-d6, 400 MHz) d (ppm): 0.89 (t,
J ¼ 7.2 Hz, 3H, H-18), 1.84–1.91 (m, 2H,
H-19), 3.70 (s, 3H, CH3OZ), 3.93 (d,
J ¼ 5.8 Hz, 2H, CH3OCOCH2NHCOOZ),
5.30 (s, 2H, H-5), 5.43 (s, 2H, H-17), 6.52
(s, 1H, 20-OH), 7.35 (s, 1H, H-14), 7.64–
7.66 (m, 1H, H-11), 7.92 (s, 1H, H-9),
8.19 (d, J ¼ 9.2 Hz, 1H, H-12), 8.42 (t,
J ¼ 5.8 Hz, 1H, CH3OCOCH2NHCOOZ),
8.68 (s, 1H, H-7). ESI-MS (m/z): 480.2
[M þ H]þ.
3.3.3 Compounds 4–10
Similar procedures as for the preparation
of compound 3 were used for the synthesis
of compounds 4–10.
3.3.3.1 Camptothecin-10-yl morpholine-
4-carboxylate (4). 1H NMR (DMSO-d6,
400 MHz) d (ppm): 0.89 (t, J ¼ 7.2 Hz,
3H, H-18), 1.84–1.91 (m, 2H, H-19), 3.47
(brs, 2H, H-morpholine), 3.69 (brs, 6H, H-
morpholine), 5.29 (s, 2H, H-5), 5.42 (s, 2H,
H-17), 6.52 (s, 1H, 20-OH), 7.34 (s, 1H, H-
14), 7.67–7.70 (dd, J ¼ 9.2 and 2.4 Hz,
1H, H-11), 7.92 (d, J ¼ 2.4 Hz, 1H, H-9),
8.17 (d, J ¼ 9.2 Hz, 1H, H-12), 8.65 (s,
1H, H-7). ESI-MS (m/z): 478.2 [M þ H]þ.
3.3.3.5 N-[(10-Camptothecinyloxy) car-
1
bonyl]-alanine methyl ester (8). H NMR
(DMSO-d6, 400 MHz) d (ppm): 0.89 (t,
J ¼ 7.2 Hz, 3H, H-18), 1.40 (d, J ¼ 7.6 Hz,
3H, CH3CHZ), 1.84–1.91 (m, 2H, H-19),

Journal of Asian Natural Products Research
3.70 (s, 3H, CH3OZ), 4.22–4.25 (m, 1H, References
873
CH3CHZ), 5.30 (s, 2H, H-5), 5.43 (s, 2H,
H-17), 6.52 (s, 1H, 20-OH), 7.34 (s, 1H, H-
14), 7.63–7.65 (dd, J ¼ 9.2, 2.4 Hz, 1H, H-
11), 7.90 (d, J ¼ 2.4 Hz, 1H, H-9), 8.18 (d,
J ¼ 9.2 Hz, 1H, H-12), 8.49 (d, J ¼ 7.2 Hz,
1H, ZNHCOOZ), 8.68 (s, 1H, H-7). ESI-
MS (m/z): 494.2 [M þ H]þ.
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J ¼ 7.2 Hz, 3H, H-18), 0.98 (t, J ¼ 6.4 Hz,
6H, (CH3)2CHCHZ), 1.84–1.91 (m, 2H,
H-19), 2.12–2.18 (m, 1H, (CH3)2-
CHCHZ), 3.70 (s, 3H, CH3OZ), 4.02–
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H-5), 5.43 (s, 2H, H-17), 6.52 (s, 1H, 20-
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J ¼ 2.8 Hz, 1H, H-9), 8.19 (d, J ¼ 9.2 Hz,
1H, H-12), 8.44 (d, J ¼ 8.0 Hz, 1H,
ZNHCOOZ), 8.69 (s, 1H, H-7). ESI-MS
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Acknowledgments
This work was financially supported by Tianjin
Municipal Science and Technology Commis-
sion (11ZCGHHZ00400, 10ZCKFSY07700)
and National Natural Science Foundation of
China (81241104).

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