Design and synthesis of new 7-(N-substituted-methyl)-camptothecin derivatives as potent cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2014.06.060

A series of novel 7-(N-substituted-methyl)-camptothecin derivatives was designed, synthesized, and evaluated for in vitro cytotoxicity against four human tumor cell lines, A-549, MDA-MB-231, KB, and KBvin. All of the derivatives showed promising in vitro cytotoxic activity against the tested tumor cell lines, with IC₅₀ values ranging from 0.0023 to 1.11 μM, and were as or more potent than topotecan. Compounds 9d, 9e, and 9r exhibited the highest antiproliferative activity among all prepared derivatives. Furthermore, all of the compounds were more potent than paclitaxel against the multidrug-resistant (MDR) KBvin subline. With a concise efficient synthesis and potent cytotoxic profiles, especially significant activity towards KBvin, compounds 9d, 9e, and 9r merit further development as a new generation of camptothecin-derived anticancer clinical trial candidates.

Full text of DOI:10.1016/j.bmcl.2014.06.060

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3850–3853
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of new 7-(N-substituted-methyl)-camptothecin
derivatives as potent cytotoxic agents
Xiao-Bo Zhao a, Masuo Goto b, Zi-Long Song a, Susan L. Morris-Natschke b, Yu Zhao b, Dan Wu a, Liu Yang d,
Shu-Gang Li a, Ying-Qian Liu a, , Gao-Xiang Zhu a, Xiao-Bing Wu a, Kuo-Hsiung Lee b,c,
⇑
⇑
a School of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
b Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, United States
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
d Environmental and Municipal Engineering School, Lanzhou Jiaotong University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 7-(N-substituted-methyl)-camptothecin derivatives was designed, synthesized, and
evaluated for in vitro cytotoxicity against four human tumor cell lines, A-549, MDA-MB-231, KB, and
KBvin. All of the derivatives showed promising in vitro cytotoxic activity against the tested tumor cell
Received 9 May 2014
Revised 17 June 2014
Accepted 19 June 2014
Available online 27 June 2014
lines, with IC50 values ranging from 0.0023 to 1.11 lM, and were as or more potent than topotecan.
Compounds 9d, 9e, and 9r exhibited the highest antiproliferative activity among all prepared derivatives.
Furthermore, all of the compounds were more potent than paclitaxel against the multidrug-resistant
(MDR) KBvin subline. With a concise efficient synthesis and potent cytotoxic profiles, especially signifi-
cant activity towards KBvin, compounds 9d, 9e, and 9r merit further development as a new generation of
camptothecin-derived anticancer clinical trial candidates.
Keywords:
Camptothecin
Antiproliferative activity
Multidrug resistance
Ó 2014 Elsevier Ltd. All rights reserved.
Camptothecin (CPT, 1, Fig. 1), a natural quinoline alkaloid iso-
lated by Wall and Wani from the Chinese tree Camptotheca acumi-
nata, showed potent antiproliferative activity against a broad
spectrum of tumors.1–3 Its antitumor activity is induced by directly
binding to topoisomerase I (Topo I), which results in interference
with the catalytic cycle of DNA-Topo I and stabilization of the
DNA-Topo I binary complex.4–6 Based on CPT’s remarkable antican-
cer activity and unique cytotoxic mechanism, numerous potent
CPT analogs have been developed. Among them, two Topo I inhib-
itors, topotecan (2) and irinotecan (3), have been used successfully
in the clinic as anticancer drugs, while several other analogs are
the subjects of ongoing preclinical or clinical evaluation.7–10
Although CPT derivatives remain a promising class of antitumor
derivatives.13,14 Substitutions at the 11- or 5-position are not well
tolerated, whereas various substituents at the 7-, 9-, or 10-position
can improve the antitumor activity, as well as increase E-ring sta-
bility.11,15,16 In particular, previous studies documented that the
introduction of lipophilic substituents at the 7-position provides
favorable molecular interactions and improved pharmacological
features that could have potential therapeutic advantages.17
A
binding model of CPT with biological macromolecules also indi-
cated that the C-7 molecular area could accommodate considerable
structural diversity.18–20 On the basis of these critical clues, various
substitutions, such as ethyl, alkylsilyl, oxyiminoalkyl, and alkylsily-
lalkyl, were introduced at the 7-position of CPT to produce potent
antitumor agents. To date, 7-substituted compounds constitute
most of the second-generation CPT analogs that have reached pre-
clinical or clinical development studies. Examples include gimatec-
an (4),21 CKD-602 (5),22 and BNP-1350 (6),23 which contain highly
lipophilic substituents intended to increase antitumor activity.
These successful examples indicate the important role played by
various C-7 substitutions in the activity profiles of CPT analogs
and the feasibility of optimizing this compound class through
rational C-7 modification.
agents, the highly electrophilic a-hydroxylacetone of the E ring is
intrinsically unstable and undergoes rapid hydrolysis to the biolog-
ically inactive carboxylate form under physiological condi-
tions.11,12 This chemical feature diminishes the efficacy of various
CPT derivatives in vivo compared to the impressive results often
obtained from in vitro studies. Thus, several synthetic strategies
to overcome this challenge have been developed, resulting in a log-
ical mapping of the structure–activity relationship (SAR) of CPT
In our continuing studies on the chemistry of CPT,24–30 we
recently reported a series of 7-ketone camptothecin derivatives
with potent antitumor activity and significantly different drug-
resistance profiles from those of the parent compound.30 Some of
⇑
Corresponding authors. Tel.: +1 (919) 962 0066; fax: +1 (919) 966 3893
(K.-H.L.).
E-mail addresses: yqliu@lzu.edu.cn (Y.-Q. Liu), khlee@email.unc.edu (K.-H. Lee).
http://dx.doi.org/10.1016/j.bmcl.2014.06.060
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

X.-B. Zhao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3850–3853
3851
N
N
9
7
5
6
2
8
HO
N
O
O
O
O
10
11
4
N
N
N
16
17
O
13
N
N
3
N
12
1
O
O
15
O
O
O
14
20
21
2
3
Camptothecin (1)
Topotecan ( )
Irinotecan ( )
18
O
OH
OH
OH
19
Si
N
O
N
H
O
O
O
N
N
N
N
BNP-1350 ( )
N
N
O
O
O
O
5
CKD-602 ( )
6
Gimatecan (4)
O
O
OH
OH
OH
Figure 1. Structures of camptothecin (1), topotecan (2), irinotecan (3), gimatecan (4), CKD-602 (5), and BNP-1350 (6).
the new compounds exhibited activity comparable to that of mar-
keted CPTs, such as 2 and 3. Notably, some compounds displayed
promising cytotoxicity against KBvin cells, while 3 lost activity
completely. The encouraging preliminary results have prompted
us to extend our investigation by synthesizing a novel series of
7-(N-substituted-methyl)-camptothecin derivatives. Herein, we
describe our introduction of different nitrogen substituted groups
into the 7-position of CPT via a coupling reaction and cytotoxic
activity studies on the resulting compounds.
Target compounds 9a–s were evaluated for in vitro cytotoxicity
against a panel of human tumor cell lines, including A-549 (lung
carcinoma), MDA-MB-231 (triple-negative breast cancer), KB (orig-
inally isolated from nasopharyngeal carcinoma), and KBvin (MDR
KB subline), using a sulforhodamine B colorimetric (SRB) assay
with triplicate experiments.34,35 Paclitaxel and 2 were used as posi-
tive controls and the screening results are shown in Table 1.
As illustrated in Table 1, all new compounds exhibited signifi-
cant in vitro cytotoxic activity against the four tested tumor cell
The synthetic route to target CPT derivatives 9a–s is depicted in
Scheme 1. Briefly, treatment of 1 with hydrogen peroxide and fer-
rous sulfate in an aqueous methanol–sulfuric acid solution fur-
nished 7-hydroxymethylcamptothecin (7) in 80% yield.31
Precursor 7 was converted into the key intermediate 7-bromom-
ethylcamptothecin (8) in 66% yield by heating in hydrobromic
acid.32 Intermediate 8 was coupled with various substituted
amines in dry DMF to afford the desired derivatives 9a–s in
21–46% yields.33 All synthesized target compounds were purified
by column chromatography, and their structures were character-
ized by 1H NMR, MS, and elemental analysis.
lines, with IC50 values ranging from 0.0023 to 1.11 lM, and except
for 9k, 9q, and 9s, were mostly more active than 2, a clinically used
CPT-derived chemotherapeutic drug. Compounds 9d, 9e, and 9r
were the most potent compounds in the series and were also supe-
rior to paclitaxel against the A-549 cell line, which was generally
most sensitive to these CPT derivatives. Against the same cell line,
many compounds, including 9d–9f, 9h, 9j, 9m–9o, and 9r, also
showed better antiproliferative activity than 2. With few excep-
tions (i.e., 9k, 9q, and 9s), the tested compounds showed increased
cytotoxic potency against the triple-negative breast cancer
(MDA-MB-231) cell line compared with 2. With regard to the KB
Br
OH
O
O
O
HBr/H2SO4
reflux, 16 h
H2O2/H2SO4/FeSO4
N
N
N
N
N
MeOH-H2O
N
O
O
O
OH O
OH O
OH
O
1
7
8
R
Cl
Br
9k
9f
N
H
9p
N
N
N
R
9a
N
H
N
H
CH3
CH3
O
H2N
DMF
rt, 12 h
R
9g
9l
N
9q
N
H
N
H
N
9b
9c
N
H
N
OCH3
O
9m
N
H
9r
9h
N
N
H
N
H
H
N
OH
9a-s
O
9n
9o
9s
OH
9d
9i
N
H
Cl
N
H
N
H
OCH3
F
N
9j
N
H
9e
Cl
N
H
Scheme 1. Synthesis of target compounds 9a–s.

3852
X.-B. Zhao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3850–3853
Table 1
Antiproliferative activity of 9a–s against four human tumor cell lines
Compd
IC50 (lM) with SD
A-549
MDA-MB-231
KB
KBvin
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
0.0532 0.0226
0.0587 0.0262
0.0470 0.0191
0.0023 0.0024
0.0063 0.0027
0.0391 0.0173
0.0592 0.0252
0.0362 0.0211
0.0504 0.0206
0.0170 0.0115
0.122 0.0406
0.0514 0.0211
0.0188 0.0154
0.0219 0.0107
0.0155 0.0059
0.0622 0.0168
0.534 0.0701
0.0048 0.0042
0.150 0.0275
0.0452 0.0004
0.0057 0.0016
0.0781 0.0178
0.0927 0.0187
0.0842 0.0062
0.0264 0.0125
0.0300 0.0163
0.0644 0.0306
0.114 0.0464
0.0640 0.0025
0.0961 0.0102
0.0576 0.0514
0.337 0.0114
0.108 0.0197
0.0532 0.0006
0.0699 0.0043
0.0609 0.0096
0.110 0.0177
1.11 0.0059
0.0783 0.0272
0.0817 0.0201
0.0758 0.0107
0.0046 0.0032
0.0050 0.0013
0.0397 0.0015
0.0821 0.0093
0.0615 0.0119
0.0713 0.0139
0.0371 0.0031
0.180 0.0662
0.0786 0.0058
0.0362 0.0153
0.0499 0.0074
0.0371 0.0018
0.0895 0.0231
0.813 0.0556
0.0150 0.0102
0.208 0.0209
0.0625 0.0042
0.0039 0.0010
0.0479 0.0126
0.0795 0.0411
0.0773 0.0255
0.0149 0.0121
0.0132 0.0055
0.0129 0.0066
0.0633 0.0177
0.0780 0.0853
0.0688 0.0315
0.0128 0.0081
0.117 0.0177
0.0706 0.0217
0.0165 0.0149
0.0205 0.0141
0.0208 0.0199
0.274 0.1592
1.06 0.0115
0.0316 0.0026
0.378 0.0082
0.102 0.0055
0.0066 0.0018
0.0217 0.0157
0.183 0.0219
0.396 0.0207
1.44 0.137
9s
2
Paclitaxel
cell line, seven compounds exhibited significant cytotoxic activity
comparable to that of 2. Remarkably, except for 9q, all of the com-
showed that both the identity and substitution pattern in the R
group at C-7 could greatly influence the cytotoxicity of the new
CPT derivatives.
pounds (IC50 0.0128–0.274
lM) were more potent than paclitaxel
(IC50 1.44 M) and 2 (IC50 0.396
l
l
M) against the KBvin subline.
In summary, 19 novel 7-(N-substituted)-methyl-camptothecin
derivatives were designed, synthesized, and evaluated for antipro-
liferative activity against four human tumor cell lines (A-549,
MDA-MB-231, KB and KBvin) by using a sulforhodamine B colori-
metric assay. Most of the new derivatives showed comparable or
superior antiproliferative activity compared with 2. In particular,
compounds 9d, 9e, and 9r were the most promising derivatives
with 7- to 20-fold greater potency than 2 against the A-549 cell
line and were selected as lead molecules for further development.
These encouraging results suggested that these new derivatives
could overcome the MDR phenotype overexpressing P-glycopro-
tein. Notably, the three most promising compounds 9d, 9e, and
9r showed broad in vitro antitumor spectra and were about 7- to
20-fold more potent than 2. Further pharmacological, toxicological
evaluations and binding affinity analysis with DNA-Topo I target in
the modeling of these promising compounds are in progress.
SAR analysis of the results from the synthesized compounds
revealed several structural properties that could influence the
in vitro cytotoxicity of the new CPT derivatives. Regarding com-
pounds 9a to 9j with substituted (phenylamino)methyl groups at
C-7, derivatives with m-/p-chlorophenyl rings (9f and 9i) exhibited
lower activity than their o-substituted isomer 9j. Similarly, the
relocation of an electron-donating methoxy group from the o-posi-
Notably, with IC50 values ranging from 0.0128 to 1.06
compounds also were more potent than paclitaxel (IC50 1.44
l
M, all of the
M)
l
against KBvin cells. Furthermore, SAR study indicated that both
N-aromatic and N-aliphatic substituents at C-7 can produce potent
activity, while selected variation of these substituents can greatly
affect the activity. These findings support our further optimization
of CPT to develop potential anticancer drug candidates. Continuing
studies to substantiate and improve activity profiles are underway
in our laboratories and will be reported in due course.
tion (9d, IC50 0.0023 lM) to the p-position (9c, IC50 0.0470 lM)
also decreased the cytotoxicity remarkably. These results sug-
gested that an o-substituted phenyl group is more favorable than
m-/p-substituted rings for better activity. Moreover 9e and 9f with
p-fluoro and p-chloro substitution, respectively, on the phenyl ring
were more potent against A-549 tumor cells than 9g with p-bromo
substitution, indicating that a substituent’s size, in addition to
position, could affect the activity. However, the presence of a pyr-
idine-2-yl rather than phenyl group on the amine did not substan-
tially change the cytotoxic activity (9l vs 9a). Among compounds
9m–9s with non-aromatic amino substituents, the SAR analysis
showed that the alkyl variant had an important effect on the
in vitro cytotoxic activity. For example, the cytotoxic activity of
9r with a (propylamino)methyl group at C-7 was significantly
greater than that of compounds with a (cyclohexylamino)methyl
(9m) or (diisopropylamino)methyl (9q) moiety. These data sug-
gested that the bulkier substituents in the two latter compounds
might produce steric hindrance at the target level and thus lower
the cytotoxic activity. The polarity and electron density in the C-
7 side chain were also important, as changing the propylamino
Acknowledgements
This work was supported financially by the National Natural
Science Foundation of China (30800720, 31371975), the Funda-
mental Research Funds for the Central Universities (lzujbky-
2013-69), and the Foundation of Priority Forestry Disciplines in
Zhejiang A&F University (KF201325). Partial support was also sup-
plied by NIH Grant CA177584 from the National Cancer Institute
awarded to K.H. Lee. Thanks are also due to the support of Taiwan
Department of Health Cancer Research Center of Excellence (DOH-
100-TD-C-111-005).
Supplementary data
Supplementary data (analytical and spectroscopic data for all
target compounds) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
06.060.
group in 9r to
decreased the cytotoxic activity (e.g., IC50 0.0048
0.15 M for 9s against A-549). Taken together, these results
a
2-hydroxyethylamino group in 9s greatly
lM for 9r vs
l

X.-B. Zhao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3850–3853
3853
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33. General synthetic procedure for target compounds 9a–s. To a solution of 7-
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(0.15 mmol) dissolved in toluene (5 mL) were added and the reaction mixture
was stirred at room temperature for 12 h. After the reaction was completed,
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by chromatography on silica gel using CHCl3/MeOH as eluant to give 9a–s.
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1H NMR (400 MHz, DMSO-d6) d: 0.85 (t, J = 7.2 Hz, 3H, 19-H), 1.79–1.86 (m, 2H,
18-H), 3.85 (s, 3H, –OCH3), 5.01 (d, J = 4.8 Hz, 2H, –CH2–), 5.21 (s, 2H, 5-H), 5.43
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H), 8.17 (d, J = 8.4 Hz, 1H, 12-H), 8.49 (d, J = 8.4 Hz, 1H, 9-H); MS-ESI m/z: 484.9
[M+H]+; Anal. Calc. For C28H25N3O5: C 69.55%, H 5.21%, N 8.69%. Found: C
69.56%, H 5.23%, N 8.67%.
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35. Cytotoxic activity was determined by the sulforhodamine B (SRB) colorimetric
assay as previously described.34 In brief, the cells (4000–7500 cells/well) were
seeded with various concentrations of samples in 96-well plates filled with
RPMI-1640 medium containing 10 mM HEPES and 2 mM
(HyClone) supplemented with 10% fetal bovine serum (HyClone), 100
streptomycin, 100 IU/mL penicillin, and 0.25 g/mL amphotericin B (Cellgro).
L
-glutamine
lg/mL
l
After 72 h incubation at 37 °C with 5% CO2 in air, the living cells were fixed in
10% trichloroacetic acid for 30 min followed by staining with 0.04% SRB (Sigma
Chemical Co.) for 30 min. The bound SRB was solubilized with 10 mM Tris-base
and the absorbance was measured at 515 nm using
a Microplate Reader
ELx800 (Bio-Tek Instruments, Winooski, VT) operated by a Gen5 software. All
results were representative of three or more experiments and IC50 is expressed
as the average with standard deviation (SD).
28. Yang, L.; Zhao, C. Y.; Liu, Y. Q. J. Braz. Chem. Soc. 2011, 22, 308.