7-Cycloalkylcamptothecin derivatives: Preparation and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2009.06.010

A series of 7-cycloalkylcamptothecin derivatives were synthesized from camptothecin with two methods. Their biological activities in vitro were evaluated with sulforhodamine-B (SRB) method on four types of human tumor cell lines A549/ATCC, HT29, NCI-H460

Full text of DOI:10.1016/j.bmcl.2009.06.010

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4107–4109
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
7-Cycloalkylcamptothecin derivatives: Preparation and biological evaluation
b
Mingzong Li a, Wei Jin b, Chen Jiang c, Chao Zheng a, Weidong Tang b, Tianpa You a, , Liguang Lou
*
a Department of Chemistry, University of Science & Technology of China, Hefei, Anhui 230026, PR China
b Chinese Acad. Sci., Shanghai Inst. Mat. Med., 555 Zuchongzhi Rd, Shanghai 201203, PR China
c Department of Basic Sciences, China Pharmaceutical University, Nanjing, Jiangsu 210000, 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 7-cycloalkylcamptothecin derivatives were synthesized from camptothecin with two meth-
ods. Their biological activities in vitro were evaluated with sulforhodamine-B (SRB) method on four types
of human tumor cell lines A549/ATCC, HT29, NCI-H460 and HL60. Most of these camptothecin analogues
show higher antitumor activity than the reference compounds SN-38 and Topotecan, with the IC50 values
low to nM level. Structure–activity relationship studies of these compounds mostly match the conclusion
we achieved before from quantitative structure–activity relationship (QSAR) research.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 2 March 2009
Revised 26 May 2009
Accepted 2 June 2009
Available online 6 June 2009
Keywords:
Camptothecin
7-Cycloalkylcamptothecin derivatives
Anti-tumor
Synthesis
Camptothecin1 (CPT, 1, Fig. 1) is a famous efficient inhibitor of
Topoisomerase I (Top I).2 Thousands of its derivatives have been
prepared and their antitumor activities were evaluated. Many of
them have been developed in pre-clinic and clinic trials,3 three
compounds (Irinotecan4 2, Topotecan5 3 and Belotecan6 4) have
been approved to use in clinic as anticancer drugs in many
countries.
According to our investigation, modifications in A or B ring or
both are the most efficient way to develop potential antitumor
drugs. Most of the active derivatives of CPT are substituted in AB
rings.3 Structure–activity relationship (SAR) studies indicate that
substitutions at 7 or 10-position or both could effectively affect
the hydrolysis equilibrium (Scheme 1) of CPT E-ring in human
blood to favor the active component ‘lactone-closed form’, by
ablating high-affinity binding of the carboxylate form to human
serum albumin (HSA).7 In fact, several series of 7-substituted or
7- and 10-dual substituted CPT derivatives have been developed
as effect antitumor candidate compounds.4,6–8
R2 R1
5
9
7
R3
6
10
8
O
4
A
B
C N
16
11
17
D
13
N
2
3
12
1
E
O
21
14
15
19
20
18
OH O
1 Camptothecin (CPT): R1 = R2 = R3 = H;
1
Irinotecan: R = Et, R2 = H, R3
=
;
2
N
NCOO
3 Topotecan: R1 = H, R2 = Me2NCH2 -, R3 = OH;
Belotecan: R1 = Me2CHNHCH2CH2 -, R2 = R3 = H;
4
5 SN-38: R1 = Et, R2 = H, R3 = OH.
Figure 1. Structures of camptothecin and derivatives.
In our earlier work,9 we introduced topological molecular
descriptors to study the QSAR of two series of 7- and 10-substi-
tuted CPT analogues, and achieved a conclusion that more lipo-
philic substitutions at 7-position would lead to higher antitumor
activity. This result showed good predictive ability. Further more,
it was pointed out in a binding model of CPT with biological mac-
romolecules that there is wide space for substitutions in 7-position
of CPT without steric repulsion.10 These investigations encouraged
us to develop 7-substituted CPT analogues. In this Letter, the
O
O
N
N
N
N
E
O
OH
COO-
OH O
OH
Lactone-closed form
Carboxylate form
Biological active
Biological inactive
* Corresponding author. Tel.: +86 5513606944.
E-mail address: ytp@ustc.edu.cn (T. You).
Scheme 1. Hydrolysis equilibrium of camptothecin E-ring in human blood.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.010

4108
M. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4107–4109
R1
synthesis and biological evaluation of a series of 7-cycloalkylcam-
ptothecin derivatives are reported.
R2
R2
O
O
Our attempts to introduce the cycloalkyl groups at C-7 from
camptothecin or 10-hydroxycamptothecin with the Sawada meth-
od4,11,12 were unsuccessful except in the case of cyclohexyl deriv-
atives (CPT-c6 and CPT-c6-OH, Scheme 2). The reaction using
cyclopropanecarbaldehyde as cyclopropyl radical source afforded
the corresponding cyclopropylcabonyl derivative(CPT-c3C and
CPT-c3C-OH, Scheme 2)13 instead of the desired 7-cyclopropyl
substituted camptothecin. We also tried to introduce cyclobutyl
at C-7 by using cyclobutylmethanol, but only a complex mixture
was recovered. Finally we found that cycloalkyl bromides could
be employed as source of cycloalkyl radicals in the presence of
diphenylsilane (Ph2SiH2) and t-butoxyperoxyde. Several 7-cyclo-
alkyl derivatives were synthesized starting from camptothecin
and 10-hydroxycamptothecin (Scheme 3)13 in medial yields except
7-cyclopropyl and 7-cyclobutly CPT analogues which were gained
with low yields due to the instability of cyclopropyl and cyclobutly
radicals. The 10-hydroxycamptothecin analogs were methylated
following standard conditions to give the corresponding 7-cyclo-
alkyl-10-methoxy-camptothecins14 (Scheme 3).
The antitumor activities of these synthetic compounds were
tested on human lung cancer cell line A549/ATCC, human colorec-
tal cancer cell line HT-29 (Table 1), human lung cancer cell line
NCI-H460 and human leukemia cancer cell line HL60 (Table 2).
Most of these compounds have higher antitumor activities than
their parent compounds (without 7-substitution) and reference
drugs SN-38 (the active component of irinotecan, Fig. 1) and Topo-
tecan. The highest activities appear with 7-cyclopentyl, 7-cyclo-
hexyl and 7-cycloheptyl CPT analogs. The IC50 values of tested
compounds on NCI-H460 and HL60, the more sensitive cell lines
to CPT analogues are all low to nM level, some of them even lower
than nM, which is up to 40-folds more active than Topotecan.
However, activities of compounds with 7-cyclopropylcarbonyl
(CPT-c3C and CPT-c3C-OH) and 7-cyclooctyl (CPT-c8) are obvi-
ously lower than other analogues’. A possible explanation for lower
activity of CPT-c3C and CPT-c3C-OH is that 7-carbonyl would af-
fect the conjugated system of CPT and provide an additional hydro-
gen bond receptor, and either of the evens would influence their
N
N
a
N
N
O
O
1 or 6
11 - 18
OH
OH
O
O
11 CPT-c3: R1 =cyclopropyl, R2 = H;
: R1 =cyclobutyl, R2 = H;
: R1 =cyclopentyl, R2 = H;
12 CPT-c4
13 CPT-c5
14 CPT-c7: R1 =cycloheptyl, R2 = H;
15 CPT-c8: R1 =cyclooctyl, R2 = H;
16 CPT-c4-OH: R1 =cyclobutyl, R2 = OH;
: R1 =cyclopentyl, R2 = OH;
: R1 =cycloheptyl, R2 = OH;
17 CPT-c5-OH
18 CPT-c7-OH
R1
R1
HO
MeO
b
O
O
N
N
N
N
O
O
O
6, 8, 16 - 18
19 - 23
OH
OH
O
19 CPT-OMe: R1 =H;
: R1 =cyclobutyl;
20 CPT-c4-OMe
21 CPT-c5-OMe
: R1 =cyclopentyl;
22 CPT-c6-OMe: R1 =cyclohexyl;
23 CPT-c7-OMe: R1 =cycloheptyl.
Scheme 3. Reagents and conditions: (a) R1Br, Ph2SiH2, t-BuOOH, AcOH-acetone
H2SO4, 80 °C, 8–12 h, 15–62%; (b) MeI, K2CO3, acetone, refluxed, over night, 88–97%.
Table 1
Antitumor activity on human tumor cell lines A549/ATCC and HT-29
a
Compds
IC50
(l
M)
HT-29
Compds
IC50 (lM)
A549/ATCC
A549/ATCC
HT-29
CPT
0.047
0.12
0.05
0.066
0.024
0.015
0.36
0.179
0.088
0.36
0.12
0.12
0.61
0.094
0.02
0.14
0.45
0.098
0.17
0.41
CPT-OH
0.11
0.21
CPT-c3
CPT-c4
CPT-c5
CPT-c6
CPT-c7
CPT-c8
CPT-c3C
SN-38
CPT-c5-OH
CPT-c6-OH
CPT-c7-OH
CPT-c3C-OH
CPT-OMe
CPT-c4-OMe
CPT-c5-OMe
CPT-c6-OMe
CPT-c7-OMe
0.071
0.030
0.003
1.28
0.058
0.018
0.012
0.57
0.076
0.041
0.031
0.035
0.025
0.04
0.011
0.031
0.039
0.056
R1
R1
Topotecan
O
O
N
N
a
a
Values are measured with SRB method.
N
N
O
O
Table 2
Antitumor activity on human tumor cell lines NCI-H460 and HL60
OH O
OH O
1 CPT: R1 = H;
6 CPT-OH: R1 = OH.
7 CPT-c6: R1 = H;
8 CPT-c6-OH: R1 = OH.
Compds
IC50
(lM)
Compds
IC50
(l
M)
HL60
NCI-H460
HL60
NCI-H460
O
CPT-c5
<0.001
<0.001
<0.001
0.0045
0.028
<0.001
<0.001
<0.001
0.0024
0.0042
CPT-c6
0.0023
<0.001
<0.001
0.036
0.0016
<0.001
<0.001
0.012
R1
R1
CPT-c5-OH
CPT-c5-OMe
CPT-c3C
CPT-c6-OH
CPT-c5-OMe
Topotecan
O
O
N
N
b
N
N
CPT-c3C-OH
O
O
a
Values are measured with SRB method.
OH O
OH O
CPT-c3C: R2 = H;
10 CPT-c3C-OH: R2 = OH.
9
binding manners with enzyme and cause the reduction of activity.
The exceptional case of CPT-c8 is interesting, and the reason is not
clear to our current knowledge. The space for substitutions at 7-po-
sition of CPT was proposed to be large enough, and the more bulky
cyclooctyl substituting at 7-positon was expected to have higher
activity than the less bulky one. However, the situation turned
Scheme 2. Reagents and conditions: (a) cyclohexanecarbaldehyde, H2O2, AcOH–
H2O, H2SO4, 0–5 °C, 3 h, 49% (CPT-c6) and 19% (CPT-c6-OH); (b) cyclopropane-
carbaldehyde, H2O2, AcOH–H2O, H2SO4, 0–5 °C. 3 h, 22% (CPT-c3C) and 16% (CPT-
c3C-OH).

M. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4107–4109
4109
Chung, M. K. Regul. Toxicol. Pharm. 2004, 40, 356; (b) Graul, A. I.; Prous, J. R. Drug
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up to be opposite. Perhaps, the 7-cycloalkyl CPT analogues would
change their binding model to the enzyme at this point as the ring
side increasing, and reduced its antitumor activity.
The relationship of structure–activity presents three situations.
Activities of derivatives of 7-cycloalkyl-CPT (CPT-cn, n = 3–7) or
10-OH-CPT (CPT-cn-OH, n = 5–7) on cell line A549/ATCC and that
of CPT-cn-OH or 7-cycloalkyl-10-OMe-CPT derivatives (CPT-cn-
OMe, n = 4–7) on cell line HT-29 generally respectively follow the
regularity that more lipophilic substitutions at 7-position would
lead higher antitumor activity, match the QSAR conclusion we
achieved before. However, activity of CPT-cn derivatives on cell
line A549/ATCC displays unordered, and situation of CPT-cn-OMe
derivatives act a contrary law to the QSAR conclusion on cell line
A549/ATCC. Methoxyl at 10-position displays to reduce the activi-
ties when the groups at 7-position become larger.
8. (a) Vladu, B.; Woynarowski, J. M.; Manikumar, G.; Wani, M. C.; Wall, M. E.; Von
Hoff, D. D.; Wadkins, R. M. Mol. Pharmacol. 2000, 57, 243; (b) Yin, M. B.; Guo, B.;
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K.; Ristum, Y. M. Mol. Pharmacol. 2000, 57, 453; (c) Dallavalle, S.; Ferrari, A.;
Biasotti, B.; Merlini, L.; Penco, S.; Gallo, G.; Marzi, M.; Tinti, M. O.; Martinelli, R.;
Pisano, C.; Carminati, P.; Carenini, N.; Beretta, G.; Perego, P.; Cesare, D. M.;
Pratesi, G.; Zunino, F. J. Med. Chem. 2001, 44, 3264; (d) Dallavalle, S.; Ferrari, A.;
Merlini, L.; Penco, S.; Carenini, N.; Cesare, D. M.; Perego, P.; Pratesi, G.; Zunino,
F. Bioorg. Med. Chem. Lett. 2001, 11, 291; (e) Du, W.; Kaskar, B.; Blumbergs, P.;
Subramanian, P.-K.; Curran, D. P. Bioorg. Med. Chem. 2003, 11, 451; (f)
Manikumar, G.; Wadkins, R. M.; Bearss, D.; Von Hoff, D. D.; Wani, M. C.;
Wall, M. E. Bioorg. Med. Chem. Lett. 2004, 14, 5377; (g) Dallavalle, S.; Giannini,
G.; Alloatti, D.; Casati, A.; Marastoni, E.; Musso, L.; Merlini, L.; Morini, G.; Penco,
S.; Pisano, C.; Tinelli, S.; Cesare, M. D.; Beretta, G. L.; Zunino, F. J. Med. Chem.
2006, 49, 5177.
In summary, we reported a series of 7-cycloalkyl-CPT analogues
with their synthesis and biological evaluations. These analogues
display high antitumor activities on four human tumor cell lines
A549/ATCC, HT-29, NCI-H460 and HL60. The SAR studies mostly
support our previous QSAR conclusion. The exceptional com-
pounds might indicate differences of CPT-DNA-Top 1 complex
model from others.
9. Li, M.; Jiang, C.; Li, M.; You, T. J. Mol. St.-Th. 2005, 723, 165.
10. (a) Redinbo, R. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.; Hol, W. G. J. Science
1998, 279, 1504; (b) Fan, J.; Weinstein, J. N.; Kohn, K. W.; Shi, L. M.; Pommier, Y.
J. J. Med. Chem. 1998, 41, 2216; (c) Fan, Y.; Shi, L. M.; Kohn, K. W.; Pommier, Y.;
Weinstein, J. N. J. Med. Chem. 2001, 44, 3254.
11. Sawada, S.; Nokata, K.; Furuta, T.; Yokokura, T.; Miyasaka, T. Chem. Pharm. Bull.
1991, 39, 2574.
12. Synthesis of 7-cyclohexylcamptothecin and 7-cyclohexyl-10-hydroxylcamptothecin
with Sawada method, general procedure: To a stirred solution of CPT (35 mg,
0.1 mmol) in HoAc (1.5 ml), deionized water (1.5 ml) and concd H2SO4 (0.4 ml)
in ice bath was add aldehyde (or cyclobutylmethanol) (0.3 mmol) and 30%
H2O2 (0.3 mmol). The stirring was continued for 3 h or stop according to the
TLC analysis. The reaction mixture was then diluted with ice water (6 ml) and
extracted with CH2Cl2 (10 ml  3). Combined organic layers were dried on
Na2SO4, evaporated and purified on silica gel column chromatography (1:100
MeOH/CHCl3).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.010.
13. Synthesis of 7-alkyl-camptothecin and 7-alkyl-10-hydroxylcamptothecin with
cycloalkyl bromides, general procedure: To the suspension of CPT (17 mg
0.05 mmol) in HOAc (2 ml) and acetone (1 ml) was added concd H2SO4
(0.03 ml), cycloalkyl bromides (0.4 mmol), Ph2SiH2 (0.45 mmol) and t-BuOOH
(1.0 mmol). The reaction mixture was heated on 80 °C oil bath and stirred for
8–12 h according to the TLC analysis. Then the reaction mixture was diluted
with ice water (10 ml) and extracted with CH2Cl2 (6 ml  3). The combined
organic phase was washed with brine, dried on anhydrous sodium sulfate,
evaporated and purified on silica gel column chromatography (1:100 MeOH/
CHCl3).
14. Methylation of 10-hydroxycamptothecin derivatives, general procedure: To the
suspension of 10-hydroxy-camptothecin derivatives (6, 8, 16–18, 0.1 mmol) in
anhydrous acetone (10 ml) was added anhydrous K2CO3 (0.5 mmol). After
stirring for 10 min, MeI (0.5 mmol) was added. The reaction mixture was then
stirred over night, cooled to room temperature, diluted with CH2Cl2 (20 ml),
washed with water, brine, and dried on anhydrous sodium sulfate, evaporated
and purified on silica gel column chromatography (1:100 MeOH/CHCl3) to give
compounds 19–23.
References and notes
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H.; Kim, J. K.; Park, S. C.; Son, W. C.; Lee, H. S.; Suh, J. E.; Kim, C. Y.; Ha, C. S.;