Synthesis of 7-triazole-substituted camptothecin via click chemistry and evaluation of in vitro antitumor activity

Article abstract of DOI:10.1002/cjoc.201300703

Camptothecin (CPT) is a natural topoisomerase I inhibitor with powerful antineoplastic activity against colorectal, breast, lung and ovarian cancers. To discover more potent antitumor agents, a series of new CPT derivatives were synthesized utilizing click chemistry. All compounds were assessed for cytotoxicity against A549, HCT-116, HT-29, LoVo, MDA-MB-231 cell lines, and some compounds exhibited good in vitro potency. Furthermore, all compounds kept or enhanced Topo I inhibition. A series of novel 7-triazole substituted camptothecin via click chemistry was designed, synthesized, and evaluated for their in vitro antitumor activity. Copyright

Full text of DOI:10.1002/cjoc.201300703

FULL PAPER
DOI: 10.1002/cjoc.201300703
Synthesis of 7-Triazole-substituted Camptothecin via Click
Chemistry and Evaluation of in vitro Antitumor Activity
a
a
b
b
a
,b
Lei Wang, Wei Yuan, Jie Zhang, Linjiang Tong, Yu Luo, Yi Chen,*
,a
,a
Wei Lu,* and Qingqing Huang*
a
Institute of Drug Discovery and Development, Shanghai Engineering Research Center of Molecular Therapeutics
and New Drug Development, East China Normal University, Shanghai 200062, China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, SIBS, Chinese Academy of Sci-
b
ences, Shanghai 201203, China
Camptothecin (CPT) is a natural topoisomerase I inhibitor with powerful antineoplastic activity against colo-
rectal, breast, lung and ovarian cancers. To discover more potent antitumor agents, a series of new CPT derivatives
were synthesized utilizing click chemistry. All compounds were assessed for cytotoxicity against A549, HCT-116,
HT-29, LoVo, MDA-MB-231 cell lines, and some compounds exhibited good in vitro potency. Furthermore, all
compounds kept or enhanced Topo I inhibition.
Keywords camptothecin, 7-triazole-substituted camptothecin, click chemistry, antitumor
Introduction
2
1
R
R
R3
9
7
5
Since being isolated from Camptotheca acuminata
by Wall and Wani in 1966, camptothecin (CPT, 1, Fig-
ure 1) as the most important topoisomerase I (Topo I)
8
6
2
O
1
0
4
17
A
B
C N
R4
11
16
N
D
12 13
3
22
1
[
1]
E O
1
4
1
1
5
20
inhibitor, has aroused widespread attention. CPT is
believed to have an enormous impact on DNA replica-
tion, transcription, and DNA repair, and hence cause
cancer cell apoptosis by specifically interfering with the
catalytic cycle of DNA topoisomerase I and reversibly
stabilizing the complex DNA-Topo I covalent com-
plex. Although CPT has drawbacks, such as severe
toxicity, instability of the lactone domain, extremely
low water solubility and other limitations, its specific
MOA (mode of action) and remarkable anticancer activ-
ity urged medicinal chemists to study the struc-
ture-activity relationship (SAR) of CPT. Many CPT
analogs were synthesized in the last decades, and some
have been applied in clinical therapy of cancers.
9
21
OH O
1
8
1
2
3
4
Camptothecin (1 R = R = R = R = H)
1
4
2
.
3
topotecan (2 R = R = H, R = CH N(CH ) HCl, R = OH)
2
3 2
1
2
4
3
irinotecan (3 R = Et, R = R = H, R = OCOPipPip)
[2]
lurtotecan (4a R1=
CH2
N
N
,
2
3
4
R = H, R = R = -OCH CH O-)
2
2
1
2
3
4
gimatecan (4b R = CH=NO(CH3)3, R = R = R = H)
1
2
3
4
namitecan (4c R = CH=NO(CH ) NH , R = R = R = H)
2 2
2
1
2
3
4
karenitecin (4d R = CH CH Si(CH ) , R = R = R = H)
2
2
3 3
1
2
4
3
silatecan (4e R = Si(CH ) C(CH ) , R = R = H, R = OH)
3
2
3 3
1
2
3
4
exatecan (4f R = R = -(S)-CH(NH )CH CH -, R = CH , R = F)
2
2
2
3
To date, two water-soluble CPT analogs topotecan
hycamtin, TPT, 2) and irinotecan (camptosar, 3) were
Figure 1 CPT and its representative derivatives with substitu-
ents at 7-position.
(
successfully commercialized and used successfully in
[
3]
the clinic (Figure 1). Others, such as belotecan
CKD-602), 9-aminocamptothecin, 9-nitrocamptothecin,
According to the previous studies of CPT derivatives,
the SAR had summarized that the natural
S-configuration at 20-position and the stability of the
E-ring lactone are both essential for their antitumor ac-
tivity, and substitutions at either 5- or 11- positions lead
(
lurtotecan (4a), gimatecan (4b), namitecan (4c),
karenitecin (BNP-1350, 4d), silatecan (AR-67, DB-67,
e), exatecan (DX-8951f, 4f), and diflomotecan
4
(
BN80915), are in various stages of clinical trials (Fig-
to decrease of activity, while substitutions at 7-, 9-, or
[
3]
[3,4]
ure 1).
10-positions enhance their antitumor activity.
Espe-
*
E-mail: qqhuang@sat.ecnu.edu.cn, wlu@chem.ecnu.edu.cn, ychen@jding.dhs.org
Received September 18, 2013; accepted November 26, 2013; published online December 11, 2013.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201300703 or from the author.
Chin. J. Chem. 2014, 32, 157—162
© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
157

Wang et al.
FULL PAPER
cially, substituents at 7-position are well tolerated.
[
5-11]
Scheme 1
A number of derivatives with substituents at 7-position
O
O
O
(
e.g. compounds 4a－4f) showed enhanced biological
a
b
c
[
12]
Br
OH
7a n = 3
Br
OEt
N3
OEt
profiles.
n
n
n
7
-Alkyl-substituted CPTs were mostly reported,
8a n = 3
8b n = 4
8c n = 5
8d n = 6
9a n = 3
9b n = 4
9c n = 5
9d n = 6
7
7
7
b n = 4
c n = 5
d n = 6
while few aromatic substituted derivatives were studied.
To evaluate the influence of substituted heteroaromatic
ring at 7-position, a series of novel 7-triazole-substituted
CPT derivatives (5a－5d and 6a－6d, structures shown
in Scheme 2) was designed herein. CPT-HDAC hybrids
O
O
d
N3
NHOH
N3
OH
n
n
1
1a n = 3
11b n = 4
1c n = 5
1d n = 6
1
1
0a n = 3
0b n = 4
10c n = 5
0d n = 6
5
a－5d were designed to synergically improve the cy-
1
1
totoxicities of CPT, according to the essential pharma-
cophore (zinc binding moiety-linker-capping group) of
1
[
13,14]
traditional HDAC inhibitors,
which have demon-
O
N3
N3
N3
n
strated the potential to enhance the cytotoxicities of a
O
[
15,16]
variety of cytotoxins,
inhibitors.
including Topo
I
12b n = 2
12c n = 6
12a
12d
SO
[
17,18]
Besides, compounds bearing ester (6a),
Reagents and conditions: (a) EtOH, H
NaI, V(DMF)∶V(H
0∶1, r.t.; (d) i) chloroformic acid ethyl ester, Et
2
4
3
, refluxed; (b) NaN ,
alkyl (6b, 6c) and phenyl (6d) tails were also designed
to examine the chemical space at the 7-position of CPT.
2
O)＝10∶1, 90 ℃; (c) KOH, V(THF)∶V(H
2
O)＝
1
3
N, THF, 0 ℃; ii)
hydroxylamine, r.t.
Results and Discussion
The traditional method to prepare 7-alkyl-substituted
Scheme 2
[19-21]
CPTs is Minisci-type reaction,
which although has
R
N N
turned out to be fruitful, the radical substitution is po-
tentially reactive to many functional groups. Another
improved way to prepare 7-alkyl-substituted CPTs is
Sonogashira reaction starting from 7-halogenated
N
O
a
b
N
O
N
N
[
22]
N
CPT.
However, the hydrogen of hydroxamate in
O
O
compounds 5a－5d is not tolerated. So a modified syn-
thetic strategy needs to be developed. Click Chemistry
was introduced by K. B. Sharpless in 2001 to describe
chemistry tailored to generate substances quickly and
reliably by joining small units together bearing the fea-
tures of modular, wide in scope, high yields, few
OAcO
OAcO
13
14a 14d
15a 15d
R
N N
N
[23]
inoffensive byproducts and high stereospecificity,
O
N
wherein the azide-alkyne Huisgen cycloaddition
employing a copper catalyst at room temperature
without requirement of any special precautions is one of
N
O
[24,25]
5a 5d
6d
the most familiar reactions.
Thus, this reaction was
OH O
6
a
employed to prepare 7-triazole-substituted CPTs in this
article.
R:
O
O
The preparation of azides with different lengths of
alkyl chain terminated with a hydroxamate was shown
in Scheme 1. Typically, according to the methods de-
NHOH
O
NHOH
O
14a, 5a
14b, 5b
[26,27]
scribed previously,
azido amino acids 10a－10d
were obtained as a colorless oil from the corresponding
bromide 7a－7d respectively in three steps, which was
further transformed into hydroxamates 9a－9d accord-
NHOH
NHOH
14c, 5c
14d, 5d
[
28]
ing to the reported method. Likewise, the precursors
O
O
1
2a－12d of compounds 6a－6d were prepared.
With these azides available, the Huisgen cycloaddi-
O
O
15a
1
6a
15b, 6b
tion reaction was applied. 7-Alkyne CPT (13) was syn-
[
22]
thesized according to our previous work.
in Scheme 2, compound 13 was reacted with azides
1a－11d and 12a－12d in the presence of catalytic
amounts of CuSO (prepared in situ) and sodium ascor-
bate in a mixture of water and tert-butyl alcohol at
As shown
5c, 6c
15d, 6d
1
Reagents and conditions: (a) 11a－11d or 12a－12d, CuSO
4
,
4
sodium ascorbate, V(H O)∶V(t-BuOH)＝1∶1; (b) NaOMe, MeOH
2
158
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© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2014, 32, 157—162

Synthesis of 7-Triazole-substituted Camptothecin
6
0 ℃ to afford compounds 14a－15d and 15a－15d in
H-bond donor was required for 7-position substitution.
The potent result of compounds 6a－6d suggested that
introduction of a proper substituted heteroaromatic ring
instead of a direct alkyl group at 7-position was feasible.
Furthermore, the designed compounds were assessed
to confirm whether they bind effectively to the original
target. As shown in Figure 2, the entire novel com-
pounds were also tested for inhibitory effects on Topo I,
using SN-38 as the reference compound. The enzyme
activity was quite different from the cytotoxicity, all
compounds except 5a and 5b exhibited superior enzy-
matic activities than SN-38 at a concentration of 0.1
μmol•L . Among them, 5d, 6a and 6d displayed
strongest Topo I inhibition. The best compound 6b did
not display most potent inhibitory effect on Topo I,
suggesting that the cytotoxicity of compound 6b was
probably not only caused by inhibiting Topo I. The re-
sult suggested that substituted heteroaromatic ring in-
stead of alkyl group at 7-position of CPT could keep or
enhance Topo I inhibition.
quantitative yields, which were then hydrolyzed with
sodium methoxide in methanol to produce 7-triazole-
substituted CPTs 5a－5d and 6a－6d.
These synthesized CPT derivatives were then as-
sessed in vitro antitumor activity against human tumor
cell lines A549 (human lung adenocarcinoma epithelial
cell line), HCT-116 (human colon carcinoma cell line),
HT-29 (human colon adenocarcinoma cell line), LoVo
(
2
human colon adenocarcinoma cell line) and MDA-MB-
31 (Human breast cancer cell line), using SN-38 (the
active metabolite of prodrug 3) and SAHA (suberoyl-
anilide hydroxamic acid, the first HDAC inhibitor ap-
proved by FDA for the sole use of cancer therapy) as
the reference compounds. The results of cytotoxicities
were shown in Table 1. It was disappointed that except
compound 5b which exhibited poor cytotoxicities
against human tumor cell lines HCT-116, LoVo and
MDA-MB-231, CPT–HDAC hybrids 5a－5d were ei-
ther inactive or showed dramatically decreased
anti-tumor activity in cell assays compared to the refer-
ence compounds. Nevertheless, compounds 6a－6d
exhibited similar or more potent cytotoxicities than
SN-38 against human colon cancer cell lines except
HT-29. In particular, compound 6b bearing the smallest
substitutent on the triazole ring in this series exhibited
impressive cytotoxicity activity in the range of IC50
－
1
－
1
values of 0.081－＜0.01 μmol•L against human tu-
mor cell lines A549, HCT-116 and MDA-MB-231, even
superior to that of SN-38. In comparison with com-
pound 5a, compound 6a bearing an ester tail instead of
hydroxamate displayed marked difference, which indi-
cated that incorporating hydroxamate into the 7-position
of CPT is not an effective way to synergically enhance
the antitumor activity, suggesting less polar, few
Figure 2 Enzyme activity inhibiting Topo I.
Experimental
All reagents and solvents were purchased from the
suppliers and purified/dried if anhydrous one was nec-
1
Table 1 Cytotoxicity assay against A549, HCT-116, HT-29,
essary. The H NMR spectra were recorded on Bruker
LoVo and MDA-MB-231 cell lines
DRX-500 (500 MHz) using TMS as internal standard.
H chemical shifts were reported as δ and spin-spin cou-
1
a
－1
IC50 /(μmol•L )
Compound
pling constants as J (Hz) values. The mass spectra were
recorded on a Finnigan MAT-95 mass spectrometer.
Melting points were taken on a Fisher-Johns melting
point apparatus, uncorrected and reported in degrees
Centigrade. Purity of compounds (＞95%) was estab-
lished by HPLC.
A549 HCT-116 HT-29 LoVo
MDA-MB-231
84.1
5
5
5
5
6
6
6
6
a
b
c
36.2 22.3
19.9 2.83
62.1 61.3
28.2 3.45
0.069 0.300
＜0.01 0.014
0.754 0.900
0.089 0.507
0.259 0.166
0.664 0.099
＞100 41.7
48.0 5.21
4.01
＞100 ＞100 43.6
d
a
b
c
33.28 11.97
30.1 0.050
7.88 0.081
30.1 0.742
22.3 0.081
3.42
0.111
＜0.01
0.589
0.497
0.176
General procedure to synthesize compounds 14a－
1
4d and 15a－15d
To a solution of 13 (1.0 equiv.) in a mixture of water
and tert-butyl alcohol (1∶1, volume ratio) was added
azides (2.0 equiv.), a solution of copper sulfate (10
mmol/L, 0.01 equiv.) and sodium ascorbate (0.1 equiv.).
Then the reaction mixture was warmed to 60 ℃ for 6 h.
The mixture was poured into water and extracted with
d
SN-38
5.59 0.027
b
b
SAHA
0.625
－
－
a
In vitro antitumor activities of 7-substituted CPTs against five
cell lines, A549, HCT-116, HT-29, LoVo and MDA-MB-231,
were measured after 3 days of incubation and expressed as the
doses required to inhibit the growth of 50% of the cells cultivated
CHCl
and dried over Na
V(chloroform)∶V(acetone)＝30∶1] afforded 14a－
14d and 15a－15d.
3
. The organic layer was washed with water, brine
2
SO . Flash silica gel chromatography
4
[
－
1
b
(
IC , μmol•L ) by SRB assay. No test.
50
Chin. J. Chem. 2014, 32, 157—162
© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
159

Wang et al.
FULL PAPER
1
2
0-Acetyl-7-(1-(5-(hydroxyamino)-5-oxopentyl)-
H-1,2,3-triazol-4-yl)camptothecin (14a) Yellow
solid, yield 80%. m.p.＞280 ℃; H NMR (500 MHz,
DMSO-d δ: 0.98 (t, J＝7.5 Hz, 3H), 1.47－1.49 (m,
H), 1.95－1.99 (m, 4H), 2.14－2.17 (m, 2H), 2.24 (s,
H), 4.60 (t, J＝7.0 Hz, 2H), 5.55－5.62 (m, 4H), 7.09
H NMR (500 MHz, CDCl
3
) δ: 0.89 (t, J＝6.7 Hz, 3H),
1
0.99 (t, J＝7.5 Hz, 3H), 1.30－1.45 (m, 10H), 2.07－
2.10 (m, 2H), 2.17－2.28 (m, 2H), 2.23 (s, 3H), 4.57 (t,
J＝7.3 Hz, 2H), 5.40－5.52 (m, 3H), 5.69 (d, J＝17.1
Hz, 1H), 7.24 (s, 1H), 7.69－7.72 (m, 1H), 7.85－7.88
(m, 1H), 8.07 (s, 1H), 8.28 (d, J＝8.2 Hz, 1H), 8.43 (d,
J＝8.4 Hz, 1H).
1
6
)
2
3
(
8
(
s, 1H), 7.63 (t, J＝7.5 Hz, 1H), 7.76 (t, J＝7.5 Hz, 1H),
.15 (d, J＝8.5 Hz, 1H), 8.53 (d, J＝8.5 Hz, 1H), 8.70
s, 1H), 9.06 (s, 1H).
0-Acetyl-7-(1-(6-(hydroxyamino)-6-oxopentyl)-
H-1,2,3-triazol-4-yl)camptothecin (14b) Yellow
20-Acetyl-7-(1-benzyl-1H-1,2,3-triazol-4-yl)camp-
tothecin (15d) Yellow solid, yield 84%. m.p.＞280
1
2
℃; H NMR (500 MHz, CDCl
3
) δ: 0.98 (t, J＝7.4 Hz,
1
3H), 2.16－2.29 (m, 5H), 5.39－5.47 (m, 3H), 5.68 (d, J
＝17.1 Hz, 1H), 5.75 (s, 2H), 7.22 (s, 1H), 7.40－7.47
(m, 5H), 7.67 (t, J＝7.5 Hz, 1H), 7.83－7.86 (m, 1H),
8.00 (s, 1H), 8.26 (d, J＝8.3 Hz, 1H), 8.33 (d, J＝8.7
Hz, 1H).
1
solid, yield 85%. m.p.＞280 ℃; H NMR (500 MHz,
DMSO-d ) δ: 0.93 (t, J＝7.4 Hz, 3H), 1.33－1.35 (m,
H), 1.57－1.60 (m, 2H), 1.96－1.99 (m, 4H), 2.15－
.18 (m, 2H), 2.24 (s, 3H), 4.58 (t, J＝7.0 Hz, 2H), 5.45
6
2
2
(
1
8
s, 2H), 5.51 (s, 2H), 7.11 (s, 1H), 7.80 (t, J＝7.6 Hz,
H), 7.94 (t, J＝7.6 Hz, 1H), 8.26 (d, J＝8.5 Hz, 1H),
.63 (d, J＝8.5 Hz, 1H), 8.67 (s, 1H), 9.07 (s, 1H).
General procedure to synthesize compounds 5a－5d
and 6a－6d
To a solution of 20-acetyl camptothecins (1.0 equiv.)
in methanol was added sodium methoxide (2.0 equiv.).
After the reaction mixture was stirred at room tempera-
ture overnight, it was neutrilized to pH＝7.0 using 1
mol•L aqueous hydrochloric acid and concentrated in
vacuo. The residue was passed through a short pad of
silica gel to give 5a－5d [V(chloroform)∶V(methanol)
2
0-Acetyl-7-(1-(7-(hydroxyamino)-7-oxopentyl)-
1
H-1,2,3-triazol-4-yl)camptothecin (14c) Yellow
1
solid, yield 82%. m.p.＞280 ℃; H NMR (500 MHz,
DMSO-d ) δ: 0.93 (t, J＝7.5 Hz, 3H), 1.26－1.34 (m,
H), 1.48－1.51 (m, 2H), 1.92－1.98 (m, 4H), 2.14－
.16 (m, 2H), 2.24 (s, 3H), 4.58 (t, J＝7.0 Hz, 2H), 5.44
s, 2H), 5.50 (s, 2H), 7.10 (s, 1H), 7.79 (t, J＝7.5 Hz,
H), 7.93 (t, J＝7.5 Hz, 1H), 8.26 (d, J＝8.5 Hz, 1H),
.61－8.64 (m, 2H), 9.07 (s, 1H), 10.32 (s, 1H).
6
－
1
4
2
(
1
8
＝
25∶1] and 6a－6d [V(chloroform)∶V(methanol)＝
0∶1].
-(1-(5-(Hydroxyamino)-5-oxopentyl)-1H-1,2,3-
triazol-4-yl)camptothecin (5a) Yellow solid, yield
1
7
2
0-Acetyl-7-(1-(8-(hydroxyamino)-8-oxopentyl)-
1
H-1,2,3-triazol-4-yl)camptothecin (14d) Yellow
1
1
5
0
1
2
7
6
5%. m.p.＞280 ℃; H NMR (500 MHz, DMSO-d ) δ:
solid, yield 79%. m.p.＞280 ℃; H NMR (500 MHz,
DMSO-d ) δ: 0.93 (t, J＝7.5 Hz, 3H), 1.26－1.31 (m,
H), 1.45－1.52 (m, 4H), 1.93－1.98 (m, 4H), 2.15－
.17 (m, 2H), 2.24 (s, 3H), 4.57 (t, J＝7.0 Hz, 2H), 5.43
.91 (t, J＝7.5 Hz, 3H), 1.33－1.35 (m, 2H), 1.87－
.89 (m, 4H), 1.90－2.05 (m, 2H), 4.59 (t, J＝7.0 Hz,
H), 5.45 (s, 4H), 7.40 (s, 1H), 7.75 (t, J＝7.5 Hz, 1H),
.95 (t, J＝7.5 Hz, 1H), 8.28 (d, J＝8.0 Hz, 1H), 8.59 (d,
6
4
2
(
1
8
1
s, 2H), 5.50 (s, 2H), 7.10 (s, 1H), 7.78 (t, J＝7.5 Hz,
J＝8.0 Hz, 1H), 9.04 (s, 1H). HRMS (ESI) calcd for
H), 7.92 (t, J＝7.5 Hz, 1H), 8.24 (d, J＝8.5 Hz, 1H),
.60 (d, J＝8.5 Hz, 1H), 8.64 (s, 1H), 9.06 (s, 1H),
0.32 (s, 1H).
＋
C
27
H
7
27
N
6
O
6
[M＋H] : 531.1992, found 531.1987.
-(1-(6-(Hydroxyamino)-6-oxopentyl)-1H-1,2,3-
triazol-4-yl)camptothecin (5b) Yellow solid, yield
2
0-Acetyl-7-(1-(5-ethoxy-5-oxopentyl)-1H-1,2,3-
1
5
0
1
4
6
0%. m.p.＞280 ℃; H NMR (500 MHz, DMSO-d ) δ:
triazol-4-yl)camptothecin (15a) Yellow solid, yield
7%. m.p.＞280 ℃; H NMR (500 MHz, CDCl ) δ:
3
.99 (t, J＝7.4 Hz, 3H), 1.27 (t, J＝7.2 Hz, 3H), 1.77－
.80 (m, 2H), 2.14－2.19 (m, 3H), 2.24 (s, 3H), 2.27－
.30 (m, 1H), 2.44 (t, J＝7.2 Hz, 3H), 4.16 (q, J＝7.1
Hz, 2H), 4.61 (t, J＝7.1 Hz, 2H), 5.40－5.53 (m, 3H),
.69 (d, J＝17.1 Hz, 1H), 7.24 (s, 1H), 7.71－7.73 (m,
H), 7.85－7.88 (m, 1H), 8.14 (s, 1H), 8.28 (d, J＝8.2
1
.90 (t, J＝7.5 Hz, 3H), 1.33－1.36 (m, 2H), 1.57－
.60 (m, 2H), 1.89－1.91 (m, 2H), 1.97－2.01 (m, 4H),
.58 (t, J＝7.0 Hz, 2H), 5.45 (s, 4H), 7.40 (s, 1H), 7.80
8
0
1
2
(
8
t, J＝7.5 Hz, 1H), 7.94 (t, J＝7.5 Hz, 1H), 8.26 (d, J＝
.0 Hz, 1H), 8.63 (d, J＝8.0 Hz, 1H), 9.07 (s, 1H).
＋
29 6 6
HRMS (ESI) calcd for C28H N O [M＋H] : 545.2149,
5
1
found 545.2143.
7
-(1-(7-(Hydroxyamino)-7-oxopentyl)-1H-1,2,3-
Hz, 1H), 8.43 (d, J＝8.3 Hz, 1H).
0-Acetyl-7-(1-butyl-1H-1,2,3-triazol-4-yl)camp-
tothecin (15b) Yellow solid, yield 83%. m.p.＞280
triazol-4-yl)camptothecin (5c) Yellow solid, yield
2
1
4
0
1
2
7
6
8%. m.p.＞280 ℃; H NMR (500 MHz, DMSO-d ) δ:
1
.91 (t, J＝7.5 Hz, 3H), 1.24－1.28 (m, 4H), 1.44－
.48 (m, 2H), 1.94－2.03 (m, 6H), 4.57 (t, J＝7.0 Hz,
H), 5.45 (s, 4H), 7.40 (s, 1H), 7.80 (t, J＝7.5 Hz, 1H),
.94 (t, J＝7.5 Hz, 1H), 8.27 (d, J＝8.5 Hz, 1H), 8.60 (d,
℃
3
－
(
1
7
8
; H NMR (500 MHz, CDCl
H), 1.05 (t, J＝7.4 Hz, 3H), 1.46－1.51 (m, 2H), 2.06
2.09 (m, 2H), 2.17－2.28 (m, 2H), 2.22 (s, 3H), 4.58
t, J＝7.2 Hz, 2H), 5.40－5.52 (m, 3H), 5.68 (d, J＝
3
) δ: 0.98 (t, J＝7.5 Hz,
J＝8.5 Hz, 1H), 9.06 (s, 1H). HRMS (ESI) calcd for
7.1 Hz, 1H), 7.23 (s, 1H), 7.69－7.72 (m, 1H), 7.85－
.88 (m, 1H), 8.07 (s, 1H), 8.28 (d, J＝8.4 Hz, 1H),
.42 (d, J＝8.6 Hz, 1H).
＋
C
29
H
7
31
N
6
O
6
[M＋H] : 559.2305, found 559.2300.
-(1-(8-(Hydroxyamino)-8-oxopentyl)-1H-1,2,3-
triazol-4-yl)camptothecin (5d) Yellow solid, yield
2
0-Acetyl-7-(1-octyl-1H-1,2,3-triazol-4-yl)campto-
1
5
6
2%. m.p.＞280 ℃; H NMR (500 MHz, DMSO-d ) δ:
thecin (15c) Yellow solid, yield 89%. m.p.＞280 ℃;
160
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© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2014, 32, 157—162

Synthesis of 7-Triazole-substituted Camptothecin
0
1
2
7
.90 (t, J＝7.5 Hz, 3H), 1.22－1.34 (m, 6H), 1.48－
.51 (m, 2H), 1.88－1.98 (m, 6H), 4.57 (t, J＝7.0 Hz,
H), 5.40 (s, 2H), 5.44 (s, 2H), 6.55 (s, 1H), 7.37 (s, 1H),
.77 (t, J＝7.5 Hz, 1H), 7.91 (t, J＝7.5 Hz, 1H), 8.23 (d,
values were determined with the Logit method from at
least three independent tests.
The Topo I we used was purchased from Amersham
Biosciences. One unit is defined as the amount of en-
zyme that completely relaxed 0.5 mg of supercoiled
pBR322 DNA in 20 μL of the reaction mixture for 30
min at 37 ℃. The reaction buffer contained 35 mmol•
J＝8.0 Hz, 1H), 8.58 (d, J＝9.0 Hz, 1H), 8.66 (s, 1H),
.07 (s, 1H), 10.35 (s, 1H). HRMS (ESI) calcd for
9
＋
C
30
H
33
N
6
O
6
[M＋H] : 573.2462, found 573.2456.
-(1-(5-Methoxy-5-oxopentyl)-1H-1,2,3-triazol-4-
yl)camptothecin (6a) Yellow solid, yield 63%. m.p.
－
1
－1
－1
7
L
Tris-HCl (pH 8.0), 72 mmol•L KCl, 5 mmol•L
－
1
2
MgCl , 5 mmol•L spermidine, 0.01% bovine serum
1
＞
＝
1
280 ℃; H NMR (500 MHz, [V(CDCl
3
3
)∶V(CD OD)
albumin, 0.25 μg supercoiled pBR322, and 2 units of
Topo I in a total volume of 20 μL. Relaxation was in-
duced at 37 ℃ for 30 min and terminated by the addi-
tion of 1/10 volume of 10% SDS. The mixture was then
treated with proteinase K at a final concentration of 50
μg/mL and incubated at 37 ℃ for 30 min. An equal
volume of chloroform∶isoamyl alcohol (24∶1, vol-
ume ratio) was used to extract the reaction mixture, and
the supernatant was then removed onto 1% agarose gel
for analysis. Electrophoresis was carried out in 1×TAE
at 4 V/cm for 2 h, and the gel was stained with ethidium
bromide (0.5 μg/mL), and photographed through a gel
document system, ChemiGenius 2.
3∶1]) δ: 1.04 (t, J＝7.3 Hz, 3H), 1.78－1.82 (m, 2H),
.91－1.96 (m, 2H), 2.15－2.18 (m, 2H), 2.49 (t, J＝7.2
Hz, 2H), 3.71 (s, 3H), 4.64 (t, J＝7.1 Hz, 2H), 5.27－
5
2
8
.44 (m, 3H), 5.63 (d, J＝16.3 Hz, 1H), 7.69－7.72 (m,
H), 7.85 (t, J＝7.6 Hz, 1H), 8.20 (d, J＝8.5 Hz, 1H),
.40 (d, J＝8.5 Hz, 1H), 8.43 (s, 1H). HRMS (ESI)
＋
calcd for C28
30.2031.
-(1-Butyl-1H-1,2,3-triazol-4-yl)camptothecin (6b)
28 5 6
H N O [M ＋H] : 530.2040, found
5
7
1
Yellow solid, yield 60%. m.p.＞280 ℃; H NMR (500
MHz, [V(CDCl )∶V(CD OD)＝3∶1]) δ: 1.01－1.05
m, 6H), 1.47－1.49 (m, 2H), 1.92－1.95 (m, 2H), 2.05
3
3
(
－
4
2.08 (m, 2H), 4.60 (t, J＝7.2 Hz, 2H), 5.28－5.66 (m,
H), 7.70－7.73 (m, 2H), 7.86 (t, J＝7.5 Hz, 1H), 8.23
Conclusions
(
d, J＝8.5 Hz, 1H), 8.36 (s, 1H), 8.48 (d, J＝8.5 H＋z,
In summary, click chemistry was applied to the fac-
ile synthesis of 7-triazole-substituted CPT derivatives.
The in vitro antitumor activity results suggested that
hybrid of CPT and HDAC inhibitor with hydroxamate
tail cannot synergically increase the cytotoxicities, but
introduction of a triazole ring terminated with ester, al-
kyl chain or phenyl instead of a direct alkyl group at
1
4
H). HRMS (ESI) calcd for C26
72.1985, found 472.1979.
26 5 4
H N O [M＋H] :
7
-(1-Octyl-1H-1,2,3-triazol-4-yl)camptothecin (6c)
1
Yellow solid, yield 65%. m.p.＞280 ℃; H NMR (500
MHz, [V(CDCl )∶V(CD OD)＝3∶1]) δ: 0.90 (t, J＝
.4 Hz, 3H), 1.05 (t, J＝7.5 Hz, 3H), 1.27－1.31 (m,
0H), 1.95－2.10 (m, 4H), 4.60 (t, J＝7.2 Hz, 2H), 5.30
5.34 (m, 1H), 5.45－5.48 (m, 2H), 5.66－5.70 (m,
3
3
6
1
－
7
-positions of CPT could promote potency to some ex-
tent. Inhibitory effects on Topo I suggested 7-triazole-
substituted CPT could keep or enhance Topo I inhibi-
tion. Therefore, all these results reinforce the notion that
introduction of other heteroaromatic ring instead of a
direct alkyl group at 7-position would be feasible.
1
8
H), 7.74－7.77 (m, 2H), 7.87－7.90 (m, 1H), 8.26－
.29 (m, 1H), 8.34 (s, 1H), 8.41－8.44 (m, 1H). HRMS
＋
34 5 4
(ESI) calcd for C30H N O [M＋H] : 528.2611, found
5
28.2605.
-(1-Benzyl-1H-1,2,3-triazol-4-yl)camptothecin
7
1
(
6d) Yellow solid, yield 70%. m.p.＞280 ℃; H
NMR (500 MHz, DMSO-d ) δ: 0.91 (t, J＝6.1 Hz, 3H),
.85－1.91 (m, 2H), 5.40 (s, 2H), 5.44 (s, 2H), 5.84 (s,
H), 6.55 (s, 1H), 7.36－7.47 (m, 6H), 7.75 (t, J＝7.6
Acknowledgement
6
1
2
This work was supported by the grants of The Na-
tional Natural Science Foundation of China (Nos.
Hz, 1H), 7.90 (t, J＝7.6 Hz 1H), 8.22 (d, J＝8.5 Hz,
H), 8.58 (d, J＝8.5 Hz, 1H), 9.18 (s, 1H). HRMS (ESI)
8
1172936, 21102046), and grants of The Fundamental
1
＋
Research Funds for the Central Universities. We also
thank the Laboratory of Organic Functional Molecules,
the Sino-French Institute of ECNU for support.
calcd for C29
06.1823.
24 5 4
H N O [M ＋H] : 506.1828, found
5
Biological assay
References
Cytotoxicity assays of all the compounds were per-
formed on human tumor cell lines A549, HCT-116,
HT-29, LoVo and MDA-MB-231, which were obtained
from the American Type Culture Collection (Manassas,
VA). Cells (6000－10000) in 100 μL culture medium
per well were seeded into 96-well plates (Falcon, CA),
cultured overnight, and treated in triplicate with gradient
concentrations of test drugs for 72 h. The growth in-
hibitory effects were measured with a sulforhodamine B
[
1] Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. J. Biol. Chem.
1985, 260, 14873.
[2] Wang, J. C. Annu. Rev. Biochem. 1996, 65, 635.
[
[
[
3] Huang, Q.; Wang, L.; Lu, W. Eur. J. Med. Chem. 2013, 63, 746.
4] Oberlies, N. H.; Kroll, D. J. J. Nat. Prod. 2004, 67, 129.
5] Kawato, Y.; Terasawa, H. In Progress in Medical Chemistry, Eds.:
Ellis, G. P.; Luscombe, D. K., Elsevier, London, 1997, p. 70.
6] Fan, Y.; Weinstein, J. N.; Kohn, K. W.; Shi, L. M.; Pommier, Y. J.
Med. Chem. 1998, 41, 2216.
[
(
SRB; Sigma, St. Louis, MO) assay. The average IC50
[7] Yao, Y.-S.; Liu, J.-L.; Xi, J.; Miu, B.; Liu, G.-S.; Wang, S.; Meng,
Chin. J. Chem. 2014, 32, 157—162
© 2014 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
161

Wang et al.
FULL PAPER
L.; Yao, Z.-J. Chem. Eur. J. 2011, 17, 10462.
Bastien, S.; Hausheer, F. A., 3rd; Lush, R.; Neuger, A.; Sullivan, D.
M.; Munster, P. N. Clin. Cancer Res. 2009, 15, 2479.
[19] Sawada, S.; Okajima, S.; Aiyama, R.; Nokata, K.; Furuta, T.;
Yokokura, T.; Sugino, E.; Yamaguchi, K.; Miyasaka, T. Chem.
Pharm. Bull. (Tokyo) 1991, 39, 1446.
[
8] Christodoulou, M. S.; Zunino, F.; Zuco, V.; Borrelli, S.; Comi, D.;
Fontana, G.; Martinelli, M.; Lorens, J. B.; Evensen, L.; Sironi, M.;
Pieraccini, S.; Dalla Via, L.; Gia, O. M.; Passarella, D.
ChemMedChem 2012, 7, 2134.
[
9] Watanabe, T.; Tsuboi, Y.; Nomura, S. Chem. Asian J. 2013, 8, 630.
[20] Sawada, S.; Matsuoka, S.; Nokata, K.; Nagata, H.; Furuta, T.;
Yokokura, T.; Miyasaka, T. Chem. Pharm. Bull. (Tokyo) 1991, 39,
3183.
[21] Sawada, S.; Nokata, K.; Furuta, T.; Yokokura, T.; Miyasaka, T.
Chem. Pharm. Bull. (Tokyo) 1991, 39, 2574.
[22] Luo, Y.; Gao, H.; Li, Y.; Huang, W.; Lu, W.; Zhang, Z. Tetrahedron
2006, 62, 2465.
[23] Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004.
[24] Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596.
[25] Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057.
[
10] Cincinelli, R.; Musso, L.; Dallavalle, S.; Artali, R.; Tinelli, S.;
Colangelo, D.; Zunino, F.; De Cesare, M.; Beretta, G. L.; Zaffaroni,
N. Eur. J. Med. Chem. 2013, 63, 387.
11] Meng, G.; Li, J.; Wang, G.; Dong, M.; Zhang, Q. Chin. J. Org.
Chem. 2013, 33, DOI: 10.6023/cjoc201307049.
12] Penco, S.; Merlini, L.; Zunino, F.; Dallavalle, S. Expert. Opin. Ther.
Patents 2002, 12, 837.
13] Suzuki, T.; Kouketsu, A.; Matsuura, A.; Kohara, A.; Ninomiya, S.-i.;
Kohda, K.; Miyata, N. Bioorg. Med. Chem. Lett. 2004, 14, 3313.
14] Suzuki, T.; Nagano, Y.; Kouketsu, A.; Matsuura, A.; Maruyama, S.;
Kurotaki, M.; Nakagawa, H.; Miyata, N. J. Med. Chem. 2005, 48,
[
[
[
[
1
019.
[
[
15] Camphausen, K.; Burgan, W.; Cerra, M.; Oswald, K. A.; Trepel, J.
B.; Lee, M. J.; Tofilon, P. J. Cancer Res. 2004, 64, 316.
16] Chinnaiyan, P.; Vallabhaneni, G.; Armstrong, E.; Huang, S. M.;
Harari, P. M. Int. J. Radiat. Oncol. Biol. Phys. 2005, 62, 223.
17] Bevins, R. L.; Zimmer, S. G. Cancer Res. 2005, 65, 6957.
18] Daud, A. I.; Dawson, J.; DeConti, R. C.; Bicaku, E.; Marchion, D.;
[26] Ames, D. E.; Bowman, R. E.; Mason, R. G. J. Chem. Soc. 1950,
174.
[27] Khoukhi, N.; Vaultier, M.; Carrié, R. Tetrahedron 1987, 43, 1811.
[28] Huang, W.-J.; Chen, C.-C.; Chao, S.-W.; Yu, C.-C.; Yang, C.-Y.;
Guh, J.-H.; Lin, Y.-C.; Kuo, C.-I.; Yang, P.; Chang, C.-I. Eur. J.
Med. Chem. 2011, 46, 4042.
[
[
(Cheng, F.)
162
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Chin. J. Chem. 2014, 32, 157—162