Synthesis and antitumor activity of 10-arylcamptothecin derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.02.005

A series of 10-arylcamptothecin derivatives was designed and synthesized. The key step of the synthesis was achieved by employing Suzuki cross-coupling chemistry. All of the new derivatives were tested for cytotoxicity against three human tumor cell lines

Full text of DOI:10.1016/j.bmcl.2011.02.005

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2071–2074
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitumor activity of 10-arylcamptothecin derivatives
Yu Jiao a,1, Hongchun Liu b,1, Meiyu Geng b, , Wenhu Duan a,
⇑
⇑
a Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
b Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, 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 10-arylcamptothecin derivatives was designed and synthesized. The key step of the synthesis
was achieved by employing Suzuki cross-coupling chemistry. All of the new derivatives were tested for
cytotoxicity against three human tumor cell lines, BEL-7402, A549, and HL-60; most of the derivatives
exhibited potent cytotoxicity. The stability study showed that compound 30 was more stable than its lead
compound 10-hydroxycamptothecin under the physiological condition. Mechanistic study demonstrated
that compound 30 and its hydrochloride 31 had a pharmacological profile similar with camptothecin.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 19 November 2010
Revised 1 February 2011
Accepted 2 February 2011
Available online 16 February 2011
Keywords:
Antitumor activity
Camptothecin derivative
Lactone stability
Synthesis
Topoisomerase I inhibitor
Camptothecin (CPT, 1), an alkaloid isolated by Wall and Wani
from Camptotheca acuminate, showed potent inhibitory activity
against a broad spectrum of tumors.1,2 Clinical use of camptothecin
in cancer therapy, however, was limited by its poor water solubility
and its severe adverse effects including neutropenia, thrombocyto-
penia, hemorrhagic cystitis, and G.I. symptoms with significant diar-
rhea. Since the discovery of its antitumor mode of action which was
closely associated with inhibition of topoisomerase I (Topo I), an
essential enzyme that catalyzes the relaxation of supercoiled DNA
during DNA replication,3 intensive efforts have been devoted to
identify novel camptothecin analogues.4–8 As a result, three drugs,
topotecan (2),4 irinotecan (4),5 and belotecan (5)9,10 (Fig. 1), have
been approved in treatment of human cancers.
Structure–activity relationship (SAR) studies revealed that
modification at northwestern positions of camptothecin may result
in increase in its potency. The substitution at the positions 7–10
and fusion of an additional ring with the ring A/B have led to the
discovery of a series of potent compounds which are in clinical
studies, such as gimatecan (7),6 silatecan (8),7 lurtotecan (9),8 exa-
tecan (10)11,12 (Fig. 1). These successful examples imply that those
N
R2
9
R3
7
NH2
N
R1
10
O
O
O
O
O
A
B
C
N
N
N
11
D
N
F
N
12
N
1
E
O
O
O
O
OH O
Belotecan (5): R1 = OH, R2 = H, R3 = CH2CH2NHCHMe2
10-Hydroxycamptothecin (6): R1=OH, R2 = R3 = H
Gimatecan (7): R1 = R2 = H, R3 = CH=N-OBu-t
Silatecan (8): R1 = OH, R2 = H, R3 = Si(Me)2Bu-t
OH O
OH
Exatecan (10)
Lurtotecan (9)
Camptothecin (1): R1 = R2 = R3 = H
Topotecan (2): R1 = OH, R2 = CH2NMe2, R3 = H
SN-38 (3): R1 = OH, R2 = H, R3 = Et
Irinotecan (4): R1
=
OCON
, R2 = H, R3 = Et
N
Figure 1. Camptothecin and its derivatives in clinic use and in clinic trial.
⇑
Corresponding authors. Tel.: +86 21 5080 6702 (M.G.); tel./fax: +86 21 5080 6032 (W.D.).
E-mail addresses: mygeng@mail.shcnc.ac.cn (M. Geng), whduan@mail.shcnc.ac.cn (W. Duan).
These authors contributed equally to this Letter.
1
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.005

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Y. Jiao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2071–2074
HO
TfO
R
O
O
O
N
N
N
a
b
N
N
N
O
O
O
O
OH
OH
O
OH
O
14
6
Cl
N
Cl
Cl
R=
18
16
15
17
COOMe
N
MeOOC
N
22
21
19
20
HOOC
HO
N
O
23
25
24
26
N
S
S
N
27
28
29
30
N
N
O
O
c
N
N
N
N
O
.
O
HCl
OH
O
OH
O
30
31
Scheme 1. The synthesis of the new derivatives. Reagents and conditions: (a) N,N-bis(trifluoromethylsulfonyl)aniline, TEA, DMF, rt; (b) Pd(PPh3)4, potassium carbonate,
RB(OH)2, 1,4-dioxane, 80 °C; (c) HCl in EtOAc.
Table 1
positions would tolerate a large group; therefore there remains
The cytotoxicity of the new derivatives against human tumor cell lines HL60, A549,
wide possibility for structural modification. On this basis we de-
signed a class of camptothecin derivatives with an aryl substituent
at the position 10. Herein we reported the synthesis of a series of
10-arylcamptothecin and their preliminary biological results.
10-Hydroxycamptothecin (6), a commercial available natural
product, was served as the starting material; the 10-hydroxyl
group was transformed into various aryl groups using a two-step
sequence (Scheme 1). Compound 6 was first converted into triflate
14 in high yield (96.7%) using the literature method.13 Then 14 was
arylated using Suzuki cross-coupling chemistry: treatment of the
triflate with Pd(PPh3)4, potassium carbonate, and an appropriate
aryl boronic acid in 1,4-dioxane afforded corresponding 10-aryl-
camptothecin (35.4–77.8% yields) (compounds 15–30).14,15 Finally,
compound 30 was treated with a hydrogen chloride solution in
ethyl acetate to give the water soluble quaternary salt 31.
The new derivatives were then subjected to testing in three can-
cer cell lines: leukemia HL60, liver cancer BEL-7402, and lung can-
cer A549, using the marketed drug topotecan as a reference drug.
The data show that most of the new 10-arylcamptothecins exhib-
ited potent cytotoxicity as shown in Table 1. The IC50 of the new
derivatives is in a broad range from 9 nM to 36.5 lM, indicating
that both aryl variants and substitution pattern might greatly
influence cytotoxicity of the new CPT derivatives.
and BEL-7402a
Compounds
In vitro cytotoxicity (IC50
A549
, l )
mol LÀ1
HL60
BEL-7402
TPT
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
0.052
0.59
0.30
0.10
0.16
0.013
2.88
0.049
0.28
0.020
0.78
1.45
0.071
1.12
0.32
0.067
0.23
0.040
2.68
0.023
0.51
0.14
0.50
11.59
5.95
0.47
2.46
3.12
0.93
1.53
0.18
36.48
5.12
0.65
1.05
5.89
13.81
6.59
0.08
0.23
0.38
0.42
0.46
1.44
0.048
0.037
0.032
0.009
0.021
0.010
0.012
0.068
0.067
0.062
a
The in vitro cytotoxicity of the CPT derivatives against three cell lines,
HL60(human leukemia), A549 (human lung cancer) and BEL-7402 (human liver
cancer) was measured by the MTT assay and SRB assay after 3 days of incubation
and expressed as the half maximal inhibitory concentration (IC50, l
mol LÀ1).
The primary structure–activity relationships were generalized
from the in vitro cytotoxicity data (Table 1). The derivatives with
m-substituted phenyl (17, 19, and 21) had a comparable activity
to topotecan; whereas o-/p-substituted phenyl derivatives
exhibited lower activity than their m-substituted isomers (15 and
16 vs. 17, 18 and 20 vs. 19, 22 vs. 21). When a bulky bicyclic aryl
group quinolyl was introduced, a sharp drop in potency was

Y. Jiao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2071–2074
2073
Figure 2. The stability of compound 30 in PBS (left panel), PBS with HSA (middle panel), and blood (right panel), HCPT (10-hydroxycamptothecin) as control. Stability profiles
were determined on HPLC with a fluorescence detector. Drug concentrations of 50
l
mol LÀ1 were used, and drug samples were incubated at pH 7.4 and 37 °C. Each data point
represents the average of three determinations with an uncertainty of 10% or less.
selective cytotoxicity against Bel-7402, a liver cancer cell line, with
IC50 of 9 nM, suggesting that compound 27 might have a potential
in treatment of the notorious liver cancers. However, the position
of heteroatom had little impact on cytotoxicity (27 vs. 28, 29 vs. 30).
Water solubility plays a very important role in improving the
potency of camptothecin derivatives. The hydrochloride salt 31,
the water soluble version of 30 was prepared to increase solubility,
its solubility was improved greatly (10 mg in 1 mL of water for 31
vs. less than 1 mg in 1 mL of water for 30).
It is well-known that only the lactone form of camptothecin
derivatives is active in vivo,16–19 the higher ratio of the lactone
form in blood, the better activity they might possess. The stability
of 30 and its lead compound 10-hydroxycamptothecin were tested
in three systems: phosphate buffered saline (PBS) at pH 7.4 (left
panel), PBS with human serum albumin (HSA) (middle panel),
and human blood (right panel) (Fig. 2). The result showed that
compound 30 displayed consistently more stable than 10-
hydroxycamptothecin in all three systems. The result indicated
that the introduction of an aryl group at the position 10 of campto-
thecin would enhance the stability of its lactone structure.
Figure 3. Topo I-mediated supercoiled DNA pBR322 relaxation assay. pBR322 DNA
was incubated with topoisomerase I (1 unit) in the presence or absence of indicated
drugs at 37 °C for 30 min and terminated by the addition of 10% sodium dodecyl
sulfate. The mixtures then were analyzed on a 1% agarose gel. CPT (camptothecin)
was used as reference drug. The position of supercoiled DNA (SC) and relaxed DNA
(RLX) were indicated.
observed (compound 26), indicating that 10-monocyclic aryl is
more favorable than 10-bicyclic aryl for maintaining the potency
of 10-aryl derivatives. Four 10-monoheteroaryl derivatives (27,
28, 29, and 30) exhibited potent cytotoxicity at sub-nanomolar
range. Moreover, 10-(2-thienyl)camptothecin (27) showed
a
Figure 4. Cell cycle alterations in response to compounds treatment. A549 cells were treated with TPT (topotecan), compounds 30 and 31 for 24 h. Cell cycle profiles were
analyzed by flow cytometry, and the percentage of the cells in G0/G1, S, and G2 /M phases were calculated by the ModFit program A.

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Y. Jiao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2071–2074
Two separate assays were carried out to explore the mode of
References and notes
action of the new derivatives. Compounds 30 and 31 were prefer-
entially tested for Topo I inhibitory activity using camptothecin as
a reference drug. As shown in Figure 3, both two compounds
exhibited strong Topo I inhibitory activity with an almost equal
potency as camptothecin. A typical characteristic of camptothecin
is its ability to induce cell cycle arrest at G2/M phase; we there-
fore assessed perturbation of the cell cycle using flow cytometry.
We found that treatment of compounds 30 and 31, at concentra-
tion from 12.5 nM to 50 nM, caused a marked increase in the pro-
portion of G2/M phase cells from 10% to 63% and 80%,
respectively (Fig. 4). Moreover, compounds 30 and 31 showed
better inhibitory effect than topotecan at the concentration of
25 nM.
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In summary, seventeen 10-arylcamptothecins were designed
and synthesized. Preliminary in vitro biological evaluation demon-
strated that some of the derivatives showed very potent cytotoxic-
ity with IC50 up to 9 nM. In particular, 10-(4-pyridyl)camptothecin
(30) and its water soluble hydrochloride 31 displayed comparable
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0.29 mmol), and 14 (100 mg, 0.20 mmol) were added to a sealed tube that had
been purged with argon. A solution of Pd(PPh3)4 (10 mg) in degassed dioxane
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The crude product was purified by flash chromatography (silica gel: 1.5–2.5%
MeOH in CH2Cl2) to give compounds 15–30 (35.4–77.8%).
15. The analytical data of 30 and 31. Compound 30: mp 287–288 °C.1H NMR
(300 MHz CDCl3) d 1.06 (3H, t, J = 7.5 Hz), 1.85-1.95 (2H, m), 5.34 (2H, s), 5.32
(1H, d, J = 14.4 Hz), 5.68 (1H, d, J = 14.4 Hz), 7.67 (2H, d, J = 6.0 Hz), 7.71 (1H, s),
8.10 (1H, d, J = 7.8 Hz), 8.19(1H, s), 8.36 (1H, d, J = 7.8 Hz), 8.43 (1H, s), 8.78(2H, d,
J = 6.0 Hz).13C NMR (100 MHz DMSO-d6) 175.5, 156.7, 153.2, 150.5(2), 149.9,
148.0, 145.7, 145.2, 135.7, 132.1, 130.4, 129.9, 128.8, 128.0, 126.8(2), 121.5,
119.3, 97.0, 72.4, 65.3, 50.3, 30.3, 7.8. MS (EI): m/z 425. HRMS (EI) calcd for
Acknowledgments
We are grateful for financial support from Major Project of Chi-
nese National Programs for Fundamental Research and Develop-
ment (No. 2009CB918404), National Science & Technology Major
Project ‘‘Key New Drug Creation and Manufacturing Program’’
(Nos. 2009ZX09301-001 and 2009ZX09103-065), the Natural Sci-
ence Foundation of China (Nos. 30725046, 81021062 and
90813034), Program of Shanghai Subject Chief Scientist (No.
10XD1405100).
C
25H19N3O4 425.1376; found 425.1383. Compound 30 has >98% chemical purity
as measured by HPLC.31: mp >300 °C. 1H NMR (300 MHz, DMSO-d6) d 0.89 (3H, t,
J = 7.5 Hz), 1.86–1.90 (2H, m), 5.35 (2H, S), 5.45 (2H, S), 8.01 (2H, d, J = 6.0 Hz),
8.23 (1H, s), 8.34 (1H, d, J = 7.8 Hz), 8. 45 (1H, s), 8.50 (1H, d, J = 7.8 Hz), 8.65 (1H,
s), 8.95 (2H, d, J = 6.0 Hz). MS (EI): m/z 425. Anal. Calcd for C25H19N3O4ÁHCl: C,
65.01; H, 4.36; Cl, 7.68; N, 9.10. Found: C, 64.89; H, 4.47; Cl, 7.79; N, 8.98.
16. Hertzberg, R. P.; Caranfa, M. J.; Holden, K. G.; Jakas, D. R.; Gallagher, G.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.005.