Novel hexacyclic camptothecin derivatives. Part 1: Synthesis and cytotoxicity of camptothecins with an A-ring fused 1,3-oxazine ring

Article abstract of DOI:10.1016/j.bmcl.2008.05.103

A novel series of A-ring modified hexacyclic camptothecin derivatives containing a 1,3-oxazine ring were first designed and synthesized. All of the hexacyclic camptothecins were assayed for in vitro cytotoxicity against nine human cancer cell lines. Among these compounds, 9b and 9c showed most potent cytotoxicity against several cell lines. Particularly, 9c was about 13-fold more potent than camptothecin, and about sixfold more potent than topotecan toward HEPG-2. Furthermore, it was also found that the N-alkyl substituted derivatives were more potent than the N-aryl and N-benzyl substituted compounds against most cell lines.

Full text of DOI:10.1016/j.bmcl.2008.05.103

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4095–4097
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel hexacyclic camptothecin derivatives. Part 1: Synthesis and cytotoxicity
of camptothecins with an A-ring fused 1,3-oxazine ring
a,
Sheng Wang a, Yuyan Li a, Yonghui Liu a, Aijun Lu b, , Qidong You
*
*
a Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, Jiangsu 210009, PR China
b JiangSu Simcere Pharmaceutical R&D Co., Ltd, Nanjing, Jiangsu 210042, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 January 2008
Revised 1 May 2008
Accepted 24 May 2008
Available online 12 June 2008
A novel series of A-ring modified hexacyclic camptothecin derivatives containing a 1,3-oxazine ring were
first designed and synthesized. All of the hexacyclic camptothecins were assayed for in vitro cytotoxicity
against nine human cancer cell lines. Among these compounds, 9b and 9c showed most potent cytotox-
icity against several cell lines. Particularly, 9c was about 13-fold more potent than camptothecin, and
about sixfold more potent than topotecan toward HEPG-2. Furthermore, it was also found that the N-alkyl
substituted derivatives were more potent than the N-aryl and N-benzyl substituted compounds against
most cell lines.
Keywords:
Camptothecin
10-Hydroxycamptothecin
1,3-Oxazine
Ó 2008 Elsevier Ltd. All rights reserved.
Camptothecin (CPT, 1), a pentacyclic alkaloid isolated by Wall in
1966 from the Chinese tree Camptotheca cuminate (Nyssaceae), was
reported to possess excellent antitumor activity.1 Clinical trials of
camptothecin derivatives were discontinued in the early 1970s be-
cause of severe and unpredictable toxicity, in particular hemor-
rhagic cystitis.2 In 1985, it was reported by Liu et al. that the
cytotoxic activity of CPT was attributed to a novel mechanism of
action involving the nuclear enzyme topoisomerase I (Topo I).3 It
was believed that camptothecin impacted upon replication, tran-
scription, and the repair of DNA and caused cancer cell death by
interfering with the catalytic cycle of DNA Top I and stabilizing
the DNA–Topo I binary complex. Since elucidation of the unique
mechanism of action, many derivatives have been synthesized
and some of them are in various stages of preclinical and clinical
development. Among them, two derivatives, topotecan4 (TPT, 2)
and irinotecan5 (3) (Fig. 1), have successfully entered into the mar-
ket and are used as Top I inhibitors in clinical practice.6
cyclic camptothecins, 4 and 5 are, respectively, studied in the clin-
ical phase II and phase III stages.
In order to further explore the potential of hexacyclic camptot-
hecins, we designed a novel series of hexacyclic camptothecin
derivatives containing a 1,3-oxazine ring fused with positions 9
and 10. We hypothesized that the novel hexacyclic camptothecins
could exhibit potent antitumor activity for the two following rea-
sons. First, it has a relatively rigid structure which could, in some
sense, be regarded as the closed ring structure of TPT, 2 (Fig. 3).
The flexible group at position 9 of TPTs molecule is fixed, which
may be favorable to the interaction with the binary DNA–Top I
complex. Second, their structure is very close to the known high
antitumor activity compounds, hexacyclic derivatives 6. Mean-
while, compared with 6, our designed structure of hexacyclic
camptothecins, in some sense, possesses an alkyl group in position
Structure–activity relationship (SAR) studies suggested that
substitution at the 7-, 9-, or 10-positions of most camptothecin
derivatives enhances their antitumor activity, but at the 11- or 5-
position usually leads to activity decrease.7 In particular, many
derivatives with an additional ring combined with the 10- and
11-positions, 7- and 9-positions, or 9- and 10-positions showed
potent antitumor activity superior to those of original pentacyclic
camptothecins, such as lurtotecan (4),8 exatecan (5),9 and other
hexacyclic derivatives 6 and 7,10,11 probably due to the extended
planarity exerted by an additional ring (Fig. 2). Among these hexa-
R2 R3
9
7
5
R1
10
O
A
N
C
B
11
D
N
12
1
E
O
OH O
Camptothecin 1: R1 = R2 = R3 = H
1
2
3
2:
Topotecan R = OH,
R = CH2NMe2, R = H
R2 = H, R3 = Et
,
1
Iri notecan 3:
OOC N
R =
N
* Corresponding authors. Tel./fax: +86 25 83271351 (Q. You).
E-mail addresses: youqidong@gmail.com, youqidongcpu@gmail.com (Q. You).
Figure 1. Structures of CPT, TPT, and irinotecan.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.103

4096
S. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4095–4097
R
N
N
NH2
N
HO
O
O
O
O
O
O
O
N
N
N
N
a
N
F
N
N
N
O
O
O
O
O
O
8
OH O
OH
9
OH
O
OH O
Lurtotecan 4
Exatecan 5
R
R
R
R
N
R
O
9a
9g
CH3-
9m
9n
OMe
O
O
N
N
N
N
9b CH3CH2-
9h
9i
O
O
Cl
Me
OH O
OH O
Hexacyclic derivatives 6
9C CH3(CH2)2-
9o
9p
Hexacyclic derivatives 7
Cl
F
Figure 2. Structures of lurtotecan, exatecan, and hexacyclic derivatives 6 and 7.
Me
9d
9e
CH3(CH2)3-
9j
Me
F
F
R
N
9q
9r
F
F
9k
N
HO
O
MeO
O
O
N
N
F
9f
9l
N
N
F
OMe
O
O
O
O
Cl
HO
HO
2
Scheme 1. Reagents and conditions: (a) 1—primary amine or aniline derivatives,
paraformaldehyde, 1,4-dioxane, 2 h; 2—8, 6–36 h.
9
Figure 3. The design of hexacyclic camptothecins 9.
9 which can enhance the liposolubility, antitumor activity, and sta-
bility of the lactone of camptothecins according to the literature.12
Moreover, our reported compounds could be synthesized in one
step with good yields from 10-hydroxycamptothecin, while the
hexacyclic derivatives 6 were synthesized in relatively low yields
in five steps from 10-hydroxycamptothecin. In this letter the syn-
thesis and in vitro cytotoxicity of the novel series of hexacyclic
camptothecins are reported.
The target hexacyclic camptothecins 9a–9r were prepared from
a Mannich-type reaction as shown in Scheme 1. Various primary
amines were treated with excess paraformaldehyde in 1,4-dioxane
at 70–105 °C under nitrogen for 2 h. Then 10-hydroxycamptothe-
cin was added to the mixture and heated for another 6–36 h to give
compounds 9a–9r. It was noteworthy that the solvent played a key
role in the reaction. The reaction did not take place when using
acetic acid or CH2Cl2 as a solvent and only trace product was affor-
ded in ethanol or DMF. However, good yields (60–85%) were
achieved using the optimal solvent 1,4-dioxane.
Three types of primary amines (alkyl, benzyl or phenyl) were
used as starting materials to give the corresponding hexacyclic
camptothecins in good yields (71–85%) except for the aniline
derivatives with electron-withdrawing substituted groups (com-
pounds 9n–9r, 60–67%), particularly, only trace product being ob-
tained using 4-nitroaniline. This was probably because of the lower
nucleophilicity of the aniline, which had a critical effect on the for-
mation of imine from paraformaldehyde and amine in the Man-
nich-type reaction. The IR, 1H NMR, ESI–MS, and HR–MS spectra
of these novel hexacyclic camptothecin analogs were consistent
with their structures and are listed in Supporting Information.
Cytotoxicity of these novel hexacyclic camptothecins was eval-
uated on nine human cancer cell lines (BXPC-3, NCI-446, MCF-7,
HEPG-2, A549, A2780, Bel7402, HT-29, and KB) using MTT assay.10
CPT and TPT were used as reference compounds. The results of the
cytotoxicity studies are shown in Table 1.
Most of novel hexacyclic camptothecins showed comparable or
superior cytotoxic activities to TPT, and five compounds (9b, 9c, 9e,
9m, and 9q) exhibited comparable cytotoxicities compared with
CPT against several cell lines. Among the four N-alkyl substituted
hexacyclic camptothecins, 9b and 9c showed more potent cytotox-
icities than CPT and TPT against BXPC-3, HEPG-2, and A2708 cell
lines. Particularly, 9c was about 13-fold more potent than CPT,
and about sixfold than TPT toward HEPG-2 cell line. This result
indicated that the relatively smaller substituted groups at N atom
of 1,3-oxazine ring could enhance cytotoxicity and it was consis-
tent with the SAR of CPT that large substituted groups at C-9 and
C-10 positions reduce the cytotoxicity of CPT.7 However, com-
pound 9a with an N-methyl showed less cytotoxicity. This was
probably due to its poor solubility in the solvents of DMSO and cul-
ture medium used in MTT assay. Cytotoxic activities of compounds
9b and 9c were comparable to the reported hexacyclic camptothe-
cin derivatives 6 with a small alkyl group at N atom of 1,4-oxazine
ring against A549 cell line, which also conformed to our primary
hypothesis of the similar antitumor activity based on their close
structures. The benzyl or phenyl with electron-withdrawing or
electron-donating substituted groups at N atom of 1,3-oxazine ring
showed similar cytotoxicity. For instance, compound 9f showed
similar cytotoxicity to 9g and cytotoxic activity of compound 9j
was comparable to 9o against most cell lines. The phenyl with
o-, m-, or p-substituted groups also exhibited similar cytotoxic
activity such as compounds 9i, 9j, and 9k. These results indicated
the substituted groups’ electronegativity and positions at benzyl
or phenyl groups slightly affected the cytotoxicity. Preliminary
study of SAR of this novel series of hexacyclic camptothecins indi-
cated that antitumor activities of N-alkyl substituted compounds
(9b and 9c) were better than those of N-benzyl and N-aryl substi-
tuted compounds (9e–9g and 9h–9r) against most cell lines. This
result was probably a result of the relatively smaller and more flex-
ible substituted groups at N atom of 1,3-oxazine ring, which could

S. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4095–4097
4097
Table 1
Cytotoxicity of hexacyclic camptothecins against nine human cancer cell linesa
Compound
In vitro cytotoxicity (IC50, l )
mol LÀ1
BXPC-3
NCI-446
MCF-7
HEPG-2
A549
A2780
Bel7402
HT-29
KB
CPT
TPT
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
0.24
0.62
0.48
0.19
0.15
0.97
0.21
1.73
1.24
0.53
0.58
2.05
3.34
0.42
0.19
0.89
0.51
1.57
0.12
0.92
3.28
10.10
37.33
0.23
3.90
10.10
6.23
43.23
56.83
1.14
7.03
9.39
—
14.45
—
19.22
—
—
—
0.20
1.20
4.52
0.21
0.13
0.06
0.64
0.05
0.01
0.35
0.11
0.14
1.47
0.18
0.93
—
0.13
0.12
0.03
—
0.17
0.55
—
0.01
0.22
0.41
0.16
0.11
0.20
0.08
0.12
0.03
1.21
0.42
0.57
0.72
0.27
0.77
0.45
0.31
0.39
0.28
0.56
0.20
3.35
0.53
0.12
0.09
3.19
0.91
11.62
17.58
4.26
0.85
2.16
2.99
1.12
3.43
4.10
0.82
2.07
1.07
3.10
0.17
3.13
1.67
0.81
1.43
4.01
1.58
5.22
9.58
6.00
5.43
3.86
2.23
4.99
7.13
15.17
16.27
4.70
13.08
3.06
0.09
0.88
0.69
0.14
0.18
0.13
0.10
1.19
1.68
0.22
0.34
0.53
0.31
0.31
0.48
1.05
0.21
0.62
0.62
1.13
0.003
0.22
0.67
0.12
0.67
0.09
0.04
0.06
0.08
0.06
0.04
0.34
0.29
0.23
0.67
0.33
0.23
0.66
0.28
0.62
b
—
0.55
1.60
0.65
—
—
—
1.64
1.40
—
2.75
2.00
5.00
4.48
2.59
9.51
—
0.18
a
In vitro cytotoxicity of the hexacyclic camptothecins against nine cell lines, BXPC-3 (human pancreatic cancer), NCI-446 (human lung cancer), MCF-7 (human breast
cancer), HEPG-2 (human livercancer), A549 (human lungcancer), A2780 (human ovariancancer), Bel7402 (human livercancer), HT-29 (human coloncancer), and KB (human
epidermoid carcinoma of the nasopharynx), were measured by the MTT assay after 4 days of incubation and expressed as the doses required to inhibit the growth of 50% of
the cells cultivated (IC50
,
l
mol LÀ1).
b
Not tested.
be more favorable to the interaction with the binary DNA–Top I
complex in three-dimension, and may also more readily make
the N atom form a hydrogen bond with receptor.
References and notes
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taining a 1,3-oxazine ring were first designed and synthesized. The
in vitro cytotoxicity was preliminarily evaluated. Several derivatives
showed very impressive cytotoxicity, especially compounds 9b and
9c, which showed great potency against several cell lines. Further
biological evaluation of compounds 9b and 9c is currently underway
in our laboratory.
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Acknowledgment
This work was supported in part by the Natural Science Founda-
tion of Jiangsu Province, China (BK2007168).
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.05.103.