Synthesis and evaluation of 9-benzylideneamino derivatives of homocamptothecin as potent inhibitors of DNA topoisomerase I

Article abstract of DOI:10.1016/j.ejmech.2010.01.063

A series of novel 9-benzylideneamino derivatives of homocamptothecin were synthesized via Friedlaender cyclization from our obtained intermediate 5. All the compounds were evaluated for in vitro cytotoxicity against three cancer cell lines (A549, LOVO and MDA-MB-435). Most of these derivatives possessed potent growth inhibitory effect on all the tested cell lines and four compounds (6d, 6f, 6i, 6k) showed higher inhibitory activities with the IC₅₀ values of 2.3 nM-9.8 nM against breast cancer cell than topotecan. As compared to CPT, compound 6f revealed higher topoisomerase I inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2010.01.063

European Journal of Medicinal Chemistry 45 (2010) 2223–2228
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of 9-benzylideneamino derivatives of
homocamptothecin as potent inhibitors of DNA topoisomerase I
**
*
Wei Guo, Zhenyuan Miao , Chunquan Sheng, Jianzhong Yao, Hao Feng, Wannian Zhang , Lingjian Zhu,
Wenfeng Liu, Pengfei Cheng, Jing Zhang, Xiaoying Che, Wenya Wang, Chuan Luo, Yulan Xu
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of 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 9-benzylideneamino derivatives of homocamptothecin were synthesized via Fried-
laender cyclization from our obtained intermediate 5. All the compounds were evaluated for in vitro
cytotoxicity against three cancer cell lines (A549, LOVO and MDA-MB-435). Most of these derivatives
possessed potent growth inhibitory effect on all the tested cell lines and four compounds (6d, 6f, 6i, 6k)
showed higher inhibitory activities with the IC50 values of 2.3 nM–9.8 nM against breast cancer cell than
topotecan. As compared to CPT, compound 6f revealed higher topoisomerase I inhibitory activity.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 24 October 2009
Received in revised form
5 January 2010
Accepted 27 January 2010
Available online 1 February 2010
Keywords:
Homocamptothecin
9-Benzylideneamino
Antitumor activity
Synthesis
1. Introduction
lactone and hence decreases the rate of lactone hydrolysis to the
corresponding inactive carboxylate form. Moreover, hCPT is found to
20(S)-Camptothecin (CPT), an antitumor alkaloid, was first iso-
lated by Wall and Wani in 1966 from Camptotheca acuminate a tree
native to China. The cellular target of CPT is DNA topoisomerase I,
a nuclear enzyme which is required for topological manipulation of
DNA during cellular events such as replication, transcription and
repair. CPT exhibits potent antitumor activity and has been evalu-
ated in clinic [1]. However, the therapeutic application of unmod-
ified CPT is hindered due to its poor aqueous solubility and high in
vivo hepatotoxicity. Subsequently, the structural modification of
natural CPT has generated many new CPT derivatives with
improved pharmacological or pharmacokinetic profile [2,3].
Among those, topotecan [4], belotecan [5] and irinotecan [6] have
entered clinical use and several drug candidates, such as rubitecan
[7], lurtotecan [8] and exatecan [9], are currently in different stages
of the clinical trials.
be much more stable than CPT inplasma because the affinity of hCPTs
to human serum albumin is lower than that of the corresponding
CPTs. As compared to CPT, hCPT exhibits better biological and phar-
macological properties [11]. The in vivo evaluation showed that hCPT
exhibited superior antitumor activities compared to CPT in the HT-29
xenograft model [12]. On the basis of these encouraging results,
several hCPTanalogues with the modified A and/or B rings have been
synthesized and evaluated in vitro and in vivo as the antitumor agents
[13–17]. Twoofthem, BN80915 (Diflomotecan) [18]andBN80927[19]
are in clinical trial and encouraging results have been obtained. The
exploration of new hCPT derivatives has recently emerged as
a promising field to find better antitumor agents.
In our laboratory, a series of homocamptothecins substituted in
position 7 with lipophilic moieties have been synthesized based on
our synthetic route which couples ring A with ring C, D and E.
Various groups were linked to the hCPT scaffold via arylimino-
methyl, pyridine quaternary salt and ester moieties [20]. These
compounds exhibited potent cytotoxic activity in vitro and in vivo
comparable or superior to topotecan.
In 1997, Lavergne et al. reported the insertion of a methylene
spacer between alcohol and carboxyl functions of CPT to obtain
homocamptothecin (hCPT) [10]. The replacement of the
six-membered
a-hydroxylactone with a seven-membered b-
hydroxylactone reduces considerably the electrophilicity of the
The high cytotoxic activity of the few 9-substituted CPTs such as
topotecan, chimmitecan and rubitecan which have been submitted
to preclinical and clinical studies prompted researchers to investi-
gate the introduction of other moieties in the unexplored position
of homocamptothecin. Few examples of homocamptothecins
substituted in position 9 are reported recently. One of the reasons
* Corresponding author. Tel./fax: þ86 21 81871243.
** Corresponding author. Tel./fax: þ86 21 81871233.
E-mail addresses: miaozhenyuan@hotmail.com (Z. Miao), zhangwnk@hotmail.
com (W. Zhang).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.063

2224
W. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 2223–2228
Table 1
for the limited number of studies on 9-substituted hCPTs may be
due to the scarce accessibility of this position which is similar to
CPT [21]. In addition, substituted C]N bands had been proved to be
useful to increase antitumor activity for antitumor agents such as
aryliminomethyl and oxyiminomethyl. In the light of these facts,
and as a continuation of reported work, we report here the
synthesis of a novel series of 9-substituted hCPTs derivatives and
also aim to test the influence of the substitution of the benzylide-
neamino moiety on the anticancer activity which have the reversed
C]N bands and to study their structure–activity relationships.
In vitro antitumor activity of 9-benzylideneamino derivatives of homocamptothecin
Compound
ICa50
A549
(mM)
MDA-MB-435
LOVO
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
151.15
3.55
4.56
3.29
2.92
2.84
2.39
5.58
3.01
4.18
2.28
2.55
3.60
1.87
5.44
1.09
4.32
0.11
0.27
0.0039
0.016
0.0091
0.72
1.18
0.0098
0.12
0.0022
0.011
0.36
0.018
0.014
0.079
190
10.33
4.26
1.38
2.60
0.91
8.62
5.96
3.39
5.29
1.93
1.61
4.19
5.80
1.07
1.21
2. Chemistry
Detailed synthetic pathway to novel 9-benzylideneamino
derivatives of homocamptothecin 6c–6o are depicted in Scheme 1.
According to published procedures, N-(2-acetyl-3-amino-
phenyl)acetamide 4 was prepared from 3-nitro-benzenamine 1 by
cyclization, oxidation and hydrogenation [22]. The key interme-
diate 5 was synthesized according to our reported procedure and
the synthetic process was optimized in our group’s previous work
[20]. Reaction of acetamide 4 with intermediate 5 in the presence of
p-methylbenzenesulfonic acid yielded the 7-methyl-9-acet-
aminohomocamptothecin 6a which was hydrolyzed to 6b. Treat-
ment of compounds 6b and commercially available substituted
benzaldehyde in pyridine led to the desired 9-benzylideneamino
homocamptothecin 6c–6o with the yield of 28%–62%.
Topotecan
aValues were measured with MTT method.
potent growth inhibitory effect on all the tested cell lines. The ratio
of the IC50 values of MDA-MB-435 reflects the relative sensitivity of
this series of compounds compared to the other two cells. Among
them, four compounds (6d, 6f, 6i, 6k) showed higher inhibitory
activities with the IC50 values of 2.2 nM–9.8 nM against breast
cancer cell than topotecan (TPT). Comparison of percent inhibition
revealed that compounds 6d, 6f, 6i and 6k possessed high growth
inhibitory effect at the concentration of 0.001 and 0.01 mg/mL while
very low levels of activity of TPT were observed at the same
concentration (Figs. 1 and 2).
3. Results and discussion
3.1. In vitro antitumor activities
It is interesting to note that the presence of substitution with
one methyl group on benzylideneamino results in a substantial
increase of the cytotoxic activity compared to two methyl group.
The behavior is again seen in the compounds with chlorine group.
Substitution with electron-donating groups at position 4 of phenyl
ring showed a distinct tendency to increase in vitro antitumor
activity. For example, 4-methyl compound 6f was shown to be 100
The synthesized compounds 6a–6o were evaluated for their in
vitro cytotoxic activity against human lung cancer (A549), breast
cancer (MDA-MB-435) and colon cancer (LOVO) cell lines. To obtain
more meaningful comparisons of relative potencies, topotecan was
tested as a positive control. From the data reported in Table 1, it
appears that most of 9-substituted hCPT derivatives possessed
O
N
O
O
O
O
O
NO2
NH2
NO2
NO2
5
a
b
c
OH
d
N
NHAc
NHAc
NH2
H
4
1
2
3
R
Ac
NH
N
NH2
O
O
e
f
O
N
N
N
N
N
N
O
O
O
O
O
O
6a
6b
6c-6o
OH
OH
OH
Me
Me
Me
Me
Me
Me
6c
6g
6d
Me
Me
Cl
6e
6f
MeO
Me
Me
N
6i
6j
6h
NO2
NO2
6k
6l
Cl
6m
6n
Br
6o
Cl
Scheme 1. Reagents and conditions: (a) butanone, t-BuOK, DMSO, 20 ꢀC, 24%; (b) CrO3, AcOH/H2O, 35 ꢀC, 42%; (c) H2, Pd–C, ethyl acetate, 30 ꢀC, 3 bar, 3 h, 91%; (d) p-TSA, toluene,
120 ꢀC, 51%; (e) 50% HCl, 90 ꢀC, 91%; and (f) benzaldehyde derivatives, Yb(OTf)3, pyridine, rt, 12–48 h, 28–62%.

W. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 2223–2228
2225
Fig. 1. Chemical structures of camptothecin analogues.
times as active as 4-nitro compound 6j in the assay of cytotoxic
4. Conclusion
activity against MDA-MB-435 cancer cell.
In summary, we have synthesized 13 novel 9-benzylideneamino
derivatives of homocamptothecin based on our synthetic route.
When evaluated for cytotoxicity against three human cell lines, all
of the compounds indicated potent growth inhibitory effect and
eight compounds were shown more efficient against breast cancer
cell than TPT. The SAR analysis of the synthesized compounds
revealed that substitution with electron-donating groups at posi-
tion 40 of benzylideneamino exhibited a distinct tendency to
increase in vitro antitumor activity. Four compounds 6b, 6d, 6f and
6k were also very potent as a topoisomerase I poison and
compounds 6f was found to exhibit more reasonably strong
topoisomerase I-dependent cytotoxic activity than CPT at the
3.2. Topoisomerase I-mediated DNA cleavage
The ability of selected homocamptothecin derivatives 6b, 6d, 6f,
6i and 6k to inhibit topoisomerase I was investigated in the
cleavable complex assay. The results presented in Fig. 3 indicated
that compounds 6b, 6d, 6f and 6k as cytotoxic agents were also
very potent as a topoisomerase I poison. It is evident that the
presence of an electron-donating group at position 40 of benzyli-
deneamino favors tertiary complex stability excepted compound 6i.
To our surprise, compound 6b with a low potency in the anti-
proliferative assay was potent in the DNA cleavage assay.
As compared to CPT, compound 6f revealed a more stable
ternary complex. For example, both CPT and 9-amino-
homocamptothecin 6b were inactive in this assay at concentrations
concentration of 1 mM.
5. Experimental protocols
down to 1
mM while compound 6f had modest activity at the same
concentration (Fig. 4).
5.1. General
Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker-500 spectrometer with TMS as an internal standard and
Fig. 3. Effect of compounds 6b, 6d, 6f, 6i and 6k on Topoisomerase-I mediated DNA
µg / mL
relaxation. Lane 1, supercoiled DNA pBR322; lane 2, supercoiled DNA and Topo I; lanes
Fig. 2. Antitumor activities of compounds 6d, 6f, 6i and 6k against MDA-MB-435
3–8 are compounds treated (10
mM) as follows: lane 3, CPT; lane 4, compound 6b; lane
cancer cell replication.
5, compound 6d; lane 6, compound 6f; lane 7, compound 6i; and lane 8, compound 6k.

2226
W. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 2223–2228
Fig. 4. Effect of compounds 6b and 6f on Topoisomerase I mediated DNA relaxation. Lane 1, supercoiled DNA pBR322; lane 2, supercoiled DNA and Topo I; lanes 3–5, 6–8, 9–11 are
three samples CPT, 6b and 6f at 1, 10, 50 M, respectively.
m
DMSO as solvent. Chemical shifts (
d
values) and coupling constants
5.4.1. 9-(4-Tert-butylbenzylideneamino)-7-
methylhomocamptothecin (6c)
(J values) are given in ppm and Hz, respectively. ESI mass spectra
were performed on an API-3000 LC–MS spectrometer. Elemental
analyses were performed with a MOD-1106 instrument and were
consistent with theoretical values within ꢁ0.4%. TLC analysis was
carried out on silica gel plates GF254 (Qingdao Haiyang Chemical,
China). Silica gel column chromatography was performed with
Silica gel 60 G (Qindao Haiyang Chemical, China). Commercial
solvents were used without any pretreatment.
The general synthetic method described above afforded 6c as
yellow solid (28%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.35 (s, 9H, 40-C(CH3)3), 1.86 (q, 2H,
–CH2CH3, J ¼ 7.4 Hz), 2.91 (s, 3H, 7-CH3), 3.27 (d, d, 2H, 21-CH2,
J ¼ 13.4, 13.9 Hz), 5.24 (s, 2H, 5-CH2, J ¼ 3.6 Hz), 5.47 (d, d, 2H, 17-
CH2, J ¼ 15.2, 15.1 Hz), 6.04 (s, 1H, 20-OH), 7.14 (d, 1H, 10-H,
J ¼ 7.3 Hz), 7.39 (s, 1H, 14H), 7.62 (d, 2H, 30-H, 50-H, J ¼ 8.4 Hz), 7.79
(dd, 1H, 11H, J ¼ 7.5, 8.4 Hz), 7.96 (d, 20-H, 60-H, J ¼ 8.4 Hz), 7.98 (d,
1H, 12H, J ¼ 8.5 Hz), 8.53 (s, 1H, –CH]N). ESI-MS: m/z, 534.20
[M ꢂ H]ꢂ. Anal. calcd. for C33H33N3O4: C, 74.00; H, 6.21; N, 7.84.
Found: C, 74.02; H, 6.20; N, 7.86.
5.2. 7-Methyl-9-acetaminohomocamptothecin (6a)
A solution of the tricyclic ketone 5 (0.50 g, 1.80 mmol) and
6-nitro-2-aminoacetophenone (0.50 g, 2.60 mmol) in toluene was
refluxed using a Dean–Stark trap for 30 min. p-Toluenesulfonic acid
(0.10 g, 0.52 mmol) was then added and refluxing was continued
for an additional 10 h. The solvent was removed and the crude
product was washed by acetone (10 mL) and methanol (10 mL) to
give 6a as yellow solid (0.40 g, 51%), mp > 300 ꢀC (dec). 1H NMR
5.4.2. 9-(2-Methylbenzylideneamino)-7-methylhomocamptothecin
(6d)
The general synthetic method described above afforded 6d as
yellow solid (29%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.86 (q, 2H, –CH2CH3, J ¼ 7.5 Hz),
2.64 (s, 3H, 20-CH3), 2.92 (s, 3H, 7-CH3), 3.33 (d, d, 2H, 21-CH2,
J ¼ 14.0, 13.9 Hz), 5.26 (s, 2H, 5-CH2), 5.47 (d, d, 2H, 17-CH2, J ¼ 15.1,
15.1 Hz), 6.03 (s, 1H, 20-OH), 7.19 (d, 1H, 10H, J ¼ 7.3 Hz), 7.36–7.50
(m, 4H, 14H, 30-H, 40-H0, 50-H), 7.80 (dd, 1H, 11H, J ¼ 7.5, 8.3 Hz), 8.00
(d, 1H, 60-H, J ¼ 7.7 Hz), 8.13 (d, 1H, 12H, J ¼ 8.4 Hz), 8.77 (s, 1H,
–CH]N). ESI-MS: m/z, 292.27 [M ꢂ H]ꢂ. Anal. calcd. for
C30H27N3O4: C, 73.01; H, 5.51; N, 8.51. Found: C, 73.00; H, 5.53; N,
8.50.
(500 Hz, DMSO):
d
: 0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.86 (q, 2H,
–CH2CH3, J ¼ 7.6 Hz), 2.13 (s, 3H, COCH3), 2.87 (s, 1H, 7-CH3), 3.27 (d,
d, 2H, 21-CH2, J ¼ 13.8, 13.9 Hz), 5.28 (s, 2H, 5-CH2,), 5.47 (d, d, 2H,
17-CH2, J ¼ 15.1, 15.1 Hz), 6.03 (s, 1H, 20-OH), 7.38 (s, 1H, 14H), 7.46
(d, 1H, 10H, J ¼ 8 Hz), 7.79 (dd, 1H, 11H, J ¼ 7.4, 8.3 Hz), 8.08 (d, 1H,
12H, J ¼ 8 Hz), 10.05 (s, 1H, NHAc). ESI-MS: m/z, 432.66 [M ꢂ H]ꢂ
.Anal. calcd. for C24H23N3O5: C, 66.50; H, 5.35; N, 9.69. Found: C,
66.48; H, 5.36; N, 9.67.
5.4.3. 9-(3-Methylbenzylideneamino)-7-methylhomocamptothecin
(6e)
5.3. 7-Methyl-9-aminohomocamptothecin (6b)
The general synthetic method described above afforded 6e as
yellow solid (31%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
7-Methyl-9-acetaminohomocamptothecin (0.50 g, 1.15 mmol)
was dissolved in HCl (6 mol/L, 10 mL) and the reaction mixture
refluxed at 90 ꢀC for 1 h; additional H2O (50 mL) was added and the
pH was adjusted to 8.4 with an addition of Na2CO3. The precipitate
was filtered and dried to give the crude compound 6b as yellow
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.87 (q, 2H, –CH2CH3, J ¼ 7.5 Hz),
2.50 (s, 3H, 30-CH3), 2.92 (s, 3H, 7-CH3), 3.27 (d, d, 2H, 21-CH2,
J ¼ 13.7, 13.9 Hz), 5.21 (s, 2H, 5-CH2), 5.47 (d, d, 2H, 17-CH2, J ¼ 15.1,
15.1 Hz), 6.04 (s, 1H, 20-OH), 7.15 (d, 1H, 10H, J ¼ 7.3 Hz), 7.39 (s, 1H,
14H), 7.43 (d, 1H, 40-H, J ¼ 7.5 Hz), 7.49 (t, 1H, 50-H, J ¼ 7.8 Hz), 7.78–
7.83 (m, 3H, 11H, 20,60-H), 8.53 (s, 1H, –CH]N). ESI-MS: m/z, 492.43
[M ꢂ H]ꢂ. Anal. calcd. for C30H27N3O4: C, 73.01; H, 5.51; N, 8.51.
Found: C, 73.03; H, 5.49; N, 8.53.
solid (0.41 g, 91%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.86 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.85 (q, 2H, –CH2CH3, J ¼ 7.4 Hz),
2.99 (s, 3H, 7-CH3), 3.26 (d, d, 2H, 21-CH2, J ¼ 14.1, 14.1 Hz), 5.20 (s,
2H, 5-CH2), 5.46 (d, d, 2H, 17-CH2, J ¼ 15.3, 15.3 Hz), 5.61 (s, 2H, 9-
NH2), 6.01 (s, 1H, 20-OH), 6.90 (d, 1H, 10-H, J ¼ 7.2 Hz), 7.30 (s, 1H,
14H), 7.36 (dd, 1H, 12H, J ¼ 8 Hz), 7.47 (dd, 1H, 11H, J ¼ 8.0, 7.8 Hz).
ESI-MS: m/z, 390.52 [M ꢂ H]ꢂ. Anal. calcd. for C22H21N3O4: C, 67.51;
H, 5.41; N, 10.74. Found: C, 66.48; H, 5.39; N, 10.76.
5.4.4. 9-(4-Methylbenzylideneamino)-7-methylhomocamptothecin
(6f)
The general synthetic method described above afforded 6f as
yellow solid (33%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, CDCl3):
d:
5.4. General procedure for the synthesis of 9-benzylideneamino
homo-camptothecin derivatives (6c–6o)
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.2 (q, 2H, –CH2CH3, J ¼ 7.8 Hz), 2.4
(s, 3H, 40-CH3), 2.91 (s, 3H, 7-CH3), 3.27 (d, d, 2H, 21-CH2, J ¼ 13.4,
13.9 Hz), 5.25 (s, 2H, 5-CH2), 5.47 (d, d, 2H, 17-CH2, J ¼ 15.1, 15.0 Hz),
6.03 (s,1H, 20-OH), 7.16 (d, 1H,10H, J ¼ 7.2 Hz), 7.39 (s,1H,14H), 7.41
(d, 2H, 30-H, 50-H, J ¼ 8.0 Hz), 7.79 (dd, 1H, 11H, J ¼ 7.6, 8.2 Hz), 7.92
(d, 2H, 20-H, 60-H, J ¼ 8.0 Hz), 7.98 (d, 1H, 12H, J ¼ 8.4 Hz), 8.52 (s, 1H,
–CH]N). ESI-MS: m/z, 494.65 [M þ H]þ. 13C NMR (125 MHz,
7-Methyl-9-aminohomocamptothecin (100 mg, 0.26 mmol) and
substituent benzaldehyde (0.78 mmol) were dissolved in the pyri-
dine (10 mL) with Yb(OTf)3 (16 mg, 0.03 mmol) as the catalyst. The
resulting mixture is stirred at room temperature for 12–48 h. After
filtering the sieves, the solvent was removed and the crude product
was purified by flash chromatography on the silica gel (100:1
CH2Cl2:CH3OH).
DMSO): d: 8.66, 21.19, 21.68, 36.70, 42.79, 50.92, 61.73, 73.57, 99.97,
116.49, 122.81, 123.00, 127.50, 129.43, 129.56, 129.77, 130.09, 130.42,
133.78, 141.66, 142.44, 145.42, 150.23, 151.87, 152.10, 156.23, 159.45,

W. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 2223–2228
2227
160.47, 172.29. Anal. calcd. for C30H27N3O4: C, 73.01; H, 5.51; N, 8.51.
Found: C, 72.99; H, 5.53; N, 8.50.
5.4.9. 9-(2-Methoxybenzylideneamino)-7-
methylhomocamptothecin (6k)
The general synthetic method described above afforded 6k as
yellow solid (30%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.87 (q, 2H, –CH2CH3, J ¼ 7.2 Hz),
2.88 (s, 3H, 7-CH3), 3.29 (d, d, 2H, 21-CH2, J ¼ 13.8, 13.9 Hz), 3.92 (s,
3H, 20-OCH3), 5.22 (s, 2H, 5-CH2), 5.48 (d, d, 2H, 17-CH2, J ¼ 15.1,
15.1 Hz), 6.03 (s, 1H, 20-OH), 7.09–7.17 (m, 2H, 30-H, 50-H), 7.22 (d,
1H, 10H, J ¼ 8.4 Hz), 7.39 (s, 1H, 14H), 7.58 (dd, 1H, 40-H, J ¼ 7.4 Hz),
7.77 (dd, 1H, 11H, J ¼ 7.4, 8.3 Hz), 7.96 (d, 1H, 12H, J ¼ 8.4 Hz), 8.10
(dd, 1H, 60-H, J ¼ 1.7, 7.7 Hz), 8.80 (s, 1H, –CH]N). ESI-MS: m/z,
510.78 [M ꢂ H]ꢂ. Anal. calcd. for C30H27N3O5: C, 70.71; H, 5.34; N,
8.25. Found: C, 70.69; H, 5.36; N, 8.23.
5.4.5. 9-(2,4-Dimethylbenzylideneamino)-7-
methylhomocamptothecin (6g)
d:
The general synthetic method described above afforded 6g as
yellow solid (31%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.87 (q, 2H, –CH2CH3, J ¼ 7.2 Hz),
0.25 (s, 3H, 40-CH3), 2.50 (s, 3H, 20-CH3), 2.89 (s, 3H, 7-CH3), 3.27 (d,
d, 2H, 21-CH2, J ¼ 13.8, 14.0 Hz), 5.22 (s, H, 5-CH2), 5.46 (d, d, 2H, 17-
CH2, J ¼ 15.1, 15.0 Hz), 6.03 (s, 1H, 20-OH), 7.17 (m, 2H, 30-H, 50-H),
7.21 (d, 1H, 10H, J ¼ 8.0 Hz), 7.38 (s, 1H, 14H), 7.78 (dd, 1H, 11H,
J ¼ 7.6, 8.1 Hz), 7.97 (d, 1H, 60-H, J ¼ 8.1 Hz), 8.00 (d, 1H, 12H,
J ¼ 7.9 Hz), 9.50 (s, 1H, –CH]N). ESI-MS: m/z, 506.34 [M ꢂ H]ꢂ.
Anal. calcd. for C31H29N3O4: C, 73.35; H, 5.76; N, 8.28. Found: C,
73.33; H, 5.78; N, 8.27.
5.4.10. 9-(4-Chlorobenzylideneamino)-7-methylhomocamptothecin
(6l)
The general synthetic method described above afforded 6l as
5.4.6. 9-(3,5-Dimethylbenzylideneamino)-7-
methylhomocamptothecin (6h)
yellow solid (36%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.87 (q, 2H, –CH2CH3, J ¼ 7.1 Hz),
2.89 (s, 3H, 7-CH3), 3.27 (d, d, 2H, 21-CH2, J ¼ 13.8, 13.8 Hz), 5.25 (s,
2H, 5-CH2), 5.47 (d, d, 2H, 17-CH2, J ¼ 15.0, 15.1 Hz), 6.03 (s, 1H, 20-
OH), 7.18 (d, 1H, 10H, J ¼ 7.2 Hz), 7.39 (s, 1H, 14H), 7.67 (d, 2H, 20-H,
60-H, J ¼ 8.4 Hz), 7.80 (dd, 1H, 11H, J ¼ 7.7, 8.1 Hz), 7.98 (d, 1H, 12H,
J ¼ 8.3 Hz), 8.03 (d, 2H, 30-H, 50-H, J ¼ 8.4 Hz), 8.58 (s, 1H, –CH]N).
ESI-MS: m/z, 512.35 [M ꢂ H]ꢂ. Anal. calcd. for C29H24N3O4: C, 67.77;
H, 4.71; N, 8.18. Found: C, 67.75; H, 4.70; N, 8.19.
The general synthetic method described above afforded 6h as
yellow solid (39%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.84 (q, 2H, –CH2CH3, J ¼ 7.4 Hz),
2.50 (s, 6H, 30-CH3, 50-CH3), 3.27 (d, d, 2H, 21-CH2, J ¼ 13.9, 13.1 Hz),
5.23 (s, H, 5-CH2) 5.47 (d, d, 2H, 17-CH2, J ¼ 15.1, 15.0 Hz), 6.04 (s, 1H,
6-OH), 7.12 (d, 1H, 10H, J ¼ 7.2 Hz), 7.25 (s, 1H, 14H), 7.39 (s, 1H, 40-H)
7.62 (s, 2H, 10-H, 50-H), 7.79 (dd, 1H, 11H, J ¼ 7.6, 8.1 Hz), 7.97 (d, 1H,
12H, J ¼ 8.3 Hz), 8.48 (s, 1H, –CH]N). ESI-MS: m/z, 506.52
[M ꢂ H]ꢂ. 13C NMR (125 MHz, DMSO):
d
: 8.67, 21.14, 21.30, 36.71,
5.4.11. 9-(2,4-Dichlorobenzylideneamino)-7-
methylhomocamptothecin (6m)
41.63, 42.79, 51.00, 61.71, 73.57, 99.95,116.54,122.84,123.02,127.20,
127.42, 127.57, 130.51, 131.13, 133.78, 134.59, 136.33, 138.15, 138.63,
141.77, 145.47, 150.29, 152.20, 159.48, 160.88, 167.92, 172.27. Anal.
calcd. for C31H29N3O4: C, 73.35; H, 5.76; N, 8.28. Found: C, 73.36; H,
5.75; N, 8.29.
The general synthetic method described above afforded 6m as
yellow solid (29%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.97 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 2.03 (q, 2H, –CH2CH3, J ¼ 7.4 Hz),
2.88 (s, 3H, 7-CH3), 3.31 (d, d, 2H, 21-CH2, J ¼ 13.6, 13.6 Hz), 4.30(s,
1H, –OH) 5.08 (d, d, 2H, 5-CH2, J ¼ 18.4, 18.4 Hz), 5.46 (d, d, 2H, 17-
CH2, J ¼ 15.5, 15.6 Hz), 6.95 (d, 1H, 10H, J ¼ 7.2 Hz), 7.49 (s, 1H, 30-H),
7.50 (d, 1H, 50-H, J ¼ 1.7 Hz), 7.57 (s, 1H, 14H), 7.66 (dd, 1H, 11H,
J ¼ 7.6, 8.1 Hz), 7.93 (d, 1H, 12H, J ¼ 8.3 Hz), 8.22 (d, 1H, 60-H,
J ¼ 8.4 Hz), 8.75(s, 1H, –CH]N). ESI-MS: m/z, 546.60 [M ꢂ H]ꢂ. Anal.
calcd. for C29H23N3O4: C, 63.51; H, 4.23; N, 7.66. Found: C, 63.53; H,
4.25; N, 7.65.
5.4.7. 9-[4-(N,N-dimethylamino)benzylideneamino]-7-
methylhomocamptothecin (6i)
The general synthetic method described above afforded 6i as
yellow solid (35%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.87 (q, 2H, –CH2CH3, J ¼ 7.6 Hz),
2.93 (s, 3H, 7-CH3), 3.05 (s, 6H, N(CH3)2), 3.27 (d, d, 2H, 21-CH2,
J ¼ 13.3, 13.8 Hz), 5.24 (s, 2H, 5-CH2) 5.47 (d, d, 2H, 7-CH2, J ¼ 15.1,
14.9 Hz), 6.03 (s, 1H, 20-OH), 6.86 (d, 2H, 30-H, 50-H, J ¼ 9.0 Hz), 7.01
(d, 1H, 10H, J ¼ 7.3 Hz), 7.38 (s, 1H, 14H), 7.76 (dd, 1H, 11H, J ¼ 7.4 Hz,
8.3 Hz), 7.82 (d, 2H, 20-H, 60-H, J ¼ 9 Hz), 7.92 (d, 1H, 12H, J ¼ 8.4 Hz),
8.33 (s, 1H, –CH]N). ESI-MS: m/z, 521.26 [M ꢂ H]ꢂ. 13C NMR
5.4.12. 9-(2-Bromobenzylideneamino)-7-
methylhomocamptothecin (6n)
The general synthetic method described above afforded 6n as
yellow solid (56%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
(125 MHz, DMSO):
d
: 8.58, 9.05, 21.17, 36.65, 40.50, 42.78, 46.28,
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.87 (q, 2H, –CH2CH3, J ¼ 7.4 Hz),
2.90 (s, 3H, 7-CH3), 3.27 (d, d, 2H, 21-CH2, J ¼ 13.9, 13.6 Hz), 5.26 (s,
2H, 5-CH2), 5.47 (d, d, 2H, 17-CH2, J ¼ 15.1, 15.2 Hz), 6.03 (s, 1H, 20-
OH), 7.19 (d, 1H, 10H, J ¼ 7.3 Hz), 7.39 (s, 1H, 14H), 7.79–7.83 (m, 3H,
11H, 30-H, 50-H), 7.96–8.02 (m, 3H, 12H, 20-H, 60-H), 8.58 (s, 1H,
–CH]N). ESI-MS: m/z, 588.89 [M ꢂ H]ꢂ. Anal. calcd. for
C29H24N3O4: C, 62.37; H, 4.33; N, 7.52. Found: C, 62.38; H, 4.31; N,
7.53.
50.86, 61.72, 73.52, 99.83, 111.46, 112.02, 116.23, 122.64, 123.19,
124.12, 126.57, 130.12, 130.37, 130.94, 141.85, 145.78, 150.34, 151.64,
152.92, 153.03, 156.21, 159.42, 159.58, 172.20. Anal. calcd. for
C31H30N4O4: C, 71.25; H, 5.79; N, 10.72. Found: C, 71.23; H, 5.81; N,
10.71.
5.4.8. 9-(4-Nitrobenzylideneamino)-7-methylhomocamptothecin
(6j)
The general synthetic method described above afforded 6j as
5.4.13. 9-(3-Nitrobenzylideneamino)-7-methylhomocamptothecin
(6o)
yellow solid (30%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d
:
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.87 (q, 2H, –CH2CH3,
The general synthetic method described above afforded 6o as
J ¼ 7.6 Hz), 2.92 (s, 3H, 7-CH3), 3.27 (d, d, 2H, 21-CH2, J ¼ 13.8,
13.9 Hz), 5.31 (s, 2H, 5-CH2), 5.47 (d, d, 2H, 17-CH2, J ¼ 15.2,
15.0 Hz), 6.03 (s, 1H, 10-OH), 7.27 (d, 1H, 10H, J ¼ 7.4 Hz), 7.40 (s,
1H, 14H), 7.83 (dd, 1H, 11H, J ¼ 7.4, 8.3 Hz), 8.04 (d, 1H, 12H,
J ¼ 8.3 Hz), 8.28 (d, 2H, 20-H, 60-H, J ¼ 8.8 Hz), 8.45 (d, 2H, 30-H, 50-
H, J ¼ 8.7 Hz), 8.76 (s, 1H, –CH]N). ESI-MS: m/z, 523.07 [M ꢂ H]ꢂ.
Anal. calcd. for C29H24N4O6: C, 66.41; H, 4.61; N, 10.67. Found: C,
66.43; H, 4.60; N, 10.69.
yellow solid (62%), mp > 300 ꢀC (dec). 1H NMR (500 Hz, DMSO):
d:
0.87 (t, 3H, –CH2CH3, J ¼ 7.4 Hz), 1.87 (q, 2H, –CH2CH3, J ¼ 7.2 Hz),
2.92 (s, 3H, 7-CH3), 3.27 (q, 2H, 21-CH2, J ¼ 13.9,13.7 Hz), 5.26 (s, 2H,
5-CH2), 5.47 (d, d, 2H, 17-CH2, J ¼ 15.5, 15.1 Hz), 6.03 (s, 1H, 20-OH),
7.24 (d, 1H, 10H, J ¼ 7.3 Hz), 7.39 (s, 1H, 14H), 7.84 (dd, 1H, 11H,
J ¼ 7.5, 8.3 Hz), 7.91 (d, 1H, 50-H, J ¼ 7.9 Hz), 8.04 (d, 1H, 12H,
J ¼ 8.4 Hz), 8.44–8.49 (m, 2H, 40-H, 60-H), 8.77 (s, 1H, –CH]N), 8.82
(s, 1H, 20-H). ESI-MS: m/z, 523.66 [M ꢂ H]ꢂ. Anal. calcd. for

2228
W. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 2223–2228
C29H24N4O6: C, 66.41; H, 4.61; N, 10.68. Found: C, 66.43; H, 4.60; N,
10.70.
Science and Technology of Shanghai (No. 09431901700), Major
Special Project for the Creation of New Drugs (Grant Nos.
2009ZX09301-011) and Shanghai Leading Academic Discipline
Project (Project Nos. B906).
5.5. Cytotoxicity
One thousand two hundred cells per well were plated in 96-well
plates. After culturing for 24 h, test compounds were added onto
triplicate wells with different concentration, and 0.1% DMSO for
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Acknowledgement
This research was supported in part by the National Natural
Science Foundation of China (No. 30371689), the Key Project of