Synthesis and pharmacological evaluation of novel homocamptothecin- dihydropyridine derivative conjugates as potent topoisomerase i inhibitors

Article abstract of DOI:10.1071/CH11091

Homocamptothecins (hCPT) represent a new generation of antitumour agents targeting DNA topoisomerase I. The expanded seven-membered lactone E-ring that characterizes hCPT enhances the plasma stability of the drug and reinforces the inhibition of topoisomerase I (Topo I) compared with conventional six-membered CPT. In an attempt to improve the antitumour activity of hCP, a series of novel hCPT derivatives conjugating with dihydropyridine derivates were designed and synthesized based on a synthetic route that couples 7-formylhomocamptothecin with different dihydropyridine derivates. Most of the synthesized compounds exhibited good cytotoxic activity on tumour cell line A549, MDA-MB-435, and HCT116. Furthermore, this class of compounds showed superior Topo I inhibition activity comparable to or higher than CPT. CSIRO 2011.

Full text of DOI:10.1071/CH11091

Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2011, 64, 1390–1396
www.publish.csiro.au/journals/ajc
Synthesis and Pharmacological Evaluation of Novel
Homocamptothecin-Dihydropyridine Derivative
Conjugates as Potent Topoisomerase I Inhibitors
A
A
B
A C
,
Ling-Jian Zhu, Chun-Lin Zhuang, Ning Lei, Chun-Quan Sheng,
A
A C
,
A
A
Wei Guo, Zhen-Yuan Miao,
and Wan-Nian Zhang
Wen-Feng Liu, Jian-Zhong Yao,
A C
,
A
School of Pharmacy, Second Military Medical University, 325 Guohe Road,
Shanghai 200433, China.
B
Department of Quality Assurance, Lian Yun Gang JARI Pharmaceutical Co., Ltd,
18 Zhenhua Road, Lianyungang 222100, China.
C
Corresponding authors. Email: zhangwnk@hotmail.com; miaozhenyuan@hotmail.com;
shengcq@hotmail.com
Homocamptothecins (hCPT) represent a new generation of antitumour agents targeting DNA topoisomerase I. The
expanded seven-membered lactone E-ring that characterizes hCPT enhances the plasma stability of the drug and reinforces
the inhibition of topoisomerase I (Topo I) compared with conventional six-membered CPT. In an attempt to improve the
antitumour activity of hCP, a series of novel hCPT derivatives conjugating with dihydropyridine derivates were designed
and synthesized based on a synthetic route that couples 7-formylhomocamptothecin with different dihydropyridine
derivates. Most of the synthesized compounds exhibited good cytotoxic activity on tumour cell line A549, MDA-MB-435,
and HCT116. Furthermore, this class of compounds showed superior Topo I inhibition activity comparable to or higher
than CPT.
Manuscript received: 25 February 2011.
Manuscript accepted: 3 June 2011.
Published online: 23 August 2011.
with conventional six-membered CPT.[7] On the basis of these
encouraging results, many hCPT analogues have been synthe-
sized and evaluated in vitro and in vivo as the antitumour
agents.[8–12] Among them, diflomotecan (BN80915) and
elomotecan (BN80927) (Fig. 2) are representative ones that
have been in clinical trial, and encouraging results have been
obtained.[13–15] Furthermore, a series of novel hCPT derivatives
have been synthesized in our laboratory and several compounds
that showed superior activity had been chosen to conduct
preclinical studies.[16–19]
Structural diversity plays a prominent role in medicinal and
combinatorial chemistry for a faster and efficient lead genera-
tion towards the new drug discovery. Dihydropyridine (DHP)
and its derivatives of the Hantzsch-type, known as ‘privileged
structures’,[20] have attracted immense attention of medicinal
chemists owing to their pharmacological properties including
antihypertensive, antidiabetic, vasodilator, antitumour, and
bronchodilator effects.[21,22] The best known members of the
DHP class are the calcium channel blockers,[23] exemplified
by nifedipine and filodipine, which are widely prescribed for a
variety of cardiovascular conditions. Interestingly, the past
research has revealed that there was a potential correlation
between cancer and hypertension, which is actually a significant
risk factor for cancer.[24,25] Therefore, we supposed that intro-
ducing the organic molecules with hypotensive properties into
Introduction
Camptothecin (CPT), a cytotoxic quinoline alkaloid, is a potent
anticancer agent that was isolated from the Chinese tree
Camptotheca acumunata by Wani and Wall in 1966.[1] As a
consequence of its broad-spectrum antitumour activity and
cytotoxic mechanism, there has been a substantial effort toward
the development of clinical CPT analogues. Topotecan and
irinotecan are two water-soluble analogues of CPT that are now
being marketed in several countries, and several other analogues
are the subject of ongoing clinical or preclinical evaluation
(Fig. 1).[2,3]
Even though CPT show significant antitumour activity,
their clinical potential is limited by severe side effects, such as
myelosuppression, nausea, vomiting, and gastrointestinal dis-
orders and stomatitis with topotecan and irinotecan,[4,5] which
have promoted intensive efforts to increase the efficacy of these
agents, reduce their toxicity in patients, and overcome the
tumour drug resistance for this class of compounds. A novel
and interesting option would be to modify the metabolically
liable CPT lactone ring from a six-membered a-hydroxylactone
to a seven-membered b-hydroxylactone ring, which leads to
the development of the homocamptothecin (hCPT) family of
compounds.[6] The expanded seven-membered lactone E-ring
that characterizes hCPT enhances the plasma stability of the
drug and reinforces the inhibition of topoisomerase I compared
Ó CSIRO 2011
10.1071/CH11091

Homocamptothecin-dihydropyridine Conjugates
1391
9
7
O
10
N
A
B
N
C
D
O
E
OH O
HO
CPT
N
N
N
O
O
O
N
N
O
N
N
O
O
O
Topotecan
OH O
OH
Irinotecan
Fig. 1. Camptothecin (CPT) and representative analogues.
O
N
N
O
CH3
O
N
OH
hCPT
• HCI
Me
Cl
O
F
F
O
N
N
N
N
O
O
O
BN80927
O
OH
Diflomotecan
OH
Fig. 2. Homocamptothecin (hCPT) and representative analogues.
antitumour molecules may be an effective measure to improve
the therapeutic effect of antitumour agents for special popula-
tions in the future.
Owing to acetoxymethyl-hCPT being the main by-product,
SeO2 was used as the oxidant instead of previously-used acetic
acid in this reaction (Scheme 2). Scheme 3 shows the synthetic
strategy for our targeted compounds 4a–j based on the coupling
of various DHP derivatives and 7-formylhomocamptothecin.
The C¼N bond was selected as a linker between the two units, as
it was successfully adopted in our previous drug design.[17] The
Schiff base was formed under catalytic reaction using Yb(OTf)3
in CHCl3.
Owing to the above pharmacological potency of the DHP
scaffold, we were interested in studying the incorporation of a
DHP unit into the hCPT skeleton in order that we could discover
novel hCPT analogues with higher Topo I inhibition efficacy,
better therapeutic effect, and lower toxicity. Although the
volume of DHP derivatives is relatively big, based on the ternary
complex of DNA-Topo I-CPT,[26] we presented a reasonable
binding mode of this class of compounds with DNA-Topo I
complex. In this model, we found a large space around the C-7
position of hCPT that allowed the introduction of DHP deriva-
tives, and two key hydrogen bonds were reserved (Fig. 3).
Result and Discussion
The in vitro antitumour activities of these DHP-hCPT deriva-
tives against solid tumour cell lines A549 (for non-small cell
lung cancer), HCT116 (for colon cancer), and MDA-MB-435
(for breast cancer), were evaluated. Topotecan was used as a
reference compound since it is a clinically effective CPT and
does not require metabolic activation.
As expected, the results (Table 1) suggested that these DHP
derivatives could promote the antitumour activity of hCPT,
which highlights the advantage of introducing a lipophilic chain
to position 7 of the hCPT derivative. The results also revealed
that these compounds were more sensitive against the HCT116
and MDA-MB-435 cell lines than against the A549 cell line.
For the A549 cell line, with few exceptions (4b), the tested
compounds showed increased cytotoxic potency compared with
TPT. According to the HCT116 cell line, all tested com-
pounds showed higher in vitro inhibitory activities than TPT.
Chemistry
The chemical synthesis of the targeted compounds was based
on two key intermediates, DHP derivatives and 7-formyl-
homocamptothecin. We employed the classical Hantzsch
reaction[27] to synthesize the nitro-substituted DHP derivatives
2a–j, then reduced them to yield the amino-substituted DHP
derivatives 3a–j (Scheme 1). 7-Formylhomocamptothecin
was prepared according to the method reported by Sawada
et al.[28] Treatment of hCPT with hydrogen peroxide and
ferrous sulfate in an aqueous methanol-sulfuric acid solution
furnished 7-hydroxymethylhomocamptothecin, which was
subsequently oxidized to form 7-formylhomocamptothecin.

1392
L.-J. Zhu et al.
Arg364
Asp533
Fig. 3. Hypothetical model for the binding of homocamptothecin- dihydropyridine conjugate (4a) with the DNA-TopoI complex.
The figure was prepared from PDB entry: 1T8I, using PyMol (http://www.pymol.org/; verified July 2011).
R
O
O
NH
O
R
O
O
NH
O
CHO
O
O
(ii)
(i)
O
NH3
ϩ
R
O
ϩ
R
O
R
NO2
NH2
NO2
1a p
1b m
2a–j
3a–j
Scheme 1. Reagents and conditions: (i) EtOH, reflux, 3 h; (ii) H2/Pd-C, EtOH, r.t.
HO
CHO
N
O
O
N
O
(i)
(ii)
N
N
N
O
N
O
O
O
OH
O
O
OH
OH
Scheme 2. Reagents and conditions: (i) 75 % H2SO4, FeSO4ꢁ7H2O, 30 % H2O2, CH3OH, H2O, 12 h, 79 %; (ii) SeO2/dioxane, 1008C,
8 h, 85 %.
In particular, the IC50 values of compounds 4a and 4h reached
21 and 14 nM respectively. Comparison of the inhibition ratio
revealed that compounds 4a and 4h possessed high growth
inhibitory effect at the concentrations of 0.01, 0.1, 1, 10, and
100 mg mLꢀ1, while relatively low activity of TPT was observed
at the same concentrations (Fig. 4). With regard to the MDA-
MB-435 cell line, compounds 4a, 4c, 4g, 4h, and 4i exhibited
antitumour activities comparable to or higher than TPT. In
particular, the activity of the most promising compounds, 4a
and 4h, showed broad in vitro antitumour spectrum. Further
pharmacological and toxicological evaluation of these promis-
ing compounds is in progress.
Generally, CPT and its clinically used derivatives act by
stabilizing a covalent topoisomerase I-DNA complex called the

Homocamptothecin-dihydropyridine Conjugates
1393
NH
O
R
O
O
O
N
R
O
O
NH
O
R
CHO
(i)
O
N
N
CH
ϩ
O
R
N
O
O
NH2
N
O
O
OH
3a–j
O
4a–j
OH
H2C
O
R ϭ
CH3CH2
H3C
p
4a
4b
H2C
p
p
4f
H2
C
p
H3C
H3C
C
4g
4h
4i
H2
CH3
H3C
H3C
C
p
p
4c
m
m
CH3
H3C
H3C
CHCH2
CHCH2
4d
4e
H3C
H3C
CH3CH2
m
4j
H2C CH2
p
Scheme 3. Reagents and conditions: (i) Yb(OTf)3, CHCl3, r.t.
100
Table 1. In vitro antitumour activity of homocamptothecin-
dihydropyridine derivative conjugatesA
80
60
40
20
0
Compounds
IC50 [mM]
HCT116
A549
MDA-MB-435
4a
4b
4c
4d
4e
4f
1.508
3.255
2.046
1.981
1.975
1.859
1.958
0.406
1.502
3.152
3.159
0.0217
0.313
0.143
0.208
0.158
0.332
0.134
0.0145
0.178
0.289
1.10
0.181
0.579
0.156
0.733
0.978
1.067
0.120
0.393
0.353
0.815
0.448
4g
4h
4i
4j
TPT
0.01
0.1
1
10
100
µg mLϪ1
ACancer cells: A549, lung cancer cell; HCT116, colon cancer cell; MDA-
MB-435, breast cancer cell.
4a
4h
TPT
Fig. 4. Comparison of the inhibition ratio of compounds 4a, 4h, and TPT
cleavable complex. Therefore, topoisomerase I-mediated DNA
cleavage assays were used to investigate the ability of the
prepared compounds to inhibit Topo I. Cleavable complexes
were revealed by the appearance of short DNA fragments when
the samples were analyzed by gel electrophoresis under dena-
turing conditions. The presented results (Fig. 5) indicated that all
compounds as cytotoxic agents were also very potent as Topo I
inhibitors. In particular, compounds 4a, 4d, 4h, and 4i showed
excellent enzyme inhibitory activity. Furthermore, most com-
pounds indicated a good correlation between the inhibitory
against cancer cell line HCT116.
activity and cytotoxic activity. For example, compounds 4a,
4c, 4d, 4e, 4h, and 4i with good antitumour activity also
displayed superior Topo I inhibitory activity. Furthermore,
the addition of increasing concentrations of CPT, 4a, or 4h
is accompanied by a dose-dependent increase in the level of
cleavable complexes. Importantly, even in low concentration

1394
L.-J. Zhu et al.
DNA Topo I CPT 4j
4h
4g
4c
4a
4b
4e
4d
4f
4i
Fig. 5. Effect of the selected compounds on Topoisomerase I-mediated DNA relaxation in single concentra-
tion. Topo I was Topoisomerase I and DNA without drugs. The samples were reacted with 100 mM drugs at
378C for 15 min. Reactions were then ceased by adding 0.5 % SDS. Gels were photographed under a UV
transilluminator.
DNA Topo I
CPT
4a
4h
Fig. 6. Effect of compounds camptothecin (CPT), 4a, and 4h on Topoisomerase I-mediated DNA relaxation
in concentration gradient. Topo I was Topoisomerase I and DNA without drugs; the samples were reacted with 1,
50, and 100 mM drugs at 378C for 15 min. Reactions were then ceased by adding 0.5 % sodium dodecyl sulfate.
Gels were photographed under a UV transilluminator.
(1 mM), 4a and 4h showed obvious Topo I inhibition activity,
while CPT exhibited no inhibition activity (Fig. 6). Based on the
binding mode (Fig. 3), there is a large cavity that can accommo-
date relatively large groups (such as DHP). The main interaction
between DHP and Topo I was van der Waals’ force, which could
facilitate the combination between hCPT and Topo I. Therefore,
these compounds showed good Topo I inhibitory activity.
until TLC showed complete disappearance of aldehyde. The
reaction mixture was poured into cold water (25 mL) and
extracted using EtOAc. The organic layer was separated, dried
over anhydrous Na2SO4, filtered, and evaporated to dryness
under reduced pressure. The residue was purified by flash
chromatography (EtOAc/petroleum ether 1:8) on silica gel to
yield target compounds 2 as yellow solids.
Typical Experimental Procedure for the Synthesis of 3a–j
Conclusions
Compound 2 (0.5 g) was dissolved in ethanol (15 mL), then
dry 10 % palladium on activated carbon (20 mg) was carefully
added to the solution and the dissolved oxygen was removed
under vacuum. The mixture was then stirred vigorously for 12 h
under a hydrogen atmosphere. The catalyst was removed by
filtration of the reaction mixture through a pad of Celite. The
filtrate was concentrated under reduced pressure to give crude
compounds 3, which were taken onto the next reaction without
further purification.
In summary, a series of novel hCPT-DHP derivate conjugates
have been synthesized based on our synthetic route. The results
presented in this study clearly indicate that hCPT-DHP
conjugates as effective cytotoxic agents also showed superior
Topo I inhibition activity. It is likely that the improvement of
cytotoxic activity is the result of multiple favourable events,
including DHP’s biological diversity and increased drug uptake
(as expected on the basis of increased lipophilicity). A better
understanding of this feature could provide meaningful insights
for the development of more effective topoisomerase I
inhibitors.
General Procedure for the Synthesis of 4a–j
A mixture of 3 (100 mg) and 7-formylhomocamptothecin
˚
(100 mg) was suspended in anhydrous CHCl3 containing 4 A
Experimental
molecular sieves. After 10 min, Yb(OTf)3 (10 mg) was added to
the solution. The resulting mixture was stirred at room tem-
perature until the reaction was complete. After removal of the
sieves via filtration, the solvent was evaporated to dryness and
the crude material purified by flash chromatography (CH2Cl2/
MeOH 99:1) on silica gel to yield target compound 4.
General
All reagents and solvents were reagent grade or were purified
by standard methods before use. The melting points were deter-
mined using an electrothermal apparatus and are uncorrected.
1H and 13C NMR spectra were recorded at 500 MHz with a
Bruker instrument, and reported with TMS as the internal
standard and CDCl3 or DMSO-d6 as solvent. Chemical shifts
(d) and coupling constants (J) are given in ppm and Hz,
respectively. Elemental analyses were performed with a MOD-
1106 instrument. TLC analysis was carried out on silica gel
plates GF254 (Qindao Haiyang Chemical, China). Flash column
chromatography was carried out on silica gel 200–300 mesh.
Anhydrous solvents and reagents were all analytically pure and
dried using routine protocols. All reactions requiring anhydrous
conditions were performed under a positive nitrogen flow, and
all glassware was oven-dried and/or flame-dried.
4a: Yellow power; yield 72 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.88 (t, J 7.0, 3H, CH3CH2), 1.18 (t, J 7.0, 3H,
CH3CH2O), 1.88 (q, J 7.7, 2H, CH3CH2), 2.29 (s, 6H,
C¼C-CH3), 3.06–3.48 (q, J 13.8, 2H, CH2CO), 4.03 (q, J 7.0,
4H, CH3CH2O), 4.94 (s, 1H, NHCH), 5.39–5.53 (q, J 15.0, 2H,
OCH2), 5.55 (s, 2H, NCH2), 6.04 (s, 1H, OH), 7.29–7.44 (dd,
J 8.2, 4H, Ar-H), 7.42 (s, 1H, C14-H), 7.80 (t, J 7.4, 1H, C10-H),
7.93 (t, J 7.8, 1H, C11-H), 8.25 (d, J 8.4, 1H, C12-H), 8.87 (s, 1H,
NH), 8.96 (d, J 8.6, 1H, C9-H), 9.65 (s, 1H, N¼CH). dC
(500 MHz, DMSO-d6) 8.56, 14.63, 18.62, 36.36, 39.15, 42.25,
52.91, 59.45, 61.70, 73.49, 99.86, 102.16, 121.79, 123.06,
125.11, 126.78, 128.67, 128.84, 130.15, 130.74, 134.89,
144.42, 145.85, 148.20, 149.05, 149.49, 153.50, 156.10,
159.37, 167.31, 172.16. m/z (ESI) 715 (M-H). Anal. Calc. for
C41H40N4O8: C 68.70, H 5.62, N 7.82. Found: C 68.54, H 5.64,
N 7.78 %.
Typical Experimental Procedure for the Synthesis of 2a–j
A solution of aldehyde 1 (5 mmol) and 1,3-dione (10 mmol)
were dissolved in ethanol (20 mL). Concentrated aqueous
ammonia (10 mmol) was added and the mixture was refluxed

Homocamptothecin-dihydropyridine Conjugates
1395
4b: Yellow power; yield 69 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.88 (t, J 7.0, 3H, CH3CH2), 1.87 (q, J 7.7, 2H,
CH3CH2), 2.29 (s, 6H, C¼C-CH3), 3.06–3.48 (q, J 13.8, 2H,
CH2CO), 3.58 (s, 6H, CH3O), 4.96 (s, 1H, NHCH), 5.39–5.54 (q,
J 15.0, 2H, OCH2), 5.55 (s, 2H, NCH2), 6.03 (s, 1H, OH), 7.28–
7.42 (dd, J 8.2, 4H, Ar-H), 7.44 (s, 1H, C14-H), 7.81 (t, J 7.4, 1H,
C10-H), 7.94 (t, J 7.8, 1H, C11-H), 8.25 (d, J 8.4, 1H, C12-H), 8.93
(s, 1H, NH), 8.96 (d, J 8.6, 1H, C9-H), 9.65 (s, 1H, N¼CH). dC
(500 MHz, DMSO-d6) 8.54, 18.62, 36.36, 39.16, 42.27, 54.45,
59.44, 61.77, 73.49, 99.88, 102.16, 121.79, 123.06, 125.12,
126.74, 128.67, 128.83, 130.15, 130.76, 134.89, 144.45,
145.86, 148.25, 149.07, 149.44, 153.50, 156.10, 159.37,
167.31, 172.17. m/z (ESI) 687 (M-H). Anal. Calc. for
C39H36N4O8: C 68.01, H 5.27, N 8.13. Found: C 68.15, H 5.26,
N 8.15 %.
4c: Yellow power; yield 77 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.88 (t, J 7.0, 3H, CH3CH2), 1.38 (s, 18H, C(CH3)3),
1.87 (q, J 7.7, 2H, CH3CH2), 2.25 (s, 6H, C¼C-CH3), 3.06–3.48
(q, J 13.8, 2H, CH2CO), 3.58 (s, 6H, CH3O), 4.87 (s, 1H,
NHCH), 5.40–5.56 (q, J 15.0, 2H, OCH2), 5.56 (s, 2H,
NCH2), 6.04 (s, 1H, OH), 7.29–7.48 (dd, 4H, J 8.2, 4H, Ar-H),
7.45 (s, 1H, C14-H), 7.81 (t, J 7.4, 1H, C10-H), 7.94 (t, J 7.8, 1H,
C11-H), 8.25 (d, J 8.4, 1H, C12-H), 8.66 (s, 1H, NH), 8.98 (d,
J 8.6, 1H, C9-H), 9.68 (s, 1H, N¼CH). dC (500 MHz, DMSO-d6)
8.57, 18.61, 30.11, 36.37, 39.14, 42.25, 59.44, 61.71, 73.48,
82.09, 99.86, 102.16, 121.79, 123.06, 125.11, 127.74, 128.67,
128.84, 130.15, 130.77, 134.89, 144.42, 145.86, 148.22, 149.15,
149.49, 153.50, 156.10, 159.41, 167.31, 172.22. m/z (ESI) 771
(M-H). Anal. Calc. for C45H48N4O8: C 69.93, H 6.26, N 7.25.
Found: C 69.87, H 6.24, N 7.23 %.
4d: Yellow power; yield 48 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.88 (t, J 7.0, 3H, CH3CH2), 0.91 (d, J 16.5, 12H, CH
(CH3)2), 1.87 (q, J 7.7, 2H, CH3CH2), 1.89 (m, 2H, CH(CH3)2),
2.31 (s, 6H, C¼C-CH3), 3.06–3.48 (q, J 13.8, 2H, CH2CO), 3.79
(m, 4H, OCH2CH(CH3)2), 5.03 (s, 1H, NHCH), 5.39–5.54 (q,
J 15.0, 2H, OCH2), 5.57 (s, 2H, NCH2), 6.04 (s, 1H, OH), 7.30–
7.44 (dd, J 8.2, 4H, Ar-H), 7.44 (s, 1H, C14-H), 7.81 (t, J 7.4, 1H,
C10-H), 7.93 (t, J 7.8, 1H, C11-H), 8.25 (d, J 8.4, 1H, C12-H), 8.95
(s, 1H, NH), 8.96 (d, J 8.6, 1H, C9-H), 9.65 (s, 1H, N¼CH). dH
(500 MHz, DMSO-d6) 8.56, 18.62, 19.45, 27.49, 36.25, 38.83,
42.69, 52.90, 61.71, 69.71, 73.49, 99.87, 102.08, 121.91,
123.06, 125.14, 126.79, 128.43, 128.72, 128.85, 130.74,
131.25, 134.09, 144.41, 146.18, 148.15, 149.09, 149.49,
153.50, 156.09, 159.37, 167.28, 172.17. m/z (ESI) 771 (M-H).
Anal. Calc. for C45H48N4O8: C 69.93, H 6.26, N 7.25. Found:
C 69.88, H 6.25, N 7.27 %.
4e: Yellow power; yield 68 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.88 (t, J 7.0, 3H, CH3CH2), 0.91 (d, J 16.5, 6H,
CH2CH2CH3), 1.60 (m, 4H, CH2CH2CH3), 1.87 (q, J 7.4, 2H,
CH3CH2), 2.30 (s, 6H, C¼C-CH3), 3.06–3.93 (q, J 13.7, 2H,
CH2CO), 3.94 (t, 4H, J 6.4, 4H, CH2CH2CH3), 4.98 (s, 1H, CH),
5.39–5.54 (q, J 15.0, 2H, OCH2), 5.55 (s, 2H, NCH2), 6.03
(s, 1H, OH), 7.29–7.44 (dd, J 8.2, 4H, Ar-H), 7.44 (s, 1H,
C14-H), 7.81 (t, J 7.4, 1H, C10-H), 7.94 (t, J 7.8, 1H, C11-H), 8.25
(d, J 8.4, 1H, C12-H), 8.90 (s, 1H, NH), 8.97 (d, J 8.6, 1H, C9-H),
9.66 (s, 1H, N¼CH). dC (500 MHz, DMSO-d6) 8.56, 11.22,
18.62, 23.69, 36.36, 39.15, 42.25, 59.45, 61.70, 67.25, 73.49,
99.89, 102.16, 121.80, 123.06, 125.11, 126.78, 128.66, 128.89,
130.16, 130.74, 134.89, 144.42, 145.86, 148.20, 149.05, 149.44,
153.50, 156.10, 159.35, 167.28, 172.19. m/z (ESI) 743 (M-H).
Anal. Calc. for C43H44N4O8: C 69.34, H 5.95, N 7.52. Found:
C 69.20, H 5.94, N 7.55 %.
4f: Yellow power; yield 56 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.87 (t, J 7.0, 3H, CH3CH2), 1.88 (q, J 7.7, 2H,
CH3CH2), 2.28 (s, 6H, C¼C-CH3), 3.06–3.48 (q, J 13.8, 2H,
CH2CO), 4.50 (d, J 4.5, 4H, CH2CH¼CH2), 4.82–4.87 (m, 2H,
CH2CH¼CH2), 4.91 (s, 1H, CH), 5.15 (m, 4H, CH2CH¼CH2),
5.39–5.55 (q, J 15.0, 2H, OCH2), 5.75 (s, 2H, NCH2), 6.03 (s,
1H, OH), 7.29–7.44 (dd, J 8.2, 4H, Ar-H), 7.44 (s, 1H, C14-H),
7.81 (t, J 7.4, 1H, C10-H), 7.94 (t, J 7.8, 1H, C11-H), 8.25 (d,
J 8.4, 1H, C12-H), 8.80 (s, 1H, NH), 8.97 (d, J 8.6, 1H, C9-H),
9.66 (s, 1H, N¼CH). dC (500 MHz, DMSO-d6) 8.56, 18.62,
36.36, 39.15, 42.25, 59.45, 61.70, 66.89, 73.49, 99.86, 102.16,
118.29, 121.79, 123.06, 125.11, 126.78, 128.67, 128.84, 130.15,
130.74, 132.44, 134.89, 144.43, 145.85, 148.20, 149.06, 149.51,
153.50, 156.10, 159.37, 167.31, 172.16. m/z (ESI) 739 (M-H).
Anal. Calc. for C43H40N4O8: C 69.72, H 5.44, N 7.56. Found:
C 69.61, H 5.42, N 7.59 %.
4g: Yellow power; yield 86 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.86 (t, J 7.0, 3H, CH3CH2), 1.24 (s, 6H, OCH3),
1.87 (q, J 7.5, 2H, CH3CH2), 2.49 (s, 6H, C¼C-CH3), 3.06–3.50
(q, J 13.7, 2H, CH2CO), 3.54 (t, J 4.7, 4H, CH2CH2O), 4.10 (t,
J 4.7, 4H, CH2CH2O), 4.96 (s, 1H, CH), 5.39–5.54 (q, J 15.0,
2H, OCH2), 5.55 (s, 2H, NCH2), 6.03 (s, 1H, OH), 7.32–7.42
(dd, J 8.2, 4H, Ar-H), 7.44 (s, 1H, C14-H), 7.81 (t, J 7.4, 1H,
C10-H), 7.94 (t, J 7.8, 1H, C11-H), 8.25 (d, J 8.4, 1H, C12-H), 8.93
(s, 1H, NH), 8.96 (d, J 8.6, 1H, C9-H), 9.65 (s, 1H, N¼CH). dC
(500 MHz, DMSO-d6) 8.56, 18.62, 36.36, 39.15, 42.25, 59.05,
59.45, 61.70, 64.78, 68.65, 73.49, 99.87, 102.16, 121.79,
123.06, 125.12, 126.78, 128.59, 128.85, 130.15, 130.74,
134.90, 144.42, 145.87, 148.20, 149.05, 149.49, 153.50,
156.11, 159.35, 167.31, 172.18. m/z (ESI) 775 (M-H). Anal.
Calc. for C43H44N4O8: C 66.48, H 5.71, N 7.21. Found: C 66.34,
H 5.70, N 7.24 %.
4h: Yellow power; yield 45 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.88 (t, J 7.5, 3H, CH3CH2), 1.87 (q, J 7.7, 2H,
CH3CH2), 2.30 (d, J 1.6, 6H, C¼C-CH3), 3.06–3.49 (q, J 13.9,
2H, CH2CO), 3.61 (s, 6H, CH3O), 5.03 (s, 1H, CH), 5.40–5.57
(q, J 14.8, 2H, OCH2), 5.57 (s, 2H, NCH2), 6.03 (s, 1H, OH),
7.15 (d, J 6.9, 1H, Ar-H), 7.25 (s, 1H, Ar-H), 7.37–7.39 (m, 2H,
Ar-H), 7.45 (s, 1H, C14-H), 7.83 (t, J 6.9, 1H, C10-H), 7.95 (t,
J 7.3, 1H, C11-H), 8.25 (d, J 8.6, 1H, C12-H), 8.94 (d, J 8.6, 1H,
C9-H), 8.96 (s, 1H, NH), 9.62 (s, 1H, N¼CH). dC (500 MHz,
DMSO-d6) 8.57, 18.62, 36.36, 39.15, 42.25, 55.16, 59.45, 61.70,
73.49, 99.86, 102.16, 119.89, 121.79, 123.06, 123.48, 125.11,
126.78, 128.67, 128.84, 130.15, 130.74, 134.89, 144.42, 145.85,
148.20, 149.05, 149.49, 153.50, 156.10, 159.37, 167.31, 172.16.
m/z (ESI) 687 (M-H). Anal. Calc. for C39H36N4O8: C 68.01,
H 5.27, N 8.13. Found: C 68.05, H 5.29, N 8.16 %.
4i: Yellow power; yield 42 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.88 (t, J 7.5, 3H, CH3CH2), 0.91 (d, J 16.5, 12H, CH
(CH3)2), 1.84–1.91 (m, 4H, CH3CH2, CH(CH3)2), 2.32 (s, 6H,
C¼C-CH3), 3.08–3.49 (q, J 13.8, 2H, CH2CO), 3.81 (d, J 6.2,
4H, OCH2CH(CH3)2), 5.07 (s, 1H, CH), 5.41–5.57 (q, J 15.5,
2H, OCH2), 5.49 (s, 2H, NCH2), 6.05 (s, 1H, OH), 7.19 (d, J 7.4,
1H, Ar-H), 7.29 (s, 1H, Ar-H), 7.34–7.40 (m, 2H, Ar-H), 7.44 (s,
1H, C14-H), 7.79 (t, J 7.41, 1H, C10-H), 7.92 (t, J 7.3, 1H, C11-H),
8.23 (d, J 8.4, 1H, C12-H), 8.87 (d, J 8.5, 1H, C9-H), 8.96 (s, 1H,
NH), 9.56 (s, 1H, N¼CH). dC (500 MHz, DMSO-d6) 8.57,
18.63, 19.43, 27.80, 36.23, 39.13, 42.56, 52.80, 61.72, 69.75,
73.50, 99.88, 102.04, 117.83, 122.73, 123.12, 124.98, 125.87,
126.45, 128.75, 128.95, 129.58, 130.14, 130.73, 134.58, 144.38,
146.32, 149.47, 149.74, 150.79, 154.04, 156.10, 156.29, 159.35,
167.30, 172.17. m/z (ESI) 771 (M-H). Anal. Calc. for

1396
L.-J. Zhu et al.
C45H48N4O8: C 69.93, H 6.26, N 7.25. Found: C 69.87, H 6.28,
N 7.27 %.
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4j: Yellow power; yield 35 %; mp .3008C. dH (500 MHz,
DMSO-d6) 0.89 (t, J 7.4, 3H, CH3CH2), 1.19 (t, J 7.0, 3H,
CH3CH2O), 1.88 (q, J 7.5, 2H, CH3CH2), 2.29 (s, 6H, C¼C-CH3),
3.07–3.48 (q, J 13.8, 2H, CH2CO), 4.03 (m, 4H, CH3CH2O),
4.99 (s, 1H, CH), 5.40–5.55 (q, J 15.0, 2H, OCH2), 5.56 (s, 2H,
NCH2), 6.03 (s, 1H, OH), 7.18 (d, J 7.1, 1H, Ar-H), 7.28 (s, 1H,
Ar-H), 7.36–7.39 (m, 2H, Ar-H), 7.45 (s, 1H, C14-H), 7.82 (t,
J 7.3, 1H, C10-H), 7.93 (t, J 7.6, 1H, C11-H), 8.26 (d, J 8.4, 1H,
C12-H), 8.86 (s, 1H, NH), 8.93 (d, J 8.4, 1H, C9-H), 9.61 (s, 1H,
N¼CH). dC (500 MHz, DMSO-d6) 8.55, 14.59, 18.62, 36.36,
39.15, 42.25, 52.95, 59.45, 61.71, 73.49, 99.88, 102.16, 119.56,
121.81, 123.06, 124.54, 125.11, 126.78, 128.67, 128.84, 130.15,
130.75, 134.89, 144.43, 145.86, 148.20, 149.09, 149.49, 153.51,
156.10, 159.37, 167.32, 172.15. m/z (ESI) 715 (M-H). Anal.
Calc. for C41H40N4O8: C 68.70, H 5.62, N 7.82. Found: C 68.54,
H 5.63, N 7.80 %.
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Cytotoxicity
A total of 1200 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 control.
After 3 days of incubation, 20 mL MTT (3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyl- tetrazolium bromide) solution (5 mg mLꢀ1
)
was added to each well, and after shaking for 1 min the plate was
incubated further for 4 h. Formazan crystals were dissolved with
100 mL DMSO. The absorbance (OD) was quantitated using a
microplate spectrophotometer at 570 nm. Wells containing no
drugs were used as blanks for the spectrophotometer. The sur-
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control wells.
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Topoisomerase I Inhibition Activity Assays
CPT was obtained from the company of Tianzunzezhong in
China. Topo I (calf thymus), buffer, bovine serum albumen
(BSA), loading buffer and supercoiled DNA pBR322 were all
sourced from TaKaRa Biotechnology CO., Ltd.
All reactions were carried out in 20 mL volumes (16 mL
double distilled water, 2 mL DNA Topo I buffer, 2 mL 0.1 %
BSA) including 0.25 mg supercoiled DNA, 0.5 U Topo I with or
without drug. The reaction was incubated at 378C for 15 min.
Reactions were ceased by adding sodium dodecyl sulfate (0.5 %
final concentration). To the reaction mixtures, 3.5 mL 6 ꢂ load-
ing buffer (0.1 mM EDTA, 7 % Glycerol, 0.01 % Xylene Cyanol
FF, Bromopenol Blue 0.01 %) was added. The mixtures were
electrophoresed in 0.8 % agarose gel in Tris base, acetic acid,
and EDTA buffer for 40 min at 120 V. The gel was stained with
ethidium bromide at room temperature and photographed with a
UV transilluminator.
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Acknowledgement
152. doi:10.1016/S0165-1838(00)00128-4
This research was supported in part by the Key Project of 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).
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