Synthesis and biological evaluation of N-(carbobenzyloxy)-L-phenylalanine and N-(carbobenzyloxy)-L-aspartic acid-β-benzyl ester derivatives as potent topoisomerase IIα inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.03.065

A new series of thirteen N-(carbobenzyloxy)-L-phenylalanine and N-(carbobenzyloxy)-L-aspartic acid-β-benzyl ester compounds were synthesized and evaluated for antiproliferative activity against four different human cancer cell lines: cervical cancer (HeLa

Full text of DOI:10.1016/j.bmc.2017.03.065

Accepted Manuscript
Synthesis and biological evaluation of N-(carbobenzyloxy)-l-phenylalanine and
N-(carbobenzyloxy)-l-aspartic acid-β-benzyl ester derivatives as potent topoi-
somerase IIα inhibitors
Xiaoyan Han, Yifan Zhong, Guan Zhou, Hui Qi, Shengbin Li, Qiang Ding,
Zhenming Liu, Yali Song, Xiaoqiang Qiao
PII:
S0968-0896(17)30012-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.065
BMC 13663
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Accepted Date:
4 January 2017
28 March 2017
Please cite this article as: Han, X., Zhong, Y., Zhou, G., Qi, H., Li, S., Ding, Q., Liu, Z., Song, Y., Qiao, X., Synthesis
and biological evaluation of N-(carbobenzyloxy)-l-phenylalanine and N-(carbobenzyloxy)-l-aspartic acid-β-benzyl
ester derivatives as potent topoisomerase IIα inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.03.065
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Bioorganic & Medicinal Chemistry
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Synthesis and biological evaluation of N-(carbobenzyloxy)-l-phenylalanine and
N-(carbobenzyloxy)-l-aspartic acid-β-benzyl ester derivatives as potent
topoisomerase IIα inhibitors
Xiaoyan Hana, Yifan Zhonga, Guan Zhoua, Hui Qia, Shengbin Lia, Qiang Dinga, Zhenming Liuc, Yali Songa,
*, Xiaoqiang Qiaoa, b, †
aKey Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding, 071002, China
bKey Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Chemistry and Environmental Science, Hebei University,
Baoding, 071002, China
cDrug Design Center, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
ARTICLE INFO
ABSTRACT
Article history:
Received
A new series of thirteen N-(carbobenzyloxy)-l-phenylalanine and N-(carbobenzyloxy)-l-aspartic
acid-β-benzyl ester compounds were synthesized and evaluated for antiproliferative activity
against four different human cancer cell lines: cervical cancer (HeLa), lung cancer (A549),
gastric cancer (MGC-803) and breast cancer (MCF-7) as well as topoisomerase I and IIα
inhibitory activity. Compounds (5a, 5b, 5e, 8a, 8b) showed significant antiproliferative activity
with low IC50 values against the four cancer cell lines. Equally, compounds 5a, 5b, 5e, 5f, 8a,
8d, 8e and 8f showed topoisomerase IIα inhibitory activity at 100 µM with 5b, 5e, 8f exhibiting
potential topoisomerase IIα inhibitory activity compared to positive control at 100 µM and 20
µM, respectively. Conversely compounds 5e, 5f, 5g and 8a showed weaker topoisomerase I
inhibitory activity compared to positive control at 100 µM. Compound 5b exhibited the most
potent topoisomerase IIα inhibitory activity at low concentration and better antiproliferative
activity against the four human cancer cell lines. The molecular interactions between compounds
5a-5g, 8a-8f and the topoisomerase IIα (PDB ID: 1ZXM) were further investigated through
molecular docking. The results indicated that these compounds could serve as promising leads
for further optimization as novel antitumor agents.
Received in revised form
Accepted
Available online
Keywords:
N-(carbobenzyloxy) derivatives
Anticancer activity
Topoisomerase IIα inhibitor
Molecular docking
2017 Elsevier Ltd. All rights reserved.
1. Introduction
DNA topoisomerases have an important role in cell
proliferation and differentiation.6 These are nuclear enzymes that
Cancer represents a group of more than hundred diseases
characterized by uncontrolled proliferation of the cells.1 It is one
of the leading diseases claiming numerous lives and consequently
responsible for high mortality rates across the globe.2 Although a
number of anticancer drugs are known, most of them have failed
to yield the desired result. In most cases, the reason for such
failure can be attributed to the lack of selectivity, less
efficaciousness, more side effects and multi-drug resistance
(MDR).3, 4 Hence, there is an increasing need to identify new and
novel highly potent anticancer drugs with alternative modes of
action. Human DNA topoisomerases (topo) have been
established as one of the most important molecular targets for the
development of anticancer agents.5
transiently break one or two strands of DNA allowing to solve
various DNA topological problems associated with DNA
replication, transcription, recombination, and other vital cellular
processes.7, 8 DNA topoisomerases are generally classified into
topoisomerase I (topo I) and topoisomerase II (topo II) depending
on their mechanism of action, making either single- or double-
stranded breaks, respectively.9 Moreover, human topoisomerase
II is further categorized into two subclasses: topoisomerase IIα
and topoisomerase IIβ. Unlike topoisomerase IIβ, topoisomerase
IIα is a cell cycle dependent enzyme which is overexpressed in
proliferating cells.10 Therefore, topoisomerase IIα has been
considered as more attractive target than topoisomerase IIβ for
the development of chemotherapeutic agents.11
———
*Corresponding author. Tel/Fax: 13483283338, E-mail: yalisong@hbu.edu.cn (Yali Song)
†Corresponding author. Tel/Fax: 15903120430, E-mail: xiaoqiao@hbu.edu.cn (Xiaoqiang Qiao)

Topoisomerase II inhibitors act in two different pathways: (1)
the distinct character of topoisomerase II poison is to bind and
stabilize transiently and covalently formed DNA-enzyme
complex and (2) topoisomerase II catalytic inhibitor does not
stabilize the DNA-enzyme complexes but eliminates essential
catalytic activities of topoisomerase II.11 During the last few
years, many researchers in medicinal chemistry seek to develop
selective topoisomerase IIα inhibitors as anticancer agents.12, 13
Pogorelčnik14 et al. reported that 4,6-substituted-1,3,5-triazin-
2(1H)-ones were novel topoisomerase IIα catalytic inhibitors that
bind to the topoisomerase IIα ATP binding site. Jha15 et al.
investigated that 3,5-bis(arylmethylene)-1-(N-(ortho-substituted
aryl)maleamoyl)-4-piperidones exhibited strong inhibitory
activity towards topoisomerase IIα in vitro. Qiu16 et al. prepared a
series of novel terphenyls and evaluated their biological activity
in vitro. It was found that some compounds had the most potent
anticancer activity and strong inhibitory effect on topoisomerase
IIα, but not on topoisomerase I, which was consistent with the
docking analysis results. Furthermore, it is unmet to design novel
topoisomerase IIα inhibitors with high selectivity, ideally
targeting topoisomerase IIα.17
lead compound. Guided by the binding model of compound
PKU10001674 with ATPase domain of topoisomerase IIα, the
ester group and the phenyl group were introduced in the target
compounds because they can accommodate in tight and deep
cavities, as well as providing in hydrophobic pockets of the
biological targets which contribute to improve the potency,
stability and specificity of the binding site. The results promoted
us to design and synthesize the target compounds which
contained the above two kinds of hydrophobic group in the
skeleton and side chain. First, N-(carbobenzyloxy)-l-
phenylalanine and N-(carbobenzyloxy)-l-aspartic acid-β-benzyl
ester were chosen as starting material. Second, starting material
connected with 2-amino-3-phenylpropan-1-ol which was
regarded as a linker. Third, alcoholic hydroxyl group was
modified with substituted carboxylic acid to find out whether
these groups were necessary for the antiproliferative activity and
topoisomerase I & IIα inhibitory activity (Figure 1).
2.2. Chemistry
N-(carbobenzyloxy)-l-phenylalanine and N-(carbobenzyloxy)-
l-aspartic acid-β-benzyl ester derivatives (5a-5g, 8a-8f) were
synthesized in three steps as illustrated in Scheme 1. At first, l-
phenylalanine (1.0 equiv), sodium borohydride (NaBH4) (4.0
equiv) and anhydrous aluminum chloride (AlCl3) (2.5 equiv) in
an anhydrous tetrahydrofuran (THF) solution were heated at
reflux for 12 h to obtain compound 2 in 86% yield. Secondly,
intermediate 4 or 7 was prepared by condensation of compound 2
(1.2 equiv) with compound 3 or 6 (commercial source) (1.0
equiv) in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU) (1.1 equiv), 4-
The identification of a proper lead compound for a given
molecular target is a critical step in the process of drug discovery.
Virtual screening is bringing a more cost-effective approach to
drug discovery by using high-performance computing to analyze
chemical databases and prioritize compounds for synthesis and
assay.18 In continuation of our earlier attempts toward the
discovery of novel topoisomerase inhibitors,19 we described the
rapid and successful identification of novel topoisomerase IIα
inhibitors using virtual screening in this work. The glide program
was applied to screen the PKU-CNCL database with
topoisomerase IIα (ID: 1ZXM) as the receptor for docking. By
using Glide-SP docking, the top 5% of the ranked compounds
were considered for Glide-XP docking, and the top 5% of the
ranked compounds were selected in the end. After observing how
these compounds interact with the protein, we screened 20
compounds for topoisomerase IIα inhibitory activity testing. The
results showed that compound PKU10001674 (Figure 1)
exhibited the highest inhibitory activity (IC50=26.95 µM). Thus,
this compound was structurally modified and engineered as a
lead compound. By analyzing the binding pocket of
topoisomerase IIα, compounds 5a-5g, 8a-8f were designed and
synthesized based on the structure of PKU10001674. Their
antiproliferative activity against four human cancer cell lines,
topoisomerase I & IIα inhibitory activity were evaluated by
assessing MTT assay and the relaxation of supercoiled pBR 322
plasmid DNA.
dimethylaminopyridine
diisopropylethylamine (DIPEA) (2.0 equiv) and anhydrous
dichloromethane (DCM) for at room temperature.
(DMAP)
(0.2
equiv),
N,N-
7
h
Intermediates 4 and 7 were obtained in the yield of 75%-78%.
Finally, intermediate 4 or 7 (1.0 equiv) and various acid (1.5
equiv)
dicyclohexylcarbodiimide
dimethylaminopyridine
were reacted in the presence of N,N'-
(DCC)
(0.2 equiv)
(1.1
equiv),
4-
anhydrous
and
dichloromethane for 4 h at reflux to give final compounds 5
(R=a-g) or 8 (R=a-f) in 65%-86% yield. After completion of the
reaction as indicated by TLC, the reaction mixture was purified
by column chromatography. (Scheme 1)
OH
COOH
NH2
NH2
(i)
2
1
O
O
O
RCOOH
(iii)
O
O
O
2
B
O
O
O
H
N
H
N
O
O
(ii)
OH
N
N
O
N
O
O
H
H
H
OH
O
O
O
O
OH
O
O
O
R
H
N
O
4
5
(R=a-g)
3
OH
O
N
H
O
N
H
C
A
A
O
O
O
RCOOH
(iii)
2
O
O
O
H
N
H
N
O
R
(ii)
OH
PKU10001674
5a-5g
O
N
H
N
H
O
O
N
H
O
O
O
OH
O
R'
O
7
6
8
(R=a-f)
Figure 1. Design of compounds 5a-5g.
Cl
CH3
2. Results and Discussion
CH3
CH2CH3
R =
Cl
OCH3
OCH3
e
2.1. Design and Development
g
a
c
b
d
f
By using virtual screening in this work, compound
PKU10001674 was structurally modified and engineered as a

Scheme 1. Synthetic route of compounds 5a-5g, 8a-8f. Reagents and
conditions: (i) NaBH4, AlCl3, THF, reflux, 12 h, 86% yield; (ii) TBTU,
DMAP, DIPEA, DCM, rt, 7 h, 75%-78% yield; (iii) DCC, DMAP, DCM,
reflux, 4 h-5 h, 65%-86% yield.
compounds may be potential candidates for new anticancer
agents.
Table 2
The structures of all compounds were shown in Table 1. All
the compounds were elucidated by 1H NMR, 13C NMR and
HRMS.
The IC50 values of compounds 5a-5g, 8a-8f against various human cancer
cells.
IC50 (µM)
Table 1
Compounds
Synthesis of compounds 5a-5g, 8a-8f.
HeLa
MGC-803
MCF-7
A549
5a
27.94 0.41
17.6 0.64
>50
32.53 1.47
28.14 0.71
>50
40 1.25
22.07 0.47
>50
46 1.26
29.92 1.07
>50
O
O
5b
O
O
H
N
H
N
O
N
O
N
H
5c
H
O
O
O
O
5d
44.7 1.22
39.64 1.85
49.92 0.80
>50
>50
41.69 1.65
27.87 0.83
40.56 0.92
47.06 2.05
41.06 1.39
36.2 2.65
43.04 0.75
39.25 1.06
41.54 3.01
>50
>50
O
R
O
R'
5 (R=a-g)
8 (R=a-f)
5e
42.3 0.55
>50
47.63 2.81
>50
No.
R
Time (h)
Yield (%)
69
m.p.(℃)
5f
5a
4.0
110-112
5g
>50
>50
5b
4.5
65
181-183
8a
30.17 0.84
22.64 1.34
>50
38.74 1.76
38.43 0.98
43.08 1.17
42.81 2.22
40.31 0.58
>50
48.77 0.74
42.44 1.53
>50
8b
5c
5d
5e
4.5
5.0
5.0
73
80
78
117-119
131-133
178-180
8c
8d
8e
39.38 0.68
34.47 2.05
>50
>50
>50
5f
4.0
4.0
82
79
109-110
83-85
8f
>50
5g
Etoposide
15.93 0.56
20.91 0.58
24.01 1.86
19.83 0.80
8a
8b
4.0
4.5
76
70
178-179
183-184
Camptothecin
10.1 0.62
9.45 1.38
28.66 1.17
13.64 0.39
Each value represents the Mean S.D. from three different experiments,
performed in triplicate. Camptothecin and etoposide were used as positive
controls.
8c
4.5
5.0
79
85
146-148
159-160
8d
8e
8f
5.0
4.0
83
86
170-172
143-144
2.4. Topoisomerase I and IIα inhibitory activity
The conversion of supercoiled plasmid DNA to relaxed DNA
by recombinant topoisomerase I and IIα in the presence of each
5a-5g and 8a-8f was evaluated as shown in Figure 2 and Table 3.
Well-known topoisomerase I and IIα inhibitors, camptothecin
and etoposide were used as positive controls. The reaction
products of topoisomerase I and IIα relaxation assays were
analyzed by electrophoretic mobility and developed in ethidium
bromide in the presence of UV light. The inhibitory activities
were evaluated at 100 µM for all compounds. Only those
compounds with moderate to significant activity were tested
further at 20 µM.
2.3. Growth inhibitory effect
The antitumor potency of target compounds against four
human cancer cell lines including HeLa (cervical cancer), A549
(lung cancer), MGC-803 (gastric cancer) and MCF-7 (breast
cancer) cells were evaluated by MTT assay. Camptothecin and
etoposide were used as positive controls. The results were shown
in Table 2. By comparing the IC50 values, compounds 5a, 5b, 5e,
8a and 8b showed good inhibitory activity for all the tested
human cancer cell lines. Especially, compound 5b exhibited the
highest and most uniform level of antiproliferative activity, with
an IC50 value<30 µM for all tested cell lines. Compound 5d
showed good inhibitory activity selectivity for HeLa as well as
MCF-7, and compounds 5f, 5g selectivity for MCF-7. The others
also showed good inhibitory except compounds 8d and 8e for
A549, 8c for HeLa and A549. Furthermore, compounds 5c and 8f
had relatively weak inhibitory activity in the four cancer cell
lines. The preliminary results are promising and some of these
2.4.1. Topoisomerase I inhibitory activity
As shown in Figure 2 and Table 3, most of the compounds
did not exhibit topoisomerase I inhibitory activity at 100 µM,
while compounds 5e, 5f, 5g, 8a showed weak topoisomerase I
inhibitory activity of 20.2%, 45.2%, 15.0%, 29.5%, respectively
(as compared to 58.2a, 79.8b of camptothecin) at 100 µM and did
not show topoisomerase I inhibitory activity at 20 µM.

2.4.2. Topoisomerase IIα inhibitory activity
Compounds
Topo I (% inhibition)
Topo IIα (% inhibition)
As shown in Figure 2 and Table 3, most of the compounds
(5a, 5b, 5e, 5f, 8a, 8d, 8e, 8f) showed significant topoisomerase
IIα inhibitory activity at 100 µM. Noteworthy, some compounds
such as 5b (92.9% and 51.2% at 100 and 20 µM, respectively),
5e (84.6% and 49.8% at 100 and 20 µM, respectively) and 8f
(86.1% and 45.6% at 100 and 20 µM, respectively) showed
higher topoisomerase IIα inhibitory activity than positive control
etoposide (82.6% and 34.7% at 100 µM and 20 µM, respectively)
at both concentrations. Moreover, compound 8e (83.2% as
compared to 78.0% of etoposide) showed better topoisomerase
IIα inhibition than etoposide at 100 µM and compound 8d
(42.1% as compared to 34.7% of etoposide) showed better
topoisomerase IIα inhibition than etoposide at 20 µM. In
addition, compounds 5a, 5f and 8a showed moderate
topoisomerase IIα inhibitory activity. Overall, compound 5b was
selected to further investigate the molecular interactions through
molecular docking due to its better antiproliferative activity
against tested cancer cell lines and topoisomerase IIα inhibitory
activity.
Concentration
Camptothecin
100 µM
20 µM
100 µM
20 µM
58.2a
79.8b
19.6
82.6a
78.0b
34.7
Etoposide
5a
5b
5c
5d
5e
5f
0.0
9.5
NA
NA
NA
NA
0.0
0.0
0.0
0.0
NA
NA
NA
NA
NA
68.4
92.9
41.4
28.2
84.6
63.8
6.7
20.1
51.2
NA
0.0
0.0
NA
20.2
45.2
15.0
29.5
0.0
49.8
19.7
NA
5g
8a
8b
8c
8d
8e
8f
Topoisomerase IIα inhibition
75.7
3.2
25.6
NA
0.0
4.1
NA
7.3
62.8
83.2
86.1
42.1
28.1
45.6
5.4
0.0
NA: not applicable.
a Value for compounds 5a-5g, 8a.
b Value for compounds 8b-8f.
Topoisomerase I inhibition
2.5. Molecular docking
2.5.1. The binding modes of compounds-topoisomerase IIα
We checked whether compounds 5a-5g, 8a-8f could bind to
the ATPase domain with molecular docking study using
coordinates (PDB ID: 1ZXM) previously reported for the co-
crystal structure of the adenosine 5’-(β,γ-imido) triphosphate
tetralithium salt hydrate (AMP-PNP) bound to the ATP-binding
domain of human topoisomerase II.13, 20 AMP-PNP is a structural
analog of ATP that has comparable binding affinity for the ATP-
binding pocket of topoisomerase II but is non-hydrolyzable
which enables to co-crystallize with topoisomerase II. AMP-PNP
was used as a reference ligand to identify the active site and to
validate our docking process by re-docking.
Lane D: pBR322 only,
Lane T: pBR322 + topoisomerase I or IIα,
Lane C: pBR322 + topoisomerase I + Camptothecin,
Lane E: pBR322 + topoisomerase IIα + Etoposide,
Lane 5a-5g: pBR322 + topoisomerase I or IIα + compounds 5a-5g,
Lane 8a-8f: pBR322 + topoisomerase I or IIα + compounds 8a-8f.
As shown in Figure 3A, the binding model of ANP with
ATPase domain of topoisomerase IIα revealed that the amino
group of adenine moiety showed hydrogen bonding with Asn120
and the two hydroxyl groups of ribose unit showed hydrogen
bond interaction with Ser149. The oxygen atoms in the three
phosphate groups displayed hydrogen bond interactions with the
nitrogen atom of the Walker A motif residues: Asn150, Asn163,
Tyr165 and Gly166. In addition, these phosphate groups showed
hydrogen bonding with Asn91, Ser148, Ala167, Lys168, and
Gln376. The highly conserved Walker A motif in the ATP
binding site of topoisomerase IIα includes the residues 161-166
and these are considered crucial for the binding of ATP and other
catalytic inhibitors acting on ATPase domain.21
Figure 2. Topoisomerase I and IIα inhibitory activity of compounds 5a-5g,
8a-8f.
Table 3
Recombinant topoisomerase I and IIα inhibitory activity of compounds 5a-5g,
8a-8f.

More insights revealed that compound 5b might bind at the
ATP site of topoisomerase IIα. The interaction model of the most
active compound 5b at the ATPase domain (Figure 3B) showed
that the carbonyl group near R formed two H-bonds with the
important residues Ser149 and Lys157. Further, ring-A and ring-
C (Figure 1) could show π-interactions with Phe142 and Arg98.
The carbamate group near ring-A has H-bonds with residues of
Asn91, Ser148 and Asn150. The interaction with Mg2+ ion has
been considered essential for the catalytic activity at the ATPase
binding domain.22 Additionally, the oxygen atom near ring-A
showed interaction with Mg2+ ion. Compared with compound 5b,
compound 8b showed weak inhibitory activity. As shown in
Figure 3C, the oxygen atom of compound 8b could only form
two H-bonds with residues of Ser149 and Asn150. Then ring-B
could show π-interaction with Arg98. Compound 8b showed
weak interactions with the key amino acid residues of the ATP
binding pocket. Thus, the molecular modeling studies suggest
that compound 5b may act as an inhibitor by occupying the ATP
binding pocket of topoisomerase IIα and showing favorable
interactions with the key amino acid residues.
2.5.2. The binding modes of compounds-topoisomerase I
In an attempt to explain the possible reason for selectivity to
topoisomerase IIα inhibition, Topotecan which was reported as
topoisomerase I inhibitor by Staker23 and compounds 5b were
docked into the crystal structure of topoisomerase I (PDB ID:
1K4T). The docking results were shown in Figure 4.
The overall conformation of Topotecan in the active site of
topoisomerase I was shown in Figure 4A. Topotecan was located
at the center of the active site, surrounded by some hydrophobic
residues, such as Asn722, Lys425 and Arg364. The phenyl and
pyrrolyl part of the Topotecan could form π-π interactions with
Da113, Tgp11. The 20(S)-hydroxyl group showed hydrogen
bonding with Asp533 and the 21-carboxylate oxygens would be
positioned for ionic interactions with Lys532.
The overall conformation of compound 5b in the active site of
topoisomerase I was shown in Figure 4B. Ring-A and ring-B
(Figure 1) could show π-π interactions with Trp416 and Lys425.
Further, the carbonyl group near R formed one H-bond with the
residue Asn722. Therefore, it could be found that the π-π
interactions and hydrogen bonding with residues of compound 5b
and Topotecan were completely different. No same interaction is
observed in compound 5b which may relate to the weaker
topoisomerase I inhibition. However, the molecular modeling
studies suggest that compound 5b may act as an inhibitor by
occupying the ATP binding pocket of topoisomerase IIα and
showing favorable interactions with the key amino acid residues.
Overall, by analyzing the binding pocket of topoisomerase IIα
and topoisomerase I, it was observed that all of the prepared
compounds exhibited weaker topoisomerase I inhibitory activity
indicating selectivity to topoisomerase IIα inhibition.
Figure 3. The predicted binding modes of ANP/5b/8b-topoisomerase IIα. (A)
Predicted binding mode of ANP-topoisomerase IIα. (B) Predicted binding
mode of 5b-topoisomerase IIα. (C) Predicted binding mode of 8b-
topoisomerase IIα.

potential topoisomerase IIα inhibitory activity and moderate
antiproliferative activity.
3. Conclusion
Figure 4. The predicted binding modes of Topotecan/5b-topoisomerase I. (A)
Predicted binding mode of Topotecan-topoisomerase I. (B) Predicted binding
mode of 5b-topoisomerase I.
In summary,
a series of novel N-(carbobenzyloxy)-l-
phenylalanine and N-(carbobenzyloxy)-l-aspartic acid-β-benzyl
ester derivatives were designed and synthesized as antitumor
agents. These compounds were evaluated for their
antiproliferative activity and topoisomerase I and IIα inhibitory
activity. Compounds 5b, 5e, 8f exhibited potential topoisomerase
IIα inhibitory activity compared to positive control at
concentration of 100 µM and 20 µM, respectively. In contrast, all
of the compounds did not exhibit moderate topoisomerase I
inhibitory activity, which indicated that these compounds were
potential and selective for topoisomerase IIα inhibition.
Compounds (5a, 5b, 5e, 8a, 8b) showed significant
antiproliferative activity at low IC50 values against the four
different human cancer cell lines. No direct correlation between
antiproliferative activity and topoisomerase inhibitory activity
was observed but compounds possessing significant antitumor
activity displayed moderate topoisomerase IIα inhibition.
Furthermore, the molecular modeling studies showed that
compound 5b may act as an inhibitor by occupying the ATP
binding pocket of topoisomerase IIα and showing favorable
interactions with the key amino acid residues. Therefore, on
overall comparison, compound 5b showed best antitumor activity
and topoisomerase IIα inhibitory activity, suggesting its potential
and viability as a good starting point for further optimization.
Importantly, this study may provide valuable information to
researchers working on the development of selective
topoisomerase IIα inhibitors, which will facilitate the
development of novel anticancer agents.
2.6. Structure activity relationships study
The structural activity relationships (SARs) was analyzed
based on the results of antiproliferative activity against tested
cancer cell lines, topoisomerase I and IIα inhibitory activity of
the evaluated compounds as well as the binding modes of
compounds with topoisomerase IIα and topoisomerase I. It was
observed that all of the prepared compounds exhibited weaker
topoisomerase I inhibitory activity indicating selectivity to
topoisomerase IIα inhibition. From the prepared compounds, we
were able to find a trend according to the different R in the
skeleton of compounds 5a-5g, 8a-8f. On the one hand, when
compounds contained N-(carbobenzyloxy)-l-aspartic acid-β-
benzyl ester, the order of topoisomerase IIα inhibitory activity
was 5b (R=4-chlorophenyl) >5e (R=3,4-dimethoxyphenyl) >5a
(R=phenyl) >5f (R=ethyl) >5c (R=2-chlorophenyl) >5d (R=2-
methylphenyl) >5g (R=methyl). The superior inhibitory activity
of 5a-5g can be explained by analyzing the binding modes of
compounds with ATPase domain of topoisomerase IIα. When R
was 4-chlorophenyl, 3,4-dimethoxyphenyl or phenyl in the
skeleton of compounds 5a-5g, the carbonyl group near R formed
one or two H-bonds with the important residues Asn120, Ser148,
Ser149, Asn150, respectively. Additionally, the oxygen atom
near R showed interaction with Mg2+ ion. However, when R was
2-chlorophenyl or 2-methylphenyl, no interaction is observed in
the carbonyl group near R. Therefore, to some extent, we could
predict that R=4-chlorophenyl, 3,4-dimethoxyphenyl or phenyl in
the skeleton of compounds 5a-5g were likely important for
significant topoisomerase IIα inhibitory activity.
4. Experimental
4.1. Chemistry
On the other hand, when compounds contained N-
(carbobenzyloxy)-l-phenylalanine, the order of topoisomerase IIα
inhibitory activity was 8f (R=ethyl) >8e (R=3,4-
dimethoxyphenyl) >8a (R=phenyl) >8d (R=2-methylphenyl) >8c
(R=2-chlorophenyl) >8b (R=4-chlorophenyl). The most active
compounds 8e, 8f showed several strong interactions with the
key amino acid residues Arg91, Ser149, Asn150, Lys168. Then,
the ring-C and carbonyl group near R showed interactions with
Mg2+ ion. Compounds 8b, 8c showed weak interactions with the
key amino acid residues of the ATP binding pocket. This
indicated that the more hydrogen bonding interactions in
compounds may relate to topoisomerase IIα inhibitory activity.
All the chemicals required for synthesized reactions were
obtained from Aladdin Chemical Co., Ltd of Shanghai, and all
other organic reagents were purchased from Kemiou Chemical
Reagent Co., Ltd of Tianjin. 1H NMR and 13C NMR were
measured on a Bruker AVANCE III 600 (600 MHz, Switzerland)
spectrometer using TMS as the internal standard. High
Resolution Mass Spectral (HRMS) data were determined on a
Thermo Scientific LTQ Orbitrap XL mass spectrometer. Melting
points were determined with a SGW X-4microscopic. The
melting point apparatus (Shanghai Precision
& Scientific
Instrument Co., Ltd, China) were in open capillaries and
uncorrected. Chemical reactions were followed by thin layer
chromatography with detection under ultraviolet light. The
reaction mixtures were separated by column chromatography
silica gel (200-300 mesh) eluted with a mixture of petroleum
ether and ethyl acetate.
In addition, from in vitro assays, we found that compounds
(5a, 5b, 5e, 8a, 8b, 8e) had moderate inhibitory activity for all
the tested cancer cell lines. Therefore, with some exceptions,
structure activity relationships study revealed that R=4-
chlorophenyl, 3,4-dimethoxyphenyl or phenyl in the skeleton of
compounds 5a-5g, 8a-8f were more favorable for topoisomerase
IIα inhibitory activity and antiproliferative activity with other
substituents. Moreover, in consideration of the preference from
the results of the docking study, antiproliferative activity and
topoisomerase IIα inhibitory activity, compound 5b was chosen
as a representative compound to determine whether it was ATP
competitive inhibitors. Taken together, the interaction model of
compounds 5a-5g, 8a-8f at the ATPase domain validated their
4.1.1. Synthesis of compound 2
l-Phenylalanine (1.0 equiv), sodium borohydride (4.0 equiv)
and anhydrous aluminum chloride (2.5 equiv) in an anhydrous
tetrahydrofuran solution were heated at reflux for 12 h to yield
compound 2. After completion of the reaction as indicated by
TLC, the reaction mixture was purified by column
chromatography.
4.1.2. Synthesis of intermediates 4 and 7

4.1.3.1.(S)-2-((S)-4-(Benzyloxy)-2-
(((benzyloxy)carbonyl)amino)-4-oxobutanamido)-3-
phenylpropyl benzoate (5a)
Compound 3 or 6 (1.0 equiv), compound 2 (1.2 equiv), 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(1.1 equiv), 4-dimethylaminopyridine (0.2 equiv), N,N-
diisopropylethylamine (2.0 equiv) were mixed together in
anhydrous dichloromethane (6 mL) for 7 h at room temperature.
After completion of the reaction as indicated by TLC, the
reaction mixture was diluted with H2O and extracted three times
with ethyl acetate. The combined organic layers were dried over
anhydrous magnesium sulfate and filtrated under suction. Then
the solvent from the extract was evaporated under reduced
pressure to give the residue, which was purified by column
chromatography on silica gel (200-300 mesh), eluted with a
mixture of petroleum ether/ethyl acetate.
White solid; m.p. 110-112 ℃. Yield 69%. 1H NMR (600 MHz,
CDCl3) δ 8.03 (d, J = 7.6 Hz, 2H, Ar-H), 7.57 (t, J = 7.5 Hz, 1H,
Ar-H), 7.44 (t, J = 7.8 Hz, 2H, Ar-H), 7.36-7.26 (m, 12H, Ar-H),
7.23-7.19 (m, 3H, Ar-H), 6.90 (d, J = 7.8 Hz, 1H, CO-NH), 5.90
(d, J = 8.4 Hz, 1H, CO-NH), 5.08 (s, 2H, Ar-CH2-O), 5.03 (d, J =
12.2 Hz, 1H, O-CH2-Ar), 4.97 (d, J = 12.2 Hz, 1H, O-CH2-Ar),
4.57-4.45 (m, 2H, N-CH, N-CH-CO), 4.29 (dd, J = 11.4, 3.6 Hz,
1H, O-CH2), 4.24 (dd, J = 10.8, 4.8 Hz, 1H, O-CH2), 3.01 (dd, J
= 17.4, 3.6 Hz, 1H, Ar-CH2), 2.94 (dd, J = 13.8, 6.6 Hz, 1H, CO-
CH2), 2.86 (dd, J = 13.8, 7.8 Hz, 1H, CO-CH2), 2.69 (dd, J =
17.4, 7.2 Hz, 1H, Ar-CH2). 13C NMR (150 MHz, CDCl3) δ
171.77, 169.91, 166.42, 155.98, 136.77, 136.00, 135.22, 133.26,
129.73, 129.29, 128.70, 128.60, 128.50, 128.42, 128.34, 128.26,
128.13, 126.85, 67.29, 66.90, 64.86, 50.87, 50.01, 37.54, 36.18.
HRMS (ESI+) [M+H]+ : C35H35N2O7: 595.2439; Found: 595.2440.
4.1.2.1. Benzyl (S)-3-(((benzyloxy)carbonyl)amino)-4-(((S)-1-
hydroxy-3-phenylpropan-2-yl)amino)-4-oxobutanoate
(intermediate 4)
White solid; m.p. 116-117 ℃. Yield 75%. 1H NMR (600 MHz,
CDCl3) δ 7.40-7.29 (m, 10H, Ar-H), 7.28 (d, J = 7.2 Hz, 2H, Ar-
H), 7.21 (d, J = 7.2 Hz, 1H, Ar-H), 7.18 (d, J = 7.8 Hz, 2H, Ar-
H), 6.57 (d, J = 7.2 Hz, 1H, CO-NH), 5.80 (d, J = 7.8 Hz, 1H,
CO-NH), 5.16-4.98 (m, 4H, Ar-CH2-O, O-CH2-Ar), 4.54-4.51 (m,
1H, N-CH-CO), 4.11-4.09 (m, 1H, N-CH), 3.60 (dd, J = 11.4, 5.6
Hz, 1H, O-CH2), 3.55-3.47 (m, 1H, O-CH2), 3.01 (dd, J = 17.4,
4.2 Hz, 1H, Ar-CH2), 2.86-2.77 (m, 2H, CO-CH2), 2.73 (dd, J =
4.1.3.2.(S)-2-((S)-4-(Benzyloxy)-2-
(((benzyloxy)carbonyl)amino)-4-oxobutanamido)-3-
phenylpropyl-4-chlorobenzoate (5b)
White solid; m.p. 181-183 ℃. Yield 65%. 1H NMR (600 MHz,
CDCl3) δ 7.93 (d, J = 7.8Hz, 2H, Ar-H), 7.39 (d, J = 8.4 Hz, 2H,
Ar-H), 7.37-7.24 (m, 12H, Ar-H), 7.22 (d, J = 6.6 Hz, 1H, Ar-H),
7.19 (d, J = 7.2 Hz, 2H, Ar-H), 6.85 (d, J = 6.6 Hz, 1H, CO-NH),
5.89 (d, J = 6.6 Hz, 1H, CO-NH), 5.08 (s, 2H, Ar-CH2-O), 5.02
(q, J = 12.0 Hz, 2H, O-CH2-Ar), 4.57-4.46 (m, 2H, N-CH, N-CH-
CO), 4.31-4.20 (m, 2H, O-CH2 ), 3.01 (dd, J = 17.4, 4.2 Hz, 1H,
Ar-CH2), 2.92 (dd, J = 13.8, 6.0 Hz, 1H, CO-CH2), 2.87 (dd, J =
13.8, 7.8 Hz, 1H, CO-CH2), 2.68 (dd, J = 17.4, 6.8 Hz, 1H, Ar-
CH2). 13C NMR (150 MHz, CDCl3) δ 171.73, 169.95, 165.55,
156.02, 139.74, 136.65, 135.95, 135.20, 131.11, 129.25, 128.85,
128.72, 128.62, 128.45, 128.37, 128.20, 128.15, 128.11, 126.90,
67.32, 66.91, 65.20, 50.92, 49.87, 37.58, 36.09. HRMS (ESI+)
[M+H]+ : C35H34ClN2O7: 629.2049; Found: 629.2049.
17.4, 6.6 Hz, 1H, Ar-CH2), 2.38 (t, J = 6.0 Hz, 1H, CH2-OH). 13
C
NMR (150 MHz, CDCl3) δ 171.65, 170.51, 169.87, 137.41,
135.92, 135.28, 129.22, 128.64, 128.62, 128.48, 128.40, 128.31,
128.21, 126.67, 67.38, 66.98, 63.58, 53.16, 51.20, 36.78, 36.22.
HRMS (ESI+) [M+H]+ : C28H31N2O6: 491.2104; Found: 491.2107.
4.1.2.2. Benzyl ((S)-1-(((S)-1-hydroxy-3-phenylpropan-2-
yl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate
(intermediate 7)
White solid; m.p. 125-127 ℃. Yield 78%. 1H NMR (600 MHz,
DMSO) δ 7.83 (d, J = 7.8 Hz, 1H, CO-NH), 7.42 (d, J = 8.4 Hz,
1H, CO-NH), 7.33 (t, J = 7.3 Hz, 2H, Ar-H), 7.30 (d, J = 7.0 Hz,
1H, Ar-H), 7.26-7.22 (m, 10H, Ar-H), 7.21-7.16 (m, 2H, Ar-H),
4.98-4.92 (m, 2H, Ar-CH2-O), 4.81 (t, J = 5.4 Hz, 1H, N-CH-
CO), 4.25-4.19 (m, 1H, CH2-OH), 3.92-3.86 (m, 1H, N-CH),
3.34-3.29 (m, 1H, O-CH2), 3.28-3.23 (m, 1H, O-CH2), 2.91 (dd, J
= 13.8, 4.5 Hz, 1H, Ar-CH2), 2.86 (dd, J = 13.8, 6.0 Hz, 1H, Ar-
CH2), 2.74-2.69 (m, 1H, Ar-CH2), 2.67 (dd, J = 13.8, 6.0 Hz, 1H,
Ar-CH2). 13C NMR (150 MHz, DMSO) δ 170.65, 166.63, 136.47,
136.10, 129.50, 129.25, 128.83, 128.69, 128.53, 128.44, 128.20,
127.33, 126.75, 63.06, 62.83, 56.42, 48.65, 37.64, 36.53. HRMS
(ESI+) [M+H]+ : C26H29N2O4: 433.2049; Found: 433.2051.
4.1.3.3.(S)-2-((S)-4-(Benzyloxy)-2-
(((benzyloxy)carbonyl)amino)-4-oxobutanamido)-3-
phenylpropyl-2-chlorobenzoate (5c)
White solid; m.p. 117-119 ℃. Yield 73%. 1H NMR (600 MHz,
CDCl3) δ 7.87 (d, J = 7.2 Hz, 1H, Ar-H), 7.46-7.39 (m, 2H, Ar-
H), 7.37-7.27 (m, 13H, Ar-H), 7.22 (d, J = 7.3 Hz, 1H, Ar-H),
7.20 (d, J = 7.1 Hz, 2H, Ar-H), 6.85 (d, J = 7.8 Hz, 1H, CO-NH),
5.88 (d, J = 7.8 Hz, 1H, CO-NH), 5.13-5.00 (m, 4H, Ar-CH2-O,
O-CH2-Ar), 4.57-4.45 (m, 2H, N-CH, N-CH-CO), 4.32-4.24 (m,
2H, O-CH2), 3.05 (dd, J = 17.4, 4.5 Hz, 1H, Ar-CH2), 2.93 (dd, J
= 13.8, 6.6 Hz, 1H, CO-CH2), 2.88 (dd, J = 13.8, 6.2 Hz, 1H,
CO-CH2), 2.70 (dd, J = 17.4, 6.6 Hz, 1H, Ar-CH2). 13C NMR
(150 MHz, CDCl3) δ 171.71, 169.89, 165.59, 156.01, 136.81,
135.94, 135.26, 133.69, 132.90, 131.94, 131.17, 129.65, 129.28,
128.70, 128.61, 128.59, 128.43, 128.35, 128.27, 128.18, 126.85,
126.78, 67.36, 66.90, 65.37, 51.01, 49.82, 37.54, 36.04. HRMS
(ESI+) [M+H]+ : C35H34ClN2O7: 629.1976; Found: 629.1976.
4.1.3. Synthesis of compounds 5a-5g and 8a-8f
Intermediate 4 or 7 (1.0 equiv), various acid (1.5 equiv), N,N'-
dicyclohexylcarbodiimide (1.1 equiv), 4-dimethylaminopyridine
(0.2 equiv) were mixed in a 100 mL round flask. The mixture
was stirred in anhydrous dichloromethane for 4 h-5 h at reflux to
give final compounds 5 (R=a-g) or 8 (R=a-f). After completion
of the reaction as indicated by TLC, the reaction mixture was
diluted with H2O and extracted three times with ethyl acetate.
The combined organic layers were dried over anhydrous
magnesium sulfate and filtrated under suction. And then the
solvent from the extract was evaporated under reduced pressure
to give the residue, which was purified by column
chromatography on silica gel (200-300 mesh), eluted with
petroleum ether or mixture of petroleum ether/ethyl acetate. Then
the solid was washed with ethyl acetate to collect solid.
4.1.3.4.(S)-2-((S)-4-(Benzyloxy)-2-
(((benzyloxy)carbonyl)amino)-4-oxobutanamido)-3-
phenylpropyl-2-methylbenzoate (5d)
White solid; m.p. 131-133 ℃. Yield 80%. 1H NMR (600 MHz,
CDCl3) δ 7.93 (d, J = 7.5 Hz, 1H, Ar-H), 7.41 (t, J = 7.2 Hz, 1H,
Ar-H), 7.36-7.27 (m, 11H, Ar-H), 7.25-7.22 (m, 3H, Ar-H), 7.21
(t, J = 6.6 Hz, 3H, Ar-H), 6.86 (d, J = 7.8 Hz, 1H, CO-NH), 5.89
(d, J = 7.8 Hz, 1H, CO-NH), 5.10-4.95 (m, 4H, Ar-CH2-O, O-
CH2-Ar), 4.59-4.44 (m, 2H, N-CH, N-CH-CO), 4.27 (dd, J =
11.4, 3.6 Hz, 1H, O-CH2), 4.21 (dd, J = 11.4, 5.4 Hz, 1H, O-CH2),

3.02 (dd, J = 17.4, 3.6 Hz, 1H, Ar-CH2), 2.93 (dd, J = 13.8, 6.0
Hz, 1H, CO-CH2), 2.86 (dd, J = 13.8, 8.4 Hz, 1H, CO-CH2), 2.69
4.1.3.8.(S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-3-phenylpropyl benzoate (8a)
(dd, J = 17.4, 6.6 Hz, 1H, Ar-CH2), 2.60 (s, 3H, Ar-CH3). 13
C
White solid; m.p. 178-179 ℃. Yield 76%. 1H NMR (600 MHz,
CDCl3) δ 7.98 (d, J = 7.8 Hz, 2H, Ar-H), 7.59 (t, J = 7.4 Hz, 1H,
Ar-H), 7.46 (t, J = 7.8 Hz, 2H, Ar-H), 7.36-7.28 (m, 5H, Ar-H),
7.24 (d, J = 8.4 Hz, 2H, Ar-H), 7.20 (t, J = 6 Hz, 1H, Ar-H),
7.18-7.10 (m, 6H, Ar-H), 7.07 (t, J = 6 Hz, 1H, Ar-H), 5.98 (d, J
= 8.4 Hz, 1H, CO-NH), 5.37 (d, J = 5.4 Hz, 1H, CO-NH), 5.06 (s,
2H, Ar-CH2-O), 4.51-4.43 (m, 1H, N-CH-CO), 4.39-4.29 (m, 1H,
N-CH), 4.13 (dd, J = 11.4, 4.8 Hz, 1H, O-CH2), 4.02 (dd, J =
11.4, 3.6 Hz, 1H, O-CH2), 3.08 (dd, J = 13.2, 6.2 Hz, 1H, Ar-
CH2), 2.94 (dd, J = 13.8, 7.8 Hz, 1H, Ar-CH2), 2.87 (dd, J = 13.2,
6.0 Hz, 1H, Ar-CH2), 2.78 (dd, J = 13.8, 8.4 Hz, 1H, Ar-CH2).
13C NMR (150 MHz, CDCl3) δ 170.35, 166.33, 155.87, 136.67,
136.36, 136.14, 133.31, 129.70, 129.22, 129.14, 128.73, 128.70,
128.57, 128.52, 128.26, 128.04, 127.13, 126.86, 67.08, 64.93,
56.45, 49.61, 38.64, 37.53. HRMS (ESI+) [M+H]+ : C33H33N2O5:
537.2311; Found: 537.2308.
NMR (150 MHz, CDCl3) δ 171.76, 169.90, 167.26, 156.01,
140.51, 136.81, 135.96, 135.24, 132.32, 131.83, 130.78, 129.29,
129.01, 128.70, 128.60, 128.43, 128.36, 128.26, 128.17, 126.85,
125.87, 67.31, 66.89, 64.61, 50.93, 50.01, 37.59, 36.11, 21.93.
HRMS (ESI+) [M+H]+ : C36H37N2O7: 609.2595; Found: 609.2595.
4.1.3.5.(S)-2-((S)-4-(Benzyloxy)-2-
(((benzyloxy)carbonyl)amino)-4-oxobutanamido)-3-
phenylpropyl-3,4-dimethoxybenzoate (5e)
White solid; m.p. 178-180 ℃. Yield 78%. 1H NMR (600 MHz,
CDCl3) δ 7.67 (d, J = 7.8 Hz, 1H, Ar-H), 7.54 (s, 1H, Ar-H),
7.37-7.26 (m, 12H, Ar-H), 7.24-7.19 (m, 3H, Ar-H), 6.89 (d, J =
8.4 Hz, 1H, CO-NH), 6.86 (d, J = 7.8 Hz, 1H, Ar-H), 5.90 (d, J =
8.4 Hz, 1H, CO-NH), 5.09 (s, 2H, Ar-CH2-O), 5.04 (d, J = 12.4
Hz, 1H, O-CH2-Ar), 4.99 (d, J = 12.4 Hz, 1H, O-CH2-Ar), 4.57-
4.48 (m, 2H, N-CH, N-CH-CO), 4.24 (d, J = 4.8 Hz, 2H, O-CH2),
3.91 (d, J = 5.7 Hz, 6H, Ar-OCH3), 3.01 (dd, J = 17.4, 3.6 Hz,
1H, Ar-CH2), 2.93 (dd, J = 13.8, 6.0 Hz, 1H, CO-CH2), 2.84 (dd,
J = 13.8, 7.8 Hz, 1H, CO-CH2), 2.68 (dd, J = 17.4, 6.6 Hz, 1H,
Ar-CH2). 13C NMR (150 MHz, CDCl3) δ 171.79, 169.91, 166.23,
155.97, 153.25, 148.73, 136.78, 135.98, 135.20, 129.28, 128.69,
128.60, 128.43, 128.34, 128.22, 128.08, 126.85, 123.76, 122.15,
112.12, 110.31, 67.27, 66.94, 64.83, 56.03, 50.86, 50.04, 37.57,
36.21. HRMS (ESI+) [M+H]+ : C37H39N2O9: 655.2650; Found:
655.2650.
4.1.3.9.(S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-3-phenylpropyl-4-chlorobenzoate (8b)
White solid; m.p. 183-184 ℃. Yield 70%. 1H NMR (600 MHz,
CDCl3) δ 7.88 (d, J = 8.4 Hz, 2H, Ar-H), 7.41 (d, J = 8.4 Hz, 2H,
Ar-H), 7.36-7.28 (m, 5H, Ar-H), 7.26-7.23 (m, 2H, Ar-H), 7.22-
7.15 (m, 3H, Ar-H), 7.14-7.07 (m, 5H, Ar-H), 5.96 (d, J = 7.8 Hz,
1H, CO-NH), 5.33 (s, 1H, CO-NH), 5.05 (s, 2H, Ar-CH2-O),
4.52-4.42 (m, 1H, N-CH-CO), 4.38-4.26 (m, 1H, N-CH), 4.12
(dd, J = 11.4, 4.8 Hz, 1H, O-CH2), 4.03 (dd, J = 11.4, 4.2 Hz, 1H,
O-CH2), 3.11-3.04 (m, 1H, Ar-CH2), 2.94 (dd, J = 13.8, 7.8 Hz,
1H, Ar-CH2), 2.85 (dd, J = 13.8, 6.0 Hz, 1H, Ar-CH2), 2.76 (dd, J
= 13.8, 8.4 Hz, 1H, Ar-CH2). 13C NMR (150 MHz, CDCl3) δ
170.37, 165.47, 155.91, 139.80, 136.54, 136.34, 136.09, 131.07,
129.18, 129.13, 128.87, 128.75, 128.72, 128.58, 128.29, 128.11,
128.02, 127.14, 126.91, 67.12, 65.27, 56.43, 49.48, 38.51, 37.57.
HRMS (ESI+) [M+H]+ : C33H32ClN2O5: 571.1994; Found:
571.1994.
4.1.3.6.Benzyl(S)-3-(((benzyloxy)carbonyl)amino)-4-oxo-4-
(((S)-1-phenyl-3(propionyloxy)propan-2-yl)amino)butanoate
(5f)
1
White solid; m.p. 83-85 ℃. Yield 79%. H NMR (600 MHz,
CDCl3) δ 7.38-7.31 (m, 10H, Ar-H), 7.28 (d, J = 7.8 Hz, 2H, Ar-
H), 7.21 (t, J = 7.3 Hz, 1H, Ar-H), 7.16 (d, J = 7.2 Hz, 2H, Ar-H),
6.69 (d, J = 8.4 Hz, 1H, CO-NH), 5.87 (d, J = 8.4 Hz, 1H, CO-
NH), 5.14-5.07 (m, 4H, Ar-CH2-O, O-CH2-Ar), 4.53 (m, 1H, N-
CH-CO), 4.34 (m, 1H, N-CH), 4.04 (dd, J = 11.4, 3.6 Hz, 1H, O-
CH2), 4.00 (dd, J = 11.4, 5.7 Hz, 1H, O-CH2), 3.04 (dd, J = 17.4,
4.2 Hz, 1H, Ar-CH2), 2.84 (dd, J = 13.8, 6.3 Hz, 1H, CO-CH2),
2.75 (dd, J = 13.8, 7.8 Hz, 1H, CO-CH2), 2.70 (dd, J = 17.4, 6.6
Hz, 1H, Ar-CH2), 2.33 (q, J = 7.2 Hz, 2H, O-CO-CH2), 1.13 (t, J
= 5.4 Hz, 3H, CH2-CH3). 13C NMR (150 MHz, CDCl3) δ 174.34,
171.68, 169.86, 156.02, 136.81, 135.99, 135.27, 129.23, 128.63,
128.61, 128.46, 128.37, 128.28, 128.15, 126.79, 67.33, 66.92,
64.20, 50.96, 49.87, 37.41, 36.08, 27.43, 9.08. HRMS (ESI+)
[M+H]+ : C31H35N2O7: 547.2366; Found: 547.2365.
4.1.3.10.(S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-3-phenylpropyl-2-chlorobenzoate (8c)
White solid; m.p. 146-148 ℃. Yield 79%. 1H NMR (600 MHz,
CDCl3) δ 7.82 (d, J = 7.2 Hz, 1H, Ar-H), 7.49-7.43 (m, 2H, Ar-
H), 7.35-7.29 (m, 6H, Ar-H), 7.25 (d, J = 8.4 Hz, 2H, Ar-H), 7.20
(d, J = 7.2 Hz, 1H, Ar-H), 7.17 (t, J = 7.8 Hz, 2H, Ar-H), 7.15-
7.07 (m, 5H, Ar-H), 6.02 (d, J = 9.0 Hz, 1H, CO-NH), 5.38 (d, J
= 6.6 Hz, 1H, CO-NH), 5.05 (m, 2H, Ar-CH2-O), 4.51-4.42 (m,
1H, N-CH-CO), 4.39-4.30 (m, 1H, N-CH), 4.17 (dd, J = 11.4, 4.8
Hz, 1H, O-CH2), 4.00 (dd, J = 11.4, 4.2 Hz, 1H, O-CH2), 3.07
(dd, J = 12.6, 5.4 Hz, 1H, Ar-CH2), 2.95 (dd, J = 13.8, 7.8 Hz,
1H, Ar-CH2), 2.87 (dd, J = 13.8, 6.0 Hz, 1H, Ar-CH2), 2.81 (dd, J
= 13.8, 8.4 Hz, 1H, Ar-CH2). 13C NMR (150 MHz, CDCl3) δ
170.31, 165.41, 155.88, 136.73, 133.67, 133.00, 132.00, 131.24,
129.50, 129.21, 129.17, 128.73, 128.70, 128.55, 128.25, 128.06,
127.11, 126.87, 126.81, 67.10, 65.45, 56.47, 49.37, 38.66, 37.57.
HRMS (ESI+) [M+H]+ : C33H32ClN2O5: 571.1922; Found:
571.1924.
4.1.3.7.Benzyl(S)-4-(((S)-1-acetoxy-3-phenylpropan-2-
yl)amino)-3-(((benzyloxy)carbonyl)amino)-4-oxobutanoate
(5g)
White solid; m.p. 109-110 ℃. Yield 82%. 1H NMR (600 MHz,
CDCl3) δ 7.36-7.32 (m, 10H, Ar-H), 7.27 (t, J = 7.8 Hz, 2H, Ar-
H), 7.21 (t, J = 7.2 Hz, 1H, Ar-H), 7.16 (d, J = 7.2 Hz, 2H, Ar-H),
6.67 (d, J = 7.8 Hz, 1H, CO-NH), 5.86 (d, J = 7.8 Hz, 1H, CO-
NH), 5.13-5.07 (m, 4H, Ar-CH2-O, O-CH2-Ar), 4.53 (m, 1H, N-
CH-CO), 4.35 (m, 1H, N-CH), 4.03 (dd, J = 11.4, 4.2 Hz, 1H, O-
CH2), 3.99 (dd, J = 11.4, 5.8 Hz, 1H, O-CH2), 3.03 (dd, J = 17.4,
4.2 Hz, 1H, Ar-CH2), 2.84 (dd, J = 13.8, 6.3 Hz, 1H, CO-CH2),
2.76 (dd, J = 13.8, 7.8 Hz, 1H, CO-CH2), 2.70 (dd, J = 17.4, 6.6
Hz, 1H, Ar-CH2), 2.05 (s, 3H, O-CO-CH3). 13C NMR (150 MHz,
CDCl3) δ 171.68, 170.99, 169.89, 156.03, 136.77, 135.99, 135.27,
129.23, 128.64, 128.61, 128.47, 128.38, 128.28, 128.17, 126.81,
67.33, 66.93, 64.35, 50.97, 49.82, 37.40, 36.08, 20.77. HRMS
(ESI+) [M+H]+ : C30H33N2O7: 533.2210; Found: 533.2211.
4.1.3.11.(S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-3-phenylpropyl-2-methylbenzoate (8d)
White solid; m.p. 159-160 ℃. Yield 85%. 1H NMR (600 MHz,
CDCl3) δ 7.84 (d, J = 7.8 Hz, 1H, Ar-H), 7.43 (t, J = 7.2 Hz, 1H,
Ar-H), 7.36-7.28 (m, 5H, Ar-H), 7.25 (d, J = 7.8 Hz, 4H, Ar-H),
7.20 (t, J = 6.9 Hz, 1H, Ar-H), 7.16 (t, J = 7.4 Hz, 2H, Ar-H),
7.14-7.06 (m, 5H, Ar-H), 5.98 (d, J = 8.4 Hz, 1H, CO-NH), 5.36
(d, J = 6.0 Hz, 1H, CO-NH), 5.10-5.00 (m, 2H, Ar-CH2-O), 4.50-
4.43 (m, 1H, N-CH-CO), 4.37-4.31 (m, 1H, N-CH), 4.12 (dd, J =

11.4, 4.8 Hz, 1H, O-CH2), 4.00 (dd, J = 11.4, 4.2 Hz, 1H, O-CH2),
3.07 (dd, J = 13.8, 6.0 Hz, 1H, Ar-CH2), 2.95 (dd, J = 13.8, 7.8
Hz, 1H, Ar-CH2), 2.86 (dd, J = 13.8, 6.0 Hz, 1H, Ar-CH2), 2.79
values were determined as the drug and sample concentration at
50% inhibition of the cell growth. Briefly, cell growth inhibition
rate (%) was calculated using the following equation: Inhibitory
rate (%) = (1-Atreatment/Acontrol) × 100%.
(dd, J = 13.8, 7.8 Hz, 1H, Ar-CH2), 2.58 (s, 3H, Ar-CH3). 13
C
NMR (150 MHz, CDCl3) δ 170.32, 167.13, 155.87, 140.49,
136.70, 132.38, 131.86, 130.73, 129.22, 129.16, 128.92, 128.73,
128.69, 128.56, 128.26, 128.05, 127.11, 126.86, 125.86, 67.08,
64.68, 56.44, 49.62, 38.60, 37.59, 21.96. HRMS (ESI+) [M+H]+ :
C34H35N2O5: 551.2541; Found: 551.2545.
4.3. Human Topoisomerase I and Topoisomerase IIα
relaxation assay
DNA topoisomerase I and IIα inhibition assay was determined
following the previously reported method.25-27 The inhibition of
compounds to topoisomerase I and topoisomerase IIα were
determined by assessing the relaxation of supercoiled pBR322
plasmid DNA. The assay was performed in a final volume of 5
µL reaction volume containing 500 ng of supercoiled DNA
pBR322 (Takara Biotechnology, Japan) and 1 unit of human
topoisomerase I (Takara Biotechnology, Japan) or topoisomerase
IIα (USB Corp, USA) with or without our compounds in the
reaction buffer. The reaction mixtures were incubated at 37 ℃
for 30 min and terminated by the addition of 1 µL of 0.77%
sodium dodecyl sulfate, 77 mM EDTA. Samples were added 1
µL of 6 × DNA loading buffer. Electrophoresis was performed on
a 1% agarose gel at 83 V for 60 min in TBE buffer (Tris-borate
and EDTA). Gels were stained for 30 min in an aqueous solution
of ethidium bromide (0.5 µg/mL) and photographed under UV
light (Tanon-1600 Gel Imaging System). UV-transilluminated
gels were documented by Gel Image System Ver 4.00 software.
Camptothecin (Sigma, USA) was used as a positive control as
topoisomerase I inhibitor and etoposide (Sigma, USA) was used
as a positive control as topoisomerase IIα inhibitor, respectively.
4.1.3.12.(S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-3-phenylpropyl-3,4-dimethoxybenzoate
(8e)
White solid; m.p. 170-172 ℃. Yield 83%. 1H NMR (600 MHz,
CDCl3) δ 7.61 (d, J = 7.8 Hz, 1H, Ar-H), 7.52 (s, 1H, Ar-H),
7.37-7.28 (m, 5H, Ar-H), 7.25 (d, J = 8.4 Hz, 3H, Ar-H), 7.22 (d,
J = 7.2 Hz, 1H, Ar-H), 7.18 (t, J = 7.2 Hz, 2H, Ar-H), 7.12 (d, J
= 7.2 Hz, 5H, Ar-H), 6.89 (d, J = 8.4 Hz, 1H, CO-NH), 5.96 (d, J
= 8.4 Hz, 1H, CO-NH), 5.06 (s, 2H, Ar-CH2-O), 4.51-4.43 (m,
1H, N-CH-CO), 4.38-4.30 (m, 1H, N-CH), 4.10 (dd, J = 11.4, 5.4
Hz, 1H, O-CH2), 4.02 (dd, J = 11.4, 4.2 Hz, 1H, O-CH2), 3.94 (d,
J = 10.2 Hz, 6H, Ar-OCH3), 3.12-3.03 (m, 1H, Ar-CH2), 2.94 (dd,
J = 13.2, 7.8 Hz, 1H, Ar-CH2), 2.87 (dd, J = 13.8, 6.0 Hz, 1H,
Ar-CH2), 2.77 (dd, J = 13.2, 8.4 Hz, 1H, Ar-CH2). 13C NMR (150
MHz, CDCl3) δ 170.31, 166.21, 155.88, 153.30, 148.76, 136.64,
129.22, 129.13, 128.75, 128.69, 128.56, 128.26, 128.01, 127.13,
126.86, 123.72, 122.13, 112.12, 110.30, 67.08, 65.30, 64.86,
56.38, 56.07, 49.71, 38.57, 37.53. HRMS (ESI+) [M+H]+
C35H37N2O7: 597.2523; Found: 597.2523.
:
4.4. Molecular docking
4.1.3.13.(S)-2-((S)-2-(((Benzyloxy)carbonyl)amino)-3-
phenylpropanamido)-3-phenylpropyl propionate (8f)
3D co-crystal structures of human topoisomerase IIα (PDB ID:
1ZXM)
was
obtained
from
protein
data
bank
a
White pink solid; m.p. 143-144 ℃. Yield 86%. 1H NMR (600
MHz, CDCl3) δ 7.35 (d, J = 6.6 Hz, 2H, Ar-H), 7.32 (d, J = 6.6
Hz, 3H, Ar-H), 7.26-7.22 (m, 4H, Ar-H), 7.21-7.14 (m, 4H, Ar-
H), 7.08 (d, J = 7.2 Hz, 2H, Ar-H), 5.88 (d, J = 7.8 Hz, 1H, CO-
NH), 5.30 (d, J = 7.8 Hz, 1H, CO-NH), 5.08 (s, 2H, Ar-CH2-O),
4.37-4.28 (m, 2H, N-CH-CO, N-CH), 3.92 (dd, J = 11.4, 4.8 Hz,
1H, O-CH2), 3.78 (dd, J = 11.4, 4.2 Hz, 1H, O-CH2), 3.11-3.02
(m, 1H, Ar-CH2), 2.96 (dd, J = 13.8, 7.8 Hz, 1H, Ar-CH2), 2.77
(dd, J = 13.8, 6.0 Hz, 1H, Ar-CH2), 2.68 (dd, J = 13.8, 8.4 Hz,
1H, Ar-CH2), 2.28 (q, J = 7.8 Hz, 2H, O-CO-CH2), 1.12 (t, J =
7.8 Hz, 3H, CH2-CH3). 13C NMR (150 MHz, CDCl3) δ 174.21,
170.25, 155.86, 136.73, 136.41, 136.15, 129.26, 129.17, 128.74,
128.63, 128.57, 128.27, 128.05, 127.08, 126.81, 67.09, 64.25,
56.36, 49.49, 38.59, 37.42, 27.39, 9.06. HRMS (ESI+) [M+H]+ :
C29H33N2O5: 489.2384; Found: 489.2388.
(http://www.rcsb.org). Human topoisomerase IIα was
homodimer consisting of two identical subunits and each
contained AMP-PNP and Mg2+ ion at the ATP binding pocket.
GLIDE 9.6 module of Schrodinger Suite was used for molecular
docking study. The ligands were prepared by LIGPREP module.
The protein preparation was carried out using Prep Wiz. The
protein was first pre-processed and the missing side chain and
loops were filled using PRIME. The metal binding states were
generated and the protein was then optimized and minimized.
The grid box was generated around the centroid of the co-
crystallized ligand at the ATP binding site. The docking was
carried out using GLIDE XP module.
Similarly, 3D co-crystal structures of human topoisomerase I
(PDB ID: 1K4T) was obtained from protein data bank
(http://www.rcsb.org). GLIDE 9.6 module of Schrodinger Suite
was used for molecular docking study. The ligands were prepared
by LIGPREP module. The protein preparation was carried out
using Prep Wiz. The protein was first pre-processed and the
missing side chain and loops were filled using PRIME. The
protein was then optimized and minimized. A receptor grid was
generated on the center of the co-crystallized ligand, which was
defined as the ligand-binding site search region. The docking was
carried out using GLIDE XP module. All the
remaining parameters were kept as default.
4.2. Growth inhibitory effect
The anticancer activities of target compounds were evaluated
against four human cancer cell lines (HeLa, MGC-803, MCF-7
and A549) by measuring cell viability according to the MTT
method described in the literature24 with camptothecin and
etoposide as the positive controls. The cells were seeded into 96-
well plates at a concentration of 5×104 cells per well, and
incubated for 12 h, then treated by adding serial dilutions of
testing chemicals or camptothecin and etoposide (last
concentrations: 0, 5.0, 10.0, 20.0, 40.0, 80.0 and 100.0 µM).
After 48 h incubation, 10.0 µL MTT (5.0 mg/mL) was added to
each well and cells were incubated for additional 4 h. The
medium was removed and 100.0 µL of DMSO was added to
dissolve the formazan crystals. Subsequently, the absorbance at a
wavelength of 490 nm was read with Bio-Tek microplate Reader.
All experiments were performed in triplicate and each
experiment was repeated at least three times. Each value
represents the mean SD of three independent experiments. IC50
Acknowledgments
We gratefully acknowledge the generous support provided by
National Natural Science Foundation of China (No. 21675039);
China Postdoctoral Science Foundation (No. 2016M591401);
Young Talent of Hebei Province; Natural Science Foundation of
Hebei Province (No. B2015201016); Hebei University Science
Fund for Distinguished Young Scholars (No. 2015JQ06); Hebei

13. Jun, K. Y.; Lee, E. Y.; Jung, M. J.; Lee, O. H.; Lee, E. S.; Park
Choo, H. Y.; Na, Y.; Kwon, Y. Eur. J. Med. Chem. 2011, 46,
1964-1971.
University Laboratory Open Project (No. sy201617); Hebei
University Innovative Funding Projects for Graduate Students
(No. X2016070); The Innovative Training Program for College
Students of Hebei University (No. 2016007).
14. Pogorelčnik, B.; Janežič, M.; Sosič, I.; Gobec, S.; Solmajer, T.;
Perdih, A. Bioorg. Med. Chem. 2015, 23, 4218-4229.
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Supplementary data
Supplementary data (1H NMR, 13C NMR, HRMS spectra of
the compounds 5b, 5e, 8d, 8f and the binding modes of all
compounds-topoisomerase IIα) associated with this article can be
found, in the online version.
16. Qiu, J.; Zhao, B.; Zhong, W.; Shen, Y.; Lin, H. Eur. J. Med.
Chem. 2015, 94, 427-435.
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Graphical Abstract.
Synthesis and biological evaluation of N-
(carbobenzyloxy)-l-phenylalanine and N-
(carbobenzyloxy)-l-aspartic acid-β-benzyl
ester derivatives as potent topoisomerase IIα
inhibitors
Xiaoyan Hana, Yifan Zhonga, Guan Zhoua, Hui Qia, Shengbin Lia, Qiang Dinga, Zhenming Liuc, Yali Songa, *,
Xiaoqiang Qiaoa, b, *
aKey Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences,
Hebei University, Baoding, 071002, China
bKey Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of
Chemistry and Environmental Science, Hebei University, Baoding, 071002, China
cDrug Design Center, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Scienc
es, Peking University, Beijing 100191, China

Highlights
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
13 compounds as topoisomerase IIα inhibitors were designed and synthesized.
Evaluated for topo I and IIα inhibitory activity, antiproliferative activity.
Molecular docking studies were investigated.
Compounds showed selective topoisomerase IIα inhibition.
5b showed best antiproliferative activity, topoisomerase IIα inhibitory activity.