Design and synthesis of the novel DNA topoisomerase II inhibitors: Esterification and amination substituted 4′-demethylepipodophyllotoxin derivates exhibiting anti-tumor activity by activating ATM/ATR signaling pathways

Article abstract of DOI:10.1016/j.ejmech.2014.03.082

According to the structure-activity relationship, drug combination principle and bioisosterism, a series of the novel esterification and amination 4′-demethylepipodophyllotoxin derivates were rationally designed in order to discover the potential antitumor prodrug. And then these compounds were tested by the drug-topoisomerase II docking models for virtual screening. Thus, twelve target compounds were screened out and synthesized. Most of compounds exhibited promising in vitro anti-tumor activity, particularly 4-N-tris(hydroxymethyl)metylaminomethane-4-deoxy-4′- demethylepipodophyllotoxin (Compound 1). The anti-tumor activity of Compound 1 against the tumor cell lines BGC-823 (i.e., the IC₅₀ value of 5.35 ± 0.77 μM), HeLa (i.e., the IC₅₀ value of 160.48 ± 14.50 μM), and A549 (i.e., the IC₅₀ value of 13.95 ± 5.41 μM) was significantly improved by 706%, 31% and 900% than that of etoposide (i.e., the IC₅₀ values of 43.74 ± 5.13, 209.90 ± 13.42, and 139.54 ± 7.05 μM), respectively. Moreover, the IC₅₀ value of Compound 1 against the normal human cell line HK-2 (i.e., 16.3 ± 3.77 μM) was 78% lower than that of etoposide (i.e., 9.17 ± 1.58 μM). Compound 1 could diminish the relaxation reaction topoisomerase II DNA decatenation at a concentration of 10 μM and induce BGC-823 apoptosis by breaking DNA double-strand and activating ATM/ATR signaling pathways.

Full text of DOI:10.1016/j.ejmech.2014.03.082

Accepted Manuscript
Design and synthesis of the novel DNA topoisomerase II inhibitors: esterification and
amination substituted 4′-demethylepipodophyllotoxin derivates exhibiting anti-tumor
activity by activating ATM/ATR signaling pathways
Wei Zhao, Li Xiao, Hong-Mei Li, Duan-Ji Wang, Dong-Sheng Li, Tao Chen, Zhan-
Peng Yuan, Ya-Jie Tang
PII:
S0223-5234(14)00304-3
10.1016/j.ejmech.2014.03.082
EJMECH 6866
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 November 2013
Revised Date: 26 March 2014
Accepted Date: 29 March 2014
Please cite this article as: W. Zhao, L. Xiao , H.-M. Li , D.-J. Wang , D.-S. Li , T. Chen , Z.-P. Yuan,
Y.-J. Tang , Design and synthesis of the novel DNA topoisomerase II inhibitors: esterification and
amination substituted 4′-demethylepipodophyllotoxin derivates exhibiting anti-tumor activity by activating
ATM/ATR signaling pathways, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.03.082.
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Graphical Abstract

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Highlights



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Twelve novel 4′-demethylepipodophyllotoxin derivates had been synthesized.
The compounds were evaluated for DNA topoisomerase II inhibitory activity.
Most of compounds exhibited promising in vitro anti-tumor activity, e.g., Compound 1.
Compound 1 induced BGC 823 apoptosis by activating ATM/ATR signaling pathways.

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Design and synthesis of the novel DNA topoisomerase II inhibitors: esterification
and amination substituted 4′-demethylepipodophyllotoxin derivates exhibiting
anti-tumor activity by activating ATM/ATR signaling pathways
1
, a
1, a
a
a
a
b
Wei Zhao , Li Xiao , Hong-Mei Li , Duan-Ji Wang , Dong-Sheng Li , Tao Chen ,
c
a,
Zhan-Peng Yuan , and Ya-Jie Tang *
a
Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of
Technology, Wuhan 430068, China
b
Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical
Engineering and Technology, Tianjin University, Tianjin 300072, China
c
School of Public Health, Wuhan University, Wuhan 430071, China
*
Corresponding author. Tel. & Fax: +86-27-5975.0491 Email: yajietang@hotmail.com
Equally contributed to this work
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ABSTRACT
According to the structure-activity relationship, drug combination principle and bioisosterism, a
series of the novel esterification and amination 4′-demethylepipodophyllotoxin derivates were
rationally designed in order to discover the potential antitumor prodrug. And then these compounds
were tested by the drug-topoisomerase II docking models for virtual screening. Thus, twelve target
compounds were screened out and synthesized. Most of compounds exhibited promising in vitro
anti-tumor
activity,
particularly
4-N-tris(hydroxymethyl)metylaminomethane-4-deoxy-4′-demethylepipodophyllotoxin (Compound
1). The anti-tumor activity of Compound 1 against the tumor cell lines BGC-823 (i.e., the IC50
value of 5.35 ± 0.77 µM), HeLa (i.e., the IC value of 160.48 ± 14.50 µM), and A549 (i.e., the IC
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value of 13.95 ± 5.41 µM) was significantly improved by 706%, 31% and 900% than that of
etoposide (i.e., the IC values of 43.74 ± 5.13, 209.90 ± 13.42, and 139.54 ± 7.05 µM), respectively.
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Moreover, the IC value of Compound 1 against the normal human cell line HK-2 (i.e., 16.3 ± 3.77
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µM) was 78% lower than that of etoposide (i.e., 9.17 ± 1.58 µM). Compound 1 could diminish the
relaxation reaction Topo II DNA decatenation at a concentration of 10 µM and induce BGC-823
apoptosis by breaking DNA double-strand and activating ATM/ATR signaling pathways.
Keywords: 4′-Demethylepipodophyllotoxin derivates, anti-tumor activity, topoisomerase II,
structure-activity relationships, apoptosis, ATM/ATR signaling pathway
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1. Introduction
4′-demethylepipodophyllotoxin (DMEP) is one of the best-known naturally occurring aryltetralin
lignans exhibiting anti-tumor activity [1], which can bind at the pocket between the link site of
DNA and topoisomerase II (Topo II) and then diminishing the relaxation reaction of Topo II-DNA
decatenation. The clinical success of etoposide and teniposide has triggered the search for
compounds with a similar mechanism of action but without their inconveniences (anti-tumor
activity need to be further improved) [2-4]. This has resulted in the discovery of many compounds
with very different chemical structures. So, most of currently numerous studies focus on the
structure modification of DMEP for generate derivatives with superior pharmacological profiles and
broader therapeutic scope [5].
To discover a series of the novel 4′-demethylepipophyllum Topo II inhibitors, a rational prodrug
design strategy should be developed to the structure modification of DMEP with the superior
inhibition on Topo II, the higher antitumor activity and the lower cytotoxicity on the human normal
cells. The steric clash and electrostatic contour plots of the comparative molecular field analysis
models [6, 7] shows that the carbon atom at the 4 position of cycloparaffin (C ring) of DMEP is
more reactive by comparing other carbon atoms in the tetranap skeleton of DMEP, and is easier
nucleophillic attacked by the molecule with the electro negativity. Thus, from the structure-activity
relationship point of view, the structure modifications at the 4 position of cycloparaffin (C ring) of
DMEP maybe a very effective pathway for improving the antitumor activity. According to drug
combination principle, the combination of two kinds of drugs through the ester bond can gain some
new compounds with better bioactivity. Many famous drugs in clinic use such as sultamicillin and
benorilate were discovered through this way. So, DMEP could be combined with other compounds
through ester bond for the better bioactivity and attenuated toxicity. From the bioisosterism point of
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view [8, 9], bioisosteres are substituents with similar physical or chemical properties which produce
broadly similar biological properties to a chemical compound for the activity improvement and the
toxicity reduction of the lead compound. Both of the ester bond (—COO—) and the —NH— bond
are the bioisosteres with the same numbers of the outermost electrons. So, the —NH— bond at the
C-4 position of DMEP may be also a potential modification direction for improving the antitumor
activity of DMEP. On the other hand, the prodrug approach is a very versatile strategy to increase
the utility of pharmacologically active compounds. According to the prodrug principle [10, 11],
both of esters (—COOR) or amides (—NHCOR) are commonly used ionizable groups, which can
be introduced into the hydroxyl, thiol, amine, or carboxylic acid functionalities of the parent drug
molecule to compensate for poor aqueous solubility. A variety of esterases, amidases, and/or
peptidases in plasma or in other tissues can bioconvert these prodrugs to their active counterparts.
Quite often, esters and amides can also be used to enhance absorption and consequently oral drug
delivery of parent drugs, because the brush-border membrane of intestinal epithelium possesses a
considerable number of transporters for amino acids and peptides. Take these into consideration,
many famous drugs in clinic were discovered by using esterification and amidation such as
sultamicillin and benorilate. So, esterification and amidation of DMEP at the C-4 position were
permitted for two useful DMEP structure modifications. Finally, from an antitumour mechanistic
point of view, substituents with the high electron density at the C-4 position of DMEP derivatives
(i.e., etoposide) has been demonstrated to be bound via the hydrogen bonding interactions with the
tyrosine residue of Topo II and the bases of DNA breakage [12-14] . A series of heterocyclic with
pharmacological activity and the high electron density were chosen as module at the C-4 position of
DMEP for improving the stabilization and inhibition of the complex of Topo II-DNA cleavage
complex. In order to identify molecular properties which have the largest affinity with DNA Topo II,
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dock the designed DMEP derivates in the active site of DNA Topo II for virtual screening.
This work demonstrated that a series of DMEP derivates were rationally designed, and then the
interactions between DMEP derivates and DNA Topo II were investigated by molecular docking for
screening target compounds. The biologic activity evaluation indicates that the anti-tumor activities
of them were stronger than those of etoposide. Moreover, the DNA Topo II inhibition of
4-N-(tris(hydroxymethyl)metylaminomethane)-4-deoxy-4′-demethylepipodophyllotoxin
(Compound 1) was significantly higher than that of etoposide. These results provided the
determinants of DNA Topo II binding affinity for this important class of anti-tumor agents and pave
the way for further rational structural modification.
2. Results and Discussion
2.1 Drug molecular design
The process of drug molecular design was performed in four steps. Firstly, numerous studies on
4′-demethylepipodophyllotoxin (DMEP) currently focus on its structure modification of the
cycloparaffin (C-ring) in the tetranap skeleton and the steric clash and electrostatic contour plots of
the comparative molecular field analysis models shows that the electron-withdrawing substituents at
the C-4 position would be a great modification direction to improve antitumour activity of DMEP
[15].
Meanwhile,
in
our
previous
work,
the
DMEP
derivate
4β-S-(1,2,4-triazole-3)-4-deoxy-podophyllotoxin that structurally modified at C-4 position of
DMEP against tumor cell line BGC-823 (IC values of 0.28 µM), A549 (IC values of 0.76 µM)
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and HepG2 (IC values of 0.42 µM) were significantly improved by 91, 221 and 2.73 times than
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those of etoposide (IC50 values of 25.72, 167.97 and 1.15 µM), respectively [16]. Thus, the C-4
position of DMEP was ensured for an effective modification site. Secondly, both of the chemical
group NH and the oxygen atom have the same numbers of the outermost electrons, but the electron
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cloud of the NH with the greater electronegativity was denser than that of the oxygen atom.
According to bioisosterism, the O-linked (ethers, glycoside) compounds could be less active in
comparison to the NH-linked congeners. So, the sulfur atom preferentially integrated with the large
biological protein molecule with the hydrogen bond in tumor cell, and the sulfur-substituted is an
effective pathway to improve anti-tumor activity and reduce toxic side-effects of the leading
compounds. Moreover, the esterification was one of the most effectual reactions in drug combine
principle, which was an effectual strategy for designing new compounds with absolute priority over
than the substitution or substrate [17]. So, esterification was ensured another direction of
modification. In this way, we ensured alkane amination and esterification for two modification
mode. Thirdly, structure of the Topo II-DNA cleavage complex finding that electrophilic group in
the C-4 position of DMEP rests in a spacious binding pocket with relatively few interactions, so it
can be either modified or replaced to produce derivatives with enhanced Top-poisoning activities, it
appears that those non-C4 substitutions would cause steric conflicts and impair the drug-binding
site. Therefore, we favor the use of C-4 substitutions for generating new bioactive DMEP
derivatives. Because the substituent with high electron density in favor of stabilization the complex
of Topo II -DNA cleavage complex, a series of heterocyclic contain electronegative atom (i.e.,
oxygen, nitrogen or sulphur) were chosen as module. Such as benzimidazole [18, 19], imidazole [20,
21], thiophene [22] and Tris(hydroxymethyl)aminomethane, whose derivatives possessed
anti-tumor activity. Taking these factors into account, nine new alkane amination podophyllum
derivates were preliminary designed. Furthermore, according to drug combine principle some of
groups and compounds with pharmacological activity were chosen as function module. Such as
adamantane nucleus [23, 24], phenylalanine nucleus, benzene ring and pyrimidine complexes which
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exhibited a high index of anti-tumor activity against a broad spectrum of cancers and a low
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associated nephrotoxicity [25]. So, eight esterification podophyllum derivates was preliminary
designed. In conclusion, according to the bioisosterism and combine principle, seventeen novel
esterification and alkane amination DMEP derivates were preliminary designed. At last, these
compounds were tested by the interplay of the topoisomerase II docking models for virtual
screening. In this study, a Topo II-DNA cleavage complex was obtained from the Brookhaven
Protein Data Bank (PDB code: 3QX3) [14] and used as the target protein in molecular docking for
virtual screening. As shown in Figure 1, all of the designed DMEP derivates showed good
interaction with DNA Topo II and shared a conformation similar to etoposide in the active site. The
binding energy of seventeen virtual compounds with DNA Topo II was shown in Table 1. The result
indicated that those new DMEP derivates affinity with Topo II as better as etoposide (interaction
energy -9.07 kcal/mol). The binding energy of twelve compounds from -8.87 to -13.20 kcal/mol,
E-4′ hydroxyl oxygen atom forming hydrogen bond with Asp 479, B ring formed π-π bond
interaction with DG-13 or DT-9; in addition the oxygen and nitrogen atom from substituent group
can form hydrogen bond with amino acid residue or basic group. Furthermore, the Ki values in the
in silico are another important parameter for the potential bioactivity evaluation of the designed
compounds [26-29]. Arif’s [26] study describes in silico validation of the wet lab data from
aeroplysinin-1, curcumin and halofuginone using Autodock Tools 4.0. And Successful validation of
the wet lab results of the selected angiogenic targets by in silico method has led to exploit the
hidden potential of in silico tools in preliminary screening of the unknown compounds for
anti-angiogenic potential in a cost-effective manner. Al-Khodairy et al’s [29] in silico results
revealed significant interaction and better inhibition of erysolin to CDK2, CDK6, Bcl2, ER-α and
other apoptosis-related proteins compared to the standard reference compounds, and then elucidated
and validated the potential erysolin-mediated apoptosis mechanism. So, the in silico protocol may
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be a useful mother for the virtual screening of unknown compounds. As shown in Table 1, the in
silico results revealed significant interaction and better inhibition of the designed Compound 1-12
to Topo II compared to the well-known Topo II inhibitor and clinically important podophyllum
anticancer drug etoposide. Compared with etoposide (i.e., the Ki value of 10.35 µM), the inhibited
Topo II activity of Compound 1-12 (i.e., the Ki values rang from 7.45 to 4.12 µM) was significantly
enhanced 25%-100%. Moreover, in silico results revealed no significant interaction and inhibition
of the designed compounds to the apoptosis-related proteins but showed feeble interactions with
pro-apoptotic protein ATR indicate possible involvement of the ATR-dependent DNA damage
response pathway. It has been known ATR pathway is the primary pathway for activating G /M
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checkpoint induction after re-replicat [30]. The results showed that the designed Compound 1-12
are promising natural anticancer. In this effort, twelve novel esterification and alkane amination
DMEP derivates were rationally designed as target compounds by virtual screening, the structure of
target compounds were shown in Schemes 1.
2.2 Molecular modification
The
target
compounds
were
directional
synthesized
by
traditional
cicyclohexylcarbodiimide/4-dimethylamino pyridine reaction and nucleophilic substitution
respectively. Compound 1-5 were prepared according to the reported procedures [31], following
sulfonylation of the hydroxyl groups at the C-4 position of DMEP, reaction with the substituent in
o
acetonitrile at 80 C. Compound 6-12 were also prepared according to the published procedures
[32], DMEP reaction with the substituent in dichloromethane at the normal temperature. The details
were shown in Schemes 1.
2.3 Activity evaluation
(i) Effect of compounds on the Topo II DNA decatenation assay
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DMEP derivates have been reported to induce cell death by inhibiting DNA Topo II activity. So we
investigated the efficacy of the newly synthesized compounds for human DNA Topo II enzyme
inhibiting ability. Kinetoplast DNA (kDNA), a catenated network of mitochondrial DNA seen in
trypanosomes; For DNA Topo II can catalyze the decatenation of kDNA in the DNA Topo II
presence of ATP, the kDNA decatenation assay has been utilized to test drug potential to inhibit
DNA Topo II activity in a cell-free system. As shown in Figure 2. Except Compound 4, all of the
other compounds diminished the relaxation reaction at a concentration of 10 μM. These results
demonstrate that the newly synthesized DMEP derivates could inhibit the human Topo II in vitro.
On the basis of our experimental results, we propose that, like the other DNA Topo II poisons (e.g.,
etoposide), the newly synthesized compounds may ligate and stabilize the DNA Topo II cleavage
complex, which in turn cause permanent double-strand breakage of kDNA. However, in cells, such
a double-strand breakage leads to apoptosis or necrosis [33]. The correctness of Topo II inhibitor
rational design was demonstrated by the Topo II DNA decatenation assay.
(ii) Effect of compounds on the proliferation of human tumor and normal cell lines
In the present study, the antitumor activities in vitro of twelve novel esterification and alkane
amination DMEP derivates on the several human tumor cell lines were investigated. The
antineoplastic drug etoposide (VP-16) was used as reference standard. Cytotoxicity value for DMEP
is included in Table 2 for comparative purposes. When these cells treated with different
concentrations of DMEP derivatives cultured up to 48 h, there was an increase in the percentage of
cells being shrunk observed with inverted microscope. The results of inhibitory proliferation
activity of three human tumor cell lines (BGC 823, A549 and HeLa) were listed in vitro in Table 2.
With few exception, the active analogs showed a remarkable potential antitumor activity,
suggesting that most of twelve novel esterification and alkane amination DMEP derivates could
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enhance antitumor potency. As shown in Table 2, the compounds were in general fairly cytotoxic,
with IC values under the micromolar level, while some of them displayed values close to, or even
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better than, those of the reference DMEP and VP-16. Although the growth of all cell lines was
inhibited by any of the compounds used independently in a dose-dependent manner, the data
demonstrated that different cells display different sensitivity to the same compound. Obviously,
BGC-823 cell lines were the most sensitive to compounds than that of A549 and HK-2 cell lines.
The following apoptosis-inducing research findings revealed that the average apoptotic ratio of
BGC-823 was almost 2 times than A549 or HK-2 cell lines. Furthermore, the results obtained from
Western blotting experiments also supported that DMEP derivatives are able to evoke a widely
apoptotic response in BGC-823 cell lines. So, the activity of inducing tumor cells apoptosis in
BGC-823 cell lines was significantly higher than those of A549 and HeLa cell lines. The above
information may be the reason why BGC-823 cells showed more antitumor activity than other
tumor cells.
As shown in Table 2, the antitumor activity of compounds on tumor cells BGC-823, A549 and
HeLa cell lines was significantly enhanced and the cytotoxicity of compounds on human normal
cell
4-N-(Tris(hydroxymethyl)metylaminomethane)-4-deoxy-4′-demethylepipodophyllotoxin
(Compound 1), 4-N-((3-Aminopropyl)imidazole)-4-deoxy-4′-demethylepipodophyllotoxin
HK-2
cell
lines.
Especially,
the
(Compound 2), 4-N-(Cyclopentylamine)-4-deoxy-4′-demethylepipodophyllotoxin (Compound 5),
and 4-O-(4-Methoxyphenylacetic acid)-4-deoxy-4′-demethylepipodophyllotoxin (Compound 9)
exhibited the better antitumor activity and the lower cytotoxicity on human normal cell. The
anti-tumor activity of Compound 1 against tumor cell line BGC-823 (i.e, the IC value of 5.35 ±
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0.77 µM), HeLa (i.e, the IC value of 160.48 ± 14.50 µM), and A549 (i.e, the IC value of 13.95 ±
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5.41 µM) was significantly improved by 706%, 31% and 900% than that of etoposide (i.e., the IC50
value of 43.74 ± 5.13, 209.90 ± 13.42, and 139.54 ± 7.05 µM), respectively. Moreover, the IC50
value of Compound 1 against the normal human cell line HK-2 (i.e, 16.3 ± 3.77 µM) was 78%
lower than that of etoposide (i.e, 9.17 ± 1.58 µM). Activity evaluation indicates that the substitute
with the long alkyl groups was well tolerated for alkane amination and esterification. Such as the
4-nitrogen-substituted 4′-demethylepipodophyllotoxin derivates with the long alkyl groups,
Compound 1, 2 and 5. Moreover, the regularity was fit for esterification, such as the esterification
4′-demethylepipodophyllotoxin derivates with the long alkyl groups, Compound 9. This result
suggests that the long alkyl group substitution at 4 position of the cycloparaffin ring was beneficial
to enhance DMEP antitumor activity. For DMEP structure modification, the functional modules
with greater electronegativity were well talented because they were preferentially integrated with
the large biological protein molecule (e.g., Topo II) with the hydrogen bond in tumor cell, such as
Compound 1 and 2 which were more potent than Compound 5. This regularity was also fit for
esterification, such as the esterification DMEP derivates Compound 9 and 8 were more potent than
Compound 6. Considering combine principle, all the substituent we chose with some antitumor
activity, but some of them failed in structure modification, These indicates the choice of substituent
group was very important in structure modification and simply combine two different
pharmacoactivity together was not enough.
(iii) Effect of Compound 1 on the BGC-823 cells apoptosis
To understand whether the compounds can induce BGC 823 cell apoptosis or not, an annexin
V-FITC/propidium iodide (PI) binding assay was performed. Because annexin V-FITC has a high
2
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Ca -dependent affinity for phosphatidylserine (PS) residues, the elements of phospholipid on the
inner leaflet locations of PS residues are easily externalized during the progression of apoptosis.
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Three cell populations, including viable (annexin V-FITC, negative; PI, negative), early apoptotic
(annexin V-FITC, positive; PI, negative), and late apoptotic cells or dead cells (annexin V-FITC,
positive; PI, positive), were utilized for this purpose. As shown in Figure 3, the early apoptosis (Q4)
and late apoptosis (Q2) was not observed when a 5 µM of Compound 1 was added at the beginning
of apoptosis process, and then the apoptosis cells reached 8.3% after 6 h of drug treatment. After 12,
24, and 36 h of drug treatment, the apoptosis cells continuously increased and reached its maximal
value of 42.2% after 48 h, and then maintain a stable. The result clearly shows that Compound 1
could significantly induce apoptosis BGC-823 cells at a concentration of 5 µM. Meanwhile, the
early apoptosis (Q4) and late apoptosis (Q2) of BGC 823 cells which treated with Compound 1
increased gradually in a time-dependent manner. The above results showed that Compound 1 could
induce BGC 823 apoptosis. However, the significant of apoptosis-inducing and time-dependent
manner were not observed in HeLa or A549 cell lines (data not shown). This may be the reason
why BGC-823 cells showed more antitumor activity than HeLa and A549 cell lines.
(iv) Effect of Compound 1 on the expression of ATM/ATR in BGC-823 cells
DMEP derivates belongs to the family of Topo II inhibitors. Topo II targeted agents interfere with
the binding between DNA and Topo II or act by increasing the concentration of Topo II-DNA
covalent complexes. These agents shifting the equilibrium of the cleavage/religation reaction can
provoke permanent DNA double strand breaks (DSBs) triggering cell death and/or causing
chromosomal aberrations [34]. DSBs are generally recognized by sensors and transducers,
containing signaling kinases, which can signal repair mechanisms or, if this fails, they could lead to
the activation of ATM/ATR damage signaling pathway and trigger apoptosis. One of the most
important proteins is ataxia-telangiectasia mutated (ATM), a crucial tumour suppressor, which is
activated by DSBs though autophosphorylation at Serine-1981 (ATM-pSer 1981) and Serine-428
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(ATR-pSer 428).
To understand whether the DNA damage induces by Compound 1 could lead to the activation of
ATM/ATR damage signaling pathways, the crucial tumour suppressor were detected by western
blot. At first, the concentration-dependent manner of Compound 1 on Topo II inhibition were
studied and kDNA were treated by Compound 1 at a concentration of 5 µM, 1 µM and 0.1 µM
respectively, as shown in Figure 4a, Compound 1 inhibited the catalytic activity of topoisomerase II
at the concentration of 0.1 to 10 µM but to a lesser extent and totally inhibited the Topo II mediated
relaxation at 10 µM. As shown in Figure 4b, Compound 1 and etoposide could active ATM and
ATR, P-Ser1981 ATM, P-Ser428 ATR and caspase 3 were detected after 48 h of treatment at a
concentration of 10 μM. The above results show that Compound 1 could lead to the activation of
ATM/ATR damage signaling pathways, which consequently increases the activated caspase 3.
(v) The diagram of docking simulations of Compound 1, 2, 5 and 9 with DNA Topo II
Molecular docking was a computational method that predicts how a ligand interacts with a receptor,
which was performed to study the molecular basis of interaction and affinity of binding of the
4′-demethylepipodophyllotoxin derivates with the complex of DNA Topo II [35]. The highly
predictive molecular docking models were derived and molecular affinity that has the highest
impact on anti-tumor activity of more number of hydrogen bond and π-π bond were identified. The
results of docking analysis nicely agree with the results obtained for the in vivo activity propensities
and Topo II inhibition measurements. In general, drugs targeting Topo II act impairing the ability of
the enzyme to religate cleaved DNA or enhancing the forward rate of DNA cleavable complex
formation [36]. The promising anti-tumor agent: Compound 1, 4 and 9 can form more hydrogen
bond as well as π-π bond with Topo II. These compounds can interact with both DNA and Topo II
molecules to form non-covalent complexes thus stabilization the complex of DNA and Topo II.
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On the basis of the docking results and SAR, we identified the binding mode and the detailed
interactions responsible for stabilizing Compound 1, 2, 5 and 9 in the DNA Topo II model. Figure 5
shows the lowest-energy conformations of Compound 1, 2, 5 and 9 in the DNA Top II active center,
calculated using the Autodock program. The results of the docking simulations were self-consistent
in the sense that Compound 1, 2, 5 and 9 were positioned in similar configurations with comparable
interactions with the amino acid residues in the active center. The bound Compound 1, 2, 5 and 9
interact extensively with both protein and DNA (Figure 5). All parts of the function module core
contribute to drug-DNA interaction by being located between base pairs. The drug’s polycyclic
aglycone core (rings A to D) sitting between base pairs, and the function module and the E ring
protruding toward the DNA major and minor grooves, respectively. The E ring is anchored both by
interacting with Asp479 and Arg 503 and the function module were more closely directed toward
the pocket around Gln 778.
4. Conclusion
This work presents the rational design of the novel DNA topoisomerase (Topo II) inhibitors,
esterification and amination substituted 4′-demethylepipodophyllotoxin (DMEP) derivates
exhibiting anti-tumor activity. The active pockets of Topo II and DMEP structure-function
relationship suggested that long alkyl group (e.g., tris(hydroxymethyl)metylaminomethane,
cyclopentylamine, (3-aminopropyl)imidazole, and (4-pyridyl)thioglycolic acid) substitution at 4
position of the cycloparaffin ring was crucial to improve the anti-tumor activity against the sensitive
tumor cell lines and to reduce the cytotoxicity on normal human cell. The antitumour mechanism
indicated that Compound 1 could damage DNA by inhibiting Topo II and then activate ATM/ATR
signaling pathways to induce apoptosis. The correctness of rational drug design was strictly
demonstrated by the anti-tumor activity tests.
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5. Experimental section
5.1 Chemical
Standard 4′-demethylepipodophyllotoxin (98%) was purchased from Shanxi Huisheng Medicament
Technology Company, Ltd (Shanxi, China). N, N'-dicyclohexylcarbodiimide (98%),
4-dimethylaminopyridine (98%), all carboxylic acid (98%), triethylamine (98%), 4-toluenesulfonyl
chloride (98%), acetonitrile (98%), N-(3-aminopropyl)-imidazole (98%), benzimidazole (98%),
2-thiophenemethylamine (98%), tris(hydroxymethyl)metyl aminomethane (98%), and
cyclopentanamine (98%) were purchased from Shanghai Jingchun Medicament Technology
Company, Ltd (Shanghai, China). Precoated silica gel G plates for TLC were purchased from
Merck Inc. (Darmstadt, Germany). Column chromatography was performed with Sephadex LH-20
gel (20–150 µm, Pharmacia & Upjohn Co., Switzerland). Flash column chromatography (FC) silica
gel (SiO ; 200–300 meshes) was purchased from Qing Dao Haiyang Chemical Group Co.
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(Shandong, China). Methanol and acetonitrile were of high-performance liquid chromatography
(HPLC) grade, and all other chemicals used for extraction and isolation were of analysis grade and
commercially available. Deionized water was used throughout the study.
5.2 Computational docking simulations
Molecular docking of compounds into the X-ray structure of the human topoisomerase II, DNA and
VP-16 complex (PDB: 3QX3) from the RCSB Protein Data Bank was carried out by using the
Auto-Dock software (version 4.2) via the graphic user interface Auto-Dock Tool Kit (ADT 1.5.4).
The grid maps of docking studies were computed by using AutoGrid 4, which was included in the
Autodock4 software. The graphical user interface ADT was employed to set up the enzymes; in that,
all hydrogen atoms were added, the Gasteiger charges were calculated, and nonpolar hydrogen
atoms were merged with the carbon atoms. For macromolecules, the generated pdbqt files were
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saved. Then, based on the Auto-Dock software, a docking procedure was applied to position the
conformation of these compounds correctly with regard to their active sites. Grid maps representing
the topoisomerase II protein in the actual docking process were constructed with Auto Grid. The
points of the grids were thus set at 60 × 60 × 60 with a grid spacing of 0.375 Å to ensure that it was
sufficiently large to include not only the active site but also significant portions of the surrounding
surface.
5.3 Analytical method
The chemical reaction solution was alternately washed in deionized water and saturated NaHCO3.
After removing the deionized water layer, drying the AcOEt layer by NaSO , and removing AcOEt
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by rotary evaporation, the residue was ground by aether to obtain a white powder product. The
powder product (2 mg) dissolved in 1 mL methanol/water (50:50 v/v) as pre-separated sample.
Pre-separated sample was filtered (0.45-µm-micropore filter) and transferred into a sampling vial
for HPLC analysis. The samples were filtered with a 0.45 µm micropore filter and transferred to a
sampling vial for HPLC analysis. HPLC analysis was carried out on a Waters 600 Series HPLC
system, equipped with 2487 UV detector. An Akasil C18 column (5 µm, 4.6 mm × 150 mm) was
used. Mobile phase was methanol/water (50:50 v/v) for the detection of alkane amination DMEP
derivatives. Mobile phase was methanol/water (40:60 v/v) for the detection of esterification DMEP
derivatives. The pH of mobile phase was adjusted to 3.00 with formic acid. The HPLC oven
temperature was maintained at 45 °C, and the detection wavelength was 230 nm or 219 nm. The
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flow rate was 0.8 ml/min. All H and C NMR spectra were recorded using a mercury-300BB
spectrometer (Varian, USA). Chemical shifts (δ) are reported in ppm relative to the TMS internal
standard. Abbreviations are as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q
(quartet) and m (multiple). The synthesized compounds are at least 95% pure according to HPLC
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analysis. ESI-MS spectra were obtained with an agility MSD trap mass spectrometer. The
separation was carried on a reversed-phase column with dimensions 150 × 4.6 mm and a particle
size of 5 µm. Thin layer chromatography (TLC) analysis was carried out with a precoated silica gel
G plate, and a small spot of solution containing the samples was applied onto the plate,
approximately 1.5 cm from the bottom edge. The plate was then placed in a chamber with a solvent
system consisting of chloroform/acetone (10:1 to 1:1, v/v). TLC spots were observed under an
ultraviolet light (UV ) and then observed after spraying with H SO /MeOH (10:1 v/v) and heating
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to 110 C.
5.4 General procedure for synthesis of alkane amination 4′-demethylepipodophyllotoxin derivatives
(1-5)
DMEP (400 mg, 5 mmol), triethylamine (1.68 mL), 4-toluenesulfonylchloride (0.96 mL) were
mixed in 40 mL dichloromethane. All reactions were stirred under nitrogen at room temperature for
3 h and monitored by TLC (20 cm × 20 cm glass plates coated with a 0.5 mm layer of Merck silica
gel GF 254). The reaction mixture was washed with saturated aqueous sodium bicarbonate (40 mL
× 2), and deionized water (40 mL × 2). The organic layer was dried over anhydrous Na SO , and
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then the liquid was removed by rotary evaporation in crude solid. Then, the crude solid (1 mmol),
module (3 mmol) were mixed in 10 mL acetonitrile. All reactions were stirred under nitrogen at 80
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C for 48 h and monitored by TLC and then the liquid was removed by rotary evaporation. The
product was purified by silica gel column chromatography to give as a white solid.
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4-N-Tris(hydroxymethyl)metylaminomethane-4-deoxy-4′-demethylepipodophyllotoxin (1). H
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NMR (300 MHz, DMSO, 25 C, TMS) and C NMR (75 MHz, DMSO, 25 C, TMS) H NMR: δ =
6.74 (s, 1H; Ar-H), 6.48 (s, 1H; Ar-H), 6.22 (s, H; Ar-H), 6.04 (s, H; Ar-H), 5.93 (d, J = 6 Hz, 2H;
OCH2O), 4.62 (d, J = 4.8 Hz, 1H; CH ), 4.30 (m, J = 8.4 Hz, 2H; CH ), 3.83 (t, J = 9.9 Hz, 1H,
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CH), 3.60 (t, J =5.1 Hz, 9H, -OCH ), 3.17 (d, J = 9 Hz, 1H, CH), 2.91 (m, 1H, CH), 1.70 (m, 6H,
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CH ), 1.35 (m, J = 11.1 Hz, 3H, OH). C NMR: δ = 180.8 (C-12), 157.1 (C-6), 157.0 (C-7),
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55.531 (3’, 5’-OCH ), 44.321 (C-1), 39.717 (C-2), 39.034 (C-3), 29.591 (C-1’’), 23.516 (C-2’’, 3”,
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4”). EI MS: m/z: 504.0 [M +H], HRMSEI(+) calcd for C H NO , [M] 503.4985 found
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503.1791.
4-N-(3-Aminopropyl)imidazole-4-deoxy-4′-demethylepipodophyllotoxin (2). H NMR (300
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MHz, CDCl , 25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 7.19 (s, 1H;
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CH), 6.76 (s, 2H; CH), 6.47 (s, 2H; Ar-H), 6.22 (s, 2H; Ar-H), 5.93 (d, J = 8.1 Hz, 1H; OCH2O),
4.56 (d, J = 2.7 Hz, 2H; CH ), 4.29 (d, J = 4.5 Hz, 1H; CH), 4.23 (s, 1H; CH), 3.40 (s, 6H; -OCH ),
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3.19 (s, 1H; CH), 1.185 (s, 6H; CH ). C NMR: δ = 175.101 (C-12), 152.631 (C-6, 7), 148.722
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(C-3’), 147.076 (C-5’), 139.833 (C-4’’, 5’’), 131.827 (C-9), 129.340 (C-1’,4’), 127.428 (C-10),
111.144 (C-6’’), 109.875 (C-5,8), 107.990 (C-2’,6’), 101.798 (-OCH O), 67.679 (C-11), 56.521
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(3’,5’-OCH ), 44.249 (C-4), 41.160 (C-1), 40.018 (C-2), 38.533 (C-3),29.917 (C-1”,2”,3”). EI MS:
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m/z: 531.0 [M +Na+H], HRMSEI(+) calcd for C H N O , [M] 507.5351 found 507.5226.
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MHz, DMSO, 25 C, TMS) and C NMR (75 MHz, DMSO, 25 C, TMS) H NMR: δ = 6.95 (s, 1H;
CH), 6.52 (s, 2H; CH), 6.33 (s, 2H; Ar-H), 6.00 (s, 2H; Ar-H), 5.49 (d, 1H; OCH2O), 4.74 (t, J =
1.8 Hz, 1H; CH ), 4.37 (t, J = 4.2 Hz, 1H; CH), 4.21 (m, J = 4.5 Hz, 1H; CH) , 3.67 (s, 6H; -OCH3),
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3.34 (s, 1H; CH), 3.31 (s, 1H; CH), 3.30 (s, 1H; CH), 2.50 (s, 2H; CH2). C NMR: δ=175.547
(C-12), 152.413(C-3’,5’), 147.8 (C-7), 147.1 (C-6), 140.8 (C-4’’), 134.0 (C-9,4’), 131.3 (C-10,1’),
126.8 (C-2’’,3’’), 110.6 (C-1’’), 109.9 (C-5,8), 108.0 (C-2’,6’), 101.8 (-OCH O), 68.4 (C-11), 65.5
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(C-4), 56.6 (3’,5’-OCH3;), 43.8 (C-5’’), 39.8 (C-1), 39.7 (C-2), 38.9 (C-3). EI MS: m/z: 496.1
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25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 8.05 (m, J = 2.7 Hz, 1H;
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CH), 7.79 (d, J = 2.1 Hz, 2H; Ar-H), 7.40 (m, J = 5.7 Hz, 2H, Ar-H), 7.19 (s, 2H, Ar-H), 6.64 (s, 1H,
Ar-H), 6.50 (s, 1H, Ar-H), 6.31 (s, 2H, Ar-H), 5.90 (s, 1H; OCH O), 4.80 (d, J = 4.8 Hz, 1H; CH),
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CH), 2.98 (s, 1H; CH). C NMR: δ = 173.517 (C-12), 153.125 (C-6,7), 149.573 (C-3’), 148.425
(C-5’), 146.529 (C-1’’), 144.849 (C-7’’), 138.782 (C-2’’), 133.735 (C-9), 132.801 (C-4’), 129.436
(C-10,1’), 128.756 (C-4’’), 128.609 (C-5’’), 125.391 (C-3’’), 124.746 (C-6’’), 110.743 (C-5),
109451 (C-8), 109.248 (C-2’), 107.922 (C-6’), 102.246 (-OCH O), 67.545 (C-11), 56.364
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(3’,5’-OCH ), 44.060 (C-4), 42.667 (C-1), 38.249 (C-2), 31.213 (C-3; CH). EI MS: m/z: 502.1
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[M +H], HRMSEI(+) calcd for C H N O , [M] 501.4874 found 501.1536.
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CDCl , 25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 6.86 (s, 1H; Ar-H),
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1H; NH), 4.37 (m, 2H, J = 8.7 Hz, CH ), 3.76 (s, 6H; -OCH ), 3.28 (m, J = 1.5 Hz, 1H; CH), 3.24
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(m, J = 3.9 Hz, 1H; CH), 2.83 (m, J = 9 Hz, 1H; CH), 1.63 (m, 1H; CH), 1.24 (m, 4H; CH ), 0.87
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(m, J = 6.6 Hz, 4H; CH ). C NMR δ = 175.2 (C-12), 146.6 (C-3’,5’), 134.4 (C-4’), 132.4 (C-9),
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5.5 General procedure for synthesis of esterification 4′-demethylepipodophyllotoxin derivatives
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DMEP (400 mg, 1 mmol), carboxylic acid (3 mmol), N, N'-dicyclohexylcarbodiimide (6 mmol) and
4-dimethylaminopyridine (0.05 mmol) were mixed in 5 mL dichloromethane. All reactions were
stirred under nitrogen at room temperature for 24 h and monitored by TLC (20 cm × 20 cm glass
plates coated with a 0.5 mm layer of Merck silica gel GF 254). The reaction mixture was diluted
with dichloromethane (100 mL), washed with saturated aqueous sodium bicarbonate (40 mL × 2),
and water (40 mL × 2). The organic layer was dried over anhydrous Na SO , and then the liquid
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was removed by rotary evaporation in crude solid. The product was purified by silica gel column
chromatography to give as a white solid.
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MHz, CDCl , 25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 7.24 (m, J =
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4.8 Hz, 1H; Ar-H), 6.85 (m, J = 4.8 Hz, 1H; Ar-H), 6.62 (d, J=27.9 Hz, 2H; Ar-H), 5.97 (d, J = 4 Hz,
1H; OCH O), 4.82 (m, J = 13.5 Hz, 1H; CH), 4.35 (m, J = 5.1 Hz, 2H, CH ), 3.64 (s, 6H; -OCH ),
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3.42 (m, 1H; CH), 3.25 (m, J = 2.4 Hz, 1H; CH); 3.14 (m, 1H; CH), 2.06 (s, 4H; CH), 2.04 (s, 4H;
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CH). C NMR δ = 174.9 (C-12,1’’), 152.5 (C-6), 151.8 (C-7), 148.7 (C-3’), 147.6 (C-5’), 140.0
(C-4’) , 137.5 (C-9), 132.0 (C-10,1’), 110.7 (C-5), 110.1 (C-8), 109.0 (C-2’), 108.1 (C-6’), 101.7
(-OCH O), 68.0 (C-11), 66.9 (C-2’’), 56.5 (3’,5’-OCH ), 46.6 (C-1), 44.1 (C-3), 41.2 (C-3), 40.6
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NH), 7.51 (d, 1H; NH), 6.86 (s, 1H; CH-N), 6.55 (d, J = 8.1 Hz, 1H; Ar-H), 6.52 (s, J = 8.1 Hz, 1H;
Ar-H), 6.25 (s, 2H; Ar-H), 5.86 (d, 1H; Ar-H), 5.25 (s, 2H; OCH O), 4.63 (d, 1H; CH), 4.29 (t, J =
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36
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66.8 (C-4), 56.4 (3’,5’-OCH ), 43.1 (C-1), 40.6 (C-2), 38.2 (C-3). EI MS: m/z: 539.2 [M +H],
3
+
HRMSEI(+) calcd for C H N O , [M] 538.4597 found 538.1224.
2
6
22
2
11
1
4-O-((4-Pyridyl)thioglycolic acid)-4-deoxy-4′-demethylepipodophyllotoxin (8). H NMR (300
o
13
o
1
MHz, CDCl , 25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 8.323 (s, 2H;
3
3
CH-N), 7.24 (s, 2H; CH), 6.84 (s, 1H; Ar-H), 6.47 (s, 1H; Ar-H), 6.27 (d, J = 18.8 Hz, 2H; Ar-H),
5.95 (d, J = 5.4 Hz, 1H; OCH O), 4.81 (s, 1H; CH), 4.58 (s, 1H; CH), 4.32 (s, 2H; CH ), 3.98 (s, 6H;
2
2
1
3
-OCH ), 3.73 (s, 1H; CH),, 3.29 (d, J = 11.1 Hz, 1H; CH), 2.76 (d, J = 8.4 Hz, 1H; CH). C NMR δ
3
= 175.3 (C-12), 166.8 (C-1’’), 151.3 (C-6,7), 149.1 (C-5’’,6’’), 148.6 (C-4’’), 148.1 (C-3’), 147.7
(C-5’), 138.7 (C-4’), 132.3 (C-9), 131.5 (C-10), 127.4 (C-1’), 121.0 (C-3’’,7’’), 110.5 (C-5), 109.4
(C-8), 107.6 (C-2’,6’), 101.8 (-OCH O), 67.9 (C-11), 56.2 (3’,5’-OCH ), 44.1 (C-1), 40.6 (C-2),
2
3
+
+
38.3 (C-3), 33.1 (C-2’’). EI MS: m/z: 552.1 [M +H], HRMSEI(+) calcd for C H NO S, [M]
2
8
25
9
551.5644 found 551.1250.
4-O-(4-Methoxyphenylacetic acid)-4-deoxy-4′-demethylepipodophyllotoxin (9). H NMR (300
1
o
13
o
1
MHz, CDCl , 25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 7.24 (s, 1H;
3
3
Ar-H), 7.16 (s, 1H; Ar-H), 6.85 (s, 1H; Ar-H), 6.80 (s, 1H; Ar-H), 6.53 (s, 1H; Ar-H), 6.24 (s, 2H;
Ar-H), 6.08 (s, 1H; Ar-H), 5.98 (d, J = 2.4 Hz, 1H; OCH O), 5.92 (s, 1H), 4.83 (d, J = 2.4 Hz, 1H;
2
CH), 4.18 (m, J = 3.9 Hz, 2H; CH ), 3.78 (d, J = 9.6 Hz, 6H; -OCH ), 3.57 (m, J = 2.1 Hz, 1H; CH),
2
3
13
3.55 (m, J = 5.7 Hz, 1H; CH), 3.14 (d, J = 10 Hz, 1H; CH), 2.81(d, 2H; CH ). C NMR δ = 174.4
2
(C-12), 171.8 (C-1’’), 160.9(C-6’’), 149.1 (C-6,7), 146.6 (C-3’,5’), 134.2 (C-4’), 133.2 (C-9), 130.4
(C-10), 130.2 (C-1’), 127.8 (C-4’’,8’’), 125.4 (C-3’’), 114.4 (C-5’’,7’’), 110.4 (C-5), 109.8 (C-8),
107.8 (C-2’,6’), 101.9 (-OCH O), 68.6 (C-11), 56.6 (3’,5’-OCH ), 55.5 (C-4), 43.8 (C-1), 41.7
2
3
+
(C-2), 40.8 (C-3), 36.9 (C-2’’), 36.9 (C-9’’). EI MS: m/z: 549.1 [M +H], HRMSEI(+) calcd for
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C H O , [M] 548.5378 found 548.1682.
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4-O-(3,4-Dimethoxybenzoic acid)-4-deoxy-4′-demethylepipodophyllotoxin (10). H NMR (300
o
13
o
1
MHz, CDCl , 25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 8.42 (m, J =
3
3
4 Hz, 1H; Ar-H), 8.26 (s, 1H; Ar-H), 8.00 (s, 1H; Ar-H), 7.69 (s, 1H; Ar-H), 7.37 (s, 1H; Ar-H), 7.13
(s, 1H; Ar-H), 6.75 (d, J = 9.9 Hz, 1H; OCH O), 5.53 (d, J = 3.6 Hz, 1H; CH), 5.16 (t, J = 8.7 Hz,
2
1H; CH), 4.68 (m, J = 10.2 Hz, 2H; CH ), 4.64 (d, J = 3 Hz, 6H; -OCH ), 4.44 (d, 6H; -OCH ), 4.13
2
3
3
13
(s, 1H; CH), 4.09 (s, 1H; CH), 3.86 (s, 1H; CH). C NMR δ = 174.5 (C-12), 166.2 (C-1’’), 164.5
(C-5’’), 153.9 (C-6), 153.6 (C-7), 152.2 (C-3’), 148.9 (C-5’), 147.8 (C-6’’), 137.6 (C-3’’), 133.0
(C-4’), 128.2 (C-9), 124.9 (C-10), 124.2 (C-1’), 121.8 (C-4’’), 121.7 (C-7’’), 110.6 (C-5,8), 108.0
(C-2’,6’), 102.0 (-OCH O), 68.7 (C-4), 67.9 (C-11), 57.8 (C-8’’,9’’), 56.4 (3’,5’-OCH ), 44.3 (C-1),
2
3
+
+
42.1 (C-2), 37.3 (C-3). EI MS: m/z: 565.1 [M +H], HRMSEI(+) calcd for C H O , [M]
3
0
28 11
564.5367 found 564.1632.
4-O-(Cyclohexane carboxylic acid)-4-deoxy-4′-demethylepipodophyllotoxin (11). H NMR (300
1
o
13
o
1
MHz, CDCl , 25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 6.74 (s, 1H;
3
3
Ar-H), 6.48 (s, 1H; Ar-H), 6.22 (s, 2H; Ar-H), 6.03 (s, 1H; Ar-H), 5.91 (s, 1H; OCH O),, 4.61 (s, 1H,
2
CH), 4.26 (s, 1H, CH), 3.80 (t, 2H, CH ), 6.60 (s, 6H; -OCH ), 3.17 (d, J = 11.4 Hz, 1H, CH), 3.13
2
3
1
3
(d, J = 11.4 Hz, 1H, CH), 2.90 (d, 1H, CH), 3.67 (s, 7H; CH), 2.55 (m, 1H; CH). C NMR δ =
175.8 (C-12), 174.4 (C-1’’), 151.8 (C-6,7), 148.8 (C-3’), 147.7 (C-5’), 137.2 (C-4’), 132.8 (C-9,10),
128.3 (C-1’), 110.5 (C-5,8), 108.6 (C-2’), 107.8 (C-6’), 101.8 (-OCH O), 67.7 (C-11,4), 56.4
2
(3’,5’-OCH ), 44.1 (C-1), 43.5 (C-2), 43.0 (C-3), 41.8 (C-2’’), 29.4 (C-3’’), 29.2 (C-6’’), 25.7
3
+
+
(C-4’’), 25.5 (C-5’’). EI MS: m/z: 497.1 [M +H], HRMSEI(+) calcd for C H O , [M] 496.5058
27
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found 496.1733.
1
4-O-(Boc-L-2-phenylalanine)-4-deoxy-4′-demethylepipodophyllotoxin (12). H NMR (300 MHz,
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CDCl , 25 C, TMS) and C NMR (75 MHz, CDCl , 25 C, TMS) H NMR: δ = 7.27 (s, 2H; Ar-H),
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7.04 (s, 1H; Ar-H), 6.62 (s, 1H; Ar-H), 6.53 (s, 1H; Ar-H), 6.27 (s, 1H; Ar-H), 6.00 (s, 2H; Ar-H),
4.85 (s, 1H; OCH O), 4.61 (s, 1H; CH), 4.47 (s, 1H; CH), 4.95 (t, J = 15.4 Hz, 1H; CH), 3.97 (t, 1H;
2
CH), 3.66 (m, J = 15 Hz, 6H; -OCH ), 3.31 (d, J = 14.1 Hz, 1H; CH), 3.22 (d, J = 9.9 Hz, 1H; CH),
3
1
3
1.39 (s, 9H; CH ). C NMR δ = 175.9 (C-12), 174.2 (C-1’’), 174.0 (C-9’’), 151.8 (C-6,7), 149.0
3
(C-3’), 147.7 (C-5’), 137.2 (C-4’,9,3”), 132.8 (C-10.1), 128.3 (C-4’’,5”,6”,8”), 110.5 (C-5,8), 109.6
(C-7”), 107.8 (C-2’,6’), 101.8 (-OCH O), 67.7 (C-11,10”), 56.4 (3’,5’-OCH ), 44.0 (C-4), 43.5
2
3
+
(C-1), 42.9 (C-2), 41.6 (C-3), 31.3 (C-11”,12”,13”). EI MS: m/z: 648.1 [M +H], HRMSEI(+) calcd
+
for C H NO , [M] 647.6684 found 647.2400.
3
5
37
11
5.6 Cytotoxicity Assay
Cytotoxicity assay was performed on human lung adenocarcinoma epithelial cell line (A549),
henrietta lacks strain of cancer cells (HeLa), human gastric carcinoma cell line (BGC 823), and
normal human proximaltubular epithelial cell line (HK-2). Cells (3500-13,000) in a 100 µL culture
medium per well were seeded into 96-well microtest plates (Falcon, CA). A549 cell lines, HeLa cell
lines and HK-2 cell lines were cultured in DMEM supplemented with 10% CS, 100 mg/L penicillin
G, 100 mg/L streptomycin. BGC 823 cell lines were cultured in RPMI Medium 1640 supplemented
with 10% CS, 100 mg/L penicillin G, and 100 mg/L streptomycin. The cells were incubated at 37
o
C, in a humidified atmosphere with 5% CO for 48 h. For all cell lines, the microculture
2
tetrazolium [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, MTT; Sigma, St.
Louis, MO] assay was performed to measure the cytotoxic effects. Drug stock solutions (the each
drug concentration was 0.5, 1, 10, 20, 50, 100, 200, and 500 µM, respectively) were prepared in
DMSO and the final solvent concentration was ≤ 2% DMSO (v/v), a concentration without effect
on cell replication. Initial seeding densities varied among the cell lines to ensure a final absorbance
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reading in control (untreated) cultures in the range 0.6-0.8 A492 units. Drug exposure was for 2 days,
and the IC50 value, the drug concentration that reduced the absorbance by 50%, was interpolated
from dose-response data. Each test was performed in triplicate, and absorbance readings varied no
more than 5%. Values were averaged from six treated wells and values for medium alone (lane 1)
were subtracted. These values were then normalized to control (unstimulated cells, lane 2) and used
to calculate the effective half-maximal concentrations for each drug as well as the percentage of cell
death (treated values, normalized to control × 100) induced at ICmax [37].
5.7 Statistical evaluation
Data presented as means ± SD. Statistical analyses were performed by the analysis of variance
(ANOVA). All statistical analyses were performed using Origin version 8.0 (GraphPad Software,
OriginLab Corp., Northampton, MA, USA). Sigmoidal dose responses and non-linear regression
analyses were undertaken to identify half-maximal concentrations for each of the drugs. To evaluate
differences in IC concentrations, analysis of variance combined with Tukey’s multiple range test
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was used.
5.8 Annexin V/PI apoptosis assay
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An amount of 2 × 10 cells/mL was plated in 6 well plates, treated with the test compounds at a
range of concentrations, and incubated for 0, 6, 12, 24, 36, and 48 h. The cells were centrifuged at
1000 rpm for 10 min. The supernatant was discarded, and the cells were resuspended in 100 μL
PBS. Apoptosis using a MultiSciences Biotech kit (Lot: 1200945) kit, and 10 μL annexin V and 5
μL PI was added. The samples in the labeling solution were transferred into Falcon tubes and
o
incubated in a water bath at 37 C for 20 min. The samples were then analyzed using a Becton
Dickinson FAC scan flow cytometer with Cell Quest software. The results were tabulated as % of
annexin V-FITC positive apoptotic cells. The experiments were performed in three replicates and
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repeated at least twice for each compound.
5.9 kDNA decatenation assay
Topoisomerase II activity was determined using kit (Topogen Inc., USA, Cat no. 2000H). The
substrate kDNA (134 ng) and 10 μM drug were combined in assay buffer (5 × Topo II assay buffer
(A+B)) and incubated for 10 min on ice. Next, 4 U of topoisomerase II was added and the reaction
o
was allowed to proceed for 60 min at 37 C. The reaction was stopped by the addition of 1/10
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volume of 1 mg/ml proteinase K and incubated for 15 min at 37 C. The reaction was quenched via
the addition of loading buffer (1% sarkosyl, 0.025% bromophenol blue, and 5% glycerol) and was
then analyzed by electrophoresis on a 1% agarose gel in TBE buffer (89 mM Tris, 89 mM borate,
and 2 mM Na-EDTA, pH 8.3) for 0.5 h at 140 V. The gel was visualized under UV illumination and
photographed on an AlphaImager.
5.10 Immunoblot analysis
o
Total protein was extracted by Mammalian Protein Extraction Kit and stored at -80 C, for
immunoblot analysis, supernatants were treated with sodium dodecyl sulfate (SDS) sample buffer
consisting of 50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, and 0.01%
bromophenol blue, equal amounts of protein were loaded (40μl of protein/lane) into individual lanes,
separated on 6% or 12% gels via SDS-PAGE, and transferred onto PVDF membranes via
electrophoresis (Immobilon-P, Millipore). Membranes were blocked with 5% BSA in TBS-T (for
anti-Bcl), or 5% skim milk in TBS-T (for other antibodies) for 2-4 h at room temperature;
membranes were then incubated with primary antibodies diluted with blocking solution over night
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at 4 C. After three washed with TBS-T, the membranes were then incubated with
peroxidase-conjugated secondary antibodies diluted with blocking solution for 2 h at room
temperature. Then, after three washes with TBS-T, the immunoreactive bands were visualized using
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ECL plus (GE He althca re, Little Chalfont, Buckinghamshire, UK).
ACKNOWLEDGMENT
Financial supports from the National Natural Science Foundation of China (NSFC, Project Nos.
21176059, 21206035, and 21376066), Hubei Provincial Natural Science Foundation for Agriculture
(2012DBA20001), High-Tech Industry Development Program for Innovative Research Team in
Wuhan Municipality (2013070204020049) are gratefully acknowledged. Ya-Jie Tang also thanks
the Chutian Scholar Program (Hubei Provincial Department of Education, China) (2006), Training
Program for the Youth Leading Talents by Ministry of Science & Technology, Program for New
Century Excellent Talents in University (NCET-11-0961), and Training Program for Top Talents in
Hubei Province.
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