One-pot synthesis of podophyllotoxin-thiourea congeners by employing NH2SO3H/NaI: Anticancer activity, DNA topoisomerase-II inhibition, and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bmcl.2015.07.100

A facile one-pot method for the synthesis of novel podophyllotoxin-thiourea congeners has been developed by using NH₂SO₃H/NaI system. Interestingly, 4β-azido podophyllotoxin reduction with concomitant aryl isothiocyanates coupling un

Full text of DOI:10.1016/j.bmcl.2015.07.100

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
One-pot synthesis of podophyllotoxin–thiourea congeners by
employing NH2SO3H/NaI: Anticancer activity, DNA topoisomerase-II
inhibition, and apoptosis inducing agents
Nagula Shankaraiah a, , Niggula Praveen Kumar a,c, Suresh Babu Amula a, Shalini Nekkanti a,
⇑
Manish Kumar Jeengar b, V. G. M. Naidu b, , T. Srinivasa Reddy c, Ahmed Kamal a,c,
⇑
⇑
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
b Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
c Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A facile one-pot method for the synthesis of novel podophyllotoxin–thiourea congeners has been devel-
oped by using NH2SO3H/NaI system. Interestingly, 4b-azido podophyllotoxin reduction with concomitant
aryl isothiocyanates coupling under mild reaction conditions has been achieved. These compounds have
been investigated for their in vitro cytotoxicity against A549, MDA MB-231, DU-145, LNCaP, and HGC-27
cancer cell lines. Some of the representative compounds have selectively exhibited cytotoxicity on
Received 15 May 2015
Revised 27 July 2015
Accepted 30 July 2015
Available online xxxx
DU-145 (human prostate cancer) cells and the most potent compound was 4a (IC50 of 0.50 0.03 lM)
Keywords:
Azido reduction
Sulfamic acid
Sodium iodide
Podophyllotoxin
Thiourea
Cell cycle analysis
Cytotoxicity
with optimal safety therapeutic window (81.7 fold) on normal human prostate cell line (RWPE-1, IC50
of 40.85 0.78). The flow-cytometric analysis of the compound 4a in prostate cancer cells indicated a
strong G2/M-phase arrest and significant topoisomerase II inhibition activity. Furthermore, these com-
pounds induce apoptosis as observed by Acridine Orange and Ethidium Bromide (AO/EB) staining and
Annexin V binding assay. Molecular docking results of the title compounds with topoisomerase-II
presented as theoretical support for the experimental data.
a were
Ó 2015 Elsevier Ltd. All rights reserved.
Topoisomerase-II inhibition
Molecular modeling
Carbamoylation/thiocarbamoylation of nucleophilic com-
pounds plays a significant role for the synthesis of various biolog-
ically important scaffolds.1 A variety of methods for the conversion
of azides into amines have been reported by using radical initia-
tors,2 metal-catalyzed hydrogenations,3 lithium aluminum
hydride,4 borohydrides,5 triphenylphosphine,6 etc. Recently,
Azhayev and co-workers have reported a direct method for the
conversion of azides to urea derivatives by employing trialkylam-
monium hydrogen carbonate via a significant in situ generation
of isocyante.7 However, to the best of our knowledge, the direct
method for the synthesis of thiourea derivatives from organic
azides has not been explored. In the literature, very few protocols
are available for the synthesis of urea/thiourea derivatives by
employing highly toxic phosgene/thiophosgene reagents or addi-
tion of nucleophilic compounds to isocyanates.8 These methods
are applicable to many synthetic conditions, but the majority of
them have several drawbacks, including tedious work-up, long
reaction times, toxic, harsh reaction conditions, and poor selectiv-
ity. In continuation of our earlier efforts in the development of
novel approaches for the azido reductive cyclization towards
bioactive pyrrolobenzodiazepines, podophyllotoxin, and fused
quinazolinones,9 herein we have reported a facile one-pot azido
reduction with concomitant isothiocyanate coupling for the syn-
thesis of novel podophyllotoxin–thiourea congeners by employing
NH2SO3H/NaI reagent system.
Podophyllotoxin (A, Fig. 1) is an antimitotic aryltetralinlignane
isolated from podophyllin, a resin produced by species of the gen-
era Podophyllum such as P. hexandrum and P. peltatum.10 It has dis-
played potent antimitotic activity against various cancer cell lines
but the clinical use was complicated by severe gastrointestinal side
effects.11 Efforts made towards the discovery of less toxic
podophyllotoxin congeners led to the development of several
semi-synthetic derivatives such as etoposide (B, VP-16), teniposide
(C, VM-26) and etopophos (D) as potent anticancer drugs, currently
in clinical use against various malignancies including leukemia,
neuroblastoma, non-Hodgkin’s lymphoma, small-cell lung cancer,
⇑
Corresponding authors. Tel.: +91 40 23073741; fax: +91 40 23073751 (N.S);
tel.: +91 40 27193157; fax: +91 40 27193189 (A.K.).
E-mail addresses: shankar@niperhyd.ac.in (N. Shankaraiah), vgmnaidu@
niperhyd.ac.in (V.G.M. Naidu), ahmedkamal@iict.res.in (A. Kamal).
http://dx.doi.org/10.1016/j.bmcl.2015.07.100
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Shankaraiah, N.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.100

2
N. Shankaraiah et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
H
MeOH, CH2Cl2, DMF and acetonitrile) but no conversion was
R
R1
O
observed. Interestingly, when we attempted the same reaction
with NH2SO3H in combination with NaI, the reactions proceeded
smoothly in 5–6 h at room temperature and acetonitrile solvent
gave the best conversion. Finally, the podophyllotoxin–thiourea
congeners 4a–u was successfully synthesized by reaction with var-
ious isothiocyanates in the presence of NH2SO3H and NaI in good
yields (60–70%).34 The synthetic plan for 4a–u is depicted in
Scheme 1 and the list of the synthesized analogues was shown in
Table 1.
These compounds 4a–u were evaluated for their in vitro
cytotoxicity on A549 (human lung cancer), MDA MB-231 (human
breast carcinoma), DU-145 (human prostate cancer), LNCaP
(androgen-sensitive human prostate adenocarcinoma) and HGC-
27 (Human gastric carcinoma) cell lines by employing 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT
assay). The concentrations of the compounds which produce 50%
inhibition of cell growth (IC50) were compared with that of the
standard drug, that is, etoposide. From close analysis of the IC50
values (Table 2), it is evident that the compounds 4a, 4p, 4s and
4t have selectively exhibited anticancer activity against the human
prostate cancer (DU-145) cell line and the most potent compound
O
O
HO
O
R2
OH
HN
O
O
O
O
O
O
4
1
3
2
O
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
OCH3
A: Podophyllotoxin
H3CO
OCH3
OR3
OH
: R = F; NPF
F: R = NO2; GL-331
: R1 = CH3, R2 = OH, R3 = H;
Etoposide
B
E
C: R1 = thiophenyl, R2 = OH, R3 = H;
Teniposide
D: R1 = CH3, R2 = OH, R3 = -H2PO3;
Etopophos
Figure 1. Representative structures of podophyllotoxin (A) and its semi-synthetic
derivatives (B–F).
soft tissue sarcoma and Kaposi’s sarcoma.12–16 However, side
effects associated with the clinical use of these drugs including
poor bioavailability and the development of drug resistance by
cancer cells has led to the need for the intensification of the
research on novel anticancer drugs based on podophyllotoxin scaf-
fold.17–19 Studies have shown that podophyllotoxin blocks cell
division by inhibition of tubulin assembly into microtubules
through tubulin binding at the colchicine site,20 whereas etopo-
side, teniposide and etopophos were potent irreversible inhibitors
of DNA topoisomerase II.21
was 4a which has shown IC50 0.50 0.03 lM. In the androgen-
sensitive human prostate adenocarcinoma cell line (LNCaP), the
compounds 4a, 4d, 4e, 4h, 4j, 4p, 4s, and 4t have exhibited remark-
able inhibitory activity with the compound 4p being the most
active (IC50 value of 3.56 0.76 lM). Thus, it can be affirmed that
the synthesized compounds are much effective towards human
prostate cancer.
Based on the results of previous structure–activity relation-
ship (SAR) studies on podophyllotoxin, 4b-stereochemistry and
4-N-linkage are essential for topoisomerase II inhibitory activity
and the introduction of bulky groups at C4-position significantly
potentiates the antitumor activity.22 It was observed that, the
sugar moiety of etoposide is not crucial for topoisomerase II
inhibition.23 Various research groups have carried out and reported
extensive structural modifications at C4-position of podophyllo-
toxin,12,24,25 which has led to the development of several N-linked
congeners, such as NPF (E)26 and GL-331 (F),27 among which the
compound GL-331 is currently in phase-II clinical trials against
various resistant malignancies. Recently, we have reported a
series of podophyllotoxin congeners as potential inhibitors of
topoisomerase and tubulin polymerization.28–30 On the whole,
these compounds was found to overcome the limitations of
Etoposide, with superior pharmacological profiles, suggesting the
prospect of further optimization through rational C4 modifications.
On the other hand, thiourea derivatives possess good protein
tyrosine kinases (PTKs) inhibitory activity, which results in
cytotoxic activity.31,32 Novel aroylthiourea–podophyllotoxin
derivatives have been reported and were found to induce G2/M cell
cycle arrest in human colon cancer cells through ATR-Chk1-cdc25C
and Weel signal pathways.33 In order to investigate the propensity
of thiourea moiety to enhance the cytotoxicity of podophyllotoxin
scaffold by tethering, we have herein reported the design and a
direct one-pot synthesis and cytotoxic evaluation of the new
podophyllotoxin–thiourea derivatives 4a–u by employing
NH2SO3H–NaI reagent system.
Fascinatingly, compound 4a has exhibited significant cytotoxic-
ity against all the five cell lines compared to the standard etopo-
side. However, the compounds 4b, 4f, 4g, 4k, 4m, 4n, 4o, 4r, and
4u were found to be inactive in all the tested cancer cell lines. It
is interesting to observe that compound 4p exhibited potent
growth inhibition activity on human lung cancer cell line (A549),
with IC50 value of 14.84 2.46
shown good growth inhibition effect against this cell line with
IC50 values of 37.6 17.1 M and 35.14 5.31 M, respectively,
in comparison with etoposide (38.5 3.6 M) and podophyllotoxin
lM. Similarly, 4a and 4e have also
l
l
l
(128.61 15.1
lM). Compound 4a was only found to display
OH
OH
O
O
O
i) CH3SO3H, NaI
O
O
rt, 1 h
O
ii) BaCO3, acetone/water
(10:1), rt, 15 min, 76%
O
O
H3CO
OCH3
H3CO
OCH3
OCH3
OCH3
1
2
TFA, NaN3
R2
rt, 15 min, 90%
R1
HN
S
R3
R4
NCS
N3
R5
R4
R1
R2
O
O
R5
O
NH
R3
Our synthetic strategy has been started from the preparation of
4b-deoxy-epipodophyllotoxin (2) stereoselectively from the com-
mercially available podophyllotoxin (1) through iodination with
CH3SO3H and NaI followed by nucleophilic substitution in the pres-
ence of BaCO3/H2O. 4b-Azido intermediate 3 has been obtained
from podophyllotoxin (2) by employing NaN3 in the presence of
CF3COOH. At this stage, we have screened the one-pot azido reduc-
tion of intermediate 3 with isothiocyanate by using different
reagents such as ZnBr2 and NH2SO3H in different solvents (THF,
O
O
O
NH2SO3H/NaI
CH3CN, rt, 5-6 h, 60−70%
O
O
H3CO
OCH3
OCH3
3
H3CO
OCH3
OCH3
4a u
−
Scheme 1. Synthesis of novel podophyllotoxin–thiourea (4a–u) congeners.
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N. Shankaraiah et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
trifluoromethyl (4p) at R3 position, has led to the most potent com-
pounds with IC50 of 0.50 0.03 M and 1.96 0.19 M, respec-
Table 1
A series of novel podophyllotoxin–thiourea derivatives 4a–u
l
l
tively. Compound 4p possessing trifluoromethyl substituent at R3
position was found to be the most active against A549 (human
lung cancer), LNCaP (androgen-sensitive human prostate adeno-
carcinoma) and MDA MB-231 (human breast carcinoma) cell lines
with IC50 of 14.84 2.46, 3.56 0.76 and 7.58 0.26, respectively.
The synthesized compounds 4a, 4c, 4p, 4s and 4t which showed
Entry
R1
R2
R3
R4
R5
Yielda (%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
H
H
CH3
NO2
H
OCH3
H
H
H
OCH3
F
H
OCH3
H
H
H
H
H
H
H
H
H
OCH3
H
OCH3
CN
NO2
CH3
OCH3
H
NO2
Cl
OCH3
H
OCH3
H
H
H
F
CF3
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
60
67
70
62
67
64
60
65
62
66
68
60
60
67
62
69
65
62
61
64
60
IC50 values less than 10 lM in the human prostate cancer (DU-145)
cell line were further assayed for cytotoxicity on normal human
prostate cell line (RWPE-1) with etoposide as reference standard,
to determine the specificity towards cancer cells. From the results,
compound 4a was found to be highly specific towards DU-145 cell
line (IC50 values are 81.7 fold higher in RWPE-1 cells) when com-
pared to normal prostate epithelial cells; whereas the IC50 value
for the standard drug etoposide was only 5.3 fold higher in
RWPE-1 cells. Interestingly, the IC50 values for the compounds
4c, 4d, 4e, 4p, 4s and 4t are also 7.2, 6.8, 6.4, 8.6, 19 and 27 fold,
respectively higher in RWPE-1 cells when compared to DU-145
cells. Hence, all these new compounds were found to be more
specific towards prostate cancer cells than the normal prostate
cells in comparison to the standard drug etoposide, with optimal
safety window.
Cl
OCH3
CF3
H
H
F
H
H
H
H
H
H
H
OCH3
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
CH(CH3)2
OCH2C6H5
H
COOC2H5
NO2
CF3
H
H
H
H
H
4u
a
Isolated yields.
On the contrary, compound 4a was 2.3 fold more specific
towards androgen-sensitive human prostate adenocarcinoma
(LNCaP) cell line when compared to normal prostate epithelial
cells; whereas the IC50 value for the standard drug etoposide was
only 1.8 fold higher in RWPE-1 cells. Interestingly, compound 4c
has the similar safety window as that of etoposide while the IC50
values for the compounds 4d, 4e, 4p, 4s and 4t are 1.8, 3.1, 4.7,
3.4 and 11.6 fold, respectively, higher in RWPE-1 cells when com-
pared to LNCaP cells.
The morphological changes in DU-145 cells after treatment
with the compound 4a for 48 h were studied under a phase-con-
trast microscope. Cells treated with 125, 250 and 500 nM of com-
pound 4a for 48 h showed obvious morphological changes, with
chromatin condensation, fragmentation and formation of apoptotic
bodies. However, the control group (without test compound)
significant cytotoxicity in the human gastric carcinoma (HGC-27)
cell line with IC50 value of 3.85 0.27 M. Likewise, compounds
4a, 4c, 4d, 4e, 4h, 4j and 4p were found to be effective in the
human breast carcinoma (MDA MB-231) cell line when compared
to etoposide, with 4p being the most active showing IC50 value of
7.58 0.26.
It is our conjecture to explain the SAR, where the substitution at
R3 position is significant for anticancer activity. The presence of
methoxy substitution at either R1 (4g, 4k and 4n, Table 1) or R2
(4i and 4u) or R4 (4n) or both R2 and R4 (4f) resulted in the loss
of cytotoxicity. Bulky substituents at the R3 position of phenyl ring
as seen in 4q and 4r resulted in no cytotoxic activity. In the human
prostate cancer (DU-145) cell line, presence of small groups
capable of hydrogen-bonding such as methoxy (4a) and
l
Table 2
IC50 (lM) values for the new podophyllotoxin–thiourea derivatives 4a–u by MTT assay
Compounds
A549
DU-145
RWPE-1
LNCaP
HGC
MDA MB-231
4a
4b
37.6 17.1
NA
0.50 0.03*
NA
40.85 0.78
–
17.56 1.45
NA
3.85 0.27
NA
18.0 0.97
NA
4c
4d
4e
4f
44.01 3.15
NA
35.14 5.31
NA
7.89 1.18
10.59 2.39
12.24 0.68
NA
56.69 4.31
71.67 4.7
78.44 3.6
NA
53.15 1.11
39.76 0.77
25.6 1.61
NA
69.61 1.34
60.33 0.03
33.37 3.18
NA
31.77 3.11
19.38 0.03
39.76 0.77
NA
4g
NA
NA
NA
NA
NA
NA
4h
4i
4j
4k
NA
14.13 0.56
58.76 1.02
20.96 1.61
NA
>100
>100
>100
NA
20.02 0.97
>100
22.23 2.11
NA
86.14 8.02
23.67 1.43
12.91 1.15
NA
11.86 0.57
>100
17.92 3.42
NA
65.53 9.35
NA
NA
4l
4m
NA
NA
63.33 1.87
NA
>100
NA
>100
NA
lM
22.43 1.04
NA
>100
NA
4n
NA
NA
NA
NA
NA
NA
4o
NA
NA
NA
NA
NA
NA
4p
4q
4r
14.84 2.46
61.97 9.75
NA
1.96 0.19
59.94 1.23
NA
16.94 1.93
>100
NA
3.56 0.76
84.78 2.33
NA
56.83 3.88
>100
NA
7.58 0.26
>100
NA
4s
4t
4u
>100
>100
NA
2.34 0.04
2.37 0.11
NA
44.50 0.30
64.01 3.79
NA
12.93 1.07
5.49 0.17
NA
86.02 0.23
45.38 0.18
NA
85.66 2.18
>100
NA
Etoposide
Podophyllotoxin
38.5 3.6
128.61 5.1
5.21 0.02
ND
27.64 2.76
ND
50.33 1.04
ND
10.90 1.37
ND
49.35 1.6
ND
NA: Compounds that have not shown 50% inhibition at 100 lM concentration.
ND: Cytotoxicity studies were not done.
*
p < 0.05, when compared to IC50 values with RWPE cells.
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N. Shankaraiah et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
showed normal healthy shape with intact nuclei and without any
abnormalities (Fig. 2a). Most of the treated cells in all the test con-
centrations exhibited similar morphological changes of apoptosis
but the damage was severe in the cells exposed to the highest con-
centration of 500 nM (Fig. 2d).
The results of light microscopy were consistent with that of
fluorescence microscopy using Acridine Orange and Ethidium
Bromide (AO/EB) staining (Fig. 3a–d). The bright condensed chro-
matin identified by the staining was a clear indication of early
apoptosis leading to margination of chromatin into a horseshoe
shaped structure. Cells treated with high concentration, that is,
500 nM of compound 4a exhibited intense nuclear fragmentation
followed by the formation of apoptotic bodies.
In tumors, there are many more cells that are in active cell divi-
sion compared with the normal tissues. As a result, cell cycle arrest
becomes a critical target in oncotherapy. The effect of compound
4a on cell cycle progression was examined by propidium iodide
staining method at concentrations of 125, 250 and 500 nM for
48 h. Compound 4a has shown significant increase (1.85 fold) in
the accumulation of cells at G2/M phase at concentration of
500 nM when compared to the untreated cells. These results indi-
cated that the compound 4a controlled the growth of the prostate
cancer cells by inhibiting the cell cycle at G2/M-phase as shown in
Figure 4.
Figure 3. Morphological changes in DU-145 cells treated with and without
compound 4a for 48 h. Fluorescence microscopy: (a) green live cells show normal
morphology of control; (b) early signs of apoptosis characterized at 125 nM by
extremely condensed chromatin, which marginated into
a horseshoe-shaped
structure and cell membrane blebbing, (c) destructive fragmentation of the nuclei
at 250 nM and irregular distribution of chromatin, (d) late apoptotic cells at 500 nM
exhibited condensed chromatin and their nuclei, stained red with EB.
Apoptosis, the process of programmed cell death (PCD), is an
important target for cancer chemotherapy. The biochemical events
lead to characteristic cell changes, including nuclear fragmenta-
tion, chromatin condensation and fragmentation of chromosomal
DNA. Externalization of phosphatidyl serine is the characteristic
feature of cell undergoing apoptosis which was confirmed by
Annexin V FITC staining technique. After treatment with 0, 125,
250 and 500 nM different concentrations of compound 4a for
48 h to the DU 145 cells, there is an increase in the percentage of
apoptotic cells (Annexin V positive only) in a dose dependent man-
ner with % percentage population of 1.42%, 5.31%, 15.35%, 44.38%,
respectively (Fig. 5). These results confirm that compound 4a inhi-
bits cell proliferation by inducing apoptosis in prostate cancer cells.
Topoisomerase II inhibition assay was performed by using
Topoisomerase II Drug Screening Kit (TG 1009, Topogen, USA) with
etoposide as the standard. From the results, it was found that
catenated DNA treated with topoisomerase II (5 units) has shown
Figure 4. Effect of compound 4a on cell cycle progression of DU-145 cells,
(a) control cells; (b–d) cells treated with 125, 250 and 500 nM for 48 h followed by
analysis of cell cycle distribution using propidium iodide cell staining method and
analyzed by Muse cell analyser. All assays were done in duplicate.
nicked circular and relaxed circular DNA in the Lane E (Fig. 6).
Catenated DNA in the presence of topoisomerase II incubated with
etoposide at a concentration of 100 lM (Lane D) has shown clear
linear DNA formation, indicating that it acts as Interfacial poison
(IFP) which blocks the resealing of the dsDNA. Catenated DNA in
the presence of topoisomerase II incubated with compound 4p at
100
lM, moderately inhibited the topoisomerase II activity
whereas that incubated with 4a at 50 and 100
lM strongly inhib-
ited the catalytic activity, as evident from the highly super coiled
catenated DNA in the wells.
To visualize the binding mode of the compounds 4a–u, molec-
ular docking studies were performed with ATP-binding domain
of human topoisomerase-IIa. The docking analysis has shown that
the scaffold of all the synthesized compounds occupied the ATP-
binding active site with similar orientations, formed hydrogen-
bonding with several surrounding residues and the results are in
Figure 2. Morphological changes observed in light microscopy in DU-145 cells
treated with and without compound 4a for 48 h. (a) Control, cells with intact nuclei;
(b) early signs of apoptosis characterized at 125 nM by extremely condensed
chromatin; (c) cell membrane blebbing, nuclear fragmentation and chromatin
condensation at 250 nM. (d) Decreased cell population and late apoptotic cells at
500 nM.
correlation with the DNA topoisomerase-II
a inhibition assay.
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N. Shankaraiah et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Figure 7. View of compound 4a (yellow) and etoposide (purple) docked in the ATP
binding domain of human topoisomerase-II . The amino acids involved in the
interaction are depicted in stick representation.
a
Figure 5. Annexin V binding assay by MuseTM Cell Analyzer (a) control cells; (b–d)
cells treated with 125, 250 and 500 nM of 4a after 48 h of treatment in DU-145.
Data represents one of three independent experiments.
by employing NH2SO3H/NaI reagent system. Further, these com-
pounds have been evaluated for their in vitro antitumor activity
on selected human cancer cell lines. Some of the representative
compounds have shown promising activity in comparison to eto-
poside with optimal safety window. One of the compounds 4a
exhibited broad spectrum of anticancer activity against all the
tested cancer cell lines and induced apoptosis in human prostate
cancer (DU-145) cell line with DNA–topoisomerase II inhibitory
activity and strong G2/M cell cycle arrest. The docking results have
also shown similar orientation and interactions at the ATPse
domain of topoisomerase-IIa compared to the standard etoposide.
Overall, these preliminary results of podophyllotoxin–thiourea
derivatives offer an impetus for the future endeavors in the drug
discovery as potent antitumour agents.
Figure 6. Effect of the synthesized compounds 4a and 4p on topoisomerase II. Lane
A: Linear DNA; Lane B: Decatenated DNA; Lane C: Catenated DNA; Lane D:
Acknowledgments
Catenated DNA + Topoisomerase II (5 units) + (Etoposide: 100
Catenated DNA + Topoisomerase II (5 units); Lane F: Catenated DNA +
Topoisomerase II (5 units) + (4p: 50 M); Lane G: Catenated DNA + Topoisomerase II
(5 units) + (4p: 100 M); Lane H: Catenated DNA + Topoisomerase II (5 units) +
(4a: 50 M); Lane I: Catenated DNA + Topoisomerase II (5 units) + (4a: 100 M).
lM); Lane E:
Authors are thankful to DoP, Ministry of Chemicals and
Fertilizers Govt. of India, New Delhi for the award of Research
Fellowship.
l
l
l
l
Supplementary data
Figure 7 represents the binding pose of 4a superimposed on etopo-
side in the binding pocket, where it is surrounded and forms strong
hydrophobic interactions with Asn91, Asn95, Arg98, Gly124,
Ile125, Pro126, Ile141, Gly166, and Gly164 residues similar to
etoposide. The methoxy oxygen at R3 in 4a forms hydrogen bonds
simultaneously with the back bones of three amino acid residues:
Gly166, Gly164 and Tyr165 whereas the hydroxy group of etopo-
side hydrogen bonded with only Ile141. This explains the higher
cytotoxicity of 4a in comparison with etoposide. All these strong
interactions of the ligands with the magnesium ion and residues
below the ATP binding site (Asn46, Asp73, Ile78, Arg136 and
Tyr165) suggest that the binding of our ligands with topoisomerase
II impedes the entry of ATP into it. The molecular docking here can
provide an insight for the design of the novel podophyllotoxin
derivatives in order to improve the binding affinity of the com-
pounds and to compete effectively with ATP.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07.
100.
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