A convergent synthesis of alkyne-azide cycloaddition derivatives of 4-α,β-2-propyne podophyllotoxin depicting potent cytotoxic activity

Article abstract of DOI:10.1016/j.ejmech.2014.02.030

A facile synthetic approach to construct the O-propargyl derivatives of 4α and 4β-(1,2,3-triazol-4-yl)-podophyllotoxin (9a-k & 10a-k) and 4′-Demethyl-4′-4β-(1,2,3-triazol-4-yl)-epipodophyllotoxin (12a-d) were synthesized by means of click chemistry. The c

Full text of DOI:10.1016/j.ejmech.2014.02.030

European Journal of Medicinal Chemistry 77 (2014) 47e55
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A convergent synthesis of alkyneeazide cycloaddition derivatives of
4-
-2-propyne podophyllotoxin depicting potent cytotoxic activityq
a,b
,
,
Mahesh K. Zilla a 1, Debasis Nayak b 1, Ram A. Vishwakarma a, Parduman Raj Sharma b,
Anindya Goswami b 1, Asif Ali a
,
, ,1
*
a Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
b Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A facile synthetic approach to construct the O-propargyl derivatives of 4a and 4b-(1,2,3-triazol-4-yl)-
podophyllotoxin (9aek & 10aek) and 40-Demethyl-40-4
b-(1,2,3-triazol-4-yl)-epipodophyllotoxin (12a
Received 10 October 2013
Received in revised form
31 December 2013
Accepted 12 February 2014
Available online 14 February 2014
ed) were synthesized by means of click chemistry. The chemical structures were confirmed by 1H, 13C,
2D NMR and HRMS spectral analysis and their cytotoxicities were measured against diverse human
cancer cell lines viz. PC-3, PANC-1, COLO-205 and A-549 by MTT assay. Some of the compounds were
found more potent than the parent molecule Podophyllotoxin, like; 9a & 10a, 9h & 10h, 9k & 10k, 10d, 8
and 12a. The most potent molecule discovered was compound 9k that exhibited the highest cytotoxicity
on all the four cancer cell lines with IC50 values of 3.8e22 nM. The compound further found to induce
apoptosis and strongly hindered the motility of aggressive prostate cancer PC-3 cells.
Keywords:
Podophyllotoxin
Triazole
Apoptosis
Motility
Ó 2014 Elsevier Masson SAS. All rights reserved.
Migration
1. Introduction
cytotoxic properties which bind to tubulin during mitosis and
inhibit microtubule assembly [5,6].
Natural product is the key source of drug discovery and design
almost about 25% prescribed medicines originate from of plant.
When we talk about natural products role upon anticancer,
approximately 60% of all drugs now in clinical trials for the multi-
plicity of cancers are either natural products, derived from natural
products, or contain pharmacophores derived from natural prod-
ucts [1,2]. Usually the structures of natural products are quite
complex, incorporating intricate ring systems and large numbers of
stereogenic centres. That is why the synthetic modification of
natural product-based libraries inevitably involve to achieve the
new biological knowledge.
Due to side effects of podophyllotoxin, such as nausea, vomiting,
neutropenia and damage of normal tissues, the clinical utility of
podophyllotoxin as an antitumour agent largely have been aban-
doned. Therefore, the structural modifications become a hotspot of
anticancer study [7]. So far the extensive clinical utility of podo-
phyllotoxin as an antitumour agent and the remarkable biological
activity for the development of new therapeutic agents based on
structural modifications of such compound opened a door to
develop unlimited podophyllotoxin leads with various modifica-
tions. Such as Etoposide (4), Etopophos (5), NK611 (6), Tafluposide
(7) [8,9] in (Fig. 1).
Several important anticancer natural products such as vinblas-
tine, vincristine, paclitaxel, and the semi-synthetic drugs Etoposide,
Etopophos and Teniposide [3,4] are the derivatives of podophyllo-
toxin, a potent natural product belonging to the cyclolignan which
is most abundant naturally occurring compound isolated mainly
from Podophyllum peltatum and Podophyllum hexandrum has
So far several distinct derivatives of 1 were made for the
development of new leads chemical entities indicates that the
trans-lactone, the 4
inhibitory activity [10,11]. There are several reports
Substituted derivatives of 1 owing improved cytotoxicity related to
1 [12e14]. Recently, 4 -1,2,3-triazole derivatives of 1 have been
b
-substituted moiety, are essential for TOP-II
4
b
-N-
b
shown to exhibit more potent anticancer activity and better bind-
ing ability to TOP-II than etoposide [15e17].
In literature 4b-substituted congeners is a promising source of
new anticancer agents [18e20]. This is the first report that we
introduce the O-propargyl at 4-OH group in the presence of Boron
trifluoride etherate (BF3.OEt2) afforded the distereomer 4a
q
IIIM communication number: IIIM/1611/2013.
* Corresponding author. Academy of Scientific and Innovative Research, New
Delhi, India.
E-mail addresses: asifali@iiim.ac.in, asifchem73@gmail.com (A. Ali).
Academy of Scientific and Innovative Research, New Delhi, India.
1
http://dx.doi.org/10.1016/j.ejmech.2014.02.030
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

48
M.K. Zilla et al. / European Journal of Medicinal Chemistry 77 (2014) 47e55
Fig. 1. Structure of Podophyllotoxin (1), Epipodophyllotoxin (2), 40-Demethyl Epipodophyllotoxin (3), Etoposide (4), Etopophos (5), NK-611 (6), Tafluposide (7).
(podophyllotoxin) and 4
b
(epipodophyllotoxin) which is not easily
flash column chromatography afforded 4
yl) podophyllotoxin (9e10aek) with good yield.
The other natural product that we isolated from P. hexandrum is
40-O-demethyl-4 -epipodophyllotoxin which C-40 and 4
-hydroxy
a and 4b-(1,2,3-triazol-4-
separable by column chromatography finally we develop the
separable condition in HPLC and separate the disteromeric mixture
in 55:41 ratio. Our delight, herein we found that the relative ratio of
two stereoisomers were separable by column chromatograph when
it were coupled with distinct azide derivatives. Finally we synthe-
sized the (9,10aek) distinct derivative of 1 and four different ana-
logues (12aed) of 3 (Scheme 1) were evaluated their cytotoxicities
against a panel of four human cancer cell lines. The most active
propargylated (9k) was evaluated for its apoptosis inducing, anti-
motility and antimigratory potential on human prostate cancer PC-
3 cells.
b
b
group were treated with NaH and propargyl bromide afforded the
dipropargylated derivative of 11 which were subsequently coupled
with four distinct derivatives of azide (Scheme 1) afforded (12aed)
in 80e90% yield respectively.
All the propargylated derivatives of 1 were characterized by 1H,
13C, 2D NMR spectral data and high-resolution mass spectrometry.
The 1H NMR data of podophyllotoxin (
a
) differ from that of epi-
) by chemical shift values of
4.78 (d, J ¼ 9.4 Hz),
4.73 (d, J ¼ 3.2 Hz). These differences, as well as
the multiplicity and coupling constants for H-4, indicate
orientation which is the bioconversion product (Fig. 1). The
remaining others major difference that we observed for -isomer
was H-1, H-2, H-3 protons were resonated at
4.59 (dd, J ¼ 8.6,
podophyllotoxin (
b
d
whereas -form
b
d
a
& b-
2. Results and discussion
a
2.1. Chemistry
d
7.2 Hz), 2.78 (dd, J ¼ 14.3, 4.6 Hz) and 2.83e2.96 (m) where as the
b-
Click chemistry is most fascinating and an effective approach for
the generation of drug candidates [21]. Several research groups
used the click chemistry as a synthetic tool for the generation of
valuable scaffolds to achieve the new biological knowledge [22,23].
The chemistry which involves copper(I)-catalyzed 1,2,3-triazole
formation from azides in the terminal acetylenes is a powerful
tool for the generation of privileged medicinal scaffolds, due to its
high degree of dependability, complete specificity, and the
biocompatibility of the reactants [24,25]. The synthetic route that
we follow to construct the new derivatives (9,10aek, and 12aed) is
exhibiting in (Scheme 1).
The compound 1 was dissolved in dry DCM which was treated
with BF3.OEt2 (1.2 eq) at 0 ꢀC after five minutes added propargyl
alcohol and warm to the room temperature and stirred for 1 h
afforded disteromeric mixture of propargylated podophyllotoxin in
55:41 ratio (96% yield) which we were separated by HPLC with the
great hardship (see Fig. S3 in supporting information). Finally we
dissolve the disteromeric mixture in t-BuOH:H2O (1:1) and added
sodium ascorbate followed by copper sulphate after 5 min added
distinct derivatives of azide and stirred the reaction mixture at
70 ꢀC. Herein we found that two disteromers were resolve it might
be possible due to attachment of bulky 1-azido substituents. Using
this reaction, we synthesized distinct derivatives of 8 which were
subsequently isolated the distereomeric mixture by exhaustive
isomer proton resonated as 4.54 (d, J ¼ 5.4 Hz), 3.32 (dd, J ¼ 14.0,
5.4 Hz) see the Figs. S4e6 in supporting information). Based on 1H
NMR chemical shifts were also in good agreement with those re-
ported for
a & b podophyllotoxin [26].
Furthermore the stereochemistry of 9j and 10j was authenti-
cated by the 1H NMR and HSQC correlation by the presence of a
peak at
2.95-3.07 (m, H-3) and 4.78 (d, J ¼ 11.9 Hz) for
which correlated with 71.2 (C-1), 45.48 (C-2), 37.81 (C-3) and 61.6
(C-4). The mark difference that we observed in proton and HSQC
spectrum of 10j which resonated at
4.64 (d, J ¼ 5.3 Hz, H-1), 3.45
(dd, J ¼ 14.0, 5.4 Hz, H-2), 2.90e3.01 (m, H-3) and 4.78 (d,
proton which correlated with 73.5 (C-1), 40.4 (C-
d
4.64 (d, J ¼ 7.4 Hz, H-1), 2.87 (dd, J ¼ 14.3, 4.7 Hz, H-2),
b
hydrogen of H-4
d
d
J ¼ 3.2 Hz) for
a
d
2), 37.6 (C-3) and 62.3 (C-4). The C-5 proton of the triazole ring in
the aromatic region at a chemical shift of w7.97 ppm (Figs. S52e61
in SI).
2.2. Biological activity
All the compounds; 9,10aek,12aed, 8 & 11 were screened for its
anticancer activity against a panel of four different human cancer
cell lines; PC-3, PANC-1, COLO-205 and A-549, procured from Eu-
ropean Collection of Cell Culture (ECACC).

M.K. Zilla et al. / European Journal of Medicinal Chemistry 77 (2014) 47e55
49
Scheme 1. Synthesis of O-propargyl derivatives 9,10aek and 12aed. Reagents and conditions: (i) BF3.OEt2, propargyl alcohol, DCM, 1 h (95%) (ii) Sodium ascorbate, CuSO4.5H2O, t-
BuOH/H2O (1:1), 1-azido (aek) (iii) H2O:HCl (1:1), 0 ꢀC, aq. NaNO2, aq. NaN3, 2 h (aek), 85e96% (iv) NaH, DMF, 0 ꢀC, Propargyl bromide, 80%.
Cell viability assay was performed to evaluate the cytotoxic
potential of these novel O-propargyl derivatives of Podophyllo-
toxin. We were expecting for strong activities in lower nano-molar
concentration of all these derivatives in comparison to its parent
moiety and also had a desire to explore the difference in anticancer
205 and A-549 cells respectively (Fig. 2a), was accordingly selected
for further study of its anticancer property.
Apoptosis is a form of cell death which involves the activation of
different caspases and other signalling molecules for the pro-
grammed death of the cell [27]. We performed apoptosis assay to
examine the effect of 9k on apoptosis. Interestingly, we observed a
sufficient number of cells undergoing apoptosis even at much
lower concentration (10e30 nM) of 9k in comparison to positive
control Etoposide. Upon treatment with 9k, morphological changes
like nuclear cleavage, membrane blebbing, chromatin condensation
and cell shrinkage were distinct under microscope showing hall-
marks of apoptosis (Fig. 2b).
activity among the
a and b isomers of this group of derivatives.
Interestingly, our results revealed most of the compounds exerted
their promising cytotoxicity on three different cancer cell lines; PC-
3, PANC-1 and COLO-205, whereas lung cancer A-549 cells were
found less responsive towards some of the compounds (Table 1).
Especially, the derivatives 9e10a, 9e10h, 9e10k, 10d, 8 and 12a
were found to be active against at least three of the four human
cancer cell lines tested with potency greater than or close to the IC50
value of the parent Podophyllotoxin. For the first time we have
Cell motility and migration correlates with the invasiveness and
metastatic behaviour of the cancer cells [28]. To test the effect of 9k
on cancer cell migration, colony formation assay was carried out
and it showed that compound 9k could block the colony formation
explored
showed better activity than the
a
-isomers of the compounds like; 9c, 9e, 9f, 9h, 9j and 9k
-isomers of this class of com-
b
pounds in all the four cell lines tested. Collectively, all these de-
rivatives synthesized in our laboratory demonstrated their
excellent cytotoxic potential against diverse tumours. Out of these
potent derivatives, compound 9k was active against all the four cell
lines with IC50 values 12, 3.8, 6.3 and 22 nM in PC-3, PANC-1, COLO-
ability of PC-3 cells in a dose dependent manner compared to 1 mM
Etoposide (Fig. 3a). We further performed scratch motility/wound
healing assay to assess the motility capability of 9k on PC-3 cells.
After creating the wound over the cell’s confluent monolayer, cells
were treated with or without compound 9k along with Etoposide

50
M.K. Zilla et al. / European Journal of Medicinal Chemistry 77 (2014) 47e55
Table 1
(Q-ToF) Mass Spectrometer, (LTQ XL/LTQ Orbitrap Discovery)
coupled to Agilent 1290 series LC system (Agilent PDA detector,
autosampler, and thermostat (thermostated column compartment,
binary pump)). HPLC separations were made using waters spher-
isorb reversed-phase (C18, 250 ꢁ 10 mm, L i.d.) column connected
to an Agilent 1200 series binary pump and monitored using an
Agilent photodiode array detector. Detection was carried out at 220,
230, 254, 280, and 350 nm. Optical rotations were measured on a
Perkin Elmer 341 polarimeter in a 1 dm cell at 25 ꢀC. All reactions
were monitored by thin layer chromatograph (TLC) on silica gel
GF254 (0.25 mm thick). Column chromatography (CC) was per-
formed on Silica Gel 60 (230e400 mesh, Merck Chemical Ltd., In-
Cytotoxic potential of O-propargyl 9,10aek and 12aed at drug exposure 48 h.
IC50 values (
mM)
PC-3
PANC-1
COLO-205
A-549
1
9a
10a
9b
10b
9c
10c
9d
10d
9e
10e
9f
10f
9g
10g
9h
10h
9i
10i
9j
10j
9k
10k
3
8
11
12a
12b
12c
12d
0.020 ꢄ 0.026
0.016 ꢄ 0.032
0.021 ꢄ 0.08
0.233 ꢄ 0. 25
0.023 ꢄ 0.09
0.019 ꢄ 0.12
0.224 ꢄ 0.233
0.254 ꢄ 0.221
0.0072 ꢄ 0.06
0.283 ꢄ 0.164
3.45 ꢄ 0.621
0.022 ꢄ 0.032
0.333 ꢄ 0.435
0.023 ꢄ 0.052
0.023 ꢄ 0.045
0.012 ꢄ 0.022
0.023 ꢄ 0.055
0.205 ꢄ 0.12
0.246 ꢄ 0.159
0.019 ꢄ 0.056
0.322 ꢄ 0.445
0.012 ꢄ 0.116
0.020 ꢄ 0.048
0.025 ꢄ 0.052
0.014 ꢄ 0.069
0.032 ꢄ 0.081
0.019 ꢄ 0.068
3.15 ꢄ 0.899
0.251 ꢄ 0.33
0.259 ꢄ 0.33
0.023 ꢄ 0.033
0.031 ꢄ 0.025
0.0205 ꢄ 0.13
0.017 ꢄ 0.06
0.016 ꢄ 0.16
0.0094 ꢄ 0.09
0.012 ꢄ 0.19
0.018 ꢄ 0.161
0.011 ꢄ 0.098
0.014 ꢄ 0.082
0.0206 ꢄ 0.104
0.0205 ꢄ 0.08
0.017 ꢄ 0.058
0.018 ꢄ 0.033
0.034 ꢄ 0.091
0.0043 ꢄ 0.31
0.019 ꢄ 0.048
0.0205 ꢄ 0.112
0.021 ꢄ 0.086
0.004 ꢄ 0.124
0.023 ꢄ 0.121
0.0038 ꢄ 0.045
0.0058 ꢄ 0.066
0.018 ꢄ 0.088
0.0046 ꢄ 0.09
0.0065 ꢄ 0.32
0.012 ꢄ 0.053
0.066 ꢄ 0.021
0.014 ꢄ 0.35
0.013 ꢄ 0.21
0.021 ꢄ 0.041
0.008 ꢄ 0.011
0.127 ꢄ 0.216
0.101 ꢄ 0.031
0.138 ꢄ 0.33
0.025 ꢄ 0.54
0.109 ꢄ 0.188
0.261 ꢄ 0.224
0.036 ꢄ 0.091
0.0059 ꢄ 0.061
0.077 ꢄ 0.085
0.018 ꢄ 0.028
0.179 ꢄ 0.34
0.016 ꢄ 0.034
0.017 ꢄ 0.085
0.0052 ꢄ 0.29
0.076 ꢄ 0.252
0.012 ꢄ 0.036
0.140 ꢄ 0.364
0.013 ꢄ 0.129
0.036 ꢄ 0.089
0.0063 ꢄ 0.085
0.0053 ꢄ 0.091
0.016 ꢄ 0.035
0.018 ꢄ 0.038
0.0083 ꢄ 0.4
0.024 ꢄ 0.018
0.014 ꢄ 0.026
0.011 ꢄ 0.189
0.096 ꢄ 0. 138
0.031 ꢄ 0.109
0.020 ꢄ 0.112
0.024 ꢄ 0.22
0.173 ꢄ 0.31
0.346 ꢄ 0.345
0.170 ꢄ 0.32
0.175 ꢄ 0.321
0.196 ꢄ 0.133
0.023 ꢄ 0.018
0.315 ꢄ 0.145
0.416 ꢄ 0.203
0.175 ꢄ 0.54
3.8 ꢄ 0.633
dia). Podophyllotoxin and 40-O-demethyl-4
b-epipodophyllotoxin
were isolated from Indian Medicinal herb P. hexandrum. Other
chemicals and reagent were purchase from Sigma Aldrich.
0.322 ꢄ 0.81
0.308 ꢄ 0.344
0.173 ꢄ 0.32
0.011 ꢄ 0.134
0.424 ꢄ 0.33
0.020 ꢄ 0.045
0.175 ꢄ 0.341
0.150 ꢄ 0.213
0.022 ꢄ 0.181
0.167 ꢄ 0.233
0.023 ꢄ 0.044
0.020 ꢄ 0.057
0.232 ꢄ 0.113
0.026 ꢄ 0.055
0.020 ꢄ 0.022
0.023 ꢄ 0.14
0.025 ꢄ 0.082
4.1.1. General procedure of click chemistry
Dissolve the podophyllotoxin 1 g (2.41 mmol) in dry Dichloro-
methane (DCM) cool at 0 ꢀC and added BF3.Et2O (315
ml, 2.90 mmol)
after five minutes added propargyl alcohol (145
ml, 3.6 mmol) and
stirred the reaction mixture for 1 h. After completion the reaction
mixture was diluted with 100 ml of water and extracted with DCM
(3 ꢁ 100 ml). Finally the combined extracts were dried over Na2SO4
and evaporated in vacuo afforded the inseparable mixture of 4a,b-
propargylated podophyllotoxin (1.05 g, 2.31 mmol) 95% yield. It
was not possible to isolate from flash column chromatography ul-
timately we develop the separative condition in semipreparative
HPLC using (C-18 Column, 5
m
, 10 ꢁ 250 mm, isocratic H2OeCH3CN
(1:1) for 20 min, flow rate 0.8 ml/min, UV detection at 254 nm).
Compound 8 was taken 10 mg from that we isolated the 8a (5 mg,
tR ¼ 7.9 min), 8b (4 mg, tR ¼ 8.2 min) were obtained under these
conditions (see Fig. 3SI).
IC50 values are determined as the mean ꢄ SD of three independent experiments
performed.
Bold indicated in the table were the activity profile of the parent compound.
4.1.1.1. 4
a
,
b
-(4-O-Propargyl)-podophyllotoxin (8). Yield: 95%; white
7.07 (s,
for 24 h. Images were captured for the 0 h (while creating the
wound) and 24 h (during termination), compared with untreated
and positive control. Complete healing of the wounded area was
observed in the DMSO/vehicle treated well (control), whereas
healing was strongly abrogated at the sub-lethal dose of 9k
(30 nM), thereby inhibiting the migration ability of these cells
(Fig. 3b). Collectively, these experimental findings demonstrated
that 9k effectively blocked the colony formation ability and motility
of prostate cancer PC-3 cells.
powder solid; mp: 157e159 ꢀC; 1H NMR (500 MHz, CDCl3)
d
1H), 6.95 (s, 1H), 6.56 (s, 1H), 6.51 (s, 1H), 6.38 (s, 2H), 6.25 (s, 2H),
5.99 (dd, J ¼ 12.0,1.2 Hz, 2H), 5.97 (t, J ¼ 1.6 Hz, 2H), 4.85 (d, J ¼ 9.5 Hz,
1H), 4.80 (d, J ¼ 3.2 Hz, 1H), 4.66 (dd, J ¼ 8.6,7.3 Hz, 1H), 4.60 (dd,
J ¼ 16.9, 5.0 Hz, 2H), 4.42e4.34 (m, 3H), 4.27 (d, J ¼ 2.4 Hz, 2H), 4.22e
4.19 (m,1H), 4.19e4.14 (m,1H), 3.81 (s, 3H), 3.80 (s, 2H), 3.75 (s, 6H),
3.74 (s, 4H), 3.39 (dd, J ¼ 14.0, 5.4 Hz, 1H), 3.01e2.87 (m, 2H), 2.84 (
dd, J ¼ 14.3, 4.6 Hz,1H), 2.58 (t, J ¼ 2.4 Hz,1H), 2.55 (t, J ¼ 2.4 Hz,1H);
HRMS (ESI) 453.1539 for [MþH]þ (calcd 452.1471 for C25H24O8).
3. Conclusions
4.1.1.2. 4a-(4-O-Propargyl)-podophyllotoxin (8a). Yield: 55%; white
powder solid; mp: 146e149 ꢀC; ½a D25
ꢂ
¼ ꢃ23.6 (c ¼ 0.2, CHCl3); 1H
O-propargyl derivatives of 4
a
and 4
b
-(1,2,3-triazol-4-yl) podo-
-(1,2,3-triazol-4-yl)-
NMR (400 MHz, CDCl3) d 7.00 (s, 1 H), 6.44 (s,1 H), 6.31 (s, 2 H), 5.90
phyllotoxin (9,10aek) and 40-Demthyl-40-4
b
(d, J ¼ 4.1 Hz, 2H), 4.78 (d, J ¼ 9.4 Hz, 1H), 4.59 (dd, J ¼ 8.6, 7.2 Hz,
1H), 4.32 (dd, J ¼ 16.2, 2.4 Hz, 1H), 4.15e4.06 (m, 2H), 3.74 (s, 3H),
3.68 (s, 6H), 2.96e2.83 (m,1H), 2.78 (dd, J ¼ 14.3, 4.6 Hz,1H), 2.47 (t,
J ¼ 2.3 Hz,1H); HRMS (ESI) 453.1521 for [MþH]þ (calcd 452.1471 for
epipodophyllotoxin (12aed) were made by click chemistry which
exhibiting the promising in vitro cytotoxic activity in distinct panel
of human cancer cell lines. Further, this class of compounds have
their potential to induce apoptosis and can abrogate motility and
migration in a low sub-toxic concentration, which confers their
anti-invasive and anti-metastatic behaviour towards aggressive
cancer cells. Our results suggest that these compounds have po-
tential for further development as anticancer agents.
C25H24O8).
4.1.1.3. 4
b
-(4-O-Propargyl)-epipodophyllotoxin (8b). Yield: 45%;
white powder solid; mp: 151e154 ꢀC; ½a D25
¼ ꢃ16.2 (c ¼ 0.2, CHCl3);
ꢂ
1H NMR (500 MHz, CDCl3)
d 6.88 (s, 1H), 6.49 (s, 1H), 6.18 (s, 2H),
5.92 (dd, J ¼ 12.8, 1.2 Hz, 2H), 4.73 (d, J ¼ 3.2 Hz, 1H), 4.54 (d,
J ¼ 5.4 Hz, 1H), 4.36e4.25 (m, 2H), 4.20 (d, J ¼ 2.3 Hz, 2H), 3.73 (s,
3H), 3.67 (s, 6H), 3.32 (dd, J ¼ 14.0, 5.4 Hz, 1H), 2.91e2.78 (m, 1H),
2.50 (t, J ¼ 2.3 Hz, 1H); HRMS (ESI) 453.1542 for [MþH]þ (calcd
452.1471 for C25H24O8).
4. Experimental
4.1. Chemistry
Melting points were recorded in Buchi apparatus and uncor-
rected. 1H, 13C and 2D NMR experiments were accomplished on a
Brucker, 400 & 500 MHz spectrometer. High-resolution mass
spectral data were obtained on an Agilent 6540 UHD Accurate Mass
Furthermore we made the 1-azido derivatives by aniline
substituted moiety which were dissolved in H2O:HCl (1:1) and
stirred at 0e5 ꢀC for 30 min. The amine hydrochloride was then
deazotized by the dropwise addition of aqueous NaNO2. The

M.K. Zilla et al. / European Journal of Medicinal Chemistry 77 (2014) 47e55
51
Fig. 2. Cytotoxic and apoptosis inducing potential of compound 9k towards human cancer cells. (a) Cell viability (MTT) assay was performed on four different cancer cells with
different concentrations of 9k in 96 well plates for 48 h. The compound showed its strong cytotoxic ability with IC50 values 12, 3.8, 6.3 and 22 nM in PC-3, Panc-1, COLO-205 and A-
549 cells respectively. (b) PC-3 cells (5 ꢁ 104) were treated with indicated concentrations of 9k for 24 h along with vehicle and Etoposide as positive control. Cells after fixation were
stained with DAPI nuclear stain and photographed under fluorescence microscope (100ꢁ magnifications). Data were calculated, compared with untreated control and expressed as
ꢄSEM of three independent experiments.
solution was stirred at 0e5 ꢀC and treated with aqueous NaN3. After
stirring for 1 h, the reaction mixture was extracted with DCM. The
combined organic layers were washed with water and brine, dried
over Na2SO4, and concentrated in vacuo afforded the eleven
different 1-azido derivatives with different substitutions on the
aromatic ring were prepared (aek in Scheme 1) in quantitative
yield [29].
CHCl3); 1H NMR (500 MHz, CDCl3)
d 8.07 (s, 1H), 7.78e7.73 (m, 2H),
7.56 (t, J ¼ 7.8 Hz, 2H), 7.48 (t, J ¼ 7.4 Hz, 1H), 7.06 (s, 1H), 6.53 (s,
1H), 6.41 (s, 2H), 5.97 (dd, J ¼ 5.1, 1.2 Hz, 2H), 4.87 (dd, J ¼ 10.6,
8.3 Hz, 2H), 4.79 (d, J ¼ 11.9 Hz, 1H), 4.67e4.62 (m, 1H), 4.60 (d,
J ¼ 4.6 Hz, 1H), 4.21 (dd, J ¼ 10.3, 9.0 Hz, 1H), 3.82 (s, 3H), 3.76 (s,
6H), 3.03 (ddd, J ¼ 20.0, 15.8, 10.2 Hz, 1H), 2.88 (dd, J ¼ 14.3, 4.7 Hz,
1H); 13C NMR (126 MHz, CDCl3)
d 173.9, 152.6, 147.9, 147.6, 145.2,
Finally the 1-azidobenzene (a) (15 mg, 0.12 mmol) were coupled
with the mixture of propagylated podophyllotoxin 8 (50 mg,
0.11 mmol) using the solvent 10 ml t-BuOH:H2O (1:1) ration which
was subsequently added sodium ascorbate (23 mg, 0.11 mmol),
copper sulphate (5 mg, 0.03 mmol) the reaction mixture were
stirred at 70 ꢀC for 1 h, after completing the reaction cool to room
temperature and then added 20 ml water and extracted with ethyl
acetate (20 ꢁ 3), dried over Na2SO4, and concentrated in vacuo
afforded the crude product (60 mg, 0.1 mmol). Surprisingly we
observed the resolution of disteromeric mixture which was sub-
sequently isolated by flash column chromatography 9a (30 mg,
0.05 mmol) and 10a (23 mg, 0.04 mmol).
137.1, 136.8,135.2, 131.9, 130.4, 129.8, 129.0, 120.7, 120.6, 109.7, 108.1,
107.0, 101.4, 79.5, 71.3, 61.7, 60.7, 56.2, 45.5, 43.9, 37.91; HRMS (ESI)
572.2025 for [MþH]þ (calcd 571.1955 for C31H29N3O8).
4.1.1.5. 4b-[4-O-Methyl-(phenyl-1,2,3-triazol-4yl)]-epi-podophyllo-
toxin (10a). ½a 2D5
ꢂ
¼ ꢃ19.1 (c ¼ 0.2, CHCl3); 1H NMR (500 MHz,
CDCl3)
d
7.97 (s, 1H), 7.74 (d, J ¼ 7.6 Hz, 2H), 7.55 (t, J ¼ 7.8 Hz, 3H),
7.47 (t, J ¼ 7.4 Hz, 1H), 6.89 (s, 1H), 6.57 (s, 1H), 6.25 (s, 2H), 6.02e
5.95 (m, 2H), 4.87 (q, J ¼ 12.2 Hz, 3H), 4.79 (d, J ¼ 3.1 Hz, 1H), 4.63
(d, J ¼ 5.3 Hz, 1H), 4.35 (d, J ¼ 9.9 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 6H),
3.44 (dd, ¼ 14.0, 5.4 Hz, 1H), 3.00e2.86 (m, 1H); 13C NMR (126 MHz,
CDCl3)
d 174.9, 152.5, 148.5, 146.8, 145.4, 137.1, 136.8, 135.3, 132.5,
129.8, 129.0, 128.8, 120.9, 120.6, 110.9, 109.7, 108.1, 101.5, 73.8, 67.4,
62.9, 60.7, 56.2, 43.9, 41.0, 38.1; HRMS (ESI) 572.2037 for [MþH]þ
(calcd 571.1955 for C31H29N3O8).
4.1.1.4. 4a-[4-O-Methyl-(phenyl-1,2,3-triazol-4yl)]-podophyllotoxin
(9a). White powder solid; mp: 171e173 ꢀC; ½a D25
¼ ꢃ27.3 (c ¼ 0.2,
ꢂ

52
M.K. Zilla et al. / European Journal of Medicinal Chemistry 77 (2014) 47e55
Fig. 3. Antimotility and antimigratory ability of 9k was observed on aggressive prostate cancer cells. (a) Colony formation assay was carried out taking PC-3 cells (1 ꢁ103)/well in six
well plates and treated with different concentrations of 9k along with DMSO and Etoposide as positive control for five days. The number of crystal violet stained cells per colony was
counted randomly, quantified and images were captured under inverted microscope in 100ꢁ magnifications. (b) Wound healing assay was performed taking PC-3 cells 1 ꢁ 106/well
and treated with 9k at given concentrations after creating the wound over the cells monolayer. The number of migratory cells were counted, quantified for the degrees of wound
healing and compared with untreated control. Columns mean and bars given here follow ꢄSEM of three independent experiments. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
4.1.1.6. 4
a
-[O-Methyl-(para trifluoromethyl phenyl-1,2,3-triazol-
J ¼ 9.2 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 6H), 3.44 (dd, J ¼ 14.0, 5.4 Hz,
1H), 2.96 (ddd, J ¼ 14.7, 9.3, 4.6 Hz, 1H); HRMS (ESI) 640.1905 for
[MþH]þ (calcd 639.1828 for C32H28F3N3O8).
4yl)]-podophyllotoxin (9b). White powder solid; mp: 186e189 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ26.0 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.12 (s,
1H), 7.93 (d, J ¼ 8.4 Hz, 2H), 7.84 (d, J ¼ 8.5 Hz, 2H), 7.05 (s, 1H), 6.54
(s, 1H), 6.41 (s, 2H), 5.97 (dd, J ¼ 5.6, 1.3 Hz, 2H), 4.88 (dd, J ¼ 14.0,
10.8 Hz, 2H), 4.80 (d, J ¼ 12.0 Hz, 1H), 4.68e4.59 (m, 2H), 4.21 (dd,
J ¼ 10.4, 8.9 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 6H), 3.09e2.98 (m, 1H),
4.1.1.8. 4a-[O-Methyl-(para bromo phenyl-1,2,3-triazol-4yl)]-podo-
phyllotoxin (9c). White powder solid; mp: 181e185 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ36.6 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.97 (s,
2.88 (dd, J ¼ 14.3, 4.7 Hz, 1H); 13C NMR (126 MHz, CDCl3)
d 173.8,
1H), 7.64e7.54 (m, 4H), 6.97 (s,1H), 6.46 (s, 1H), 6.34 (s, 2H), 5.89 (d,
J ¼ 2.8 Hz, 2H), 4.83e4.74 (m, 2H), 4.71 (d, J ¼ 12.0 Hz,1H), 4.54 (dd,
J ¼ 16.0, 6.6 Hz, 2H), 4.12 (dd, J ¼ 10.2, 9.0 Hz, 1H), 3.74 (s, 3H), 3.68
(s, 6H), 2.95 (dtd, J ¼ 17.0, 10.0, 7.2 Hz, 1H), 2.80 (dd, J ¼ 14.2, 4.6 Hz,
1H); HRMS (ESI) 650.1143 for [MþH]þ (calcd 649.1060 for
152.6, 147.9, 147.7,145.9,139.2,137.3, 135.2,132.0,130.2, 127.2, 127.2,
124.5, 122.3, 120.5, 109.8, 108.2, 108.1, 106.9, 101.4, 79.8, 71.3, 61.5,
60.7, 56.2, 45.5, 43.9, 37.8; HRMS (ESI) 640.1896 for [MþH]þ (calcd
639.1828 for C32H28F3N3O8).
C31H28BrN3O8).
4.1.1.7. 4b-[O-Methyl-(para trifluoromethyl phenyl-1,2,3-triazol-
4yl)]-epipodophyllotoxin (10b). White powder solid; mp: 168e
4.1.1.9. 4b-[O-Methyl-(para bromo phenyl-1,2,3-triazol-4yl)]-epi-
171 ꢀC; ½a 2D5
ꢂ
¼ ꢃ18.4 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
podophyllotoxin (10c). White powder solid; ½a D25
¼ ꢃ27.0 (c ¼ 0.2,
ꢂ
d
8.02 (s, 1H), 7.92 (d, J ¼ 8.4 Hz, 2H), 7.83 (d, J ¼ 8.6 Hz, 2H), 6.89 (s,
CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.86 (s, 1H), 7.64e7.53 (m, 4H),
1H), 6.58 (s, 1H), 6.25 (s, 2H), 5.99 (dd, J ¼ 14.0, 1.1 Hz, 2H), 4.94e
6.81 (s, 1H), 6.50 (s, 1H), 6.18 (s, 2H), 5.91 (dd, J ¼ 13.2, 1.3 Hz, 2H),
4.83 (m, 2H), 4.78 (d, J ¼ 3.1 Hz, 1H), 4.63 (d, J ¼ 5.3 Hz, 1H), 4.36 (d,
4.79 (q, J ¼ 12.2 Hz, 2H), 4.70 (d, J ¼ 3.2 Hz, 1H), 4.55 (d, J ¼ 5.4 Hz,

M.K. Zilla et al. / European Journal of Medicinal Chemistry 77 (2014) 47e55
53
1H), 4.28 (d, J ¼ 9.2 Hz, 2H), 3.73 (s, 3H), 3.67 (s, 6H), 3.36 (dd,
J ¼ 14.0, 5.4 Hz, 1H), 2.87 (ddd, J ¼ 14.6, 9.3, 4.7 Hz, 1H); HRMS (ESI)
650.1119 for [M þ H]þ (calcd 649.1060 for C31H28BrN3O8).
6.25 (s, 2H), 5.98 (dd, J ¼ 13.1,1.2 Hz, 2H), 4.91e4.81 (m, 2H), 4.77 (d,
J ¼ 3.2 Hz, 1H), 4.62 (d, J ¼ 5.4 Hz, 1H), 4.35 (d, J ¼ 9.2 Hz, 2H), 3.80
(s, 3H), 3.73 (s, 6H), 3.46e3.40 (m, 1H), 3.00e2.86 (m, 1H); 13C NMR
(126 MHz, CDCl3)
d 174.9, 152.5, 148.6, 146.8, 145.7, 137.1, 135.3,
4.1.1.10. 4
a
-[O-Methyl-(ortho fluoro phenyl-1,2,3-triazol-4yl)]-podo-
134.8, 132.6, 130.0, 128.7, 121.7, 120.7, 110.9, 109.6, 108.1, 101.5, 74.0,
67.4, 62.9, 60.7, 56.2, 43.9, 41.0, 38.1; HRMS (ESI) 606.1635 for
[MþH]þ (calcd 605.1565 for C31H28ClN3O8).
phyllotoxin (9d). White powder solid; ½a D25
¼ ꢃ29.3 (c ¼ 0.2,
ꢂ
CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.07 (s, 1H), 7.59e7.50 (m, 3H),
7.18 (ddd, J ¼ 8.0, 4.5, 2.2 Hz, 1H), 7.05 (s, 1H), 6.53 (s, 1H), 6.41 (s,
2H), 5.97 (d, J ¼ 2.8 Hz, 2H), 4.86 (dd, J ¼ 12.5, 7.7 Hz, 2H), 4.81e4.75
(m, 1H), 4.67e4.58 (m, 2H), 4.20 (dd, J ¼ 10.2, 9.0 Hz, 1H), 3.81 (s,
3H), 3.75 (s, 6H), 3.08e2.96 (m, 1H), 2.87 (dd, J ¼ 14.2, 4.7 Hz, 1H);
HRMS (ESI) 690.1978 for [MþH]þ (calcd 589.1860 for C31H28FN3O8).
4.1.1.16. 4a-[O-Methyl-(meta chloro para fluoro phenyl-1,2,3-triazol-
4yl)]-podophyllotoxin (9g). White powder solid; mp: 186e189 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ31.2 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.02 (s,
1H), 7.86 (dd, J ¼ 6.2, 2.6 Hz, 1H), 7.66 (ddd, J ¼ 8.8, 3.9, 2.7 Hz, 1H),
7.34 (t, J ¼ 8.6 Hz, 1H), 7.04 (s, 1H), 6.53 (s, 1H), 6.41 (s, 2H), 5.97 (d,
J ¼ 3.2 Hz, 2H), 4.90e4.81 (m, 2H), 4.78 (d, J ¼ 12.0 Hz,1H), 4.62 (dd,
J ¼ 14.7, 6.8 Hz, 2H), 4.20 (dd, J ¼ 10.2, 9.0 Hz, 1H), 3.82 (s, 3H), 3.76
(s, 6H), 3.02 (ddd, J ¼ 19.9, 15.7, 10.1 Hz, 1H), 2.88 (dd, J ¼ 14.2 Hz,
1H); HRMS (ESI) 624.1543 for [MþH]þ (calcd 623.1471 for
4.1.1.11. 4b-[O-Methyl-(ortho fluoro phenyl-1,2,3-triazol-4yl)]-epi-
podophyllotoxin (10d). White powder solid; ½a D25
¼ ꢃ21.5 (c ¼ 0.2,
ꢂ
CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.95 (s, 1H), 7.57e7.49 (m, 3H),
7.18 (ddd, J ¼ 9.4, 4.6, 2.5 Hz, 1H), 6.89 (s, 1H), 6.57 (s, 1H), 6.25 (s,
2H), 5.99 (dd, J ¼ 13.7, 1.2 Hz, 2H), 4.92e4.81 (m, 2H), 4.78 (d,
J ¼ 3.2 Hz, 1H), 4.63 (d, J ¼ 5.4 Hz, 1H), 4.35 (d, J ¼ 9.2 Hz, 2H), 3.80
(s, 3H), 3.74 (s, 6H), 3.44 (dd, J ¼ 14.0, 5.4 Hz,1H), 2.94 (ddd, J ¼ 14.7,
9.2, 4.6 Hz, 1H)); HRMS (ESI) 690.1979 for [MþH]þ (calcd 589.1860
for C31H28FN3O8).
C31H27ClFN3O8).
4.1.1.17. 4
b
-[O-Methyl-(meta chloro para fluoro phenyl-1,2,3-triazol-
4yl)-podophyllotoxin (10g). White powder solid; mp: 175e178 ꢀC;
a 25
½ ꢂD ¼ ꢃ22.9 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.91 (s,
1H), 7.85 (dd, J ¼ 6.2, 2.6 Hz, 1H), 7.64 (ddd, J ¼ 8.9, 3.9, 2.7 Hz, 1H),
7.36e7.30 (m, 1H), 6.87 (s, 1H), 6.57 (s, 1H), 6.25 (s, 2H), 6.02e5.96
(m, 2H), 4.86 (td, J ¼ 12.1, 5.4 Hz, 2H), 4.76 (d, J ¼ 3.1 Hz,1H), 4.63 (d,
J ¼ 5.3 Hz, 1H), 4.35 (d, J ¼ 8.7 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 6H), 3.43
(dd, J ¼ 14.0, 5.4 Hz, 1H), 2.94 (ddd, J ¼ 21.1, 13.3, 4.0 Hz, 1H); HRMS
(ESI) 624.1575 for [MþH]þ (calcd 623.1471 for C31H27ClFN3O8).
4.1.1.12. 4
a
-[O-Methyl-(paramethoxy
phenyl-1,2,3-triazol-4yl)]-
podophyllotoxin (9e). White powder solid; ½a D25
¼ ꢃ36.0 (c ¼ 0.2,
ꢂ
CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.90 (s, 1H), 7.59e7.54 (m, 2H),
6.98 (s, 2H), 6.96 (s, 1H), 6.45 (s, 1H), 6.34 (s, 2H), 5.89 (d, J ¼ 2.8 Hz,
2H), 4.79 (dd, J ¼ 10.7, 6.6 Hz, 2H), 4.71 (d, J ¼ 11.9 Hz, 1H), 4.59e
4.50 (m, 2H), 4.17e4.08 (m, 1H), 3.81 (s, 3H), 3.74 (s, 3H), 3.68 (s,
6H), 3.01e2.88 (m, 1H), 2.80 (dd, J ¼ 14.3, 4.6 Hz, 1H); 13C NMR
4.1.1.18. 4
a
-[O-Methyl-(orthohydroxy
phenyl-1,2,3-triazol-4yl)]-
(126 MHz, CDCl3)
d
172.9, 158.9, 151.6, 146.8, 146.5, 143.9, 136.0,
podophyllotoxin (9h). White powder solid; mp: 193e195 ꢀC;
134.2,130.9,129.4,129.2,121.2,119.9,113.8,108.7,107.1,106.0,100.4,
78.4, 70.3, 60.8, 59.7, 55.1, 54.6, 44.5, 42.9, 36.9; HRMS (ESI)
602.2171 for [MþH]þ (calcd 601.2060 for C32H31N3O9).
½
a 2D5
ꢂ
¼ ꢃ17.5 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 9.54 (s,
1H), 8.11 (s, 1H), 7.38 (d, J ¼ 8.0 Hz, 1H), 7.26 (t, J ¼ 7.7 Hz, 1H), 7.18
(d, J ¼ 4.1 Hz, 1H), 7.15 (d, J ¼ 8.1 Hz, 1H), 6.46 (s, 1H), 6.34 (s, 3H),
5.89 (d, J ¼ 3.5 Hz, 3H), 4.84e4.76 (m, 3H), 4.73 (d, J ¼ 12.0 Hz, 1H),
4.55 (dd, J ¼ 14.9, 6.3 Hz, 3H), 4.12 (t, J ¼ 9.6 Hz, 1H), 3.74 (s, 3H),
3.68 (s, 6H), 2.95 (dt, J ¼ 16.9, 9.8 Hz, 1H), 2.80 (dd, J ¼ 14.2, 4.5 Hz,
4.1.1.13. 4b-[O-Methyl-(paramethoxy phenyl-1,2,3-triazol-4yl)]-epi-
podophyllotoxin (10e). White powder solid; mp: 168e172 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ28.2 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d
7.88 (s,
1H); 13C NMR (126 MHz, CDCl3)
d 173.9, 152.7, 149.2, 148.0, 147.7,
1H), 7.65e7.60 (m, 2H), 7.06e7.01 (m, 2H), 6.88 (s, 1H), 6.56(s, 1H),
6.25 (s, 2H), 5.98 (dd, J ¼ 13.5, 1.3 Hz, 2H), 4.90e4.81 (m, 2H), 4.78
(d, J ¼ 3.2 Hz,1H), 4.62 (d, J ¼ 5.4 Hz,1H), 4.37e4.31 (m, 2H), 3.88 (s,
3H), 3.80 (s, 3H), 3.73 (s, 6H), 3.43 (dd, J ¼ 14.0, 5.4 Hz, 1H), 2.98e
144.8,137.2,135.2,132.1,130.2,130.0,122.7,120.7,120.4,119.8,119.5,
109.8, 108.2, 106.9, 101.5, 79.8, 71.3, 61.5, 60.7, 56.2, 45.5, 43.9, 37.8;
HRMS (ESI) 588.1988 for [MþH]þ (calcd 587.1904 for C31H29N3O9).
2.87 (m, 1H); 13C NMR (126 MHz, CDCl3)
d
174.8, 159.9, 152.5, 148.5,
4.1.1.19. 4b-[O-Methyl-(orthohydroxy phenyl-1,2,3-triazol-4yl)]-epi-
146.8, 145.2, 137.2, 135.2, 132.5, 130.3, 128.9, 122.3, 121.0, 114.8,
110.8, 109.6, 108.2, 101.4, 73.7, 67.3, 63.0, 60.6, 56.2, 55.6, 43.9, 41.0,
38.1; HRMS (ESI) 602.2131 for [MþH]þ (calcd 601.2060 for
podophyllotoxin (10h). White powder solid; ½a D25
¼ ꢃ13.8 (c ¼ 0.2,
ꢂ
CHCl3); 1H NMR (400 MHz, CDCl3)
d 9.65 (s, 1H), 8.06 (s, 1 H), 7.43
(dd, J ¼ 10.9, 9.6 Hz, 1H), 7.33 (dd, J ¼ 11.2, 4.4 Hz, 1H), 7.23 (d,
J ¼ 7.7 Hz, 1H), 7.06e7.00 (m, 1H), 6.90 (s, 1H), 6.58 (s, 1H), 6.25 (s,
2H), 5.99 (d, J ¼ 13.4 Hz, 2H), 4.89 (q, J ¼ 12.2 Hz, 2H), 4.75 (d,
J ¼ 3.0 Hz, 1H), 4.63 (d, J ¼ 5.2 Hz, 1H), 4.35 (d, J ¼ 9.2 Hz, 2H), 3.80
(s, 3H), 3.74 (s, 6H), 3.45 (dd, J ¼ 14.0, 5.4 Hz, 1H), 3.03e2.88 (m,
C
32H31N3O9).
4.1.1.14. 4
a
-[O-Methyl-(para chloro phenyl-1,2,3-triazol-4yl)]-podo-
phyllotoxin (9f). White powder solid; mp: 180e183 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ36.6 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d
7.96 (s,
1H); 13C NMR (126 MHz, CDCl3)
d 174.8, 152.5, 149.2, 148.6, 146.8,
1H), 7.67e7.61 (m, 2H), 7.49e7.42 (m, 2H), 6.98 (s, 1H), 6.46 (s, 1H),
6.34 (s, 2H), 5.90 (d, J ¼ 3.7 Hz, 2H), 4.83e4.75 (m, 2H), 4.71 (d,
J ¼ 12.0 Hz, 1H), 4.55 (dd, J ¼ 16.4, 6.1 Hz, 2H), 4.16e4.10 (m, 1H),
3.74 (s, 3H), 3.68 (s, 6H), 2.95 (ddd, J ¼ 19.9, 15.8, 10.1 Hz, 1H), 2.80
144.9, 137.2, 135.3, 132.6, 130.0, 128.5, 122.7, 120.8, 120.4, 119.8,
119.4, 111.0, 109.7, 108.2, 101.6, 74.1, 67.4, 62.7, 60.7, 56.2, 43.9, 41.0,
38.1; HRMS (ESI) 588.2012 for [MþH]þ (calcd 587.1904 for
C31H29N3O9).
(dd, J ¼ 14.2,4.6 Hz, 1 H); 13C NMR (126 MHz, CDCl3)
d 172.9, 151.5,
146.8, 146.6, 144.5, 136.0, 134.3, 134.2, 133.7, 130.9, 129.2, 129.0,
120.7, 119.6, 108.7, 107.0, 105.9, 100.4, 78.5, 70.2, 60.5, 59.7, 55.1,
44.4, 42.8, 36.8; HRMS (ESI) 606.1648 for [MþH]þ (calcd 605.1565
for C31H28ClN3O8).
4.1.1.20. 4a-[O-Methyl-(orthoiodo phenyl-1,2,3-triazol-4yl)]-podo-
phyllotoxin (9i). White powder solid; mp: 196e201 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ18.1 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.05 (d,
J ¼ 8.0, 1H), 7.98 (s, 1H), 7.58e7.54 (dd, J ¼ 8.0 Hz, 2H), 7.52e7.47 (d,
J ¼ 8.0 Hz, 1H), 7.07 (s, 1H), 6.54 (s, 1H), 6.43 (s, 2H), 5.98 (d,
J ¼ 3.0 Hz, 2H), 4.98e4.88 (m, 2H), 4.83 (d, J ¼ 12.3 Hz, 1H), 4.70e
4.60 (m, 2H), 4.29e4.20 (m, 1H), 3.83 (s, 3H), 3.78 (s, 6H), 3.06e3.02
(m, 1H), 2.95e2.85 (dd, J ¼ 14.3, 4.7 Hz, 1H); HRMS (ESI) 698.1000
for [MþH]þ (calcd 697.0921 for C31H28N3O8I).
4.1.1.15. 4b-[O-Methyl-(para chloro phenyl-1,2,3-triazol-4yl)]-epi-
podophyllotoxin (10f). White powder solid; mp: 173e177 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ24.0 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.95 (s,
1H), 7.74e7.65 (m, 2H), 7.56e7.47 (m, 2H), 6.88 (s, 1H), 6.57 (s, 1H),

54
M.K. Zilla et al. / European Journal of Medicinal Chemistry 77 (2014) 47e55
4.1.1.21. 4
b
-[O-Methyl-(orthoiodo
phenyl-1,2,3-triazol-4yl)]-epi-
4.1.1.26. 40-Demethyl-40,4
b-(O-dipropargyl)-epipodophyllotoxin (11).
podophyllotoxin (10i). White powder solid; mp: 183e187 ꢀC;
Compound 3 (500 mg, 1.25 mmol) was dissolved in 50 ml of dry
DMF and cool to ꢀC added NaH (47 mg, 1.95 mmol) then further
addition of propargyl bromide (300 mg, 2.54 mmol) warm to the
room temperature and stirred the reaction mixture for 3 h after
aqueous work up followed by the column purification afforded the
compound 11 as a colourless solid (480 mg, 80% yield) mp: 193e
½
a 2D5
ꢂ
¼ ꢃ13.8 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.04e
8.02 (dd, J ¼ 8.0, 1.2 Hz, 1H), 7.87 (s, 1H), 7.54 (dd, J ¼ 10.8, 4.4 Hz,
1H), 7.50e7.44 (m, 1H), 7.17 (d, J ¼ 6.8 Hz, 1H), 6.85 (d, J ¼ 1.9 Hz,
1H), 6.26 (s, 2H), 5.97 (dd, J ¼ 11.6, 1.6 Hz, 2H), 4.95e4.82 (m, 3H),
4.62 (d, J ¼ 5.3 Hz, 1H), 4.40e4.33 (m, 2H), 3.82 (s, 3H), 3.74 (s, 6H),
3.46e3.38 (m, 1H), 3.00e2.86 (m, 1H); HRMS (ESI) 698.1006 for
[MþH]þ (calcd 697.0921 for C31H28N3O8I).
196 ꢀC; ½a 2D5
ꢂ
¼ ꢃ37.4 (c ¼ 0.3, CHCl3); 1H NMR (400 MHz, CDCl3)
d
6.89 (s, 1H), 6.48 (s, 1H), 6.43 (s, 2H), 5.95 (dd, J ¼ 7.4, 1.3 Hz, 2H),
4.68 (d, J ¼ 2.4 Hz, 2H), 4.48e4.42 (m, 2H), 4.29 (dd, J ¼ 9.6, 3.7 Hz,
1H), 4.20 (dd, J ¼ 5.7, 3.6 Hz, 2H), 4.14e4.07 (m, 1H), 3.79 (s, 6H),
3.29 (dd, J ¼ 9.7, 4.7 Hz, 1H), 3.06e2.96 (m, 1H), 2.52 (t, J ¼ 2.3 Hz,
1H), 2.44 (t, J ¼ 2.4 Hz, 1H); HRMS (ESI) 477.1677 for [MþH]þ (calcd
476.1471 for C27H24O8).
4.1.1.22. 4
a
-[O-Methyl-(meta
bromo
phenyl-1,2,3-triazol-4yl)]
podophyllotoxin (9j). White powder solid; mp: 191e195 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ43.6 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.04 (s,
1H), 7.95 (t, J ¼ 2.0 Hz, 1H), 7.71 (ddd, J ¼ 8.1, 2.1, 0.9 Hz, 1H), 7.60
(ddd, J ¼ 8.1, 1.8, 0.9 Hz, 1H), 7.42 (t, J ¼ 8.1 Hz, 1H), 7.04 (s, 1H), 6.52
(s, 1H), 6.40 (s, 2H), 5.96 (dd, J ¼ 4.6, 1.3 Hz, 2H), 4.90e4.81 (m, 2H),
4.78 (d, J ¼ 11.9 Hz,1H), 4.66e4.58 (m, 2H), 4.19 (dd, J ¼ 10.3, 8.9 Hz,
1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.07e2.95 (m, 1H), 2.87 (dd, J ¼ 14.3,
4.1.1.27. 40-Demethyl-40,4
b-[O-dimethyl-(diphenyl-di-1,2,3-triazol-
4yl)]-epipodophyllotoxin (12a). Yield: 93%; white powder solid;
mp: 201e203 ꢀC; ½a D25
ꢂ
¼ ꢃ44.0 (c ¼ 0.3, CHCl3); 1H NMR (400 MHz,
CDCl3)
d 8.07 (s, 1H), 7.78 (s, 1H), 7.76e7.68 (m, 5H), 7.56e7.41 (m,
4.7 Hz, 1H); 13C NMR (126 MHz, CDCl3)
d 173.8, 152.6, 147.8, 147.6,
7H), 7.00 (s, 1H), 6.59 (s, 1H), 6.44 (s, 2H), 5.99 (dd, J ¼ 8.4, 1.3 Hz,
2H), 5.22 (s, 2H), 4.67 (q, J ¼ 12.0 Hz, 3H), 4.49e4.41 (m, 3H), 4.32
(d, J ¼ 3.9 Hz, 1H), 4.11 (dd, J ¼ 9.5, 4.2 Hz, 1H), 3.73 (s, 6H), 3.37 (dd,
J ¼ 9.8, 4.0 Hz, 1H), 3.15 (ddd, J ¼ 14.1, 9.5, 4.5 Hz, 1H); HRMS (ESI)
715.2511 for [MþH]þ (calcd 714.2438 for C39H34N6O8).
145.5, 137.7, 137.2, 135.1, 131.9, 131.0, 130.2, 123.6, 123.3, 120.5, 118.9,
109.7, 108.2, 106.9, 101.3, 79.6, 71.2, 61.6, 60.6, 56.1, 45.4, 43.9, 37.8;
HRMS (ESI) 650.1109 for [MþH]þ (calcd 649.1060 for
C
31H28N3O8Br).
4.1.1.23. 4
b
-[O-Methyl-(meta bromo phenyl-1,2,3-triazol-4yl)]-epi-
4.1.1.28. 40-Demethyl-40,4
b
-[O-dimethyl-(para
ditrifluoromethyl
podophyllotoxin (10j). White powder solid; mp: 183e187 ꢀC;
phenyl-di-1,2,3-triazol-4yl)]-epipodophyllotoxin (12b). Yield: 95%;
½
a 2D5
ꢂ
¼ ꢃ33.0 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d
7.98 (s,
white powder solid mp: 183e187 ꢀC; ½a D25
¼ ꢃ46.0 (c ¼ 0.3, CHCl3);
ꢂ
1H), 7.96 (t, J ¼ 1.9 Hz, 1H), 7.72 (ddd, J ¼ 8.1, 2.1, 0.9 Hz, 1H), 7.61
(ddd, J ¼ 8.1, 1.8, 0.9 Hz, 1H), 7.43 (t, J ¼ 8.1 Hz, 1H), 6.90 (s, 1H), 6.58
(s, 1H), 6.27 (s, 2H), 6.00 (dd, J ¼ 12.6, 1.3 Hz, 2H), 4.93e4.82 (m,
2H), 4.78 (d, J ¼ 3.2 Hz, 1H), 4.64 (d, J ¼ 5.3 Hz, 1H), 4.36 (d,
J ¼ 9.2 Hz, 2H), 3.81 (s, 3H), 3.75 (s, 6H), 3.45 (dd, J ¼ 14.0, 5.4 Hz,
1H NMR (400 MHz, CDCl3)
d 8.13 (s,1H), 7.92e7.86 (m, 4H), 7.77 (dd,
J ¼ 22.2, 8.6 Hz, 4H), 7.72 (s, 1H), 6.98 (s, 1H), 6.63 (s, 2H), 6.43 (s,
2H), 6.00 (dd, J ¼ 9.5, 1.3 Hz, 2H), 5.23 (s, 2H), 4.63 (dd, J ¼ 27.4,
12.0 Hz, 3H), 4.48 (dd, J ¼ 9.4, 8.1 Hz, 1H), 4.42 (d, J ¼ 4.8 Hz, 1H),
4.37 (d, J ¼ 3.7 Hz,1H), 4.07 (dd, J ¼ 9.6, 4.4 Hz,1H), 3.72 (s, 6H), 3.42
(dd, J ¼ 9.9, 3.8 Hz, 1H), 3.24e3.14 (m, 1H); HRMS (ESI) 851.2254 for
[MþH]þ (calcd 850.2186 for C41H32F6N6O8).
1H), 3.01e2.90 (m, 1H); 13C NMR (126 MHz, CDCl3)
d 174.2, 152.0,
148.0,145.2,137.2,134.7,132.0,131.4,130.5,128.2,123.1,122.8,120.1,
118.4, 110.3, 109.1, 107.7, 100.9, 73.5, 66.8, 62.3, 60.1, 55.6, 43.4, 40.4,
37.6 HRMS (ESI) 650.1125 for [MþH]þ (calcd 649.1060 for
4.1.1.29. 40-Demethyl-40,4
b-[O-dimethyl-(para dibromo phenyl-di-
C
31H28N3O8Br).
1,2,3-triazol-4yl)]-epipodophyllotoxin (12c). Yield: 96%; white
powder solid; mp: 193e197 ꢀC; ½a D25
ꢂ
¼ ꢃ32.6 (c ¼ 0.3, CHCl3); 1H
NMR (400 MHz, CDCl3) d 8.02 (s,1H), 7.63 (s,1H), 7.62e7.50 (m, 8H),
4.1.1.24. 4
a
-[O-Methyl-(orthocyano phenyl-1,2,3-triazol-4yl)]-podo-
6.90 (s, 1H), 6.53 (s, 1H), 6.36 (s, 2H), 5.92 (d, J ¼ 8.6 Hz, 2H), 5.11 (s,
2H), 4.55 (dd, J ¼ 25.1, 12.0 Hz, 2H), 4.41 (t, J ¼ 8.8 Hz, 1H), 4.34 (d,
J ¼ 4.8 Hz, 1H), 4.26 (d, J ¼ 3.3 Hz, 1H), 4.01 (dd, J ¼ 9.4, 4.1 Hz, 1H),
3.64 (s, 6H), 3.38e3.30 (m, 2H), 3.15e3.06 (m, 1H); HRMS (ESI)
871.1011 for [MþH]þ (calcd 870.0648 for C39H32Br2N6O8).
phyllotoxin (9k). White powder solid; mp: 178e182 ꢀC;
½
a 2D5
ꢂ
¼ ꢃ42.0 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.33 (s,
1H), 7.95e7.80 (m, 3H), 7.64 (t, J ¼ 7.5 Hz, 1H), 7.07 (s, 1H), 6.52 (s,
1H), 6.41 (s, 2H), 5.97 (d, J ¼ 2.3 Hz, 2H), 4.89 (dd, J ¼ 10.7, 6.9 Hz,
2H), 4.82 (d, J ¼ 12.1 Hz, 1H), 4.64 (dd, J ¼ 15.0, 7.3 Hz, 1H), 4.60 (d,
J ¼ 4.5 Hz, 1H), 4.23e4.16 (m, 1H), 3.81 (s, 3H), 3.76 (s, 6H), 3.03
(ddd, J ¼ 19.9, 15.7, 9.9 Hz, 1H), 2.88 (dd, J ¼ 14.3, 4.6 Hz, 1H); 13C
4.1.1.30. 40-Demethyl-40,4
b-[O-dimethyl-(ortho difluoro phenyl-di-
1,2,3-triazol-4yl)]-epipodophyllotoxin (12d). Yield: 92%; white
NMR (126 MHz, CDCl3) d 173.9, 152.6, 147.9, 147.7, 145.5, 138.4, 137.1,
powder solid; mp: 203e206 ꢀC; ½a D25
ꢂ
¼ ꢃ27.7 (c ¼ 0.2, CHCl3); 1H
135.2, 134.5, 134.4, 134.3, 132.0, 130.3, 129.7, 125.4, 123.1, 115.6,
109.7,108.1,108.0,107.0,106.5,101.4, 79.6, 71.3, 61.5, 60.7, 56.2, 45.5,
43.9, 37.9; HRMS (ESI) 597.2007 for [MþH]þ (calcd 596.1907 for
NMR (400 MHz, CDCl3) d 8.08 (s,1H), 7.72 (s,1H), 7.58e7.43 (m, 6H),
7.19e7.07 (m, 2H), 6.98 (s, 1H), 6.61 (s, 1H), 6.43 (s, 2H), 6.00 (dd,
J ¼ 9.2, 1.2 Hz, 2H), 5.21 (s, 2H), 4.63 (dd, J ¼ 25.6, 12.0 Hz, 2H), 4.47
(dd, J ¼ 9.4, 8.1 Hz, 1H), 4.42 (d, J ¼ 5.0 Hz, 1H), 4.34 (d, J ¼ 3.7 Hz,
1H), 4.09 (dd, J ¼ 9.6, 4.3 Hz, 1H), 3.72 (s, 6H), 3.40 (dd, J ¼ 9.9,
3.8 Hz, 1H), 3.17 (ddd, J ¼ 14.1, 9.4, 4.5 Hz, 1H); HRMS (ESI) 751.2363
for [MþH]þ (calcd 750.2250 for C39H32F2N6O8).
C
32H28N4O8).
4.1.1.25. 4
b-[O-Methyl-(orthocyano
phenyl-1,2,3-triazol-4yl)]-epi
podophyllotoxin (10k). White powder solid; mp: 161e165 ꢀC;
a 25
½ ꢂD ¼ ꢃ36.3 (c ¼ 0.2, CHCl3); 1H NMR (400 MHz, CDCl3)
d 8.21 (s,
1H), 7.92e7.80 (m, 3H), 7.63 (td, J ¼ 7.7 Hz, 1.2, 1H), 6.92 (s, 1H), 6.57
(s, 1H), 6.25 (s, 2H), 6.02e5.96 (m, 2H), 4.93e4.86 (m, 3H), 4.76 (d,
J ¼ 3.1 Hz, 1H), 4.62 (t, J ¼ 5.4 Hz, 1H), 4.35 (dd, J ¼ 9.2, 3.2 Hz, 2H),
4.2. Biology
4.2.1. Cell culture and reagents
3.80 (s, 3H), 3.73 (s, 6H), 3.48e3.40 (m, 1H), 3.00e2.86 (m, 1H); 13
C
Human cancer cell lines, viz: prostate cancer (PC-3), pancreatic
cancer (PANC-1), colon cancer (COLO-205) and lung cancer (A-549)
were purchased from European Collection of Cell Cultures (ECACC).
Cells were cultured in Minimum Essential Medium (MEM), RPMI-
1640 and DMEM (Invitrogen) with Penicillin G, Streptomycin
NMR (126 MHz, CDCl3)
d 174.9, 152.5, 148.6, 146.8, 145.6, 138.4,
137.1,135.4,134.4,134.3,132.6,129.8,128.5,125.5,123.3,115.6,111.0,
109.7, 108.1, 106.7, 101.5, 73.7, 67.4, 62.4, 60.7, 56.2, 44.0, 40.9, 38.1;
HRMS (ESI) 597.1977 for [MþH]þ (calcd 596.1907 for C32H28N4O8).

M.K. Zilla et al. / European Journal of Medicinal Chemistry 77 (2014) 47e55
55
from Sigma and 10% foetal bovine serum (FBS) from GIBCO.
Acknowledgements
Trypsin-EDTA was obtained from Sigma and cells were incubated at
37 ꢀC with 5% CO2 incubator (Eppendorf). 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Etoposide, Para-
formaldehyde, Triton X-100, UltraCruz DAPI mounting media and
Dimethylsulfoxide (DMSO) were obtained from Sigma Chemicals.
M.K.Z and D.N are thankful to the Council of Scientific and In-
dustrial Research (CSIR) for the financial assistance. We are
thankful to Dr. Arvinda and Deepika Singh for NMR spectral data
analysis.
4.2.2. Cell viability assay
The cell viability was determined by standard MTT dye uptake
method [30]. Briefly PC-3, PANC-1, COLO-205 and A-549 cells from
moderately confluent flasks were trypsinized properly and plated
into 96 well tissue culture plates at a density of 2.5 ꢁ 103 cells/well.
Treatment to the cells was given with different concentrations of
test compounds along with Podophyllotoxin (þve control) in trip-
licates so that the final concentration of DMSO solvent was 0.2%.
After 44 h incubation, MTT solution (2.5 mg/ml) was added and
further incubated at 37 ꢀC and 5% CO2 for 4 h. The amount of col-
oured formazan derivatives was determined by measuring optical
density (OD) using TECAN microplate reader (Infinite M200 PRO) at
570 nm. The percentage of cell viability was determined and the
IC50 values were calculated using GraphPad Prism software [31].
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.030.
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