4β-amidotriazole linked podophyllotoxin congeners: DNA topoisomerase-IIα inhibition and potential anticancer agents for prostate cancer

Article abstract of DOI:10.1016/j.ejmech.2017.12.050

Topoisomerases (topo-I and topo-II) have occupied a significant role in DNA replication, transcription, and are a promising set of antitumor targets. In the present approach, a series of new 4β-amidotriazole linked podophyllotoxin derivatives (10a-i and 1

Full text of DOI:10.1016/j.ejmech.2017.12.050

Accepted Manuscript
4β-amidotriazole linked podophyllotoxin congeners: DNA topoisomerase-IIα inhibition
and potential anticancer agents for prostate cancer
V. Ganga Reddy, Srinivasa Reddy Bonam, T. Srinivasa Reddy, Ravikumar Akunuri,
V.G.M. Naidu, V. Lakshma Nayak, Suresh K. Bhargava, H.M.Sampath Kumar, P.
Srihari, Ahmed Kamal
PII:
S0223-5234(17)31082-6
10.1016/j.ejmech.2017.12.050
EJMECH 10028
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 October 2017
Revised Date: 12 December 2017
Accepted Date: 13 December 2017
Please cite this article as: V.G. Reddy, S.R. Bonam, T.S. Reddy, R. Akunuri, V.G.M. Naidu, V.L. Nayak,
S.K. Bhargava, H.M.S. Kumar, P. Srihari, A. Kamal, 4β-amidotriazole linked podophyllotoxin congeners:
DNA topoisomerase-IIα inhibition and potential anticancer agents for prostate cancer, European Journal
of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.050.
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ACCEPTED MANUSCRIPT
Graphical Abstract
4β-Amidotriazole linked podophyllotoxin congeners: DNA
topoisomerase-IIα inhibition and potential anticancer agents for
prostate cancer
V. Ganga Reddy, Srinivasa Reddy Bonam, T. Srinivasa Reddy,* Ravikumar Akunuri, V. G.
M. Naidu, V. Lakshma Nayak, Suresh K. Bhargava, H. M. Sampath Kumar, P. Srihari,*
Ahmed Kamal*
Graphical Abstract:
Compounds showed < 1 µM (IC50
)
n
on tested human cancer cell lines
R
N N
N
Cell cycle arrests at G2/M phase
O
O
O
Showing DNA topo-II inihibition
HO
OH
HN
O
O
Inducing apoptosis on DU-145
cell line
O
O
O
O
O
O
O
O
ROS
loss of MMP
O
O
O
O
O
OH
Synthesized derivatives
(n=0, 10a-i and n=1, 11a-k)
Etoposide
IC50 values against selected
human cancer cell lines > 2 µM

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4β-Amidotriazole linked podophyllotoxin congeners: DNA
topoisomerase-IIα inhibition and potential anticancer agents for
prostate cancer
V. Ganga Reddy,a, b, c Srinivasa Reddy Bonam,a, d T. Srinivasa Reddy,*g Ravikumar Akunuri,e
V. G. M. Naidu,e V. Lakshma Nayak,b Suresh K. Bhargava,g H. M. Sampath Kumar,a, d P.
Srihari,*a, c Ahmed Kamal*a, b, e, f
aAcademy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical
Technology (IICT), Hyderabad, 500007, India
bMedicinal Chemistry and Biotechnology, CSIR-Indian Institute of Chemical Technology
(IICT), Hyderabad 500007, India
cDivision of Natural Products Chemistry, CSIR-Indian Institute of Chemical Technology
(IICT), Hyderabad 500007, India
dVaccine Immunology Laboratory, Natural Products Chemistry Division, CSIR-Indian
Institute of Chemical Technology (IICT), Hyderabad, 500007, India
eDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad, 500037, India
fSchool of Pharmaceutical Education and Research, Jamia Hamdard University,
New Delhi, 110062, India
gCentre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne 3001, Australia
*Corresponding authors: E-mail addresses: ahmedkamal@iict.res.in (Dr. Ahmed Kamal),
srihari@iict.res.in (Dr. P. Srihari), srinivasareddy.telukutla@rmit.edu.au (Dr. Srinivasa
Reddy Telukutla)

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Abstract: Topoisomerases (topo-I and topo-II) have occupied a significant role in DNA
replication, transcription, and are a promising set of antitumor targets. In the present
approach, a series of new 4β-amidotriazole linked podophyllotoxin derivatives (10a-i and
11a-k) were designed, synthesized by employing the click chemistry and their biological
activities were evaluated. The majority of derivatives showed promising antiproliferative
activity with IC50 values ranging from 1-10 µM on the six human cancer cell lines; cervical
(HeLa), breast (MCF-7), prostate (DU-145), lung (A549), liver (HepG2) and colon (HT-29).
Among them, some of the congeners 10b, 10g and 10i have shown remarkable cytotoxicity
with IC50 values of, < 1 µM against the tested cancer cell lines and found to be more active
than etoposide. Topoisomerase-mediated DNA relaxation assay results showed that the
derivatives could efficiently inhibit the activity of topoisomerase-II. In addition, flow
cytometry analysis on DU-145 cells revealed that these compounds arrest G2/M phase of cell
cycle. Further apoptotic studies were also performed on these DU-145 cells, which showed
that this class of compounds could induce apoptosis effectively.
Keywords: Triazole; topo-II inhibition; anticancer activity; cell cycle; apoptosis
Introduction
Chemotherapy is one of the most prominent treatment approaches for cancer [1]. Even
though a huge number of anticancer drugs are available in the market, their utility is limited
due to prominent side effects instead of their selective toxicity and outcome of resistance [2].
The ultimate goal of cancer chemotherapy is to produce a drug that can selectively destroy
cancer cells without having any significant effects on normal cells. Moreover, intense efforts
have focused on the development of novel antitumor agents with selective cytotoxicity
against resistance tumor cells.
Natural products have long served as lead compounds for the development of molecules with
optimized pharmacological properties for a variety of diseases, including cancer [3-5]. In this
context, podophyllotoxin is a well-established lead in the development of new chemical
agents for the treatment of cancer [6]. The semi-synthetic derivatives of podophyllotoxin like
etoposide and teniposide inhibit DNA topoisomerase-II by stabilizing the topoisomerase-II
via binding covalently with DNA and are used against a variety of cancers [7-8]. However,
their therapeutic use has encountered certain limitations such as acquired drug resistance [9],
cytotoxicity towards normal cells, poor aqueous solubility [10]. Extensive synthetic efforts
have been carried out by a number of research groups to improve the selectivity as well as

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DNA topoisomerase-II inhibition [11-13]. Previous reports suggested that the core structural
features like transfused γ-lactone, fused dioxole ring, and the orthogonal free-rotating 3,4,5-
trimethoxyphenyl fragment are considered essential for the cytotoxic activity of
podophyllotoxin derivatives [14-15]. It has also been indicated in the literature that bulkier
substituents at C-4 position of podophyllotoxin usually enhances the cytotoxicity and DNA
topoisomerase-II inhibition activity [16-17]. Currently, some of these synthetic
podophyllotoxin derivatives are being used in the treatment of several cancers [18-19] and
many of them have entered clinical trials for the treatment of diverse neoplasms (Figure 1). It
was found that most of these podophyllotoxin derivatives induce apoptosis by inhibiting
topoisomerase II [20]. Hence, a significant piece of attention has been diverted towards the
structural modification of podophyllotoxin to develop new therapeutic agents because of the
development of resistance towards etoposide [21-23].
On the other aspect, nitrogen containing heterocyclic motifs such as triazoles play a key role
in enhancing the cytotoxicity towards the cancer cells because of their improved solubility
and pharmacokinetic parameters at the binding site [24-25]. In addition, 1,2,3-triazoles have a
high dipole moment and are able to participate actively in hydrogen bonding as well as in
dipole-dipole and π stacking interactions, which help them in binding easily with the
biological targets to improve their solubility [26]. In continuation of our efforts to improve
the cytotoxicity of podophyllotoxin [27], herein we report the design and synthesis of a series
of 4β-amidotriazole linked podophyllotoxin derivatives to improve its potency by the
incorporation of the amido-triazolic group at the C-4 position, which can increase the
solubility and pharmacokinetic parameters of the podophyllotoxin skeleton.
< Fig. 1 >
2. Results and Discussion
2.1. Chemistry
The synthetic route adopted to obtain compounds (10a-i and 11a-k) is depicted in Scheme 1.
The main precursor’s aryl azides (1a-i), benzyl azides (2a-k) and 4β-aminopodophyllotoxin
(9) were prepared by using reported methods [28]. The triazole acid intermediates (6a-i and
7a-k) were synthesized by the reaction of ethyl propiolate with different azides by employing
the click chemistry protocol to produce triazolic esters (4a-i and 5a-k), which upon base
hydrolysis afforded triazolic acids (6a-i and 7a-k) in good yields. Subsequently, the acids

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were coupled with 4β-aminopodophyllotoxin (9) in the presence of N-(3-
dimethylaminopropyl)-Nꞌ-ethylcarbodiimide
hydrochloride
(EDCI),
and
1-
hydroxybenzotriazole (HOBT) in anhydrous DMF under nitrogen atmosphere to provided the
desired 4β-amidotriazole linked podophyllotoxin derivatives (10a-i and 11a-k) in best yields
(75-90%) and all these compounds were characterized by spectroscopic studies (NMR, MS,
and HRMS).
< Scheme 1 >
< Scheme 2 >
2.2. Antiproliferative activity
MTT assay [29] was performed to evaluate the cytotoxic effects of all newly synthesized
analogues of 4β-amidotriazole linked podophyllotoxin against the selected human cancer cell
lines, and the results were summarized as IC50 values in Table 1. As shown in Table 1,
several of the 4β-amidotriazole linked podophyllotoxin derivatives showed enhanced in vitro
cytotoxic activity with IC50 values of 1-10 µM, whereas the compounds did not show
significant cytotoxicity on NIH/3T3 skin fibroblast cells and some of the compounds 10a,
10b, 10g, 10i, 11b and 11h displayed potent broad-spectrum of activities against all the tested
cell lines. The compounds 10b, 10g, and 10i exhibited significant cytotoxicity with the IC50
values of < 1 µM and were superior to etoposide. Moreover, compound 10g exhibited
remarkable cytotoxicity with the IC50 values in the range of 0.70-4.11 µM and was most
promising compound in the series. For instance, cervical (Hela), breast (MCF-7), prostate
(DU-145), lung (A549), liver (HepG2) and colon (HT-29) were affected by 10g with IC50
values of 0.78, 0.97, 0.70, 1.20, 0.78 and 4.11 µM, respectively. Likewise, 10b exhibited
6.49, 1.10, 0.99, 1.61, 2.79 and 11.4 µM whereas 10i showed 1.21, 1.35, 0.89, 1.96, 1.21 and
4.40 µM against these human cancer cell lines (HeLa, MCF-7, DU-145, A549, HepG2 and
HT-29), respectively and the IC50 of etoposide employed as a standard drug ranges from 1.62
to 2.84 µM.
Based on the cytotoxicity results, the structure-activity relationship (SAR) was examined for
these 4β-amidotriazole linked podophyllotoxin derivatives (10a-i and 11a-k) to investigate
the effect of electron donating methoxy, methylenedioxy as well as electron withdrawing
groups like chloro, fluoro and trifluoromethyl groups on cancer cell toxicity. By comparing
the cytotoxicity values of compounds with different substitutions, it was clearly observed that

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the aryl triazolic-amide derivatives (n = 0, 10a-i) shown higher cytotoxicity in comparison to
their corresponding benzyl triazolic-amide derivatives (n = 1, 11a-k). In case of aryl
triazolic-amide derivatives, chloro substituent containing compound (10g) at position-4
exhibited high cytotoxicity (0.70-4.11 µM) against the tested cell lines and trifluoro methyl,
methoxy substituted compounds (10b; 0.99-11.40 µM and 10i; 0.89-4.40 µM) have also
shown promising inhibition values than the other substituents i.e., fluoro, 3,4-dimethoxy,
3,4,5-trimethoxy and 3,4-methylenedioxy groups baring compounds. Subsequently, the
benzyl triazolic-amide derivatives of podophyllotoxin (11a-k) displayed moderate
cytotoxicity. The cytotoxicity data indicates that the strong electron withdrawing groups like
chloro, trifluoromethyl at position-4 in aryl triazolic-amide derivatives exhibited superior
cytotoxicity than the standard.
< Table 1 >
2.3. Cell cycle analysis
To understand the mode of action of the most active compounds (10b, 10g, and 10i), we
examined the effects on cell cycle [30] by flow cytometry in DU-145 cancer cells. In this
study, DU-145 cells were treated with mentioned compounds for 24 h at indicated
concentrations. The compounds 10b, 10g, and 10i at 0.5 µM showed 56.71%, 31.94% and
52.42% and at 1 µM 64.3%, 35.61% and 56.16% of cell accumulation in G2/M phase,
whereas VP-16 (etoposide) showed 71.53% at 2 µM concentrations (Fig. 2). The data
obtained clearly indicated that these compounds including VP-16 leads to G2/M cell cycle
arrest of DU-145 cells.
< Fig. 2 >
2.4. DNA topoisomerase-II inhibition assay
Topoisomerase II inhibition assay was performed by using Topoisomerase II Drug Screening
Kit (TG1009, TopoGEN, USA) with etoposide as the standard. The DNA substrate, pHOT1
was included in the assay as supercoiled DNA, because of its small size and easy to handle
and has a large number of Topo II recognition elements. The assay system is based on
evaluating the formation of DNA cleavage products, primarily linearized DNA (linear DNA)
by topo-II enzymes [31-32].
< Fig. 3 >

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From the results, it was observed that catenated DNA treated with topoisomerase II (5units)
has shown nicked circular and relaxed circular DNA without any catenated DNA in the wells
as represented in the lane D (Fig. 3). Catenated DNA in the presence of topoisomerase II
incubated with etoposide (Lane E) has shown clear linear DNA formation, indicating that it
acts as IFP (Interfacial Poisons) which blocks the resealing of the double-stranded DNA
(dsDNA). Catenated DNA in the presence of topoisomerase II incubated with compound 10b,
10g and 10i inhibited the topoisomerase II activity represented (lane F, G, H) by the presence
of catenated DNA in the well, whereas compound 10b and 10g showed clear linear DNA
formation similar to etoposide as represented in the well (lane F and G) indicated the
inhibition of topo-II.
2.5. Apoptotic studies
2.5.1. Determination of morphological changes
To examine whether the treatment with these compounds could lead to loss of cell viability
and induce apoptosis, DU-145 cells were treated with these compounds (10b, 10g, and 10i).
Cells were observed, and photographs were taken with phase contrast microscope [33]. It can
be inferred from Fig. 4, that the treatment with these compounds resulted in reduced number
of viable cells in comparison to the vehicle-treated control cells as observed by the distinctive
morphological features such as cells detachment from substratum and shrinkage of cells.
Even though three compounds have shown morphological effects, compounds 10b and 10i
had determinant morphological changes compared to the standard VP-16 (Fig. 4).
< Fig. 4 >
2.5.2. Apoptosis by Hoechst staining
Nuclear fragmentation, chromatin condensation, and nuclear shrinkage are the foremost
characteristics of apoptosis. Morphological assessment of these characteristics for apoptosis
was performed with Hoechst staining [34]. In our present study, apoptosis-inducing the effect
of compounds VP-16, 10b, 10g, and 10i where studied in DU-145 cells. Cells treated with
compounds VP-16, 10b, 10g, and 10i at indicated concentrations for 24 h disclosed the
apoptotic characteristics such as condensed and fragmented nuclei. These results revealed
that the compounds VP-16, 10b, 10g, and 10i were effective in inducing cellular apoptosis
(Fig. 5).

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< Fig. 5 >
2.5.3. Measurement of mitochondria membrane potential (∆Ψm)
Mitochondria membrane potential (∆Ψm) was determined by the JC-1 dye. Cells with intact
mitochondrion with energized membrane potential shows red aggregates in the presence of
JC-1 dye [35]. Potent apoptotic inducers show green fluorescence by losing the membrane
potential as de-energized. Cells treated with these compounds (10b, 10g, and 10i) as well as
VP-16 showed the increased green fluorescence in VP-16, 10b, and 10i treated cells as
compared to untreated (Fig. 6), whereas 10g has not shown significant green fluorescence in
comparison with untreated cell control, indicating that compounds 10b and 10i could induce
depolarization of mitochondrial membrane potential (Fig. 6).
< Fig. 6 >
2.5.4. Measurement of reactive oxygen species (ROS)
Accumulation of ROS in the DU-145 cells treated with VP-16, 10b, 10g and 10i for 24 h was
estimated by 2’, 7’– dichlorofluorescein diacetate (DCFDA). It is a non-fluorescent substance
and it is converted to 2’, 7’–dichlorofluorescein (DCF), inside the cells through oxidation by
ROS [36]. Upon treatment with these compounds at indicated concentrations, increased ROS
production was observed in dose dependent-manner in comparison to VP-16. Whereas
compound 10g has shown equal ROS production and dose dependency was not observed
(Fig. 7).
< Fig. 7 >
2.5.5. Scratch assay
As migration is an important characteristic for metastatic cancers, the effect of these
compounds (10b, 10g, and 10i) on migration of cancer cells was investigated by scratch assay
[37]. After 0, 24 and 48 h study revealed that the treatment by 10b and 10i resulted in
decreased cell migration compared to VP-16. However, compound 10g did not show any
significant decrease in the cell migration (Fig. 8).
< Fig. 8 >
2.5.6. Annexin V/PI staining assay

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Quantification of apoptosis induced by these compounds was determined by using annexin
V-FITC-PI staining [38]. This assay is a proven method that differentiates the extent of live,
early apoptotic, late apoptotic and necrotic cells as shown in Fig. 9. The percentage of early
apoptotic cells in the increased by 6.7% and 7.01% at 0.5 µM whereas 7.0% and 17.65% at 1
µM in comparison to VP-16 that has shown 4.52% at 2 µM and 10g did not show any
significant increase in the activity. Therefore, these results have proved that the compounds
10b and 10i induced apoptosis in a dose-dependent manner (Fig. 9).
< Fig. 9 >
2.6. Molecular docking study
Cytotoxicity and enzymatic assay results of newly designed podophyllotoxin-triazole
conjugates (especially conjugates 10b, 10gand 10i) encouraged us to perform their molecular
docking studies at EVP binding pocket of DNA-topoisomerase-IIα to explore the binding
pose and protein-small molecule interactions. As similar to co-crystal ligand (EVP), chiral
centers present in these conjugates lead to attaining unique conformation to fit well into the
binding pocket (Fig. 10A) and showing intercalation with DNA structure present in target
protein (Fig. 10B).
< Fig. 10 >
These conjugates contain aromatic, acyclic ring systems along with a good number of
heteroatoms hence they show hydrogen bonding and hydrophobic interactions with the target
protein. Interactions shown by these conjugate with target protein are similar due to the
similarity in the binding pose. Oxygen atom present in the trimethoxy phenyl ring shows
hydrogen bonding (red dashed lines as shown in figure 11A, 11B and 11C) with
anamideNHgroup of Asp463. Oxygen atom present in dioxole ring shows hydrogen bonding
with side chain NH of Arg487 and amide NH present in these conjugate showing interaction
with thecarbonyl oxygen of DG13. In addition, Cl and CF3 groups present in conjugate 10g
and 10i respectively are showing interactions with side chain hydroxyl groups of Ser802.
< Fig. 11 >
In addition to the above mentioned strong interactions, these molecules also show
hydrophobic interactions (pink colored region shown in Figure 12A, 12B and 12C) with the
target protein. The core tetracyclic ring system present in these conjugates shows

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hydrophobic interaction with Arg487, Gly488, DC8, DA12, DG13 residues. Trimethoxy
phenyl ring shows interactions with Glu461, Gly462, Asp463, Ser464, Arg487, Gly488, DT9
and DG10. 1-aryl-1H-1,2,3-triazole-4-carboxamide motif showing interactions with Met762,
Ser802, Pro803, DT9, DC11, DA12.
< Fig. 12 >
3. Conclusion
In summary, we have designed and synthesized a series of 4β-amidotriazole linked
podophyllotoxin derivatives (10a-i and 11a-k) and examined their cytotoxicity on six human
cancer cell lines. The antiproliferative activity results indicated that most of these compounds
exhibit significant inhibitory activities. Compounds 10b, 10g and 10i showed potent
cytotoxic efficacy against the tested human cancer cell lines. Treatment of DU-145 cells with
these compounds resulted in G2/M cell cycle arrest. Moreover, they induce apoptosis through
depolarization of mitochondrial membrane potential and increased ROS production. The 4β-
amidotriazole linked analogues of podophyllotoxin are found to be more potent in inhibiting
human DNA topoisomerase-II. Moreover, the molecular docking study provided some
insights into the binding mode of the synthesized compounds in the active site of DNA
topoisomerase-II. As a result, it could be concluded that these compounds could be
considered as interesting lead molecules and further structural modifications may result in
promising new anticancer agents against the prostate cancer.
Acknowledgement
V.G.R acknowledges the UGC, New Delhi for the award of research fellowship and authors
thankful to CSIR, New Delhi for the financial support under the 12th Five Year Plan projects
“Affordable Cancer Therapeutics (ACT)” (CSC0301) & ORIGIN (CSC-108). S.R.B thanks
the Department of Science and Technology (DST), Government of India and the Centre
Franco-Indien pour la Promotion de la Recherche Avancée (CEFIPRA) for the award of a
Raman-Charpak fellowship.
4. Experimental Section
All reagents, starting materials, and solvents were purchased from Aldrich (Sigma-Aldrich,
St. Louis, MO, USA) or Alfa Aesar (Johnson Matthey Company, Ward Hill, MA, USA) and
used without further purification. Reactions were monitored by TLC, performed on 0.25 mm

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silica gel 60-F254 plates, and visualization on TLC was achieved by UV light or using an
iodine indicator. Column chromatography was performed using Merck silica gel of 60-120
1
13
mm with hexane and ethyl acetate as eluents. H and C NMR spectra were recorded with
75, 100, 300, 400, and 500 MHz (AVANCE) spectrometer in CDCl3 and DMSO-d6 solutions.
Chemical shifts (δ) are expressed in ppm relative to the internal standard TMS and
multiplicities of NMR signals are represented as singlet (s), broad singlet (bs), doublet (d),
triplet (t), double doublet (dd), triplet (t), q (quartet), and multiplets (m). High-resolution
mass spectra (ESI-HRMS) were obtained by using ESI-QTOF mass spectrometer (70 eV).
Melting points were determined on an electro thermal melting point apparatus and are
uncorrected.
4.1. General Procedure for the synthesis of aryl/aliphatic triazolic carboxylic acids (6a-i
and 7a-k)
The aryl/aliphatic triazolic carboxylic acids (6a-i and 7a-k) were synthesized based on a
literature method as following: By using click chemistry protocol method from the literature,
the triazolic esters (4a-i and 5a-k) were synthesized with ethyl propiolate (3) and
corresponding azides (2a-i and 3a-k). Than after, LiOH·H2O (1.5 mmol) was added in one
portion to a solution of ester (4a-i and 5a-k, 1 mmol) in THF/water (1:1, 20 mL). The
reaction mixture was stirred until the solid had dissolved and was then left overnight at room
temperature. The solvents were removed in vacuo, and the residue was dissolved in water (15
mL). The resulting solution was washed with diethyl ether (7 mL). The aqueous layer was
concentrated to half of its volume and then acidified with 30% hydrochloric acid (15 mL).
The resulting precipitate was filtered and dried to give compounds 6a-i and 7a-k as a solid
with good yields (80-90%). The spectroscopic data of the obtained triazolic acid compounds
was in agreement with the reported data [39] and few compounds are reporting for the first
time in the present work.
4.1.1. 1-(3,4-dimethoxyphenyl)-1H-1,2,3-triazole-4-carboxylic acid (6c)
1
Light brown solid, yield 82%, Mp: 194-196 ºC; H NMR (300 MHz, CDCl3+DMSO-d6): δ
8.87 (s, 1H), 7.45 (d, J = 2.47 Hz, 1H), 7.38 (dd, J = 2.47, 8.80 Hz, 1H), 7.03 (d, J = 8.80 Hz,
1H), 3.96 (s, 3H), 3.93 (s, 3H). 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 161.4, 149.1,
148.9, 140.3, 129.4, 125.9, 112.1, 111.2, 104.4, 55.7, 55.5; MS (ESI): m/z 250 [M+H]+.
HRMS (ESI) calcd for C11H11O4N3 [M+H]+ 250.08223; found: 250.08358.

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4.1.2. 1-(3,5-dimethoxyphenyl)-1H-1,2,3-triazole-4-carboxylic acid (6d)
1
Light brown solid, yield 88%, Mp: 192-194 ºC; H NMR (300 MHz, CDCl3+DMSO-d6): δ
8.61 (s, 1H), 6.94 (s, 2H), 6.53 (s, 1H), 3.87 (s, 6H). 13C NMR (75 MHz, CDCl3+DMSO-d6):
δ 161.6, 161.1, 140.6, 137.4, 125.8, 100.6, 98.6, 55.4; MS (ESI): m/z 250 [M+H]+. HRMS
(ESI) calcd for C11H11O4N3 [M+H]+ 250.08223; found: 250.08356.
4.1.3. 1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole-4-carboxylic acid (6e)
Off white solid, yield 83%, Mp: 187-189 ºC; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 8.80
13
(s, 1H), 7.09 (s, 2H), 3.95 (s, 6H), 3.87 (s, 3H). C NMR (75 MHz, CDCl3+DMSO-d6): δ
161.4, 153.2, 140.3, 137.6, 131.7, 126.0, 97.8, 60.1, 55.9; MS (ESI): m/z 280 [M+H]+. HRMS
(ESI) calcd for C12H13O5N3 [M+H]+ 280.09280; found: 280.09443.
4.1.4. 1-(benzo[d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-4-carboxylic acid (6f)
Brown solid, yield 86%, Mp: 175-177 ºC; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 8.56 (s,
13
1H), 7.34-7.30 (m, 1H), 7.26-7.20 (m, 1H), 6.95 (m, 1H), 6.12 (s, 2H). C NMR (75 MHz,
CDCl3+DMSO-d6): δ 161.6, 148.1, 147.9, 140.5, 130.2, 125.3, 113.9, 108.0, 102.1, 101.6;
MS (ESI): m/z 234 [M+H]+. HRMS (ESI) calcd for C10H7O4N3 [M+H]+ 234.05093; found:
234.05221.
4.1.5. 1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxylic acid (6i)
1
Pale brown solid, yield 81%, Mp: 180-182 ºC; H NMR (300 MHz, CDCl3+DMSO-d6): δ
8.92 (s, 1H), 8.06 (d, J = 8.52 Hz, 2H), 7.85 (d, J = 8.52 Hz, 2H). 13C NMR (75 MHz,
CDCl3+DMSO-d6): δ 161.4, 141.1, 138.5, 126.8 (d, JCF3 = 3.30 Hz), 125.4, 121.2, 120.3. MS
(ESI): m/z 258 [M+H]+. HRMS (ESI) calcd for C10H6O2N3F3 [M+H]+ 258.04849; found:
258.04992.
4.1.6. 1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (7d)
White solid, yield 90%, Mp: 196-198 ºC; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 7.99 (s,
1H), 6.94-6.80 (m, 3H), 5.51 (s, 2H), 3.88 (s, 3H), 3.85 (s, 3H). 13C NMR (75 MHz,
CDCl3+DMSO-d6): δ 161.6, 148.9, 148.8, 140.2, 126.9, 126.0, 111.0, 55.4, 53.5; MS (ESI):
m/z 264 [M+H]+. HRMS (ESI) calcd for C12H13O4N3 [M+H]+ 264.09788; found: 264.09895.
4.1.7. 1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (7e)

ACCEPTED MANUSCRIPT
White solid, yield 88%, Mp: 186-188 ºC; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 7.96 (s,
1H), 6.37-6.26 (m, 3H), 5.42 (s, 2H), 3.71 (s, 6H). 13C NMR (75 MHz, CDCl3+DMSO-d6): δ
159.9, 135.1, 130.0, 127.2, 120.1, 114.2, 113.5, 55.0, 54.0; MS (ESI): m/z 264 [M+H]+.
HRMS (ESI) calcd for C12H13O4N3 [M+H]+ 264.09788; found: 264.09922.
4.1.8. 1-(benzo[d][1,3]dioxol-4-ylmethyl)-1H-1,2,3-triazole-4-carboxylic acid (7g)
White solid, yield 85%, Mp: 170-172 ºC; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 8.13 (s,
1H), 6.88-6.78 (m, 3H), 5.99 (s, 2H), 5.48 (s, 2H). MS (ESI): m/z 248 [M+H]+. HRMS (ESI)
calcd for C11H9O4N3 [M+H]+ 248.06658; found: 248.06780.
4.1.9. 1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazole-4-carboxylic acid (7j)
1
Pale brown solid, yield 80%, Mp: 193-195 ºC; H NMR (300 MHz, CDCl3+DMSO-d6): δ
8.15 (s, 1H), 7.65 (d, J = 7.97 Hz, 2H), 7.43 (d, J = 7.97 Hz, 2H), 5.68 (s, 2H). MS (ESI): m/z
272 [M+H]+. HRMS (ESI) calcd for C11H8O2N3F3 [M+H]+ 272.06414; found: 272.06541.
4.2. General Procedure for the synthesis of 4β-amido triazole linked derivatives (10a-i
and 11a-k)
To a solution of aryl/aliphatic triazolic carboxylic acid (6a-i and 7a-k, 0.5mmol) in
anhydrous dimethylformamide, EDCI (0.6 mmol) and HOBT (0.6 mmol) were added at 0 °C
and the reaction mixture was stirred for 20 min. To the reaction mixture 4β-amino
phodophyllotoxin (9, 0.5 mmol) was added and stirred at room temperature for 12 h. The
contents of the reaction mixture were poured into ice-cold water (25 mL), extracted with
ethyl acetate (3x15 mL) and the combined organic phase was washed with brine, dried over
anhydrous sodium sulfate, filtered and concentrated in vacuo. The obtained residue was
purified by column chromatography using ethyl acetate–hexane (10-50%) as eluent to give
analytically pure compounds (10a-i and 11a-k) with finest yields (75-90%).
4.2.1. N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-phenyl-1H-1,2,3-triazole-4-
carboxamide (10a)
White solid, yield 88%, Mp: 170-172 ºC.; [α]25D: -86.0 (c: 3.6, CHCl3); 1H NMR (300 MHz,
CDCl3): δ 8.54 (s, 1H), 7.76 (d, J = 7.55, 2H), 7.64-7.50 (m, 5H), 6.84 (s, 1H), 6.49 (s, 1H),
5.99 (d, J = 3.5 Hz, 2H), 5.45 (dd, J = 3.39, 7.16 Hz, 1H), 4.58 (d, J = 3.21 Hz, 1H), 4.49 (t, J

ACCEPTED MANUSCRIPT
= 6.79 Hz, 1H), 3.98 (d, J = 8.87 Hz, 1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.10 (d, J = 3.02 Hz,
13
2H). C NMR (100 MHz, CDCl3): δ 174.2, 159.7, 152.5, 148.4, 147.5, 142.8, 137.1, 136.2,
134.7, 133.4, 132.3, 130.1, 129.6, 128.2, 123.6, 120.5, 109.6, 108.0, 101.7, 68.8, 60.7, 56.1,
54.6, 48.1, 43.6, 41.5, 37.5; MS (ESI): m/z 585 [M+H]+. HRMS (ESI) calcd for
C31H28O8N4Na [M+Na]+ 607.1805; found: 607.1805.
4.2.2. 1-(4-methoxyphenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (10b)
White solid, yield 85%, Mp: 195-197 ºC.; [α]25D: -77.6 (c: 3.2, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.43 (s, 1H), 7.65 (d, J = 9.0 Hz, 2H), 7.58 (d, J = 7.47 Hz, 1H), 7.08 (d, J = 9.0
Hz, 2H), 6.84 (s, 1H), 6.47 (s, 1H), 6.29 (s, 2H), 6.00 (d, J = 6.56 Hz, 1H), 5.45 (dd, J = 4.12,
7.47 Hz, 1H), 4.57 (d, J = 4.57 Hz, 1H), 4.49 (dd, J = 7.17, 9.30 Hz, 1H), 3.95 (t, J = 10.07
13
Hz, 1H), 3.89 (s, 3H), 3.81 (s, 3H), 3.75 (s, 6H), 3.11-3.03 (m, 2H). C NMR (100 MHz,
CDCl3): δ 174.2, 160.4, 159.8, 152.5, 148.4, 147.5, 142.5, 137.1, 134.7, 132.3, 129.5, 128.2,
123.6, 122.1, 115.0, 109.7, 109.1, 108.0, 101.6, 68.8, 60.7, 56.1, 55.6, 48.1, 43.6, 41.5, 37.5;
MS (ESI): m/z 615 [M+H]+. HRMS (ESI) calcd for C32H30O9N4Na [M+Na]+ 637.1910;
found: 637.1909.
4.2.3. 1-(3,4-dimethoxyphenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (10c)
White solid, yield 84%, Mp: 158-160 ºC.; [α]25D: -84.6 (c: 4.7, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.46 (s, 1H), 7.69 (s, 1H), 7.36 (s, 1H), 7.21 (dd, J = 2.44, 8.54 Hz, 1H), 7.01 (d, J
= 8.69 Hz, 1H), 6.85 (s, 1H), 6.43 (d, J = 4.73 Hz, 1H), 6.28 (s, 2H), 6.00 (q, J = 1.37, 3.66
Hz, 2H), 5.45 (dd, J = 4.42, 7.32 Hz, 1H), 4.53 (d, J = 4.57 Hz, 1H), 4.49 (dd, J = 7.47, 9.00
Hz, 1H), 4.00 (s, 3H), 3.97 (s, 3H), 3.94 (d, J = 3.46 Hz, 1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.18-
3.12 (m, 1H), 3.10-3.02 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 174.3, 159.7, 152.5, 150.1,
150.0, 148.3, 147.4, 142.6, 137.0, 134.7, 132.2, 129.6, 128.2, 123.3, 112.0, 111.3, 109.3,
109.1, 107.9, 104.1, 101.7, 68.8, 60.7, 56.3, 56.2, 56.0, 48.2, 43.6, 41.4, 37.5; MS (ESI): m/z
645 [M+H]+. HRMS (ESI) calcd for C33H32O10N4Na [M+Na]+ 667.2016; found: 667.2019.

ACCEPTED MANUSCRIPT
4.2.4. 1-(3,5-dimethoxyphenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (10d)
White solid, yield 87%, Mp: 145-148 ºC.; [α]25D: -78.7 (c: 3.8, CHCl3);1H NMR (500 MHz,
CDCl3): δ 8.51 (s, 1H), 7.71 (d, J = 7.47 Hz, 1H), 6.92 (d, J = 2.13 Hz, 2H), 6.84 (s, 1H),
6.58 (t, J = 2.13 Hz, 1H), 6.42 (s, 1H), 6.28 (s, 2H), 6.00 (d, J = 2.44 Hz, 2H), 5.45 (dd, J =
4.42, 7.47 Hz, 1H), 4.54 (d, J = 4.88 Hz, 1H), 4.49 (q, J = 7.47, 9.00 Hz, 1H), 3.94 (t, J =
9.46 Hz, 1H), 3.89 (s, 6H), 3.81 (s, 3H), 3.75 (s, 6H), 3.16 (dd, J = 5.18, 14.34 Hz, 1H), 3.10-
3.02 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 174.3, 161.7, 159.6, 152.5, 148.4, 147.4,
142.6, 137.6, 137.0, 134.7, 132.2, 128.1, 123.7, 109.4, 109.1, 107.9, 101.7, 101.3, 68.8, 60.7,
56.1, 55.8, 48.2, 43.6, 41.5, 37.5; MS (ESI): m/z 645 [M+H]+. HRMS (ESI) calcd for
C33H32O10N4Na [M+Na]+ 667.2016; found: 667.2017.
4.2.5.
N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazole-4-carboxamide (10e)
White solid, yield 89%, Mp: 181-183 ºC.; [α]25D: -83.1 (c: 5.9, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.49 (s, 1H), 7.69 (d, J = 7.47 Hz, 1H), 6.97 (s, 2H), 6.85 ( s, 1H), 6.43 (s, 1H),
6.28 (s, 2H), 6.01 (q, J = 1.37, 2.13 Hz, 2H), 5.45 (dd, J = 4.42, 7.47 Hz, 1H), 4.55 (d, J =
4.88 Hz, 1H), 4.49 (q, J = 7.47, 9.15 Hz, 1H), 3.97 (s, 6H), 3.94 (d, J = 1.37 Hz, 1H), 3.91 (s,
13
3H), 3.81 (s, 3H), 3.75 (s, 6H), 3.15 (dd, J = 4.88, 14.34 Hz, 1H), 3.11-3.03 (m, 1H). C
NMR (100 MHz, CDCl3): δ 174.3, 159.6, 154.1, 152.5, 148.4, 147.4, 142.7, 138.9, 137.1,
134.6, 132.3, 131.8, 128.1, 123.6, 105.9, 109.1, 108.0, 101.7, 97.9, 68.8, 61.0, 60.7, 56.5,
56.1, 54.6, 48.2, 43.7, 41.5, 37.5; MS (ESI): m/z 675 [M+H]+. HRMS (ESI) calcd for
C34H34O11N4Na [M+Na]+ 697.2122; found: 697.2116.
4.2.6. 1-(benzo[d][1,3]dioxol-5-yl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (10f)
White solid, yield 79%, Mp: 188-191 ºC.; [α]25D: -54.6 (c: 4.6, CHCl3); 1H NMR (300 MHz,
CDCl3): δ 8.42 (s, 1H), 7.46 (d, J = 7.45 Hz, 1H), 7.23 (d, J = 2.07 Hz, 1H), 7.16 (dd, J =
2.07, 8.31 Hz, 1H), 6.95 (d, J = 8.31 Hz, 1H), 6.82 (s, 1H), 6.54 (s, 1H), 6.32 (s, 2H), 6.11 (s,
2H), 5.99 (d, J = 7.21 Hz, 2H), 5.45 (dd, J = 2.81, 6.96 Hz, 1H), 4.64 (d, J = 2.07 Hz, 1H),

ACCEPTED MANUSCRIPT
4.52-4.46 (m, 1H), 3.97-3.91 (m, 1H), 3.82 (s, 3H), 3.76 (s, 6H), 3.09-3.05 (m, 2H). 13C
NMR (100 MHz, CDCl3): δ 174.2, 159.7, 152.5, 148.8, 148.7, 148.4, 147.5, 142.5, 137.1,
134.7, 132.3, 130.6, 128.2, 123.9, 114.4, 109.8, 109.1, 108.7, 108.1, 102.5, 102.3, 101.6,
68.8, 60.6, 56.1, 48.1, 43.7, 41.6, 37.5; MS (ESI): m/z 629 [M+H]+. HRMS (ESI) calcd for
C32H28O10N4Na [M+Na]+ 651.1703; found: 651.1703.
4.2.7. 1-(4-chlorophenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (10g)
White solid, yield 83%, Mp: 222-225 ºC.; [α]25D: -72.5 (c: 1.2, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.52 (s, 1H), 7.72 (d, J = 8.85 Hz, 2H), 7.57 (d, J = 8.85 Hz, 2H), 7.52 (d, J = 7.47
Hz, 1H), 6.83 (s, 1H), 6.51 (s, 1H), 6.30 (s, 2H), 6.00 (d, J = 7.47 Hz, 2H), 5.45 (dd, J = 3.35,
7.47 Hz, 1H), 4.60 (d, J = 3.35 Hz, 1H), 4.51-4.47 (m, 1H), 3.96-3.91 (m, 1H), 3.81 (s, 3H),
13
3.76 (s, 6H), 3.08-3.04 (m, 2H). C NMR (100 MHz, CDCl3): δ 174.1, 159.7, 152.5, 148.4,
147.6, 142.6, 137.1, 135.3, 134.7, 132.3, 131.9, 129.5, 128.2, 125.5, 109.8, 109.1, 108.1,
101.6, 68.8, 60.7, 56.1, 53.8, 47.9, 43.7, 41.6, 37.5; MS (ESI): m/z 619 [M+H]+. HRMS (ESI)
calcd for C31H27O8N4ClNa [M+Na]+ 641.1415; found: 641.1417.
4.2.8. 1-(4-fluorophenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (10h)
White solid, yield 81%, Mp: 228-230 ºC.; [α]25D: -52.8 (c: 3.5, CHCl3); 1H NMR (400 MHz,
CDCl3): δ 8.49 (s, 1H), 7.74 (dd, J = 4.52, 8.92 Hz, 2H), 7.49 (s, 1H), 7.31-7.27 (m, 2H),
6.83 (s, 1H), 6.55-6.51 (s, 1H), 6.31 (s, 2H), 5.99 (dd, J = 1.22, 7.21 Hz, 2H), 5.45 (dd, J =
3.30, 7.45 Hz, 1H), 4.62 (s, 1H), 4.51-4.46 (m, 1H), 3.98-3.91 (m, 1H), ), 3.82 (s, 3H), 3.76
(s, 6H), 3.10-3.04 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 174.1, 162.9 (d, JCF = 250.8 Hz),
159.6, 152.5, 148.4, 147.6, 142.8, 137.1, 134.6, 132.5 (d, JCF = 2.9 Hz), 132.3, 128.1, 123.8,
122.6 (d, JCF = 8.0 Hz), 117.1 (d, JCF = 22.7 Hz), 109.8, 109.1, 108.0, 101.6, 68.8, 60.7, 56.1,
48.1, 43.7, 41.6, 37.5; HRMS (ESI) calcd for C31H27O8N4FNa [M+Na]+ 625.1711; found:
625.1717.
4.2.9. N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(4-(trifluoromethyl)phenyl)-
1H-1,2,3-triazole-4-carboxamide (10i)

ACCEPTED MANUSCRIPT
White solid, yield 79%, Mp: 190-194 ºC.; [α]25D: -93.6 (c: 3.9, CHCl3); 1H NMR (400 MHz,
CDCl3): δ 8.62 (s, 1H), 7.93 (d, J = 8.55 Hz, 2H), 7.87 (d, J = 8.55 Hz, 2H), 7.47 (d, J = 7.45
Hz, 1H), 6.83 (s, 1H), 6.55 (s, 1H), 6.32 (s, 2H), 6.01-5.97 (m, 2H), 5.47 (dd, J = 3.42, 7.33
Hz, 1H), 4.64 (d, J = 4.89 Hz, 1H ), 4.52-4.41 (m, 2H), 3.97-3.91 (m, 1H), 3.82 (s, 3H), 3.76
(s, 6H), 3.10-3.04 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 174.2, 159.4, 152.5, 148.5, 147.6,
143.1, 138.6, 137.2, 134.6, 132.4, 128.2, 128.1, 127.4, 123.7,120.7,110.0, 109.1, 108.1,
101.6, 69.0, 68.8, 60.6, 56.1, 48.1, 43.7, 41.7, 37.4; MS (ESI): m/z 653 [M+H]+. HRMS (ESI)
calcd for C32H27O8N4F3Na [M+Na]+ 675.1679; found: 675.1677.
4.2.10. 1-benzyl-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-4-
carboxamide (11a)
White solid, yield 80%, Mp: 164-166 ºC.; [α]25D: -97.5 (c: 5.5, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 7.99 (s, 1H), 7.42-7.39 (m, 4H), 7.32-7.29 (m, 2H), 6.78 (s, 1H), 6.53 (s, 1H), 6.31
(s, 2H), 5.98 (dd, J = 1.22, 10.33 Hz, 2H), 5.57 (s, 2H), 5.40 (dd, J = 3.66, 7.47 Hz, 1H), 4.63
(d, J = 3.66 Hz, 1H), 4.46-4.42 (m, 1H), 3.89 (t, J = 9.15 Hz, 1H), 3.81 (s, 3H), 3.75 (s, 6H),
3.05-3.02 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 174.2, 159.8, 152.5, 148.4, 147.5, 142.4,
137.1, 134.7, 133.4, 132.3, 129.3, 129.2, 128.2, 125.5, 109.9, 109.1, 108.0, 101.6, 68.8, 60.7,
56.1, 54.6, 47.9, 43.6, 41.6, 37.5; MS (ESI): m/z 599 [M+H]+. HRMS (ESI) calcd for
C32H30O8N4Na [M+Na]+ 621.1961; found: 621.1963.
4.2.11. 1-(4-methoxybenzyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (11b)
White solid, yield 84%, Mp: 174-177 ºC.; [α]25D: -72.55 (c: 7.6, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 7.95 (s, 1H), 7.60 (d, J = 7.55 Hz, 1H), 7.28 (d, J = 1.88 Hz, 1H), 7.25 (s, 1H),
6.93 (d, J = 8.68 Hz, 2H), 6.80 (s, 1H), 6.44 (s, 1H), 6.30 (s, 2H), 5.98 (d, J = 4.72 Hz, 2H),
5.49 (s, 2H), 5.38 (dd, J = 3.77, 7.36 Hz, 1H), 4.5 (d, J = 4.15 Hz, 1H), 4.46-4.39 (m, 1H),
3.88 (t, J = 9.81 Hz, 2H), 3.81 (s, 6H), 3.75 (s, 6H), 3.06-3.00 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 174.2, 160.2, 159.9, 152.5, 148.4, 147.5, 142.3, 137.1, 134.7, 133.4, 132.3, 129.9,
128.2, 125.3, 114.6, 109.8, 109.1, 108.1, 101.6, 68.8, 60.6, 56.1, 55.3, 54.2, 47.9, 43.6, 41.6,
37.5; MS (ESI): m/z 629 [M+H]+. HRMS (ESI) calcd for C33H32O9N4Na [M+Na]+ 651.2067;
found: 651.2070.

ACCEPTED MANUSCRIPT
4.2.12. 1-(3-methoxybenzyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (11c)
White solid, yield 83%, Mp: 165-168 ºC.; [α]25D: -78.0 (c: 5.5, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.00 (s, 1H), 7.37 (d, J = 7.47 Hz, 1H), 7.32 (t, J = 7.93 Hz, 1H), 6.92 (dd, J =
2.44, 8.39 Hz, 1H), 6.88 (d, J = 7.62 Hz, 1H), 6.82 (t, J = 1.98 Hz, 1H), 6.78 (s, 1H), 6.54 (s,
1H), 6.31 (s, 2H), 5.98 (dd, J = 1.37, 10.98 Hz, 2H), 5.53 (s, 2H), 5.40 (dd, J = 3.81, 7.62 Hz,
1H), 4.63 (d, J = 3.96 Hz, 1H), 4.46-4.42 (m, 1H), 3.89 (t, J = 9.15 Hz, 1H), 3.81 (s, 3H),
13
3.80 (s, 3H), 3.75 (s, 6H), 3.05-3.01 (m, 2H). C NMR (100 MHz, CDCl3): δ 174.2, 160.1,
159.8, 152.5, 148.4, 147.6, 142.4, 137.1, 134.7, 132.3, 130.4, 128.2, 125.6, 120.4, 114.4,
114.1, 109.9, 109.1, 108.1, 101.6, 68.8, 60.7, 56.1, 55.3, 54.5, 47.9, 43.6, 41.6, 37.5; MS
(ESI): m/z 629 [M+H]+. HRMS (ESI) calcd for C33H32O9N4Na [M+Na]+ 651.2067; found:
651.2064.
4.2.13. 1-(3,4-dimethoxybenzyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (11d)
White solid, yield 88%, Mp: 180-183 ºC.; [α]25D: -59.6 (c: 5.6, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 7.98 (s, 1H), 7.40 (t, J = 7.32 Hz, 1H), 6.90 (d, J = 1.90 Hz, 1H), 6.88 (s, 1H), 6.82
(d, J = 1.98 Hz, 1H), 6.78 (s, 1H), 6.53 (d, J = 3.66, 1H), 6.31 (s, 2H), 5.98 (dd, J = 1.22,
10.22 Hz, 2H), 5.49 (s, 2H), 5.39 (dd, J = 3.50, 7.47 Hz, 1H), 4.63 (s, 1H), 4.46-4.42 (m, 1H),
3.89 (s, 3H), 3.87 (s, 3H), 3.81 (s, 3H), 3.75 (s, 6H), 3.05-3.01 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 174.2, 159.9, 152.5, 149.8, 149.5, 148.4, 147.4, 142.3, 137.1, 134.7, 132.3, 128.2,
125.6, 125.3, 121.2, 111.4, 111.3, 109.9, 109.1, 108.1, 101.6, 68.8, 60.7, 56.1, 55.9, 54.6,
47.9, 43.6, 41.6, 37.5; MS (ESI): m/z 659 [M+H]+. HRMS (ESI) calcd for C34H34O10N4Na
[M+Na]+ 681.2173; found: 651.2167.
4.2.14. 1-(3,5-dimethoxybenzyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (11e)
White solid, yield 86%, Mp: 245-248 ºC.; [α]25D: -70.4 (c: 4.5, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.01 (s, 1H), 7.40 (d, J = 7.47 Hz, 1H), 6.79 (s, 1H), 6.53 (s, 1H), 6.46 (s, 1H),
6.42 (s, 2H), 6.31 (s, 2H), 5.98 (d, J = 10.22, 2H), 5.47 (s, 2H), 5.40 (dd, J = 2.28, 6.71 Hz,

ACCEPTED MANUSCRIPT
1H), 4.63 (s, 1H), 4.47-4.42 (m, 1H), 3.92-3.87 (m, 1H), 3.81 (s, 3H), 3.78 (s, 6H), 3.75 (s,
13
6H), 3.06-3.01 (m, 2H). C NMR (100 MHz, CDCl3): δ 174.2, 161.4, 159.8, 152.5, 148.4,
147.5, 142.4, 137.1, 135.3, 134.7, 132.3, 128.2, 125.6, 109.9, 109.1, 108.0, 106.3, 101.6,
100.5, 68.8, 60.6, 56.1, 55.4, 54.6, 47.9, 43.6, 41.6, 37.5; MS (ESI): m/z 659 [M+H]+. HRMS
(ESI) calcd for C34H34O10N4Na [M+Na]+ 681.2173; found: 651.2173.
4.2.15.
N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(3,4,5-trimethoxybenzyl)-1H-
1,2,3-triazole-4-carboxamide (11f)
White solid, yield 85%, Mp: 189-192 ºC.; [α]25D: -87.9 (c: 6.0, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.02 (s, 1H), 7.37 (d, J = 7.47 Hz, 1H), 6.78 (s, 1H), 6.54 (s, 1H), 6.53 (s, 2H),
6.31 (s, 2H), 5.98 (dd, J = 1.22, 10.37 Hz, 2H), 5.47 (s, 2H), 5.40 (dd, J = 3.96, 7.47 Hz, 1H),
4.64 (d, J = 4.27 Hz, 1H), 4.47-4.43 (m, 1H), 3.90-3.87 (m, 1H), 3.86 (s, 6H), 3.85 (s, 3H),
13
3.81 (s, 3H), 3.75 (s, 6H), 3.06-3.01 (m, 2H). C NMR (100 MHz, CDCl3): δ 174.1, 159.8,
153.8, 152.5, 148.4, 147.6, 142.4, 138.7, 137.1, 134.7, 132.3, 128.6, 128.2, 125.5, 109.9,
109.1, 108.1, 105.6, 101.6, 68.8, 60.8, 60.7, 56.2, 56.1, 55.0, 47.9, 43.6, 41.6, 37.5; MS
(ESI): m/z 689 [M+H]+. HRMS (ESI) calcd for C35H36O11N4Na [M+Na]+ 711.2278; found:
711.2267.
4.2.16.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-
trimethoxyphenyl)-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-
yl)-1H-1,2,3-triazole-4-carboxamide (11g)
White solid, yield 83%, Mp: 170-173 ºC.; [α]25D: -50.0 (c: 6.9, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 7.98 (s, 1H), 7.39 (d, J = 7.62 Hz, 1H), 6.82 (d, J = 0.91 Hz, 2H), 6.78 (s, 1H),
6.76 (s, 1H), 6.53 (s, 1H), 6.31 (s, 2H), 5.99 (s, 2H), 5.98 (dd, J = 1.37, 11.29 Hz, 2H), 5.46
(s, 2H), 5.40 (dd, J = 3.66, 7.47 Hz, 1H), 4.63 (d, J = 3.96 Hz, 1H), 4.46-4.42 (m, 1H), 3.91-
3.87 (m, 1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.05-3.01 (m, 2H). 13C NMR (100 MHz, CDCl3): δ
174.2, 159.8, 152.5, 148.4, 147.6, 142.4, 137.1, 134.7, 132.3, 128.2, 126.9, 125.4, 122.3,
109.9, 109.1, 108.7, 108.6, 108.1, 101.6, 101.5, 68.8, 60.7, 56.1, 54.5, 47.9, 43.6, 41.6, 37.5;
MS (ESI): m/z 643 [M+H]+. HRMS (ESI) calcd for C33H30O10N4Na [M+Na]+ 665.1860;
found: 665.1861.

ACCEPTED MANUSCRIPT
4.2.17.
1-(4-chlorobenzyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (11h)
White solid, yield 78%, Mp: 180-184 ºC.; [α]25D: -87.4 (c: 3.7, CHCl3); 1H NMR (400 MHz,
CDCl3): δ 8.01 (s, 1H), 7.39 (d, J = 8.43 Hz, 2H), 7.39-7.35 (m, 1H), 7.24 (d, J = 8.43 Hz,
2H), 6.78 (s, 1H), 6.54 (s, 1H), 6.31 (s, 2H), 5.98 (dd, J = 1.2, 8.9 Hz, 2H), 5.54 (s, 2H), 5.40
(dd, J = 3.7, 7.5 Hz, 1H), 5.30 (s, 1H), 4.63 (d, J = 4.03 Hz, 1H), 4.46-4.42 (m, 1H), 3.92-
3.86 (m, 1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.06-3.01 (m, 2H). 13C NMR (100 MHz, CDCl3): δ
174.2, 159.7, 152.5, 148.4, 147.6, 142.6, 137.1, 135.3, 134.7, 132.3, 131.9, 129.5, 128.2,
125.5, 109.9, 109.1, 108.1, 101.6, 68.8, 60.6, 56.1, 53.8, 47.9, 43.6, 41.6, 37.4; MS (ESI): m/z
633 [M+H]+. HRMS (ESI) calcd for C32H29O8N4ClNa [M+Na]+ 655.1572; found: 655.1572.
4.2.18.
1-(4-fluorobenzyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-1,2,3-triazole-
4-carboxamide (11i)
White solid, yield 82%, Mp: 169-172 ºC.; [α]25D: -76.0 (c: 5.5, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.00 (s, 1H), 7.38 (d, J = 7.32 Hz, 1H), 7.32-7.29 (m, 2H), 7.10 (t, J = 8.54 Hz,
2H), 6.78 (s, 1H), 6.53 (s, 1H), 6.31 (s, 2H), 5.98 (dd, J = 1.22, 10.98 Hz, 2H), 5.55 (s, 2H),
5.40 (dd, J = 3.81, 7.62 Hz, 1H), 4.63 (d, J = 43.50 Hz, 1H), 4.46-4.42 (m, 1H), 3.91-3.87 (m,
13
1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.06-3.01 (m, 2H). C NMR (100 MHz, CDCl3): δ 174.2,
163.0 (d, JCF = 249.4 Hz), 159.7, 152.5, 148.4, 147.6, 142.5, 137.1, 134.7, 132.3, 130.2 (d,
JCF = 8.0 Hz), 129.3 (d, JCF = 2.2 Hz), 128.2, 125.4, 116.4 (d, JCF = 21.2 Hz), 109.9, 109.1,
108.1, 101.6, 68.8, 60.7, 56.1, 53.8, 47.9, 43.6, 41.6, 37.5; MS (ESI): m/z 617 [M+H]+.
HRMS (ESI) calcd for C32H29O8N4FNa [M+Na]+ 639.1867; found: 639.1871.
4.2.19.
N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(4-(trifluoromethyl)benzyl)-
1H-1,2,3-triazole-4-carboxamide (11j)
White solid, yield 80%, Mp: 171-174 ºC.; [α]25D: -93.6 (c: 3.9, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.05 (s, 1H), 7.68 (d, J = 8.08 Hz, 2H), 7.42 (d, J = 8.08 Hz, 2H), 7.37 (d, J = 7.47
Hz, 1H), 6.78 (s, 1H), 6.55 (s, 1H), 6.31 (s, 2H), 5.98 (dd, J = 1.22, 10.98 Hz, 2H), 5.65 (s,
2H), 5.40 (dd, J = 3.96, 7.47 Hz, 1H), 4.64 (d, J = 4.12 Hz, 1H), 4.46-4.43 (m, 1H), 3.92-3.87
(m, 1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.06-3.01 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 174.1,

ACCEPTED MANUSCRIPT
159.6, 152.5, 148.5, 147.6, 142.7, 137.4, 137.2, 134.7, 132.4, 128.4, 128.2, 126.3 (d, JCF3
=
3.6 Hz), 125.7, 110.0, 109.1, 108.1, 101.6, 68.8, 60.7, 56.1, 53.9, 47.9, 43.7, 41.7, 37.5; MS
(ESI): m/z 667 [M+H]+. HRMS (ESI) calcd for C33H30O8N4F3 [M+H]+ 667.2016; found:
667.2013.
4.2.20.
N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(3-phenoxybenzyl)-1H-1,2,3-
triazole-4-carboxamide (11k)
White solid, yield 87%, Mp: 148-151 ºC.; [α]25D: -74.0 (c: 4.9, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 8.02 (s, 1H), 7.38-7.33 (m, 4H), 7.16 (t, J = 7.47 Hz, 1H), 7.01 (d, J = 7.78 Hz,
2H), 6.99 (dd, J = 1.98, 8.08 Hz, 2H), 6.94 (s, 1H), 6.79 (s, 1H), 6.54 (s, 1H), 6.32 (s, 2H),
5.98 (d, J = 11.44 Hz, 2H), 5.53 (s, 2H), 5.40 (dd, J = 3.81, 7.62 Hz, 1H), 4.64 (d, J = 4.12
Hz, 1H), 4.47-4.43 (m, 1H), 3.92-3.88 (m, 1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.06-3.02 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 174.1, 159.8, 158.2, 156.1, 152.5, 148.4, 147.5, 142.4, 137.1,
135.2, 134.7, 132.3, 130.6, 129.8, 128.2, 125.6, 123.9, 122.5, 119.3, 118.8, 118.1, 109.9,
109.1, 108.0, 101.6, 68.8, 60.6, 56.1, 54.2, 47.9, 43.6, 41.6, 37.5; MS (ESI): m/z 691 [M+H]+.
HRMS (ESI) calcd for C38H34O9N4Na [M+Na]+ 713.2223; found: 713.2224.
4.4. Biology
4.4.1. Cell culture
The cell lines, Cervical (Hela), Breast (MCF-7), Prostate (DU-145), Lung (A549), Liver
(HepG2) and Colon (HT-29) which were used in this study were procured from American
Type Culture Collection (ATCC), United States and were maintained in the Dulbecco’s
modified Eagle’s medium containing 10% (v/v) fetal bovine serum (FBS; HyClone, Logan,
UT) supplemented with 100 units/mL of penicillin and streptomycin (Sigma-Aldrich, St.
Louis, MO). All cells were cultured at 37 ºC in a humidified atmosphere with 5% CO2. Test
compounds were dissolved in DMSO (Sigma-Aldrich) to prepare 10 mM stock solution for
the following experiments. The stock was diluted with culture medium to desired
concentrations for drug treatment.
4.4.2. Evaluation of anti-proliferative activity

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The cytotoxic activity of the compounds was determined by using MTT assay [40]. Cells
were seeded in 100 µL DMEM, supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h at 37 ºC in a CO2 incubator. After incubation cells
were treated with test compounds for 48 h. After 48 h of incubation, 10 µL MTT (3-(4,5-
dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to each well
and the plates were further incubated for 4 h. Then the supernatant from each well was
carefully removed, formazan crystals were dissolved in 100 µL of DMSO and absorbance at
570 nm wavelength was recorded.
4.4.3. Cell cycle analysis
Effect of compounds 10b, 10g and 10i on DNA content by cell cycle progression was
assessed using DU-145 cells. Cells (2x105/well) were incubating in a 6-well plate with
incomplete media (serum free) for 24 h; to bring all cells in a synchronized state [41]. After
24 hrs the incomplete media was replaced with complete media with or without compounds
VP-16, 10b, 10g and 10i at indicated concentrations and incubated for 24 h. The cells were
washed thrice with PBS, harvested, fixed in ice cold PBS in 70% ethanol, and stored at -20
ºC for 30 min. After fixation, these cells were incubated with RNase A (0.1 mg/mL) at 37 ºC
for 30 min, stained with propidium iodide (50 µg/mL) for 30 min on ice in dark, and then
measured for DNA content using BD FACSVerse flow cytometer (Becton Dickinson, USA).
4.4.4. DNA topoisomerase-II inhibition assay
The topoisomerase II assay was done by the protocol provided by TopoGEN, Inc. The total
reaction volume of the topoisomerase II mediated cleavage reaction was fixed at 20 µL.
Briefly, assay buffer [50 mM Tris-HCl, pH 8, 120 mM KCl, 10 mM MgCl2, 0.5 mM ATP,
0.5 mM dithiothreitol, 30 µg/mL bovine serum albumin (BSA)] containing 200 ng of KDNA
(TOPOgen), and a solution of the test drugs were added to five units of the human
recombinant topoisomerase II (the amount of enzyme which resulted in the complete
decatenation of 200 ng of KDNA). After 10 min. of incubation at 37 ºC, the reaction was
stopped by addition of 5 µL of stop buffer containing the loading dye and then the reaction
mixture was analyzed on a 1% agarose gel in TAE buffer.
4.4.5. Determination of morphological changes

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Morphological changes were assessed based on the method described by the reported method
[42] with slight modifications. DU-145 cells were seeded at a density of 2x105 cells/well in a
6-well plate and incubated overnight. Media was replaced with the fresh media containing the
compounds 10b, 10g and 10i at indicated concentrations for 24 h. After incubation
morphological changes in the cells were observed under the phase contrast microscope and
images were captured with (Olympus-1X71SIF-3), capture 7 pro software.
4.4.6. Apoptosis by Hoechst staining
An apoptotic cell staining was performed according to the literature method [43]. Briefly,
DU-145 cells were seeded in to 6-well plate at a density of 1x105 cells/well and incubated for
overnight, and media was replaced with fresh media containing with or without compounds
VP-16, 10b, 10g and 10i at indicated concentrations and further incubated for 24 hrs. Then
cells were washed three times with PBS and stained with Hoechst (1 µg/ml) for 20 min at 37
ºC. After incubation cells were washed with PBS three times to remove unstained Hoechst
and images were captured with fluorescence microscope (Olympus-1X71SIF-3), capture 7
pro software.
4.4.7. Measurement of mitochondrial membrane potential (∆Ψm)
Mitochondrial membrane potential was measured by according to the manufacturer’s
protocol BDTM Mitoscreen kit. Briefly, seed 2x105 cells/well in to the 6-well plates and
incubate for overnight. Replace the medium with fresh medium containing compounds VP-
16, 10b, 10g and 10i at indicated concentrations for 24 h. After incubation trypsinise the cells
and collect it into 15 mL tubes and centrifuge at 400 g for 5 min at room temperature. Collect
the pellet and add 0.5 mL of freshly prepared JC-1 working solution to each pellet and
incubate for 15 min at 37 ºC in CO2 incubator. After incubation wash the cells twice with 1x
assay buffer at room temperature. Resuspend the cells in 0.5 mL of 1x assay buffer and
analyze by flow cytometry [44].
4.4.8. Measurement of reactive oxygen species (ROS)
ROS production from DU-145 cells treated with compounds VP-16, 10b, 10g and 10i was
measured using 2’,7’–dichlorofluorescin diacetate (DCFDA) dye [24]. DU-145 cells were
seeded in a 6-well plate at a density of 2x105 cells/well and incubated for overnight. After
incubation media was replaced with fresh medium containing compounds VP-16, 10b, 10g

ACCEPTED MANUSCRIPT
and 10i at indicated concentrations for 24 h. After 24 h medium was replaced with fresh
medium containing DCFDA (10 µM) and incubated in dark conditions for 30 min at room
temperature. Cells were washed with PBS twice and fluorescence intensity was measured by
fluorescence microscope (Olympus-1X71SIF-3), capture 7 prosoftware.
4.4.9. Scratch assay
In vitro scratch assay is used to determine the cell migration and method was adopted from
the literature [45]. DU-145 cells were seeded in a 6-well plate at a density of 2x105 cells/well
and maintained till confluency. Small scratch was made with sterile 200 µL tip and washed
with PBS to remove debris occurred by scratch. Fresh media containing compounds VP-16,
10b, 10g and 10i at indicated concentrations was added. Cell migration was monitored with
phase contract microscopy and images were captured at 0, 24 and 48 h.
4.4.10. Annexin V/PI staining assay
Annexin V/PI assay was determined using the Annexin V FITC apoptosis detection kit
(Sigma Aldrich cat. No: APOAF) [46]. Briefly, cells were seeded in 6-well plate at a density
of 1×105 cells/well and allowed to attach overnight, followed by treatment with compounds
VP-16, 10b, 10g and 10i at indicated concentrations for 24 h. After 24 h media was discarded
and cells were washed twice with PBS. Gently trypsinize and resuspend the cells in 1x
binding buffer at a concentration of 1x106 cells/mL. Incubate the cell suspension with 5 µL
Annexin-V- FITC and 10 µL of propidium iodide for 10 min at room temperature and protect
from light. Analysis was carried out by flow cytometry (FACSVerse, Becton-Dickinson,
USA).
4.5. Molecular docking
The molecular docking studies were performed at the EVP binding site of the Topoisomerase
–IIα (PDB ID: 5GWK) [47]. The co-ordinates of the crystal structure were obtained from
RCSB-Protein Data Bank and suitable corrections to it were made by using Protein
Preparation Wizard from Schrödinger package. In regard to the ligands, molecules were
constructed using ChemBio3D Ultra 12.0 and their geometries were optimized using
molecular mechanics. Finally, docking studies were performed on the most active conjugates
(10b, 10g and 10i) by using AutoDock 4.2 docking software [48] and the results were
visualized through PyMOL [49].Conjugate 10b, 10g and 10i were shown in stick and

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coloured by the atom type. Carbon: Cyan (10b), yellow (10g), Pink (10i), salmon (EVP);
Oxygen: Red; Hydrogen: White; Nitrogen: Blue; Chlorine: Green; Fluorine: Ice blue.
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