Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin analogues as inhibitors of tubulin polymerization and apoptosis inducers

Article abstract of DOI:10.1016/j.bmc.2014.03.021

A series of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates (8a-r) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated anticancer activity against five human cancer cell lines (lung, colon, renal, prostate and cervical). All the tested compounds showed potent anticancer activity with IC₅₀ values ranging from 0.87 to 16.59 μM. Among them compounds 8n and 8p showed significant anticancer activity in lung cancer cells with IC₅₀ values 0.91 and 0.87 μM, respectively. Flow cytometric analysis revealed that these compounds induced cell cycle arrest in G₂/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay and immunofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, DNA fragmentation analysis also suggested that these compounds induced cell death by apoptosis. Overall, the current study demonstrated that the synthesis of terphenyl based 4-aza-2,3- didehydropodophyllotoxin conjugates as promising anticancer agents with G ₂/M cell cycle arrest and apoptotic-inducing activities via targeting tubulin.

Full text of DOI:10.1016/j.bmc.2014.03.021

Accepted Manuscript
Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin ana-
logues as inhibitors of tubulin polymerisation and apoptosis inducers
Ahmed Kamal, Jaki R. Tamboli, V. Lakshma Nayak, S.F. Adil, M.V.P.S.
Vishnuvardhan, S. Ramakrishna
PII:
S0968-0896(14)00194-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.03.021
BMC 11468
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 January 2014
8 March 2014
11 March 2014
Please cite this article as: Kamal, A., Tamboli, J.R., Lakshma Nayak, V., Adil, S.F., Vishnuvardhan, M.V.P.S.,
Ramakrishna, S., Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin analogues as inhibitors
of tubulin polymerisation and apoptosis inducers, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.bmc.2014.03.021
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Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin
analogues as inhibitors of tubulin polymerisation and apoptosis inducers
Ahmed Kamal,*a Jaki R. Tamboli,a V. Lakshma Nayak, a S. F. Adil, b M.V. P. S.
Vishnuvardhan, a S. Ramakrishnaa
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
bCatalytic Chemistry Chair, Chemistry Department, College of Science, King Saud University,
Riyadh 11451, Saudi Arabia.
Abstract:
A series of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates (8a–r) were
synthesized by a straightforward one-step multicomponent synthesis that demonstrated
anticancer activity against five human cancer cell lines (lung, colon, renal, prostate and cervical).
All the tested compounds showed potent anticancer activity with IC50 values ranging from 0.87
to 16.59 µM. Among them compounds 8n and 8p showed significant anticancer activity in lung
cancer cells with IC50 values 0.91 and 0.87 µM respectively. Flow cytometric analysis revealed
that these compounds induced cell cycle arrest in G2/M phase in A549 cell line leading to
caspase-3 dependent apoptotic cell death. The tubulin polymerization assay and
immunofluorescence analysis showed that these compounds effectively inhibit microtubule
assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, DNA
fragmentation analysis also suggested that these compounds induced cell death by apoptosis.
Overall, the current study demonstrated that the synthesis of terphenyl based 4-aza-2,3-
didehydropodophyllotoxin conjugates as promising anticancer agents with G2/M cell cycle arrest
and apoptotic-inducing activities via targeting tubulin.
Keywords: Multicomponent synthesis, Anticancer activity, Podophyllotoxin, Inhibition of
tubulin polymerization, G2/M cell cycle arrest, Caspase-3 activation, Hoechst staining, DNA
fragmentation, Apoptosis.
1

* Corresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189;
E-mail addresses: ahmedkamal@iict.res.in
1. Introduction:
Plants have been used for centuries in traditional medicine and currently a quarter of all
prescribed pharmaceuticals in industrialized countries contain compounds derived from plants,
directly or indirectly via semi-synthesis1. Podophyllotoxin (1) was isolated as an active antitumor
agent from the roots of Podophyllum species. Thus, the biological profile of 1 has led to the
preparation of several hundred derivatives, culminating with the clinical introduction of some
semisynthetic analogues named etoposide3 and teniposide,4 and more recently etopophos,5 a
more soluble prodrug of etoposide (Figure 1). Surprisingly, these semisynthetic derivatives and
the parent compound (podophyllotoxin), show different mechanisms of action.2 Podophyllotoxin
inhibits the assembly of tubulin into microtubules through interaction with the protein at the
colchicine binding site, preventing the formation of the spindle. Whereas the semisynthetic
derivatives have shown to inhibit DNA topoisomerase-II (topo-II) by stabilizing the covalent
topo-II DNA cleavable complex6 and are used against a variety of cancers, including germ-cell
malignancies, small-cell lung cancer, non-Hodgkin’s lymphoma, leukemia, Kaposi’s sarcoma,
neuroblastoma, and soft tissue sarcoma.7–9 Despite its extensive use, etoposide is not devoid of
toxic side effects. Bone-marrow depression is a frequent dose-limiting toxicity encountered in
patients receiving etoposide, and unfortunately the efficacy of the drug is associated with an
increased risk of secondary acute myelogenous leukemia.10,11 For this reason, the development of
more potent analogues remain highly valuable area of research and this has led to the
development of NK-611 (5), and GL-331 (6). GL-331 contains a p-nitroanilino moiety at the 4β-
position instead of a glycoside of etoposide, which proved to be more potent than etoposide,
however this did not proceed further. Recently, we have been involved in the development of
new synthetic procedures14 for the podophyllotoxin-based compounds and also in the design and
synthesis of new analogs of podophyllotoxin as potential anticancer agents.15,16
<Insert Figure 1 >
Multicomponent reactions (MCRs) are more beneficial to construct the natural product-
based libraries particularly in the area of medicinal chemistry research. In this elegant approach,
2

three or more reactants are combined together to produce a product incorporating most of the
atoms contained in the starting materials have the advantages of the intrinsic atom economy,
simpler procedures as well as equipment employed, time and energy savings, apart from
environmental concerns.17–22 Podophyllotoxin (1) structural features four chiral centers, making
it a challenge for the structure activity relationship studies. Absolute stereochemistry of
substituents at the C1 atom (the ring C) was reported to have major influence on antitumor
activity23 (Fig 1). Most of the structure-activity relationship (SAR) studies have been performed
by derivatization of parent natural product rather than by de novo chemical synthesis.24 In this
connection, Itokawa and Takeya made an important contribution for the synthesis of 4-aza-
podophyllotoxin (7a) and (7b) derivatives via three step condensation, cyclization and reduction.
Their cytotoxicity is comparable to that of podophyllotoxin.25 The removal of the stereo centers
at C-2 and C-3 solves the problem of epimerization at C-2 that has plagued the clinical growth of
podophyllotoxin and its stereochemical complex derivatives, as the rapidly formed in vivo cis-
lactone metabolite is significantly less potent.26 Tu and coworkers reported a multicomponent
reaction leading to the formation of N-substituted dihydropyridines, however no biological data
for these compounds was provided.27 Recently, Magedov and co-workers reported a
multicomponent reaction leading to the formation of didehydropyridopyrazole analogues.28
Considering synthetic feasibility, antimitotic, antiproliferative and tubulin destabilizing activity
of 4-aza-podophyllotoxin derivatives, these compounds continue to attract a considerable
interest. On the other hand, a number of terphenyl containing molecules have been synthesized
as useful therapeutic agents for various diseases over the past few years including potent
antioxidant,29 antithrombotic,30 anticoagulant,31 antibacterial32 and anticancer activity.33-36 Thus,
due to the broad range of the biological activities of terphenyl and its derivatives, it was
considered of our interest to explore various substituted terphenyl derivatives as a
pharmacophore in the design of 4-aza-2,3-didehydropodophyllotoxin.
In our previous synthetic efforts,37 we focused upon the modifications of ring B and E of
4-aza-podophyllotoxin by employing the versatility of the MCR approach to prepare different
derivatives of 4-aza-podophyllotoxin. Interestingly, in the case of the substituent effects of the
ring E, we found that the presence of fluoro substituent adds the 4-position in the pendant aryl
ring (E ring) enhances the cytotoxicity in KB cell line. It was also observed that the change of B
ring to heteroaryl or benzohetero fused system have not shown much difference in the
3

cytotoxicity. Therefore, in the present study, we have focused on making systematic E ring
modifications by using requisite aldehyde in order to enhance the potency. Herein, we report an
efficient access to the synthesis of 4-aza-2,3-didehydropodophyllotoxin congeners containing
terphenyl skeletons and this evaluated for the cytotoxic activity and their effect on the inhibition
of tubulin polymerization.
2. Chemistry
The synthetic approach of substituted terphenyl-4-aza-2,3-didehydropodophyllotoxin
analogues is outlined in Scheme 1. Biphenyl systems were prepared utilizing the chemoselective
Pd-catalyzed Suzuki cross-coupling reaction between 1-bromo-4-iodobenzene (9) and 4-formyl
phenyl boronic acid (10) in toluene/ethanol as a solvent in the presence of catalytic amount of
tetrakis(triphenylphosphine) palladium (0) using 2M sodium carbonate solution to afford the
biphenyl aldehyde (11). This undergoes Suzuki cross-coupling reaction with different boronic
acids (12a-f) by employing the above mentioned procedure to afford terphenyl aldehydes (13a-
f). The desired terphenyl based 4-aza-2,3-didehydropodophyllotoxin congeners (8a–r) were
synthesized by applying a multicomponent route involving the condensation of substituted
heteroaromatic amines, tetronic acid, and substituted terphenyl aldehydes in refluxing ethanol. In
all cases, the resulting terphenyl-4-aza-2,3-didehydropodophyllotoxins precipitated as the
reaction mixtures were allowed to cool to room temperature and isolated by simple filtration
followed by recrystallization from ethanol to afford pure compounds (Scheme 2). All the
synthesized compounds were characterized by 1H NMR, 13C NMR and mass spectral data.
<Insert Scheme 1and 2 >
3. Biological evaluation
3.1. Anticancer activity
The newly synthesized compounds (8a–r) were evaluated for their anticancer activity
against a panel of five human cancer cell lines, A-549 (lung), HT-29 (colon), ACHN (renal),
4

DU-145 (prostate) and Hela (cervical) by employing MTT assay.38 The results are summarized
in Table-1 and expressed as IC50 values. The screening results revealed that these compounds
exhibited promising anticancer activity with IC50 values ranging from 0.87 to 16.59 µM against
all the tested cell lines. Among this series, compounds 8n and 8p showed significant activity in
lung cancer cells with IC50 values 0.91 and 0.87µM respectively. From the structure–activity
relationship (SAR) studies of these compounds, some interesting trends have been observed. The
substitution pattern (R1, R2, R3) on the terphenyl ring of 4-aza-2,3-didehydropodophyllotoxin
showed no significant effect on the activity of such analogues. On the other hand, it was
observed that the replacement of B ring to heteroaryl or fused heteroaryl system plays an
important role in enhancing the cytotoxicity of these compounds. Amongst them, the presence of
fused indazole pharmacophore have shown superior effect on the cytotoxicity in comparison
with pyrazole and indole pharmacophores.
Particularly, the compounds 8m, 8n and 8p with 4-fluoro, 3,5-difluoro and 4-
trifluoromethoxy substitution pattern displayed higher anticancer potency as compared to
etoposide and podophyllotoxin. Whereas the compounds 8a, 8d, 8o and 8q have shown
moderate anticancer activity and the remaining compounds 8b, 8c, 8e, 8g, 8i, 8j and 8k
displayed lower anticancer potency. These aspects shows that the replacement of B ring by the
indazole with substitution like 4- fluoro, 3,5-difluoro and 4-trifluoromethoxy on the phenyl ring
of terphenyl of 4-aza-2,3-didehydropodophyllotoxin is important to enhance the anticancer
activity. These interesting preliminary results prompted us to investigate the detailed biological
effects of the most potent compound 8n and 8p in the A549 cell line (Table 1).
<Insert Table 1 >
3.2. Cell cycle analysis
Anticancer drugs are known to interact with cells leading to cell growth arrest or cell
death. To shed more light on the mechanism responsible for the anticancer effect of these
compounds (8n and 8p) were examined for their influence on the cell cycle progression.39 In this
study, A549 cells were treated with compounds 8n and 8p in comparison to etoposide for 48 h.
The data obtained clearly indicated that these compounds (8n and 8p) showed G2/M cell cycle
arrest as compared to the untreated control cells. The tested compound, 8n showed 24.56% and
5

52.34% of cell accumulation and compound 8p showed 32.01% and 58.23% of cell
accumulation in G2/M phase at concentrations of 0.5 and 1µM respectively, whereas etoposide
showed 20.09 and 46.14% of cell accumulation in G2/M phase at 0.5 and 1µM concentrations
respectively as shown in Figure 2 and Table 2.
<Insert Figure 2 >
<Insert Table 2 >
3.3. Effect of compounds on tubulin polymerization
One of the possibilities for these compounds to exhibit anticancer activity as well as
G2/M cell cycle arrest is by the inhibition of tubulin polymerization as this has been observed in
many antimitotic anticancer agents. These compounds showed G2/M cell cycle arrest, hence it
was considered of our interest to investigate the tubulin polymerization aspect. As tubulin
subunits heterodimerize and self-assemble to form microtubules in a time dependent manner, we
have investigated the progression of tubulin polymerization40-41 by monitoring the increase in
fluorescence emission at 420 nm (excitation wavelength is 360 nm) in 384 well plate for 1 h at
37 oC with these compounds (3 µM concentration). In tubulin polymerization assay, compounds
8n and 8p, inhibited 51.27% and 60.01% tubulin polymerization respectively, compared to the
control (Figure 3). Whereas podophyllotoxin inhibited 51.96% tubulin polymerization. Among
these two potent compounds compound, 8p showed significant inhibition than podophyllotoxin
but compound 8n showed comparable inhibition to that of podophyllotoxin.
Furthermore, these two potential compounds (8n and 8p) were evaluated for their in vitro
tubulin polymerization assay at different concentrations. The tested compounds (8n, 8p and
podophyllotoxin) showed significant inhibition on tubulin polymerization with IC50 values 2.95,
2.74 and 3.03 µM, respectively (Table 3).
<Insert Figure 3 >
<Insert Table 3 >
.
6

3.4. Immunohistochemistry of tubulin
To substantiate the inhibition of tubulin polymerization by these compounds for functional
microtubules, immunohistochemistry was carried out to examine the in situ effects on cellular
microtubules in A549 cancer cells.41 A549 cells were treated with compounds 8n and 8p at 1 µM
concentration for 48 h and the results were compared with that of podophyllotoxin. In this study,
untreated human lung cancer cells displayed the normal distribution of microtubules, whereas
cells treated with compounds 8n and 8p showed disrupted microtubule organization (Figure 4 )
and thus demonstrated the inhibition of tubulin polymerization.
<Insert Figure 4 >
3.5. Morphological analysis for apoptosis with Hoechst staining
In addition, we have examined the cellular effects of the compounds (8n and 8p) on
nuclear condensation by Hoechst staining. Cells treated with these compounds showed
significant effect on nuclear condensation in comparison to untreated control cells. The results
clearly demonstrated that these compounds (8n and 8p) are effective in inducing cellular
apoptosis as shown in Figure 5.
<Insert Figure 5 >
3.6. Activation of caspase-3
From previous reports, it is well established that molecules affecting microtubule
polymerization cause mitotic arrest and ultimately lead to apoptosis.42 It was also known that
caspases play a vital and active role for the initiation and execution of the apoptotic process.43
Among the caspases, caspase-3 is one of the key effector caspase which cleaves multiple
proteins in cells, leading to apoptotic cell death.44 In this context, A549 cells were treated with
compounds 8n and 8p (1 and 2 µM) and examined for the activation of caspase-3. Results
indicated that there was nearly 3 to 7-fold induction in caspase- 3 levels compared to control
(Figure 6).
<Insert Figure 6 >
7

3.7. DNA Fragmentation assay
DNA fragmentation is well known for a typical biochemical hallmark of apoptotic cell
death.45 From the in vitro anticancer studies it was observed that these compounds (8n and 8p)
significantly inhibited the growth of human lung cancer cell line A549. Therefore it was of our
interest to determine the mechanism of cell death in the same cell line. The DNA fragmentation
analysis revealed that these tested compounds induced a discrete ladder pattern in A549 cell line
at 1µM concentration after 48 h of the treatment, thereby showing DNA fragmentation as
outlined in Figure 7.
<Insert Figure 7 >
4. Conclusion
In summary, a new class of terphenyl substituted 4-aza-2,3-didehydropodophyllotoxins
were synthesized and tested for their anticancer potential against five human cancer cell lines.
All the compounds exhibited promising anticancer activity at micro molar (µM) concentration.
Among the series compounds, 8n and 8p showed significant anticancer activity in lung cancer
cells with IC50 values 0.91 and 0.87µM respectively. These compounds (8n and 8p) arrested the
cell cycle progression at G2/M phase and inhibited tubulin polymerization. Furthermore these
compounds induced apoptosis by caspase-3 dependent mechanism, which was further confirmed
by Hoechst staining and DNA fragmentation. This investigation revealed that synthesis of 4-aza-
2,3-didehydropodophyllotoxin incorporating a substituted terphenyl moiety enhances the
anticancer activity. Based on the results from the biological studies it is clear that compounds 8n
and 8p have the potential to be taken up for further detailed studies either individually or in
combination with existing therapies as potential tubulin polymerisation inhibitors.
5. Experimental
All chemicals and reagents were obtained from Aldrich (Sigma– Aldrich, St. Louis, MO,
USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA, USA) or
Spectrochem Pvt. Ltd (Mumbai, India) and were used without further purification. Reactions
were monitored by TLC, performed on silica gel glass plates containing 60 GF-254, and
visualization on TLC was achieved by UV light or iodine indicator. Column chromatography
was performed with Merck 60–120 mesh silica gel. 1H spectra were recorded on Gemini Varian-
VXR-unity (200 MHz) or Bruker UXNMR/XWIN-NMR (300 MHz) instruments. Chemical
8

shifts (δ) are reported in ppm downfield from internal TMS standard. ESI spectra were recorded
on Micro mass, Quattro LC using ESI+ software with capillary voltage 3.98 kV and ESI mode
positive ion trap detector. High-resolution mass spectra (HRMS) were recorded on QSTAR XL
Hybrid MS/MS mass spectrometer. Melting points were determined with an Electro thermal
melting point apparatus, and are uncorrected
5.1. Synthesis of 4'-bromobiphenyl-4-carbaldehyde (11)
To a solution of 1-bromo-4-iodobenzene 9 (564 mg, 2 mmol) in 6 mL of toluene was
added a catalytic amount (0.4% mol) of tetrakis-triphenylphosphine palladium (0) and 0.5 mL of
aqueous 2 M Na2CO3. A solution of 4-formyl phenylboronic acid 10 (300 mg 2 mmol) in 2 mL
of ethanol was added, stirred at room temperature under a nitrogen atmosphere. After 10 min, the
solution was heated to reflux for 2-3 h in an argon atmosphere. Reaction is monitored by the
TLC. After completion of the reaction, reaction mixture is cooled to room temperature and
extracted with ethyl acetate (3×30 mL) and the organic phase was washed with water and brine
solution, dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure to
obtain the crude product. This product was further purified by column chromatography using
ethyl acetate and hexane to afford the pure bromobiphenyl aldehyde (11) as a white solid, 370
mg in 71% yield. Mp: 136-137 oC; 1H NMR (300 MHz, CDCl3): δ 7.47-7.52 (m, 2H), 7.58-7.63
(m, 2H), 7.69 (d, 2H, J = 8.3 Hz), 7.93-7.97 (m, 2H), 10.05 (s, 1H); (ESI) MS: m/z 261 (M+H)+.
5.2. 4′′-Fluoro[1'',4';1′,4]Terphenyl-1-carbaldehyde (13a)
To a solution of bromobiphenyl aldehyde 11 (390 mg, 1.5 mmol) and 4-fluoro phenyl boronic
acid 12a (209.8 mg, 1.5 mmol) in 2 M aqueous sodium carbonate (2 mL) and toluene/ethanol
(12:4 mL) is added a catalytic amount (0.4% mol) of tetrakis-triphenylphosphine palladium, and
the mixture was heated to reflux under argon atmosphere for 3-4 h. After completion of the
reaction, the reaction mixture is cooled to room temprature and extracted with ethyl acetate
(3×30 mL) and the organic phase was extracted with water and brine solution, dried over
anhydrous Na2SO4 and the solvent was removed under reduced pressure to get the crude product.
This crude product was further purified by column chromatography (10%ethyl acetate and
hexane) to afford the pure terphenyl aldehydes (13a) as a white solid in 310 mg, 74% yield; Mp:
135-136 oC; 1H NMR (300 MHz, CDCl3): δ 7.12-7.19 (m, 2H), 7.57-7.73 (m, 6H), 7.78 (d, 2H, J
= 8.3 Hz), 7.96 (d, 2H, J = 8.3 Hz), 10.06 (s, 1H); (ESI) MS: m/z 277 (M+H)+.
9

5.3. 3'',5''-Difluoro[1'',4';1′,4]Terphenyl-1-carbaldehyde (13b)
The compound 13b was prepared following the method described for the preparation of the
compound 13a, employing bromobiphenyl aldehyde 11 (390 mg, 1.5 mmol) and 3,5-difluoro
phenyl boronic acid 12b (236.8 mg, 1.5 mmol) to afford the pure compound 13b as a white solid
1
in 320 mg, 72% yield; Mp: 140-141 oC; H NMR (300 MHz, CDCl3): δ 6.80-6.87 (m, 1H), 7.15
(d, 2H, J = 6.8 Hz), 7.65 (d, 2H, J = 8.7 Hz), 7.72 (d, 2H, J = 7.8 Hz), 7.78 (d, 2H, J = 8.7 Hz),
7.97 (d, 2H, J = 7.8 Hz), 10.07 (s, 1H); (ESI) MS: m/z 295 (M+H)+.
5.4. 3'', 4'', 5''-Trifluoro[1'',4';1′,4]Terphenyl-1-carbaldehyde (13c)
The compound 13c was prepared following the method described for the preparation of the
compound 13a, employing bromobiphenyl aldehyde 11 (390 mg, 1.5 mmol) and 3,4,5-trifluoro
phenyl boronic acid 12c (263.8 mg, 1.5 mmol) to afford the pure compound 13c as a white solid
1
in 355 mg, 75% yield; Mp: 143-144 oC; H NMR (300 MHz, CDCl3): δ 7.01-7.12 (m, 1H), 7.29-
7.37 (m, 1H), 7.60 (d, 2H, J = 7.5 Hz), 7.71-7.81 (m, 4H), 7.97 (d, 2H, J = 8.3 Hz), 10.07 (s,
1H); (ESI) MS: m/z 313 (M+H)+.
5.5. 4''-Trifluoromethoxy[1'',4';1′,4]Terphenyl-1-carbaldehyde (13d)
The compound 13d was prepared following the method described for the preparation of the
compound 13a, employing bromobiphenyl aldehyde 11 (390 mg, 1.5 mmol) and 4-
trifluoromethoxy phenyl boronic acid 12d (308.8 mg, 1.5 mmol) to afford the pure compound
13d as white solid in 355 mg, 69% yield; Mp: 158-159 oC; 1H NMR (300 MHz, CDCl3): δ 7.30
(d, 2H, J = 7.5 Hz), 7.63-7.76 (m, 6H), 7.79 (d, 2H, J = 8.3 Hz), 7.9 (d, 2H, J = 8.3 Hz), 10.07 (s,
1H); (ESI) MS: m/z 343 (M+H)+.
5.6. 4''-Trifluoromethyl[1'',4';1′,4]Terphenyl-1-carbaldehyde (13e)
The compound 13e was prepared following the method described for the preparation of the
compound 13a, employing bromobiphenyl aldehyde 11 (390 mg, 1.5 mmol) and 4-
trifluoromethyl phenyl boronic acid 12e (284.8 mg, 1.5 mmol) to afford the pure compound 13e
o
1
as a white solid in 383mg, 78% yield; Mp: 149-150 C; H NMR (300 MHz, CDCl3): δ 7.70-
7.77 (m, 8H), 7.80 (d, 2H, J = 8.3 Hz), 7.98 (d, 2H, J = 8.3 Hz), 10.07 (s, 1H); (ESI) MS: m/z
327 (M+H)+.
5.7. 3''-Chloro-4''-fluoro[1'',4';1′,4]Terphenyl-1-carbaldehyde (13f)
The compound 13f was prepared following the method described for the preparation of the
compound 13a, employing bromobiphenyl aldehyde 11 (390 mg, 1.5 mmol) and 3-chloro-4-
10

fluoro phenyl boronic acid 12f (261.4 mg, 1.5 mmol) to afford the pure compound 13f as a white
o
1
solid in 355 mg, 76% yield; Mp: 152-153 C; H NMR (300 MHz, CDCl3): δ 7.22 (d, 1H, J =
8.3 Hz),7.46-7.52 (m, 1H), 7.62-7.68 (m, 3H), 7.71 (d, 2H, J = 8.3 Hz), 7.78 (d, 2H, J = 8.3 Hz),
7.96 (d, 2H, J = 8.3 Hz), 10.07 (s, 1H); (ESI) MS: m/z 311 (M+H)+.
(The spectral data of final compounds can be found in supplementary data)
6. Biology
6.1. MTT Assay: The anti cancer activity of the compounds was determined using MTT assay.
1×104 cells/well were seeded in 100µl DMEM, supplemented with 10% FBS in each well of 96-
well micro culture plates and incubated for 24 h at 37°C in a CO2 incubator. Compounds, diluted
to the desired concentrations in culture medium, were added to the wells with respective vehicle
control. 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, formazon crystals were
dissolved in 100µl of DMSO and absorbance at 540 nm wavelength was recorded.
6.2. Cell cycle analysis: Flow cytometric analysis (FACS) was performed to evaluate the
distribution of the cells through the cell cycle phases. A549, lung cancer cells were incubated
with compounds 8n and 8p at concentrations of 0.5µM and 1µM for 48 h along with Etoposide
in the same concentration. Untreated and treated cells were harvested, washed with PBS, fixed in
ice-cold 70% ethanol and stained with propidium iodide (Sigma Aldrich). Cell cycle was
performed by flow cytometry (Becton Dickinson FACS Caliber) as earlier described.
6.3. Hoechst staining: Cells were seeded at a density of 10,000 cells over 18-mm cover slips
and incubated for 24 h. Then, the medium was replaced, and cells were treated with compounds
8n and 8p at 1µM for 48 h. Cells treated with vehicle (0.001% DMSO) were included as controls
for all experiments. After 48 h of treatment, Hoechst 33258 (SigmaeAldrich) were added to the
medium at a concentration of 0.5 mg/mL containing 4% paraformaldehyde. After incubation for
30 min 37°c, cells from each dish were captured from randomly selected fields under fluorescent
microscope (Leica, Germany) to qualitatively determine the apoptotic cells based on their
relative fluorescence, chromatin condensation and nuclear fragmentation.
6.4. Tubulin polymerization assay: A fluorescence based in vitro tubulin polymerization assay
was performed according to the manufacturer’s protocol (BK011, Cytoskeleton, Inc.). Briefly,
the reaction mixture in a total volume of 10 µL contained PEM buffer, GTP (1µM) in the
11

presence or absence of test compounds (final concentration of 3 µM). Tubulin polymerization
was followed by a time dependent increase in fluorescence due to the incorporation of a
fluorescence reporter into microtubules as polymerization proceeds. Fluorescence emission at
420 nm (excitation wavelength is 360 nm) was measured by using a Varioscan multimode plate
reader (Thermo scientific Inc.). Podophyllotoxin was used as positive control in each assay. The
IC50 value was defined as the drug concentration required inhibiting 50% of tubulin assembly
compared to control. The reaction mixture for these experiments include: tubulin (3 mg/ml) in
PEM buffer, GTP (1 mM), in the presence or absence of test compounds at 2.5, 5, 10, and 15 µM
concentrations. Polymerization was monitored by increase in the Fluorescence as mentioned
above at 370C.
6.5. Immunohistochemistry: A549 cells were seeded on glass cover slips and incubated for 48
h in the presence or absence of the test compounds 8n and 8p (1µM). After treatment, the cover
slips were fixed with a paraformaldehyde solution (4% in 1×PBS) for 20 min at room
temperature. Cell permeabilization was achieved by administration of a Triton X-100 solution
(0.2% in 1×PBS) for 5 min. The cover slips were left in 100% MeOH overnight at -40C.
Subsequently, the cover slips were blocked with a 1% bovine serum albumin (BSA) solution for
60 min and then incubated with anti-α-tubulin antibody (1:1000) at room temperature for 2 h.
The slides were washed three times for 5 min each with PBST. Next, the cover slips were
incubated with FITC-conjugated anti-mouse secondary antibody (Sigma–Aldrich) for 1 h and
then washed three times with PBST solution. Finally, the cells were observed under a
fluorescence microscope (Leica, Germany), and the pictures were analyzed for the integrity of
the microtubule network.
6.6. Caspase 3 activity: Caspase-3 assay was conducted for detection of apoptosis in Lung
cancer cell line (A549). The commercially available apoptosis detection kit (Sigma-Caspase 3
Assay kit, Colorimetric) was used. A549 cells were treated with compounds 8n and 8p (2µM)
for 48 h. After 48 h of treatment, cells were collected by centrifugation, washed once with PBS,
and cell pellets were collected. Suspended the cell pellet in lysis buffer and incubated for 15 min.
After incubation, cells were centrifuge at 20,000 rpm for 15 min and collected the supernatant.
12

Supernatants were used for measuring caspase 3 activity using an ELISA-based assay, according
to the manufacturer’s instructions.
6.7. DNA Fragmentation: Cells were seeded (1×106) in six-well plates. After 24 h of
incubation, cells were treated with the compounds, 8n, 8p and Etoposide at concentration of
1µM. After 48 h of treatment, cells were collected and centrifuged at 2500 rpm for 5 min at 4°C.
Pellet was collected and washed with Phosphate buffered saline (PBS). Then added 100µl of
Lysis buffer centrifuged at 3000 rpm for 5 min at 4°C and collected supernant. And added 10µl
of 10% SDS and 10µl of (50 mg/ml) RNaseA and incubated for 2 h at 56°C. After incubation
Proteinase K (25 mg/ml) was added and further incubated at 37° for 2 h. Then added 65µl of 10
M Ammonium acetate and 500µl of ice cold ethanol and mixed well. And these samples were
incubated at -80°C for 1 h. After incubation samples were centrifuged at 12000 rpm for 20 min at
4°C. After centrifuge pellet was washed with 80% ethanol and air dried for 10 min at room
temperature. Dissolved the pellet in 50µl of TE buffer and DNA laddering was determined by
using 2% agarose gel electrophoresis in TE Buffer.
Acknowledgments
J.R.T. is thankful to DST (India) for the award of research fellowship.
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16

Figures
H
R1
O
O
OH
O
R2
O
O
O
HO
R1
R2
R3
H
O
O
O
CH3
O
Etoposide
OH
2
( )
O
O
Teniposide 3
( )
H
H3CO
OH
OH
OCH3
S
OCH3
OH
P
H3CO
OCH3
Etopophos (4)
HO
OR3
CH3
O
1
( )
Podophyllotoxin
HN
5)
NK611 (
NMe2
H
NMe2
R
H
N
H
N
RO
RO
O
O
O
R
O
O
O
O
O
O
O
H3CO
O
OCH3
OH
R = CH3 (7a)
R = -CH2- (7b)
R = NO2; GL 331 (6)
8(a-r)
R3
R1
R2
Figure 1. Chemical structures of podophyllotoxin (1), etoposide (2), teniposide (3), etopophos
(4), NK611 (5), GL331 (6), 4-aza-podophyllotoxin (7a and 7b), Terphenyl substituted 4-aza-
podophyllotoxin derivatives (8a-r)
17

Figure 2: Flow cytometric analysis in A549 lung cancer cell lines after treatment with
compounds 8n and 8p at 0.5 and 1µM concentrations for 48 h, A: Control cells (A549), B:
Etoposide (0.5 µM), C: Etoposide (1µM), D: 8n (0.5 µM), E: 8n (1 µM) and F: 8p (0.5 µM)
and G: 8p (1 µM).
18

Figure 3: Effect of compounds (8n and 8p) on tubulin polymerization: tubulin polymerization
was monitored by the increase in fluorescence at 360 nm (excitation) and 420 nm (emission) for
0
1h at 37 C. The tested compounds were included at a final concentration of 3µM.
Podophyllotoxin was used as a positive control. Values indicated are the mean SD of two
different experiments performed in triplicates.
19

Figure 4: Immunohistochemistry analysis of compounds on the microtubule network. A: Control
(A549) , B: Podophyllotoxin (1µM), C: 8n (1µM) and D: 8p (1µM).
Figure 5 : Hoechst staining in A549 cell line, A: A549 control cells, B: Etoposide (1 µM), C: 8n
(1 µM) and D: 8p (1 µM).
20

Figure 6: Effect of compounds 8n and 8p on caspase-3 activity: A549 cells were treated for 48 h
with compounds 8n and 8p at 1 and 2µM concentrations. Values indicated are the mean SD of
two different experiments performed in triplicates.
Figure 7: DNA Fragmentation analysis of compounds 8n and 8p in A549 lung cancer cells.
Lane-1: Control DNA, Lane-2: 8n (1µM), Lane-3: 8p (1µM)), Lane-4: Marker (100bp) and
Lane-5: Etoposide (1µM)
21

Tables
Table 1. IC50a values (expressed in µM) of compounds 8(a-r)
Compound
A549b
HT-29c
ACHNd
DU-145e
HeLa f
1.47 0.03
6.72 1.02
4.36 1.41
0.98 0.08
5.75 2.11
2.88 0.83
4.36 1.41
5.49 1.37
5.49 0.94
5.12 1.05
10.0 3.26
2.55 0.02
0.95 0.72
0.91 0.09
3.98 1.02
0.87 0.03
1.16 0.02
7.41 2.64
2.45 0.63
3.31 0.94
5.83 1.35
6.45 0.98
1.77 0.05
8.51 1.01
6.30 0.05
8.91 0.09
6.45 1.26
6.60 0.03
9.33 2.04
7.41 1.06
8.70 2.76
1.69 0.42
1.02 0.96
8.91 3.54
1.00 0.09
2.39 0.05
9.42 3.01
1.47 0.06
2.29 0.97
2.18 0.95
14.11 2.76
7.52 0.08
4.07 0.74
5.24 1.12
3.16 0.65
5.75 0.03
5.75 1.04
6.45 0.95
5.37 0.72
7.58 1.35
2.95 0.04
1.47 0.05
0.95 0.09
7.41 0.99
0.95 0.02
2.75 0.05
9.33 1.23
2.04 0.08
1.93 0.24
4.16 0.97
10.0 1.04
6.50 1.65
1.12 0.73
6.45 1.11
5.75 0.94
7.24 1.04
8.70 2.05
13.18 2.63
6.02 0.98
12.58 2.71
4.78 0.74
1.44 0.07
1.28 0.52
6.91 0.25
0.98 0.09
4.89 0.38
12.6 1.36
3.17 0.04
3.21 0.48
3.80 0.62
8.12 1.06
7.07 0.94
2.23 0.02
6.16 0.73
6.02 0.04
9.54 1.02
8.51 0.25
10.47 3.65
7.41 1.11
16.59 2.97
3.16 0.07
1.94 0.09
1.23 0.32
5.49 0.26
1.24 0.43
7.09 1.05
7.53 0.08
2.75 0.36
2.81 0.27
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
Etoposide
Podophyllotoxin 3.09 1.00
a 50% Inhibitory concentration and the values are average of three individual experiments after
48 h of treatment. bLung cancer,c Colon cancer, d Renal cell carcinome, eProstate cancer,
f Cervical cancer
22

Table 2. Cell cycle distribution of the compounds 8n and 8p
SubG1
6.47
4.93
4.01
1.27
1.22
1.08
1.01
G0/G1
78.23
70.74
46.09
69.78
41.23
64.52
38.41
S
G2/M
11.73
20.09
46.14
24.56
52.34
32.01
58.23
A: Control (A549)
B: Etoposide (0.5 µM)
C: Etoposide (1 µM)
D: 8n (0.5 µM)
3.57
4.24
3.76
4.39
5.21
2.39
2.35
E: 8n (1 µM)
F: 8p (0.5 µM)
G: 8p (1 µM)
Table 3: Inhibition of tubulin polymerization (IC50) of compounds 8n and 8p
a
IC50 SD (in µM)
2.74 0.07
Compound
8n
2.95 0.16
3.03 0.42
8p
Podophyllotoxin
a Concentration of drug to inhibit 50% of tubulin assembly
23

Schemes
Scheme 1. Reagents and conditions: (i) Pd(PPh3)4, 2M Na2CO3, toluene/EtOH, 2-3 h, reflux (ii)
Pd(PPh3)4, 2M Na2CO3, toluene/EtOH, 3-4 h, reflux
Scheme 2. Reagents and conditions: (i) EtOH, reflux, 30 min-1h.
24

Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin
analogues as inhibitors of tubulin polymerisation and apoptosis inducers
a
a
b
S. F. Adil, M.V. P. S.
Ahmed Kamal,*a Jaki R. Tamboli,
Vishnuvardhan, a S. Ramakrishnaa
V. Lakshma Nayak,
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
bCatalytic Chemistry Chair, Chemistry Department, College of Science, King Saud University,
Riyadh 11451, Saudi Arabia.
25