Synthesis and Biological evaluation of novel 4β-[(5-substituted)-1,2,3,4-tetrazolyl] podophyllotoxins as anticancer compounds

Article abstract of DOI:10.1016/j.bmcl.2015.04.053

A series of novel 4β-[(5-substituted)-1,2,3,4-tetrazolyl] podophyllotoxin derivatives were synthesized by employing azide-nitrile click chemistry approach. All the derivatives were evaluated for their cytotoxicity against a panel of four human cancer cell lines and their IC₅₀ values were found to be in the range of 2.4-29.06 μM. The cytotoxicity exhibited by the majority of test compounds were found to comparable and often more effective than doxorubicin and all compounds exhibited higher cytotoxicity on A-549 cell lines. Cell cycle analysis showed that the novel 4β-[(5-substituted)-1,2,3,4-tetrazolyl] podophyllotoxins resulted in cell cycle arrest at G2/M phase and were also found to be the potent inhibitors of tubulin polymerization in vitro.

Full text of DOI:10.1016/j.bmcl.2015.04.053

Accepted Manuscript
Synthesis and Biological Evaluation of novel 4β-[(5-subtituted)-1, 2, 3, 4-tet-
razolyl] podophyllotoxins as Anticancer compounds
Irfan Hyder, Deepthi Yedlapudi, Shasi V. Kalivendi, Jabeena Khazir, Naresh
Nalla, Sreekanth Miryala, Halmuthur M. Sampath Kumar
PII:
S0960-894X(15)00385-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.04.053
BMCL 22636
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
3 December 2014
5 April 2015
15 April 2015
Please cite this article as: Hyder, I., Yedlapudi, D., Kalivendi, S.V., Khazir, J., Nalla, N., Miryala, S., Sampath
Kumar, H.M., Synthesis and Biological Evaluation of novel 4β-[(5-subtituted)-1, 2, 3, 4-tetrazolyl]
podophyllotoxins as Anticancer compounds, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.04.053
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Letters
Synthesis and Biological Evaluation of novel 4β-[(5-subtituted)-1, 2, 3, 4-tetrazolyl]
podophyllotoxins as Anticancer compounds
a,d
b
b,*
c
a
Irfan Hyder , Deepthi Yedlapudi , Shasi V Kalivendi , Jabeena Khazir , Naresh Nalla , Sreekanth
a.d a,d
Ma iryala , Halmuthur M. Sampath Kumar *
NPC Division, CSIR- Indian Institute of Chemical Technology, Hyderabad, 500007, India.
Centre for Chemical biology Division, CSIR- Indian Institute of Chemical Technology, Hyderabad, 500007, India.
Medicinal Chemistry Division, Indian institute of Integrative Medicine, Jammu-180001, India.
Academy of Scientific and Innovative Research, CSIR- Indian Institute of Chemical Technology, Hyderabad 500007, India.
b
c
d
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of novel 4β-[(5-susbtituted)-1,2,3,4-tetrazolyl] podophyllotoxin derivatives were
synthesized by employing azide-nitrile click chemistry approach. All the derivatives were
evaluated for their cytotoxicity against a panel of four human cancer cell lines and their IC50
values were found to be in the range of 2.4 µM to 29.06µM. The cytotoxicity exhibited by the
majority of test compounds were found to comparable and often more effective than
Doxorubicin and all compounds exhibited higher cytotoxicity on A-549 cell lines. Cell cycle
analysis showed that the novel 4β-[(5-susbtituted)-1,2,3,4-tetrazolyl] podophyllotoxins resulted
in cell cycle arrest at G2/M phase and were also found to be the potent inhibitors of tubulin
polymerization in vitro.
Revised
Accepted
Available online
Keywords:
Tetrazolyl Podophyllotoxin ,
Anticancer activity,
Tubulin polymerization
2
009 Elsevier Ltd. All rights reserved.
Podophyllotoxin (1), is a most abundant naturally occurring
cyclolignan, isolated from the different plants of the genus
approach by using azide and nitrile with a view to examine their
effect on the cytotoxicity pattern on cancer cell lines and the
interactions of these ligands with tubulins. Genesis of this
chemistry is also derived from the fact that tetrazole containing
1
Podophyllum. Over the last decade, there has been a plenty of
research on podophyllotoxin primarily for antimitotic activity,
even though wide range of other pharmacological attributes of
podophyllotoxin derivatives viz., cathartic, antirheumatic and
antiviral properties have been reported. However, the anticancer
activity of these cyclolignans proved to be promising and several
research groups worldwide engaged in the structural modification
around the podophyllotoxin scaffold resulting in the development
of clinically approved cancer drugs like etoposide, teniposide and
a soluble prodrug of etoposide-etopophos, against various types
8
moieties were found to be cytotoxic. Microtubules are the
cytoskeletal structures formed due to the self assembly of two
homologues proteins, α and β tubulin which are present in all
eukaryotic cells. Microtubules are significant target for many
natural product anticancer agents. The taxane domain, the vinca
domain and the colchicine domain are the characteristic binding
9
sites of tubulin. There are several reports proving that
podophyllotoxin derivatives were known to inhibit tubulin.
Recently Chunyan Zhao et al proved that these semi synthetic
derivatives can act as tubulin polymerization inhibitors
of cancers, including
small cell lung cancer, testicular
carcinoma, lymphoma, and Kaposi’s sarcoma and some of them
are in late stage clinical development like NK-611, NPF and
1
0
interacting with the colchicine-binding site. Keeping all these
reports in view, our attention was drawn towards tubulin
inhibition of the novel podophylloxin analogues and interestingly
these derivatives are also proved to be tubulin inhibitors.
2-4
GL-331(Figure.1).
The cytotoxic mechanism of these
semisynthetic derivatives was explained by the inhibition of
topoisomerase-II mechanism whereas its lead compound
mechanism was explained by assembly of microtubule. SAR of
5
podophyllotoxin indicates that 4-β substituted, 4’-demethyl and
trans lactone moieties are essential to maintain the antimitotic
As illustrated in Scheme-1, 4β-[(5-susbtituted)-1,2,3,4-tetrazol-1-
yl] podophyllotoxin derivatives were synthesized by the
cycloaddition reaction of C4β-azidopodophyllotoxin, 9 and C4β-
azido-4′-O-demethyl podophyllotoxin, 10 with various cyano
compounds. Compounds 9, 10 were prepared by the previous
6
activity. Previously our group designed and synthesized some
potent anilino, phenol, thiophenol, carbohydrate, aliphatic
substituted 1, 2, 3- triazole derivatives of podophyllotoxin and it
was found that some of these derivatives are more potent than
1
1
literature procedure. Compound 9 was synthesized by treating
podophyllotoxin with MeSO H and NaI in acetonitrile which
forms the corresponding iododerivative followed by hydrolysis
with H O/Me CO/BaCO then treatment with NaN , TFA in
7
etoposide. In continuation of our work, we synthesized a series
of tetrazolyl derivatives of podophyllotoxin via. click chemistry
3
2
2
3
3
chloroform. Similarly compound 10 was synthesized by treating
podophyllotoxin with MeSO H and NaI in presence
dichloromethane which forms the corresponding iododerivative
*Corresponding authors Tel.: +91-40-27193005 Fax: +91-191-2569333,
Email: sampath@iict.res.in. kalivendi@iict.res.in
3
1

followed by hydrolysis with H
2
O/Me
O-demethyl podophyllotoxin followed by treatment with NaN
2
CO/BaCO
3
which forms 4’-
Doxorubicin were taken as taken as positive controls. Majority of
compounds exhibited IC50 values less than doxorubicin across the
panel of all the four cell lines tested. While the IC50 values were
in the range of 3.44 -6.5 µM in SK-NSH, the corresponding
values were 2.4-4.29 µM, 3.05 – 5.25 µM and 4.85 – 9.44 µM in
A549, HeLa and MCF-7 cells respectively. However, compound
11h exhibited either comparable (6.48 µM in A549 and 12.43
µM in MCF-7) or greater IC50 values (25.95 µM SK-N-SH and
29.06 µM in HeLa) than doxorubicin. Since a majority of
compounds exhibited lower IC50 values in A549 cells amongst
the cell lines employed hence further studies were carried out in
this cell line.
3
,
1
2, 13
TFA in presence of chloroform.
allowed to react with various cyanides in two different methods.
Compounds 9, 10 were
0
Sulphonyl cyanides treated with compounds 9, 10 at 100 C
without solvent for 10 hours to form corresponding tetrazolyl
derivatives. Whereas acyl cyanides treated with 9, 10 at RT in
presence of ZnBr to form corresponding tetrazolyl derivatives in
2
moderate yields.
14-16
R1
O
O
HO
O
OH
O
OR2
O
O
O
IC50 values (µM)
O
O
1
2
O
Compound
R
R
I
SK-NSH
5.31±0.12
6.5±0.23
A-549
HeLa
MCF-7
6.99±0.08
6.97±0.36
7.47±0.05
6.89±0.04
4.85±0.46
5.29±0.10
5.27±0.51
12.43±1.08
7.02±0.92
9.44±0.42
3.34±0.15
12.34±2.57
O
O
1
1a
1b
Me
Me
Me
Me
Me
H
2.89±0.43
3.11±0.31
2.4±0.05
3.15±0.11
5.25±0.06
4.76±0.73
4.76±0.45
4.01±0.08
3.05±0.02
3.95±0.33
29.06±4.18
3.61±0.18
4.77±0.11
1.92±0.06
10.78±0.69
MeO
OMe
MeO
OMe
1
II
III
IV
V
OMe
OR3
1
1c
1d
1e
3.61±0.30
4.68±0.19
4.58±0.02
3.44±0.09
5.64±0.57
25.95±5.11
3.49±0.06
6.16±0.78
7.92±1.23
7.36±0.01
1
. Podophyllotoxin
R3
H
R1
R2
1
4.29±0.89
2.48±0.25
3.29±0.28
2.44±0.01
6.49±0.77
2.71±0.53
2.98±0.48
2.55±0.27
6.13±0.54
2
.
.
CH3
H
H
H
Etoposide
Teniposide
Etopophos
1
3
H
S
11f
I
O
P
CH3
4
.
11g
H
II
OH
HO
1
1h
H
III
Iv
V
5
.
CH3
NMe2
H
NK-611
1
1i
H
R
1
1j
H
HN
1
N
Me
O
O
O
O
Doxorubicin
O
O
O
Table 1.IC50 values (µM) of various 4β-[(4-Substituted)-1,2,3,4-
tetrazol-1-yl] podophyllotoxin derivatives.
O
MeO
OMe
MeO
OMe
OH
OH
Cell viability assays revealed that a majority of derivatives
exhibited effective antiproliferative activity against all the four
cell lines tested. To investigate further, the anti-tubulin activity of
the compounds (5 µM) was evaluated using podophyllotoxin as a
positive control. Results as shown in Figure.2/Table-2,
demonstrate that podophyllotoxin inhibited the polymerization of
tubulin to nearly 90% at 5 µM concentration. Under similar
experimental conditions, compound 11c inhibited in vitro tubulin
polymerization to 99%, whereas, 11d, 11e, 11f, 11g and 11j
exhibited 72, 57, 22, 28 and 29% inhibition respectively.
However, no significant anti-tubulin activity was observed with
compounds, 11a, 11b, 11h and 11i.
6
.
.
R = NO2 GL-331
R = F NPF
8. TOP-53
7
Figure.1; Structures of some podophyllotoxin derivatives
N
N
N
R2
N3
N
O
O
O
O
O
O
R2-CN
O
O
Neat reaction
O
O
O
O
Molecules that inhibit tubulin polymerization are known to
induce cell cycle arrest at G2/M or mitotic phase. To elucidate
O
O
13
R1
R1
9
.
R1=Me
0. R1=H
11a-j
the down-stream consequence of anti-tubulin effects of
derivatives, we analysed the effect of individual compounds on
cell cycle by flow cytometry, which is used to quantitatively
determine the population of cells in each phase of the cell cycle
by measuring the DNA content of individual cells. A549 cells
were treated individually with compounds (5 µM concentration)
for duration of 24 h and found that compounds that exhibited
anti-tubulin activity such as, 11c, 11d, 11e, 11f, 11g and 11j
induced cell cycle arrest at G2/M phase(Figure.3). Even, the
compounds that did not affect tubulin polymerization (11a, 11b,
11h and 11i) also induced mitotic arrest. Further, 11a and 11b
which possess either negligible / no anti-tubulin activity induced
cell cycle arrest at both G2/M to a lesser extent of 54 and 45%
respectively as compared to other derivatives, but significantly
induced arrest even at sub-G0 phase. The observed findings
could be due to the possible topoisomerase inhibition of the
derivatives as it has been shown previously that modification at
C-4 position on podophyllotoxin leads to the generation of topo
II inhibitors capable of inducing cell cycle arrest at G2/M as well
as in sub G0 phase of the cell cycle.
1
O
S
O
O
O
O
O
II
R2
=
O
O
O
,
,
,
,
V
I
III
IV
Reaction conditions: Method-I: Suphonyl cyanides, heat at 100
c; Method-II: Acyl cyanides, ZnBr , RT, 48 hrs.
2
0
Scheme-1. Click-chemistry strategy for the synthesis of 4β-[(5-
substituted)-1,2,3,4-tetrazolyl]podophyllotoxin derivatives.
1
All the synthesized compounds were characterized by IR, H and
C NMR spectroscopy and ESI-MS.
1
3
All the 4β-[(5-susbtituted)-1,2,3,4-tetrazolyl] podophyllotoxin
derivatives were screened for their cytotoxicity against a panel of
four human cancer cell lines(SK-N-SH, A549,HeLa and MCF-7)
and the results are summarized in Table1. Podophyllotoxin and
2

%
Inhibition of tubulin
Entry
Compound
polymerization
0.73
1
2
3
4
5
6
7
8
9
11a
11b
12.40
11c
99.15
11d
72.50
11e
57.47
11f
22.46
11g
28.29
11h
2.68
11i
11.52
Figure.2; Effect of tubulin Polymerization on 4β-[(5-susbtituted)
1,2,3,4-tetrazol-1-yl] podo-phyllotoxin derivatives.
1
1
1
0
1
2
11j
29.79
-
1
93.90
Nocodozole
65.27
Table 2. In vitro tubulin polymerization assay. Values are
indicates as % inhibition with respect to control.
In summary, a series of novel 4β-[(5-susbtituted)-1,2,3,4-
tetrazolyl] podophyllotoxin derivatives were synthesized and
evaluated for their cytotoxicity against a panel of four cell lines.
The IC50 values varied from 2.4µM to 29.06 µM. These
derivatives showed inhibition of tubulin polymerization in vitro
and arrested the cells at G2/M phase
Figure.3; Antimitotic activity of compounds by FACS analysis:
A549 cells were harvested after treatment with compounds at
Acknowledgments
The authors thank CSIR, New Delhi for financial support as part
of XII Five Year plan programme under title DENOVA (CSC-
0205), ORIGIN (CSC-0108). I.H thanks CSIR for the award of
fellowship.
5
µM for 12h. Untreated cells and DMSO treated cells served as
controls. The percentage of cells in each phase of cell cycle was
quantified by Flow cytometry and the values indicate the number
of cells stalled at G2/M phase.
References and notes
Most of the sulphonyl and acyl tetrazolyl podophyllotoxin
derivatives exhibited uniform cytotoxicity against four different
cell lines without profound effect of the substituion on tetrazolyl
ring on the derived cell viability with an exception of compound
1. Lin, H.W.; Kwok, K.H.; Doran P.M. Biotechnol. Prog. 2003, 19, 1417.
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1
1h bearing ethyl substitution on tetrazole moiety with free
5
1
hydroxyl on the phenyl ring of podophyllotoxin scaffold(R =H),
exhibiting least cytotoxicity amongst all the compounds studied.
Except compound 11h, all compounds shown comparable IC50
values with parent compound podophyllotoxin against human
neural bone marrow metastasis cancer cel line(SK-NSH) and
human lung adenocarcinoma epithelial cell line (A-549).
However, the observed IC50 values for the compounds were
slightly higher than that of podophyllotoxin in cervical cancer
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2002, 50, 399-402; (b) Xu, H.; Zhang, L.; Su, B.F.; Zhang, X.; Tian, X.
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(
HeLa) and breast cancer (MCF-7) cell lines. Again, except 11h,
most of the compounds exhibited lower IC50 values in all four
cell lines when compared to doxorubicin. Based on this premise,
all compounds were tested for their in vitro inhibition of tubulin
polymerization and it was observed that compound 11c shown 99
5. Macdonald, T.L.; Lehnert, E.K.; Loper, J.T.; Chow, K.C.; Ross, W.E., in
DNATopoisomerases in Cancer; M. Potmesil, K.W. Kohn, Oxford University
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Biol. Chem. 2002, 277, 16547-16552.
%
inhibition as compared to podophyllotoxin (93% inhibition)
followed by other derivatives. However, cell cycle arrest at both
G2/M phase and very significant induction of cell cycle arrest at
sub-G0 phase by the compounds viz.,11a, 11b and 11i which did
not otherwise inhibit tubulin polymerization, may be attributed
to the topoisomerase inhibition which contribute to apoptosis.
Thus, the tetrazolyl derivatives of podophyllotoxin might be
inducing apoptosis in cancer cell lines through dual target
interaction i.e., tubulin polymerization inhibition as well as
topoisomerase-II inhibition as observed previously by other C-4
substituted podophyllotixin analogues leading to induction of
apoptosis.
7
. (a) Bhat, B.A.; Reddy, P.B.; Agrawal, S.K.; Saxena, A.K.; Kumar, H.M.S.;
Qazi, G.N. Eur. J.Med. Chem. 2008, 43, 2067-2072; (b) Reddy, P.B.;
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3

Hamel, E.; Bortolozzi, R.; Basso, G.; Viola, G. J Med Chem. 2012, 55,
4
Hz, 2H), 6.20 (s, 1H), 5.98 (d, J = 1.2 Hz , 1H), 5.96 (d, J = 1.2 Hz , 1H),
4.73 (d, J = 4.4 Hz , 1H), 4.25 (m, 1H), 4.06 (s, 3H), 3.85 (s, 6H), 3.83 (s,
75−488.
1
3
3
H), 3.74-3.68 (m,1H), 3.09 (dd, J = 14.3, 4.4 Hz 1H); C NMR (75 MHz,
CDCl ): δ 37.7, 44.0, 45.4, 55.9, 60.3, 66.3, 69.4, 101.7, 106.2, 107.7, 110.3,
125.5, 128.6, 130.8, 133.1, 133.8, 134.1, 148.8, 152.9, 163.3, 172.1; IR
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Cancer Res., 1978, 38, 2688-2693.
3
-
1
(KBr): 3414, 2924, 2853, 1783, 1748, 1591, 1496, 1238, 1123 cm ; ESI–MS:
+
m/z 547 [M +Na] ;
1
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1
2
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1
1
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7. General procedure for preparation of suphonyl tetrazoles (I); A
mixture of Azido- podophyllotoxin and paratoulene sulphonyl cyanide were
o
taken in a sealed tube and stir at 100 c for 12 hours in the absence of
solvent. After completion of reaction, the reaction mixture was
chromatographed on silica gel eluting with ethyl acetate/hexane (24-30%) to
get the desired products 11a and 11f as white solids.
(
5R,5aR,8aS,9S)-9-(5-Tosyl-1H-tetrazol-1-yl)-5-(3,4,5-trimethoxyphenyl)-
,5a,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(8H)-one
11a):
5
(
This compound 11a was prepared by the method (I), employing 4β- Azido
podophyllotoxin ( 90 mg, 0.20 mmol) and para toulene sulphonyl cyanide
(0.20 mmol) The crude product was was chromatographed on a silica gel
column with ethyl acetate/hexane (24%) as eluent afford the pure compound
2
5
1
1a, 86 mg in 69 % yield as White solid. m.p: 216 ºC; [α]
D
- 29 (c 0.5,
=0.5 (50% Ethyl acetate/hexane); H NMR ( 300 MHz, CDCl ):
δ 8.01 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H), 6.64 (s, 1H), 6.55 (s,
H), 6.37 (d, J = 5.1 Hz , 1H), 6.32 (s, 2H), 6.02 (d, J = 1.2 Hz, 1H), 5.97
d, J = 1.2 Hz, 1H), 4.81 (d, J = 5.1 Hz, 1H), 4.30 (m, 1H), 3.82 (s, 3H), 3.77
1
CHCl
3
), R
f
3
1
(
(
1
3
C
s, 6H), 3.69 (dd, J = 14.4, 5.1 Hz, 1H), 3.36-3.28 (m, 1H), 2.47 (s, 3H);
) δ 21.7, 36.2, 40.9, 43.5, 56.3, 60.7, 63.2, 66.2, 102.0,
08.1, 108.2, 110.7, 123.0, 128.8, 130.3, 133.3, 134.0, 134.8, 137.5, 146.7,
47.9, 149.6, 152.7, 166.8, 172.6; IR (KBr) 3436, 2929, 1788, 1726, 1594,
NMR(75 MHz, CDCl
3
1
1
1
-
1
+
498, 1124, 1040 cm ; ESI–MS: m/z 643[M +Na] ;
1
(
8. General procedure for preparation of aromatic/aliphatic tetrazoles
II) A mixture of Azido- podophyllotoxin, acyl cyanide and zinc bromide
were taken in a round bootom flask under nitrogen atmosphere and the
reagents were stirred vigorously for 48 h at room temperature. To the reaction
mixture add ethyl acetate and water and stir for 2 h. The organic layer was
then isolated and the aqueous layer was extracted with ethyl acetate. The
combined organic layers were dried over NaSO4. After filtration and
concentration, the crude product was purified by column chromatography
with ethyl acetate/ hexane afford the pure compounds (11b, 11c, 11d, 11e,
1
1g, 11h, 11i, 11j).
Methyl1-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1H-
tetrazole-5-carboxylate (11b):
5
This compound 11b was prepared by the method (II), employing 4β- Azido
podophyllotoxin (100 mg, 0.22 mmol) and methyl cyano formate (2.86
mmol) and Zinc bromide(0.22 mmol).
chromatographed on a silica gel column with ethyl acetate/hexane (28 %) as
The crude product was
Supplementary Material:
1
eluent) afford the pure compound 11b, 93 mg in 77 % yield as White solid.
2
5
13
Copies of H, C NMR and mass spectra of all the new compounds can
be obtained free of charge from the internet.
1mp 126 ºC; [α]
D
- 37.7 (c 0.5, CHCl ); R =0.4 (50% Ethyl acetate/hexane);
): δ 6.64 (s, 1H), 6.57 (s, 2H), 6.30 (d, J = 10.8
3
f
H NMR ( 300 MHz, CDCl
3
4

Graphical Abstract
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Synthesis and Biological Evaluation of novel
Leave this area blank for abstract info.
4β-[(5-subtituted)-1,2,3,4-tetrazolyl] podophyllotoxins
as Anticancer compounds
Irfan Hyder , Deepthi Yedlapudi, Shasi V Kalivendi, Jabeena Khazir,Tabasum Ismail, Naresh Nalla, Sreekant
Miryala , Halmuthur M. Sampath Kumar*
N
N
N
R
2
N
R
1
= OMe,H
O
O
O
O
O
O
O
O
S
O
R2
=
O
O
O
O
,
,
,
,
O
O
- Most of the compounds are cytotoxic on A-549 cell line.
- cells arrested in a G2/M phase and inhibited tubulin polymerization in vitro.
O
R
1
5