Synthesis of D-ring modified acid hydrazide derivatives of podophyllotoxin and their anticancer studies as Tubulin inhibiting agents

Article abstract of DOI:10.1016/j.bioorg.2019.103384

A new series (except compound 3a) of D-ring modified acid hydrazides of podophyllotoxin were synthesized by cleaving of its D-ring with various hydrazines. Furthermore, the synthesized compounds were screened for their anticancer activity against human tu

Full text of DOI:10.1016/j.bioorg.2019.103384

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Synthesis of D-ring modified Acid hydrazide derivatives of Podophyllotoxin
and their anticancer studies as Tubulin inhibiting agents
Srinivas Nerella, Shravankumar Kankala, Suresh Paidakula, Brahmeshwari
Gavaji
PII:
S0045-2068(19)31288-X
DOI:
https://doi.org/10.1016/j.bioorg.2019.103384
YBIOO 103384
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To appear in:
Bioorganic Chemistry
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Revised Date:
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7 October 2019
21 October 2019
Please cite this article as: S. Nerella, S. Kankala, S. Paidakula, B. Gavaji, Synthesis of D-ring modified Acid
hydrazide derivatives of Podophyllotoxin and their anticancer studies as Tubulin inhibiting agents, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103384
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1
Bioorganic Chemistry
journal homepage: www.elsevier.com
Synthesis of D-ring modified Acid hydrazide derivatives of Podophyllotoxin and their
anticancer studies as Tubulin inhibiting agents
Srinivas Nerella,a,b* Shravankumar Kankalaa, Suresh Paidakulaa, Brahmeshwari Gavajia
aDepartment of Chemistry, Kakatiya University, Warangal, India
bDepartment of Chemistry, Pingle Government College for Women, Warangal, Kakatiya University, India
Corresponding Author: nerellasrinivas1@gmail.com
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A new series (except compound 3a) of D-ring modified acid hydrazides of podophyllotoxin were
synthesized by cleaving of its D-ring with various hydrazines. Furthermore, the synthesized
compounds were screened for their anticancer activity against human tumor cell lines i.e., MCF-7,
HeLa and A-549 and among the synthesized compounds 3c and 3f have shown significant anticancer
activity almost similar to that of standard drug etoposide. Molecular modelling studies were also
conducted for active compounds and found that the free energies obtained were in good agreement
with the observed IC 50 values.
Keywords:
Podophyllotoxin
D-Ring modification
Acid hydrazides
2009 Elsevier Ltd. All rights reserved.
Anti-cancer studies
Tubulin inhibiting agents
The crude extract isolated from the roots and rhizomes of the
plants of podophyllum species is called Podophyllum resin and it
has been used as a cathartic, antihelminthic agent and a remedy
for condyloma acuminatum for several years.1,2 The active
component of this resin is an aryltetralin lignan named
podophyllotoxin (1), a promising anticancer molecule. Its
analogues Etoposide (2) and Teneposide (3) were proved to be
potential antitumor drugs for various types of cancer ailments.3
Currently some of its structural analogues such as GP-11 (4),
GL-331 (5) and TOP-53 (6) are drug leads for cancer treatment
(Fig. 1).4 GL-331 is currently in phase-II clinical trials against
gastric carcinoma, colon cancer, non-small cell carcinoma and
etoposide resistant malignancies.5 However, their clinical use has
encountered certain problems, such as poor water solubility, drug
resistance, metabolic inactivation and certain toxic effects.6,7
and its binding site has been characterized recently. In the past
number of small molecules were discovered which bind to
Tubulin, interfering with the polymerization or depolymerization
of microtubules and then inducing cell cycle arrest, resulting in
cell death.9,10
H
H
OH
C
H3C
O
O
O
O
O
O
S
O
A
O
O
O
HO
HO
OH
OH
O
D
B
O
O
O
O
O
O
O
O
O
E
H3CO
OCH3
OCH3
H3CO
OCH3
H3CO
OCH3
OH
OH
Podophyllotoxin (1)
Etoposide (2)
Teniposide (3)
NO2
N
N
OH
OH
Antitumor property of podophyllotoxin and its analogues was
mainly due to its mechanism of action which involves inhibition
of Tubulin polymerization.8 Tubulin inhibiting agents interfere
with microtubule dynamics and hinder the assembly of Tubulin
into microtubules. Microtubules are vital cytoskeletal filaments
that found in eukaryotes and play key role in numerous cellular
functions, such as cell motility, vesicle transport and cell
division. Disruption of this equilibrium will lead to cell cycle
arrest or cell apoptosis. Given their significant role in the growth
and function of cells, microtubules are among the most important
molecular targets for cancer chemotherapeutic agents.
Colchicine is the first drug that is well known to bind Tubulin,
HN
O
O
O
O
O
H
O
O
O
O
N
NH
N
N
O
H
H
O
O
MeO
OMe
H3CO
OCH3
OCH3
OMe
H3CO
OCH3
OCH3
GP-11 (4)
GL-331 (5)
TOP-53 (6)
Figure 1. Podophyllotoxin, its anticancer drugs and drug lead compounds.
On the other hand, acid hydrazides have wide applications as
drugs, chemical preservers for plants, for manufacturing

2
polymers, glues, etc., in industry and for many other purposes.11
A wide variety of heterocyclic compounds like pyrroles,
pyrazoles, pyrazolines, oxadiazolethiones, thiazoles, oxadiazoles,
triazoles, thiadiazoles and tetrazines can be synthesized starting
from acid hydrazides using Gewald reaction, Curtius
rearrangement, Dimroth rearrangement, Horner-Emmons
reaction and Reid-Heindel reaction.
The NMR and HRMS techniques were used to elucidate the
structures of the synthesized acid hydrazides. The formation of
acid hydrazides from podophyllotoxin can be realised by
comparing the 1H-NMR & 13C-NMR spectra of parent compound
1
and formed acid hydrazides. The appearance of new peak in H-
NMR spectrum of acid hydrazides at around 9.16δ (corresponds
to CO-NH proton) clearly indicates the formation of acid
hydrazides from the parent compound. This peak was not there in
the 1H-NMR spectrum of parent compound. In 13C-NMR
spectrum, the peak corresponding to carbonyl carbon of lactone
ring was experienced a downfield shift due to deshielding effect
with D-ring opening and thus moved from~175 to ~172 δ. The
same downfield shift was also seen in the case of methylene
carbon of lactone ring i.e., from ~72 to 68.7 δ. Further
confirmation of acid hydrazide formation was also done by
HRMS spectra.
Based on the SAR studies, initially it was presumed that intact
trans-lactone ring of Podophyllotoxin i.e., D-ring was responsible
for its anticancer property. But reports thereafter revealed that it
may not be the case. For example GP-11(4) and other D-ring
modified derivatives have disproved it and moreover the former
is equipotent to a well-known cancer drug, etoposide.12 In
addition, the clinical success of GP-11,
a derivative of
Podophyllic acidhydrazide, encouraged us to take up synthesis
and anticancer screening of similar derivatives of
podophyllotoxin.
Table 1.
Showing the results for the synthesis of podophyllic acid hydrazides from
structurally diverse hydrazines
.
To overcome the toxic effects, poor water solubility and other
issues associated with the earlier reported podophyllotoxin
derivatives, Herein we have shown some necessary modifications
to Lactone ring (D-ring) of podophyllotoxin to synthesize
podophyllic acid hydrazides. The synthesized compounds were
also tested for their efficacy in inhibiting tumor growth of three
human tumor cell lines (MCF-7, HeLa and A-549).
Reaction time
(hour), (yield, %)b
Reaction time
(hour), (yield, %)b
Product a
Product a
Entry Hydrazine
Entry
7
Hydrazine
H
N
NH2
NH2
1
2
3
3a
6(94)
6(92)
6(94)
3g
10(75)
7(90)
8(88)
H2N
OH
H3C
NH2
H
N
3b
3c
8
9
3h
3i
N
H
NH2
NH2
NH2
H
N
H3C
N
H
HO
HO
NH2
HO
H
N
4
5
N
3d
3e
6.5(90)
8(65)
10
3j
8(85)
H
NH2
O
H
N
11c
Initially Podophyllotoxin (1) was dissolved in methanol, and
then, acetic acid (AA) and corresponding anhydrous hydrazines
(2a-j) were added with stirring.13 The reaction mixture was
refluxed for about 6-10 h to afford podophyllic acid hydrazides
(3a-j) in good to excellent yields (Scheme 1).
NH2
3k
3l
No Reaction
NH2
H2N
N
H
H
N
H
N
12c
NH2
6
3f
10(72)
No Reaction
NH2
O2N
NO2
aAll products were characterized by 1H/13C NMR and mass spectral analysis.b Isolated yields after
column chromatography. c 24 h, reflux
In vitro cytotoxicity assay: The synthesized podophyllic acid
hydrazides (3a-j) were evaluated for in vitro cytotoxic ability
against a panel of human cancer cell lines, including MCF-7,
HeLa and A549 using a MTT assay.14 The results were
summarized in Table 2 and well-known standard drug etoposide
was used as a reference. The newly synthesized podophyllic acid
hydrazides have shown moderate to good anticancer activity
against most of the cell lines in this investigation. Among them,
compounds 3c and 3f were exhibited significant anticancer
activity with IC50 values ranging from 12-20 μM and the
observed activity of these compounds was almost similar to that
of standard drug etoposide. As far as structure activity
relationship is concerned, the compounds having ethyl and
cyclohexyl substituents on hydrazine i.e., the compounds 3c and
3f have shown the most promising apoptotic activity than the
other substituents (for 3c, 15±0.04 μM against MCF-7, 18±0.18
μM against HeLa and 16±0.02 μM against A-549; for 3f,
12±0.06 μM against MCF-7, 20±0.07 μM against HeLa and
15±0.12 μM against A-549). In the case of compound 3g,
introduction of hydroxyl group next to the hydrazine of
cyclohyxyl ring significantly reduced the activity (38±0.01 μM to
45±0.25 μM). Particularly, the compounds having benzyl moiety
on hydrazine i.e., 3h, 3i and 3j have shown very moderate
activity ranging from 30±0.02 μM to 45±0.05 μM.
OH
OH
OH
O
O
O
O
D-Ring
O
NHNHR
MeOH, AA
Reflux, 6h
+
NH2-NH-R
O
O
2a-j
R= -H, -CH3,-CH2CH3, -CH2CH2OH,
-CONH2, -C6H11, -C6H11(2-OH),
-C7H7, -C7H7(3-OH), -C7H7(4-OH)
MeO
OMe
MeO
OMe
OMe
OMe
1
Podophyllotoxin
Podophyllic acid Hydrazides (3a-j)
Scheme 1. Synthesis of podophyllic acid hydrazides.
Podophyllotoxin was made to react with structurally different
hydrazine derivatives and the results obtained were summarized
in Table 1. It was clear from the table that among various
hydrazines, aliphatic hydrazines (Table 1, Entries 1-4) performed
well by giving good yields in short reaction times. Semicarbazide
and alicyclic hydrazines (Entries 5-7) were took longer times to
cleave the ring and gave lower yields than that of aliphatic
hydrazines. Even benzylic hydrazines (Entries 8-10) were also
equally good at cleaving D-ring of podophyllotoxin to give
corresponding acid hydrazides. However, aromatic hydrazines
(Entries 11 and 12) were unable to cleave the D-ring of
podophyllotoxin and there were no products formed even after
refluxing for 24 h. This inability of aromatic hydrazines to cleave
D-ring may be attributed their lower basicity than the
corresponding aliphatic and other hydrazines of present study.

3
Table 2
in vitro cytotoxic activity of podophyllic acidhydrazides (3a-j) on human
cancer cell linesa (IC50 µM) b.
Entry Compound
MCF-7(Breast)
HeLa (Cervical)
A-549 (Lung)
30±0.03
35±0.05
15±0.04
3a
3b
3c
35±0.01
40±0.13
18±0.18
30±0.02
32±0.14
16±0.02
1
2
3
2D &3D poses of 3c with the Tubulin
37±0.01
45±0.01
12±0.06
42±0.05
40±0.02
30±0.02
34±0.02
3d
3e
3f
3g
3h
3i
45±0.24
43±0.04
20±0.07
45±0.25
35±0.06
35±0.11
35±0.01
40±0.01
38±0.45
15±0.12
38±0.01
42±0.01
45±0.05
38±0.10
4
5
6
7
8
9
3j
10
10±0.32
Etoposide
15±0.37
12±0.12
11
a Data represent as mean ± SEM values. Cytotoxicity as IC50 for each cell line, is the
concentration of compound which reduced by 50% the optical density of treated cell
with respect to untreated cells using the MTT assay.
2D &3D poses of 3f with the Tubulin
b Data represent as mean ± SEM values of these independent determinations.
Figure 2. The docking poses of 3c & 3f with Human γ-Tubulin
In conclusion, we synthesized Podophyllic acid hydrazides in
good yields and estimated the relative basicities of diverse
hydrazines in cleaving lactone ring of podophyllotoxin. In future,
this chemistry would eventually lead to synthesis of
Molecular docking studies: Docking studies of the active
compounds 3c and 3f were performed with Auto Dock software
to reveal the interactions of these compounds with the active sites
of Human γ-Tubulin with PBD ID 1Z5V. The results were
impressive showing high binding affinity of 3c and 3f with the
enzyme and free energies of -7.8 & -7.6 kcal/mole were observed
for the compounds respectively (Table 3).
pharmacologically
novel
heterocyclic
analogues
of
podophyllotoxin as acid hydrazides are important synthetic
precursors. Among the synthesized compounds 3c and 3f have
shown impressive in vitro anticancer activity and thus they are
identified as promising lead compounds. Molecular docking
studies of synthesized compounds as Tubulin inhibiting agents
have also supported the observed cytotoxicity.
Table 3.
Binding energies for active molecules 3c and 3f
Supplementary data
Binding Energy
(PBD ID 1ZXN)
Sl. No. Compound
Residues
Experimental procedures, Spectral data and antitumor evaluation
methods for compounds 3a-j can be found, in the online
version.
ASP A60, GLY A129, GLY A130, THR A131,
ASN A88, VAL A164
1
2
3c
3f
-7.8
GLN A11, CYS A12, GLN A15, PHE A208
SER A126, VAL A164
-7.6
References and notes.
1.
2.
Teillac-Hamel, D.; Roux, A.; Loeb, G. Eur. J. Dermatol. 1996, 6,
437-440
Docking studies of 3c revealed that the compound docked
well with enzyme through various types of interactions such as
Hydrogen bonding, pi-alkyl and carbon hydrogen interactions
(Figure 2). The cleaved D-ring was actively involved in bonding
with GLY A130, THR A131 and ASN A88 through H-bonding.
Other notable interactions of 3c include H-bonding between
carbonyl oxygen of Asparagine and Hydrogen of C-ring OH.
Syed, A.; Lundin, S.; Ahmad M. Dermatology. 1994, 189, 142-
145.
3. Gupta, S.; Das, L.; Datta, A.B.; Poddar, A.; Janik,
M.E.; Bhattacharyya, B. Biochemistry. 2006, 45, 6467-6475.
4. Utsugi, T.; Shibata, J.; Sugimoto, Y.; Aoyagi, K.; Wierzba, K.;
Kobunai, T.; Terada, T.; Oh-hara, T.; Tsuruo, T.; Yamada, Y.
Can. Res. 1996, 56, 2809-2814.
On the other hand, compound 3f also interacted well
with amino acid residues of the enzyme through various
connections which include pi-pi stacked, pi-alkyl and hydrogen
bonding. In the case of 3f, the E-ring efficiently binds with the
amino acid residues like GLN A11 and CYS A12 through
hydrogen bonding involving oxygens of methoxy groups and
hydrogens of amino groups of respective amino acids. The other
prominent interactions include hydrogen bonds between etheric
oxygen of A-ring and GLN A 15 and between SER A 126 and
carbonyl oxygen of cleaved D- ring.
5. Chen, Y.; Lin, T.Y.; Chen, J.C.; Yang, H.Z.; Tseng, S.H.;
Antican. Res. 2006, 26, 2149-56.
6.
Chamberlain, M.C.; Tsao-Wei, DD.; Groshen, S. Cancer.
2006, 106, 2021-2027.
7. Pettit, G.R.; Searcy, J.D.; Tan, R.; Cragg, G.M.; Melody, N.;
Knight, J.C.; Chapuis, J.C. J. Nat. Prod. 2016, 79, 507-518.
8. Kamal, A.; Reddy, T.S.; Polepalli, S.; Shalini, N.; Reddy,
V.G.; Rao, A.V.S.; Jain, N.; Shankaraiah, N. Bioorg. Med.
chem. 2014, 22, 5466-5475.
9. Lavelle, F.; Bissery, M.; Combeau, C.; Riou, J.; Vrignaud, P.;
Andre, S. Semin. Oncol. 1995, 2, 3-16.
10. Dumontet, C.; Jordan, M.A.; Nat. Rev. Drug. Discov. 2010, 9,
790-803.

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11. Grekov, A.P., Organic Chemistry of Hydrazines, Technika
Publishers, Vilnius. 1996, 23, p. 75.
13. Tian, X.; Zhang, F. M.; Li, W. G.; Life Sciences. 2002, 70,
2433-2443.
12. Wang, Y.; Shao, Y.H.; Wang, Y.Y.; Fan, L.L.; Yu, X.; Zhi
X.Y.; Yang, C.; Qu, H.; Yao, X.J.; Xu, H. J. Agric. Food.
Chem. 2012, 60, 8435-8443.
14. Mosmann, T. J. Immunol. Methods. 1983, 65, 55-63.

5
Highlights
 D-ring modified Podophyllic Acid hydrazides were successfully synthesized
 Among synthesized compounds, 3c and 3f have shown potential anticancer activity
 Tubulin inhibiting efficacy of active compounds were proved by Docking studies
Graphical Abstract
OH OH
O
HumanTubulin
Inhibition
NHNHR
O
O
MeO
OMe
OMe
llic ac
Pod
ophy
id H
ydraz
ide
‘Declarations of interest: none.’