Synthesis of neolignans as microtubule stabilisers

Article abstract of DOI:10.1016/j.bmc.2013.12.067

Tubulin is a well established target for anticancer drug development. Lignans and neolignans were synthesized as tubulin interacting agents. Neolignans 10 and 19 exhibited significant anticancer activity against MCF-7 and MDAMB-231 human breast cancer cell lines. Both the compounds effectively induced stabilization of microtubule at 4 and 20 μM concentrations respectively. Neolignan 10 induced G2/M phase arrest in MCF-7 cells. Docking experiments raveled that 10 and 19 occupied the same binding pocket of paclitaxel with some difference in active site amino acids and good bioavailability of both the compounds. In in vivo acute oral toxicity 10 was well tolerated up to 300 mg/kg dose in Swiss-albino mice.

Full text of DOI:10.1016/j.bmc.2013.12.067

Bioorganic & Medicinal Chemistry 22 (2014) 1342–1354
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of neolignans as microtubule stabilisers
B. Sathish Kumar a, Aastha Singh a, Amit Kumar b, Jyotsna Singh b, Mohammad Hasanain b, Arjun Singh a,
Nusrat Masood a, Dharmendra K. Yadav a, Rituraj Konwar b, Kalyan Mitra b, Jayanta Sarkar b
Suaib Luqman a, Anirban Pal a, Feroz Khan a, Debabrata Chanda a, Arvind S. Negi a,
⇑
a CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow 226015, India
b CSIR-Central Drug Research Institute (CSIR-CDRI), B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Tubulin is a well established target for anticancer drug development. Lignans and neolignans were syn-
thesized as tubulin interacting agents. Neolignans 10 and 19 exhibited significant anticancer activity
against MCF-7 and MDAMB-231 human breast cancer cell lines. Both the compounds effectively induced
Received 31 October 2013
Revised 27 December 2013
Accepted 30 December 2013
Available online 9 January 2014
stabilization of microtubule at 4 and 20 lM concentrations respectively. Neolignan 10 induced G2/M
phase arrest in MCF-7 cells. Docking experiments raveled that 10 and 19 occupied the same binding
pocket of paclitaxel with some difference in active site amino acids and good bioavailability of both
the compounds. In in vivo acute oral toxicity 10 was well tolerated up to 300 mg/kg dose in Swiss-albino
mice.
Keywords:
Neolignans
Lignans
Anticancer
Ó 2014 Elsevier Ltd. All rights reserved.
Microtubules
In silico studies
Acute oral toxicity
1. Introduction
anticancer drug. However, two of its semisynthetic derivatives,
etoposide and teniposide are used clinically to treat small-cell lung
Lignans and neolignans are important biodynamic agents with
varied structural diversity. In plants, these are produced as second-
ary metabolites derived from phenylpropanoids (C6–C3). Both
lignans and neolignans are dimeric compounds with different link-
ages. Lignans are formed by C2–C20 linkage through carbons of
propyl chains, while other C–C linkages are known as neolignans.
Both lignans and neolignans are widespread in plants.1–4 Lignans
and neolignans have exhibited a wide range of biological activities
such as antimalarial,5,6 antitubercular,7,8 anticancer,9–14 apoptosis
inducers,15,16 antiviral17,18 and antioxidants,19 etc. More interest-
ingly, lignans and neolignans have been good ligands for estrogen
receptors,19 aldose reductase,20,21 tyrosinase,22 topoisomerase II,23
GABAA receptor,24 voltage gated K+ channels,25 etc. acting as
inhibitors by curtailing these enzymatic actions.
cancer, testicular cancer, leukemia, lymphoma, and other
cancers.26
3,4,5-Trimethoxyphenyl unit in several antitubulins plays a
crucial role in interacting with tubulin. There are antitubulin
agents like podophyllotoxin, colchicine, combretastatin A4, etc.
possessing this unit.26 We designed lignans (prototype-I) and
neolignans (prototype-II) as possible anticancer agents with this
fragment (Fig. 1). Several analogues were synthesized and evalu-
ated against human cancer cell lines. The mode of action of active
compounds was evaluated against tubulin polymerase enzyme.
The tubulin interaction was further confirmed by in silico docking
studies. The most active compound 10 was evaluated for its effect
on cell cycle phases. It was also evaluated for acute oral toxicity in
Swiss-albino mice at various doses.
Several potent leads like podophyllotoxin (PDT) as anticancer,26
silymarin, phyllanthin, hypophyllanthin and cleomiscosins as
hepatoprotectives27 have been obtained from this class of com-
pounds. Podophyllotoxin is an aryl tetralin lignan isolated from
Podophyllum spp.26 It acts as a mitotic inhibitor by binding revers-
ibly to tubulin and inhibiting microtubule assembly.26,28 Due to
toxicity reasons podophyllotoxin could not be developed as
2. Results
2.1. Chemistry
The synthetic strategy was as depicted in Scheme 1. Firstly,
3,4,5-trimethoxycinnamic acid (1) was esterified to corresponding
ethyl ester (2) by treating it with thionyl chloride and ethanol at
room temperature. The p-methoxy group of ester 2 was selectively
demethylated by treatment with anhydrous aluminium chloride in
⇑
Corresponding author. Tel.: +91 522 2718583; fax: +91 522 2342666.
E-mail address: arvindcimap@rediffmail.com (A.S. Negi).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.067

B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
1343
O
O
OH
H3CO
H3CO
R1
O
O
R
R
R2
OH
OCH3
OCH3
O
H3CO
HO
OCH3
O
O
H3CO
H3CO
R1
OH
OCH3
H3CO
H3CO
O
O
O
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
HN
OCH3
R2
OCH3
Combretastatin A4
Colchicine
Podophyllotoxin
Prototype-I
3,4,5-trimethoxyphenyl unit
Prototype-II
Figure 1. Structures of antitubulins (podophyllotoxin, colchicine, CA4), prototypes I and II, and trimethoxyphenyl fragment.
O
O
O
H3CO
H3CO
(A)
H3CO
H3CO
H3CO
HO
OH
OEt
i
ii
OEt
OCH3
OCH3
3
OCH3
2
1
iii
O
O
H3CO
RO
H3CO
OEt
OEt
OEt
HO
H3CO
EtOOC
+
OCH3
O
OCH3
OH
H3CO
OCH3
OR
OCH3
4
5a: R=H;
5b: R=Ac
iv
O
O
O
R
R
OEt
OH
(B)
R
i
OH
OEt
iii
HO
HO
HO
6: R=H;
7: R=OCH3
EtO
R
8: R=H;
O
9: R=OCH3
10: R=H;
11: R=OCH3
O
O
H3CO
H3CO
O
O
H3CO
H3CO
OEt
OEt
H3CO
CHO
OH
OH
v
OEt
OEt
(C)
+
vi
H3CO
O
R
R
O
R
R
12: R=H;
H3CO
R
OCH3
14
H3CO
13: R=OCH3
OCH3
15: R=H;
16: R=OCH3
17: R=H;
18: R=OCH3
Scheme 1. Reagents and conditions: (i) EtOH, SOCl2, RT, 2 h, 89–94%; OR concd H2SO4, EtOH, 85 °C, 4 h, 84–91%; (ii) anhydrous AlCl3, DCM, 40 °C, 2 h, 84%; (iii) aqueous KOH,
K3[Fe(CN)6], benzene, RT, 30 min, neolignan 21%, lignan 24–29%; (iv) dry pyridine, Ac2O, RT, overnight, 92%; (v) NaH, DMF, reflux, 4 h, 43–49%; (vi) Et2SO4, K2CO3, acetone/
DMSO = 3:1, reflux, 2 h, 82%.
dry methylenechloride to get 3,5-dimethoxy, 4-hydroxycinnamic
acid ethyl ester (3).29 The free radical coupling of ester 3 was done
in potassium ferricyanide solution in aqueous alkali-benzene sys-
tem. Use of phase transfer catalyst (tetrabutyl ammoniumbromide,
TBAB) was not beneficial in this reaction, as reaction failed in pres-
ence of it. In case of ester 3, two products were formed. Compound
4 was identified as neolignan and 5a was identified as lignan. Sim-
ilarly ethyl esters of 8 and 9 were processed to get neolignans 10
and 11 respectively. In case of 8 and 9, we did not get lignans, only
neolignans were obtained. But, the yields in these reactions were
poor (21–29% only). So, alternatively, we synthesized these lignans
through Stobbe’s condensation. Here, substituted aromatic alde-
hyde (12 or 13) was condensed with diethyl succinate (14) in
presence of sodium hydride in dry dimethylformamide (DMF) un-
der reflux condition in inert atmosphere (N2). The yields of the
lignans 15 and 16 as diacids were significantly higher (43–49%)
by this method. The diacids were further converted to ethyl esters
(17 and 18) by using diethyl sulfate in anhydrous potassium car-
bonate in dry acetone.
Further these neolignans 10 and 11 were modified to various
simple derivatives (Scheme 2) by acetylation (19 and 20), methyl-
ation (21 and 22) and amide formation (23 and 26), etc. using stan-
dard protocols.30 Ester 22 was hydrolysed to corresponding diacid
(25) using 5% methanolic–KOH at 60 °C. Ester 22 was treated with
30% aqueous ammonia at 0 °C to get corresponding diamide (26).
On reduction with lithium borohydride in THF ester 22 yielded a

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B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
O
R
OEt
OH
vii
AcO
EtO
R
19: R=H;
O
20: R=OCH3
O
O
O
R
OEt
OH
R
H3CO
H3CO
OEt
OH
viii
OCH3
HO
OCH3
H3CO
R
R
OCH3
EtO
EtO
x
HO
10: R=H;
21: R=H;
O
O
25
O
11: R=OCH3
22: R=OCH3
ix
O
O
H3CO
HO
H3CO
H3CO
NH2
OH
NH2
OCH3
OCH3
xi
ix
OCH3
H2N
H2N
23
O
O
26
H3CO
H3CO
OH
H3CO
H3CO
OAc
OCH3
OCH3
vii
OCH3
OCH3
HO
27
AcO
24
Scheme 2. Reagents and conditions: (vii) pyridine, Ac2O, RT, overnight, 89–92%; (viii) Me2SO4, K2CO3, DMF, reflux, 80 °C, 2 h, 87–91%; (ix) 30% aqueous NH3, 0 °C–rt, 2 h,
58–62%; (x) 5% methanolic–KOH, 60 °C, 2 h, 86%; (xi) LiBH4, THF, 0 °C, 4 h, 87%.
diol derivative 27. Diol 27 was finally acetylated with pyridine–
Ac2O to get diacetyl derivative 24. All the compounds were
characterized by spectroscopy.
2.3. Effect of lead molecule on cell cycle phases of MCF-7 and
MDA-MB-231 cells
MCF-7 cells were treated with compound 10 for 24 h and cell
cycle phase distribution was recorded with PI-staining method
(Fig. 2). Neolignan 10 showed significant increase in G2 phase at
all concentrations evaluated suggesting G2/M arrest, along with a
dose dependent increase in sub-diploid population indicating
possible apoptosis. There was also decrease in S-phase population
for all the concentrations of compound 10 in comparison to un-
treated control. However, G1 phase was not affected by any of
the concentrations of compound 10.
2.2. Biological results
All the lignans and neolignans were evaluated against MCF-7
and MDAMB-231 human breast cancer cell lines (Table 1). Only
eight of the derivatives showed some cytotoxicity against breast
cancer cell lines. Rests of the derivatives were inactive at
100
lM. The best compound of the series was 10 exhibiting IC50
12 and 15
l
M against MCF-7 and MDAMB-231, respectively. How-
ever, IC50 of 10 and 19 were much better when incubation time
was enhanced to 48 and 72 h.
In case of MDA-MB-231 cells, compound 10 did not affect cell
division cycle in treated cells, except dose-dependent increase of
Table 1
In vitro cytotoxicity of lignans and neolignans by MTT assay
#
#
#
Compound no.
Mol wt.
MCF-7 IC50
(lM)
MDAMB-231 IC50
(lM)
HEK-293 IC50 (lM)
4
502
502
586
382
74 0.012
—
—
92 0.026
82 0.037
—
Nd
Nd
Nd
*
5a
5b
10
12 0.0052 (24 h)
15 0.0085 (24 h)
12
l
M
M
0.12
9.29 0.03 (48 h)
6.47 0.03 (48 h)
3.27 0.02 (72 h)
0.45 0.02 (72 h)
11
17
18
19
442
470
530
424
—
Nd
—
Nd
—
Nd
Nd
Nd
20
100 0.0013
66 0.003 (24 h)
16 0.0025 (24 h)
l
0.233
9.65 0.02 (48 h)
3.22 0.03 (48 h)
0.82 0.06 (72 h)
0.21 0.05 (72 h)
20
21
22
23
24
25
26
27
484
410
470
384
470
414
412
386
470
414
371
—
—
—
—
—
—
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
50
100 0.0019
—
Nd
100 0.018
—
100 0.011
64.99 4.38
80 0.099
—
85 0.022
32 0.0068
—
100 0.0247
35.7 11.80
10 0.010
28
Podophyllotoxin
Tamoxifen
l
l
M
M
0.224
0.22
9
0.003
26
*
Means not active, IC50 >100
Incubation time = 24 h.
lM, Nd = not done.
#

B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
1345
groups. No changes in actin network were evident in control as
well as in treatment groups.
2.6. Molecular docking for binding studies
The modulation of anticancer activity of 10 and 19 was eluci-
dated through interaction with tubulin polymerase and identifying
binding site pocket. The molecular docking results also confirmed
that both 10 and 19 stabilize the polymerisation of tubulin. The
orientations and binding affinities (in terms of total score) of 10
and 19 were established towards tubulin (PDB ID: 1TUB). The
docking reliability was validated by using the known X-ray crystal
structure of tubulin complexed with taxol and docked conforma-
tion with the highest total score of 6.3796 was selected as the most
probable binding conformation. The low root mean-square devia-
tion (RMSD) of 0.6014 Å between the docked and the crystal con-
formations indicates the high reliability of Surflex-dock software
in reproducing the experimentally observed binding mode for
taxol. As shown in Figure 6A, redocked molecules were almost in
the same position with co-crystallized at the active site. Crystallog-
raphy data showed that the amino acid Threonine-276 is the ‘gate-
keeper’ residue, an important determinant of stabilizing specificity
in the tubulin binding pocket.
The docking results of 19 against target protein showed high
binding affinity docking score indicated by total score of 8.6059
and formation of two hydrogen bonds of length 2.0 and 1.8 Å
through hydrophobic residues THR-276 and HIS-229. In docking
pose, the conserved binding site pocket amino acid residues within
a selection radius of 4 Å from bound ligand were hydrophobic res-
idue Val-23 (Valine) PHE-272; nucleophilic (polar, hydrophobic),
for example, THR-276 (Threonine), SER-232, SER-236, SER-277
(Serine); basic LYS-218 (Lysine), ARG-320 (Arginine) CYS-213 (Cys-
teine), GLN-281(Glutamine), HIS-299 (Histidine); acidic (polar,
negative charged), for example, ASP-26 (Aspartic acid), Hydropho-
bic, for example, ALA-233, ARG-278 (Alanine), LEU-217, LEU-219,
LEU-230, LEU-275, LEU-371 (Leucine), and imino acids PRO-274,
PRO-360 (Proline), as a result, the bound compound 19 showed
strong hydrophobic interactions with tubulin, thus leading to more
stability and activity in this compound (Fig. 6B).
Figure 2. Effect of neolignan 10 in cell cycle phases of MCF-7 cells.
sub-diploid cells at higher concentrations (15 and 30 lM).
Compound 10 might have only caused apoptosis in MDA-MB-231
without causing cell cycle arrest at any phase. This also suggested
that compound 10 affects cell cycle phases of MDA-MB-231 differ-
ently than MCF-7 cells (Fig. 3).
2.4. Biochemical measure of tubulin polymerization activity of
lead molecules
Neolignans 10 and 19 were further evaluated for their effect on
tubulin polymerisation (Fig. 4). For this experiment, we incorpo-
rated tubulin destabilizing agent PDT and stabilizing agent taxol
as controls to improve reliability of our assay. As can be seen in
Figure 4, both the neolignans showed stabilization of tubulin
assembly similar to taxol at various concentrations (10, 20 and
40 lM) whereas, the standard tubulin destabilizing agent PDT,
effectively inhibited tubulin polymerization in comparison to con-
trol groups. Compounds 10 showed better stabilization of microtu-
bule polymerization in comparison to compound 19.
2.5. Effect of lead molecule on actin–tubulin cytoskeleton
structure with confocal microscopy
The binding affinity obtained in the docking study allowed a
comparison between the activities of the 10 to be compared to that
of the standard anticancer drug taxol. Compound 10 showed a
higher binding affinity against tubulin, the target protein. During
the comparison of the nature of interaction between the binding
pocket amino acid residues of target protein and compound 10, it
was found that the compound 10 showed molecular interactions
with conserved hydrophobic amino acid residues, thus leading to
more stability and potency (Table 2). The docking results for 10
showed that the compound docked on tubulin with a high binding
affinity docking score indicated by its total score of 6.3796 and also
showed the formation of a two H-bond of length 1.9 and 1.8 Å to
the acidic residues, Glu-22 and basic (hydrophobic) residues, Lys-
19. The 10 tubulin-docked complex also showed a similar type of
binding site residues within a radius of 4 Å of bound ligand such
as nucleophilic (polar, hydrophobic), for example, SER-232, SER-
236 (Serine); basic LYS-19 (Lysine), ARG-320, HIS-299 (Histidine);
acidic (polar, negative charged), for example, ASP-26 (Aspartic
acid); Hydrophobic, for example, ALA-233 (Alanine), ARG-278,
ARG-369 (Arginine) LEU-219, LEU-230, LEU-275, LEU-371
(Leucine), Val-23 (Valine), PHE-272 (Phenylalanine); imino acids
PRO-274, PRO-360 (Proline); and acidic (polar, negative charged)
residues, for example, GLU-22 (Glutamic acid), therefore, the
docked molecule also showed a strong hydrophobic interaction
with tubulin, thus leading to more stability (Fig. 6C).
In order to observe the phenotypic effect of compound 10 (as
showed better activity in tubulin polymerization assay) on cellular
cytoskeletal network of actin and tubulin, MCF-7 cells were immu-
nostained and analyzed under confocal microscope. As illustrated
in Figure 5, substantial stabilization of microtubules in the form
of bundle like appearance was observed in paclitaxel-treated cells
that were used as positive control in this assay. However, in
compound 10 treated cells, stabilization of tubulin network was
not apparent up to the level in comparison to positive control
The docking results for the negative control compound
podophyllotoxin (tubulin inhibitor) with tubulin showed a low
Figure 3. Effect of compound 10 in cell cycle phases of MDA-MB-231 cells.

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B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
Figure 4. Kinetics of tubulin polymerisation by compounds 10 and 19 at different concentrations with controls.
binding affinity, docking score indicated by a low total score of
2.9284 without any H-bond (hydrogen bond) formation (Fig. 6D),
in comparison to the docking score of anticancer known stabilizing
agent taxol, which showed a total score of 6.3796 (Table 2). Thus,
the docking procedure of Surflex-dock software (Sybyl-X 1.3) in
reproducing the experimental binding affinity seems reliable, and
therefore predicted as true positive.
Molecules with intermediate lipophilicities have a better chance
of arriving at the receptor site.32 Typically, low solubility is associ-
ated with poor absorption, so the general aim is to avoid poorly
soluble compounds. The aqueous solubility (LogS) of a compound
significantly affects its absorption and distribution characteristics.
The calculated LogS values of 10 and 19 were within the acceptable
range. Other calculations related to solubility, serum protein bind-
ing, blood–brain barrier (LogBB and apparent MDCK cell perme-
ability), gut–blood barrier (Caco-2 cell permeability), predicted
central nervous system activity, number of likely metabolic reac-
tions, hERG K+ (LogIC50) channel blockage, skin permeability
(Kp), and human oral absorption in the gastrointestinal tract
showed these values for both 10 and 19 within the standard ranges
for good bioavailable drugs (Table 3).
2.7. In silico ADMET analysis
Neolignans 10 and 19 were further evaluated for several phys-
iochemical properties (ADME) related to pharmacokinetics. The
logP value indicates about hydrophilicity and lipophilicity of the
molecule which has an important role for the transportation of
the compound in the body. Both the compounds follow Lipinski’s
rule of five for good bioavailability. Both possess molecular weights
less than 500 (mw <500 kDa), number of hydrogen bond donors
less than 5 (Hd <5), H-bond acceptors less than 10 (Ha <10) and
LogP value less than 5 (LogP <5). Low hydrophilicity and therefore
a high LogP value may lead to poor absorption or permeation. Both
the compounds showed calculated LogP values less than 5, so
these compounds will have good hydrophilicity with moderate
lipophilicity and hence should be able to gain access to membrane
surfaces. LogP value has also been linked to blood–brain barrier
penetration and utilized to predict cell membrane permeability.
The process of excretion, which eliminates the compound from
the human body, depends on its molecular weight and LogP.31
2.8. In vivo acute oral toxicity of neolignan 10
No observational changes, morbidity and mortality were ob-
served throughout the experimental period up to the dose level
of 1000 mg/kg body weight. No morbidity or any other gross obser-
vational changes could be noticed in the any group of animals trea-
ted with compound 10. Blood and serum samples upon analysis
showed non-significant changes in all the parameters studied like
total haemoglobin level, RBC count, WBC count, differential leuco-
cytes count, SGPT, ALKP, creatinine, triglycerides, cholesterol, albu-
min, serum protein (Table 4 and Fig. 7). However, SGOT showed
significant increase in group of animals treated with the test drug

B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
1347
Figure 5. Effect of compound 10 on actin–tubulin cytoskeleton structure in MCF-7 cell line with confocal microscopy.
at 1000 mg/kg body weight as compare to control groups. Animals
on gross pathological study showed no changes in any of the or-
gans studied including their absolute and relative weight (Fig. 8).
Therefore, the experiment showed that compound 10 is well toler-
ated by the Swiss albino mice up to the dose level of 300 mg/kg
body weight as a single acute oral dose. However, sub-acute and
or chronic experiment with the test drug needs to be carried out
to look for any adverse effect on repeated exposure to compound
10 for its future development.33
block the polymerisation of tubulins to microtubulin. Both types
of tubulin interacting agents have different binding sites in the
tubulin polymerase.
The lignans and neolignans synthesized were supposed to inhi-
bit tubulin polymerase enzyme similar to podophyllotoxin. But,
unexpectedly, these compounds stabilized the tubulin polymerisa-
tion process very similar to taxol. Docking experiments showed
significant binding affinities of 10 and 19 to tubulin, but both the
compounds possessed moderate cytotoxicities in in vitro experi-
ments against human breast cancer cell lines (MCF-7 and
MDAMB-231). It might be because the amino acids involved in
hydrogen bonding with 10 and 19 are different from taxol. 19
has only one amino acid (THR-276) common to taxol while, 10
has none. These bindings might be responsible for eliciting cyto-
toxicity in the molecule. Due to this, although 10 and 19 possessed
tubulin stabilization property (similar to taxol) with better binding
affinities, these could not induce better cytotoxicity against human
cancer cell lines. Anti-tubulin agents can act differently due to their
differential binding sites or other indirect mechanism leading to
modulation of microtubule dynamics. While some anti-tubulin
agents act by promoting polymerization (taxanes) or by augment-
ing depolymerisation (vincas) of microtubules, there are some
more class of microtubule inhibitors, like EM011, which do not
cause condensation of tubulin by altering total microtubule mass.
EM011, a synthetic noscapine derivative, is a well established
microtubule interfering agent which binds tubulin with high
3. Discussion
Microtubules are a component of cytoskeleton, involved in
maintaining cell structure and cell division including the formation
of spindles. These are polymers of
a- and b-tubulin dimers. The
polymerisation of tubulins to microtubulins is a reversible process
which is in a dynamic equilibrium. A number of anticancer mole-
cules bind to tubulin and modify its activation state. They can
either stabilize the polymerisation process or destabilize (inhibit)
it. In both the cases the microtubule dynamics is disturbed, which
leads to cell cycle arrest and can lead to apoptosis. Owing to their
essential role in forming dynamic spindle apparatus in mitosis,
microtubules are considered an ideal target for anticancer drug
development.34 Taxol and epothilones block dynamic instability
by stabilizing GDP-bound tubulin in the microtubule. On the con-
trary, podophyllotoxin, vincristine, vinblastine, and colchicine

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B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
Figure 6. In silico molecular docking studies elucidating the possible mechanisms of 19 and 10 induced modulation of tubulin protein. The docking studies were carried out
using SYBYL-X 1.3, Tripos International. (A) The co-crystallized taxol was redocked into the binding site of tubulin (PDB: 1TUB) with 0.6014 Å of root mean-square deviation
between docked and crystallized conformation and a total docking core of 6.3796. (B) 19 docked on to tubulin with high binding affinity, as indicated by a total docking score
of 8.6059 compared to taxol (C) 10 docked on tubulin with high binding affinity, as indicated by a total docking score of 6.4471. (D) Podophyllotoxin docked onto tubulin with
a low binding affinity, as indicated by a total docking score of 2.9284. In A, B and C structures, tubulin adopts the same active conformation and the inhibitors bind in a similar
manner, with enlarged hydrophobic pocket that is characteristic of the active conformation.
affinity,35 but does not perturb the morphology of microtubules as
visible under microscope.36 In ADME analysis both 10 and 19
exhibited good pharmacokinetics profile for better bioavailability.
Tubulin polymerization kinetics clearly show that compound 10
stabilizes polymerization reaction (Fig. 4). However, at morpholog-
ical evaluation under confocal microscopy, microtubule condensa-
tion in compound 10 treated cells was not as evident as in positive
control groups. Hence, we assume that our compound 10 may also
act in a different way leading to tubulin stabilization without any
morphological changes in microtubule architecture promoting
cancer cell death. Nonetheless, presence of dense tubulin in com-
pound 10 treated cells may denote stabilization of microtubule
network.
Compound 10 caused significant arrest at G2/M phase arrest,
S-phase decrease and increase in sub-diploid population
depending on concentration in MCF-7 cells. However, in case of
MDA-MB-231 cells, it did not affect any phase of cell cycle in
MDA-MB-231, but only increased sub-diploid population. We
assume that microtubule stabilization action of the lead compound
is responsible for subsequent cell cycle arrest and apoptosis in
breast cancer cells. Compound 10 is found to be relatively cytotoxic
in vitro against normal cells. However, it is well recognized that
toxicity in vivo reflects better profile of a compound than in vitro
model. Therefore, we carried out in vivo acute oral toxicity studies.
In in vivo acute oral toxicity compound 10 was well tolerated up to
300 mg/kg and can be declared as non-toxic up to this level.
Table 2
Comparison of binding affinities of standard drug (control) and most active compounds 10 and 19 with tubulin
Compound name
Total
score
Amino acid involved in active pocket in 4 Å
Involved
group of
amino acids
Length of No. of
H-bond Å hydrogen
bonds
Taxol (standard stabilizer, 6.3796 GLU-22, VAL-23, ILE-24, SER-25, ASP-26, LEU-42, PHE-83, LEU-217, LEU-219, ASP-
control positive) 226, HIS-229, LEU-230, SER-232, ALA-233, SER-236, PHE-272, PRO-274, LEU-275,
ASP-226
THR-276
1.9
1.8
1.9
1.8
2.0
1.8
4
THR-276, SER-277, ARG-278, GLN-281, ARG-320, PRO-360, ARG-369, GLY-370, LEU– ARG-278
371 ARG-369
19
10
8.6059 VAL-23, CYS-213, LEU-217, LYS-218, LEU-219, ASP-226, HIS-229, LEU-230, SER-232, THR-276
ALA-233, SER-236, PHE-272, PRO-274, LEU-275, THR-276, SER-277, ARG-278, GLN- HIS-229
281, ARG-320, PRO-360, LEU-371
2
6.4471 LYS-19, GLU-22, VAL-23, ASP-26, LEU-42, PHE-83, HIS-229, SER-232, ALA-233, SER- GLU-22
1.9
1.8
—
2
236, PHE-272, ARG-320, PRO-360, ARG-369, LEU-371,
2.9284 VAL-23, ASP-26, HIS-229, LEU-230, SER-232, ALA-233, SER-236, PHE-272, ARG-320,
PRO-360, ARG-369, GLY-370, LEU-371
LYS-19
—
Podophyllotoxin
(standard inhibitor,
control negative)
—

B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
1349
Table 3
Various physicochemical (ADME) parameters calculated for 10 and 19
Compound
name
LogS for LogP
aqueous
LogKhsa LogBB
No. of
metabolic CNS
Predicted HERG for Apparent
Apparent
MDCK
LogKp for
skin
% Human oral Qual.
absorption in model
for
for
K+
Caco-2
solubility
serum
protein blood
binding
brain/
reactions Activity
(LogIC50
channel (nm/s)
blockage
)
permeability
permeability
(nm/s)
permeability GI ( 20%)
for human
oral
absorption
19
10
PDT
Taxol
ꢀ5.656 4.186
ꢀ5.449 3.855
ꢀ5.358 3.317
ꢀ10.117 5.052
0.535
0.509
0.111
ꢀ2.007
ꢀ2.211
0.21
ꢀ1.984
(ꢀ3.0/
1.2)
1
2
6
8
ꢀ2
ꢀ2
1
ꢀ2
ꢀ6.278
ꢀ6.302
ꢀ3.403
ꢀ6.752
240.84
154.116
7274.037
224.696
106.187
65.541
4225.005
98.515
(<25 Poor,
>500 great)
ꢀ2.83
ꢀ3.133
ꢀ1.026
ꢀ1.889
(ꢀ8.0 to
ꢀ1.0, Kp in
cm/h)
85.866
50.034
84.428
44.589
(<25% is poor) (>80% is
high)
High
High
High
Medium
0.585
Stand. range* (ꢀ6.5/
(ꢀ2.0/ (ꢀ1.5/
(1.0/8.0) (ꢀ2
(Concern (<25 Poor,
0.5)
6.5)
1.5)
Inactive, below
+2 active) ꢀ5)
>500 great)
*
Note: for 95% of known drugs based on Schrödinger, USA—Qikprop v3.3 (2010) software results.
Table 4
Effect of 10 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body weight on body weight, haemogram and serum biochemical parameters in Swiss albino mice
Parameters
Dose of 10 at mg/kg body weight as a single oral dose
Control
24.5 0.76
11.39 0.84
5.43 0.27
5 mg/kg
50 mg/kg
300 mg/kg
1000 mg/kg
Body weight (g)
Haemoglobin (g/dL)
RBC (million/mm3)
WBC (1000*/mm3)
ALKP (U/L)
SGOT (U/L)
SGPT (U/L)
Albumin (g/dL)
24.33 1.26
12.35 0.73
5.99 0.25
24.83 0.65
10.67 0.54
5.58 0.49
25.0 1.06
10.83 1.03
5.11 0.37
13.76 1.83
217.42 15.97
37.95 3.42
15.56 5.20
2.59 0.25
26.50 0.89
13.32 0.42
5.15 0.64
15.94 1.52
143.89 9.18
53.29 3.67*
21.95 3.98
3.86 0.78
14.2 7.49
14.6 6.99
14.7 1.43
218.67 12.43
25.27 1.32
12.28 3.73
3.35 0.51
227.47 25.91
27.67 1.26
13.15 5.22
3.17 0.12
234.14 12.24
34.34 4.20
12.52 1.69
2.76 0.10
Creatinine (mg/dL)
Triglycerides (mg/dL)
Serum protein (mg/ml)
Cholesterol (mg/dL)
0.63 0.18
121.45 24.02
1.11 0.14
1.05 0.24
106.46 13.63
0.88 0.03
1.45 0.49
147.23 18.15
0.94 0.10
0.79 0.14
134.38 17.99
0.92 0.06
1.87 0.45
156.83 29.30
1.02 0.06
81.64 4.26
75.94 4.08
58.62 1.96
77.65 6.12
60.30 4.50
Mean SE; n = 6.
*
P < 0.05 compared to control, 5, 50, 300, 1000 mg/kg.
molecules may further be optimized to get better anticancer
agent.
5. Experimental section
5.1. General methods
Reagents and biological standards, MTT, podophyllotoxin, taxol,
etc. were procured from Sigma–Aldrich, USA and used as such
without purification. Melting points were determined on E-Z melt
automated melting point apparatus Stanford Research System, USA
in open capillaries. For thin layer chromatography, Merck silica gel
(TLC, UV254nm) aluminium sheets were used to monitor the reac-
tions and visualisation was done by spraying 2% ceric sulfate–
10% aqueous sulfuric acid subsequent charring at 80–100 °C. Dry
solvents were prepared as per standard methods. Purifications of
compounds were done through column chromatography over sil-
ica gel (60–120 and 100–200 mesh, Thomas Baker), evaporating
the solvents under reduced pressure. NMR experiments were car-
ried out on Bruker Avance DRX-300 MHz instrument using tetra-
methylsilane (TMS) as internal standard. Chemical shifts are
given in d ppm. NMR abbreviations for signal patterns are as; s, sin-
glet; d, doublet; t, triplet, m, multiplet and br s, broad singlet. All
1H and few 13C spectral data are reported. Electrospray ionization
mass spectra (ESI-MS) were recorded on APC3000 (Applied biosys-
tem) LC–MS–MS after dissolving compounds in methanol. FT-IR
spectra were recorded on Perkin–Elmer SpectrumBX after making
KBr pellets. Tubulin polymerisation assay kit (BK006P) was pro-
cured from Cytoskeleton, USA. The nomenclatures of lignans and
neolignans have been given as per IUPAC recommendations.37
Figure 7. Effect of 10 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body
weight on differential leucocytes counts in Swiss albino mice (n = 6, non significant
changes were found compared to control).
4. Conclusion
The lignans and neolignans have exhibited significant anti-
breast cancer activity. In cell cycle analysis, we observed that
compound 10 caused significant enhancement of sub-diploid
population that is indicative of increased apoptosis, along with
or without arrest of cell cycle phases depending on the concen-
trations. As suggested by in silico studies and biochemical tubu-
lin polymerization assay, the probable mechanism of action of
the lead compound 10 and 19 is through stabilization of micro-
tubule assembly. Compound 10 was found to be non-toxic up to
300 mg/kg dose in Swiss albino mice. These neolignan lead

1350
B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
Figure 8. Effect of 10 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on absolute and relative organ weight in Swiss albino mice (n = 6, non significant changes were
found compared to control).
5.2. Chemical synthesis
60.80, 109.85, 115.23, 115.90, 123.39, 127.35, 145.18, 147.28,
148.43, 167.81; Electrospray mass (MeOH): 245 [M+Na]+, Negative
mode: 221 [MꢀH]ꢀ; IR (cmꢀ1): 3399, 2979, 1707, 1688, 1602,
1516, 1270, 1176.
5.2.1. General procedure for the synthesis of esters 2, 8 and 9
5.2.1.1. Synthesis of 3,4,5-trimethoxycinnamic acid ethyl ester
(2).
Compound 1 (1 g, 3.94 mmol) was taken in 2 mL of thio-
nyl chloride. To this stirred solution absolute ethanol (2 mL) was
added and further stirred for 2 h at room temperature. Excess of
thionyl chloride and ethanol were removed under reduced pres-
sure and the reaction mixture was quenched with water. The prod-
uct was extracted with ethyl acetate, washed with water and dried
over anhydrous sodium sulfate. The organic layer was evaporated
to dryness to get a crude residue. It was purified through column
chromatography over silica gel (60–120 mesh) to get the desired
ester as white crystalline solid.
Alternatively, these products can also be obtained using another
protocol. Same quantity of acid (1 g) was taken in 15 mL ethanol.
To this 0.5 mL concentrated sulfuric acid was added and the reac-
tion mixture was refluxed for 4 h. The solvent was evaporated and
10 mL water was added to it. The reaction mixture was extracted
with ethyl acetate, washed with water and dried over anhydrous
sodium sulfate. The solvent was evaporated and the crude product
was purified through column chromatography over silica gel to get
the desired ester 2.
5.2.2. Synthesis of 4-hydroxy-3,5-dimethoxycinnamic acid ethyl
ester (3)
The demethylation of 2 was done as per previously reported
method.29
5.2.2.1. 4-Hydroxy-3,5-dimethoxycinnamic acid ethyl ester
(3).
Yield 84%; mp = 74–77 °C; 1H NMR (CDCl3, 300 MHz): d
1.33 (t, 3H, CH3), 3.90 (s, 6H, 2ꢁ OCH3), 4.24 (q, 2H, OCH2), 5.81
(s, 1H, exchangeable, phenolic OH), 6.27–6.32 (d, 1H, @CH,
J = 15.9 Hz), 6.76 (s, 2H, CH, aromatic), 7.56–7.61 (d, 1H, CH@,
J = 15.6 Hz). 13C NMR (CDCl3, 75 MHz): d 14.74, 56.71, 56.71,
60.80, 105.40, 105.40, 116.41, 126.34, 137.47, 145.27, 147.60,
147.60, 167.57. Electrospray mass: 253.3 [M+H]+, 275.1 [M+Na]+,
291.1 [M+K]+, 527.2 [2M+Na]+; IR (cmꢀ1): 3371, 2940, 1693,
1601, 1516, 1190, 1110.
5.2.3. General procedure for the synthesis of lignan 5a and
neolignans 4, 10 and 11
Yield 91%; mp = 68–70 °C; 1H NMR (CDCl3, 300 MHz): d 1.32 (t,
3H, CH3), 3.87 (s, 9H, 3ꢁ OCH3), 4.24 (q, 2H, OCH2), 6.31–6.36 (d,
1H, @CH, J = 15.9 Hz), 6.74 (s, 2H, CH, aromatic), 7.56–7.61 (d,
1H, CH@, J = 15.9 Hz). 13C NMR (CDCl3, 75 MHz): d 14.72, 56.52,
56.52, 60.88, 61.34, 105.56, 105.56, 117.90, 130.35, 140.42,
144.95, 153.81, 153.81, 167.33; Electrospray mass: 289.2
[M+Na]+, 305.2 [M+K]+, 555.3 [2M+Na]+. IR (cmꢀ1): 2943, 1702,
1633, 1583, 1506, 1279, 1122.
5.2.3.1. Synthesis of neolignan 4 and lignan 5a.
To a stirred
cold solution of aqueous KOH (6%, 20 mL) a solution of ester 3
(504 mg, 2.0 mmol) in benzene (15 mL) was added. After 10 min
potassium ferricyanide (329 mg, 0.1 mmol) was added to this reac-
tion mixture to give a light brown colour. After 30 min when the
reaction mixture turns to light green, it is quenched with dil HCl
(5%, 5 mL). It was extracted with ethyl acetate (3 ꢁ 20 mL), washed
with water and dried over anhydrous sodium sulfate. The organic
layer was dried in vacuo. The residue thus obtained was purified
through column chromatography over silica gel to get neolignan
(4) at 6% ethylacetate–hexane and lignan (5a) at 9% ethyl ace-
tate–hexane both as dense liquids.
5.2.1.2. 4-Hydroxy cinnamic acid ethyl ester (8).
Yield 94%;
mp = 69–71 °C; 1H NMR (CDCl3, 300 MHz): d 1.33 (t, 3H, CH3), 4.28
(m, 2H, OCH2), 6.26–6.31 (d, 1H, @CH, olefinic, J = 15.9 Hz), 6.86–
6.89 (d, 1H, CH, aromatic, J = 8.4 Hz), 7.38–7.41 (d, 1H, CH, aro-
matic, J = 8.4 Hz), 7.61–7.66 (d, 1H, CH@, olefinic, J = 15.9 Hz). 13C
NMR (CDCl3, 75 MHz): d 14.69, 61.26, 115.32, 116.41, 116.41,
127.04, 130.50, 130.50, 145.61, 158.91, 168.86. Electrospray mass
(MeOH): 193 [M+H]+, negative mode: 191 [MꢀH]ꢀ; IR (cmꢀ1):
3289, 1717, 1683, 1604, 1516, 1280.
5.2.3.2. Diethyl-4,40-dihydroxy-3,30,5,50-tetramethoxy-8,20-neo-
lign-7,70-dien-9,90-dionate (4).
Yield 21%; oil, 1H NMR
(CDCl3, 300 MHz): d 1.18 (bs, 3H, CH3), 1.34 (t, 3H, CH3), 3.85 (s,
6H, OCH3), 4.06 (s, 6H, 2ꢁ OCH3), 4.38 (m, 4H, 2ꢁ OCH2), 5.8 (bs,
1H, exchangeable, phenolic OH), 6.1 (bs, 1H, exchangeable, pheno-
lic OH), 6.28–6.33 (d, 1H, @CH, J = 15.9 Hz), 6.78 (s, 1H, CH, aro-
matic), 7.01 (s, 1H, CH, aromatic), 7.57–7.62 (d, 1H, CH@,
J = 15.9 Hz), 8.09 (s, 1H, CH, aromatic), 8.37 (s, 1H, CH, aromatic).
13C NMR (CDCl3, 75 MHz): d 14.58, 14.74, 56.71, 56.71, 60.79,
61.67, 61.87, 61.87, 102.67, 102.67, 105.46, 105.46, 116.46,
123.56, 125.18, 127.74, 127.74, 128.75, 129.19, 139.46, 139.46,
145.25, 147.63, 147.63, 168.33, 168.67. Electrospray mass: 503.4
5.2.1.3. 4-Hydroxy-3-methoxycinnamic acid ethyl ester
(9).
Yield 89%; mp = 61–63 °C; 1H NMR (CDCl3, 300 MHz): d
1.22 (t, 3H, CH3), 3.88 (s, 3H, OCH3), 4.25 (q, 2H, OCH2), 6.22 (d,
1H, OH, phenolic), 6.24–6.29 (d, 1H, @CHACOA, J = 15.9 Hz), 6.89
(d, 1H, CH, aromatic), 7.05 (d, 2H, CH, aromatic), 7.57–7.62 (d,
1H, CH@, J = 15.9 Hz). 13C NMR (CDCl3, 75 MHz): d 14.72, 56.30,

B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
1351
[M+H]+, 525.3 [M+Na]+, 541.4 [M+K]+. IR (cmꢀ1): 3401, 2933, 1718,
1628, 1477, 1272, 1107.
CH). Electrospray mass: 531.2 [M+H]+, 553.3 [M+Na]+, 569.3
[M+K]+; IR (cmꢀ1): 2925, 1772, 1764, 1601, 1458, 1256, 1187,
1129.
5.2.3.3. Diethyl-4,40-dihydroxy-3,30,5,50-tetramethoxy-lign-7,70-
dien-9,90-dionate (5a).
Yield 29%; gummy, 1H NMR (CDCl3,
5.2.4.2. Diethyl-3,30,4,40-tetramethoxy-lign-7,70-dien-9,90-diona
300 MHz): d 1.20 (t, 6H, 2ꢁ CH3), 3.63 (s, 3H, OCH3), 3.72 (s, 6H,
2ꢁ OCH3), 3.88 (s, 3H, OCH3), 4.02 (m, 4H, 2ꢁ OCH2), 4.96 (s, 2H,
CH@, olefinic), 5.36 (bs, 1H, exchangeable, OH), 5.81 (bs, 1H,
exchangeable, OH), 6.27 (s, 2H, CH, aromatic), 6.68 (s, 1H, CH, aro-
matic), 7.60 (s, 1H, CH, aromatic). 13C NMR (CDCl3, 75 MHz): d
14.44, 14.61, 56.61, 56.61, 56.61, 60.95, 61.09, 61.57, 104.81,
104.81, 107.81, 107.81, 123.66, 123.78, 124.21, 133.96, 134.11,
137.67, 137.67, 141.37, 145.39, 147.21, 147.21, 167.13, 172.40.
Electrospray mass: 525.3 [M+Na]+, 541.3 [M+K]+. IR (cmꢀ1):
3401, 2929, 1720, 1703, 1687, 1596, 1496, 1258.
te (17).
Yield 43%, oil, 1H NMR (CDCl3, 300 MHz): d 1.24 (t,
6H, 2ꢁ CH3), 3.70 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.92 (s, 3H,
OCH3), 3.94 (s, 3H, OCH3), 4.24 (m, 4H, 2ꢁ OCH2), 6.77–6.88 (m,
6H, aromatic), 7.56 (s,1H, olefinic, CH@), 7.82 (s, 1H, olefinic,
CH@). Electrospray mass: 493.3 [M+Na]+, 509.2 [M+K]+; IR
(cmꢀ1): 2933, 1702, 1596, 1514, 1257.
5.2.5. Synthesis of 5b, 19, 20, 28 were carried out as per reported
method30
5.2.5.1. Diethyl-4,40-diacetoxy-3,30,5,50-tetramethoxy-lign-7,70-
dien-9,90-dionate (5b).
Yield 92%, oil, 1H NMR (CDCl3,
5.2.3.4. Diethyl-4,40-dihydroxy-8,30-neolign-7,70-dien-9,90-dio-
300 MHz): d 1.27 (bs, 6H, 2ꢁ CH3), 2.27 (s, 3H, OAc), 2.29 (s, 3H,
OAc), 3.64 (s, 3H, OCH3), 3.68 (s, 6H, 2ꢁ OCH3), 3.88 (s, 3H,
OCH3), 4.16 (bm, 4H, 2ꢁ OCH2), 6.30 (s, 2H, 2ꢁ CH, aromatic),
6.77 (s, 1H, @CH), 7.28 (bs, 1H, CH@), 7.61 (s, 2H, 2ꢁ CH, aromatic).
13C NMR (CDCl3, 75 MHz): d 14.44, 14.63, 20.86, 23.09, 56.45 (ꢁ3),
56.60, 61.38, 61.76, 104.52 (ꢁ4), 108.55, 114.47, 123.13, 126.63,
127.80, 130.28, 136.58, 136.58, 140.90, 151.34, 152.27, 152.27,
166.69, 168.48, 169.12, 171.93. Electrospray mass: 609.2
[M+Na]+, 625.3 [M+K]+. IR (cmꢀ1): 2926, 1766, 1720, 1705, 1601,
1188.
nate (10).
Yield 24%, oil, 1H NMR (CDCl3, 300 MHz): d 1.30
(t, 6H, 2ꢁ CH3), 4.28 (q, 4H, 2ꢁ OCH2), 6.07 (d, 1H, CH, aromatic,
J = 7.5 Hz), 6.27–6.32 (d, 1H, @CH, olefinic, J = 16.2 Hz), 6.82 (m,
3H, 3ꢁ CH, aromatic), 7.23 (d, 2H, 2ꢁ CH, aromatic), 7.44 (d, 1H,
CH, aromatic, J = 9.6 Hz), 7.57 (s, 1H, CH, olefinic), 7.59–7.64 (d,
1H, CH@, olefinic, J = 16.2 Hz). 13C NMR (CDCl3, 75 MHz): d 14.67,
14.73, 61.00, 62.37, 110.71, 115.89, 116.11, 116.11, 125.31,
125.67, 127.92, 127.92, 128.20, 130.26, 131.14, 132.27, 145.05,
156.75, 156.75, 161.56, 168.13, 170.89; Electrospray mass (MeOH):
421 [M+K]+, Negative mode: 381 [MꢀH]ꢀ; ESI-HRMS: 383.1489
(calcd for C22H23O6, 383.1495), 405.1307 (calcd for C22H22O6Na,
405.1314), 421.1052 (calcd for C22H22O6K, 421.1054); IR: 3393,
2980, 2928, 1733, 1706, 1602, 1511, 1245.
5.2.5.2.
9,90-dionate (19).
Diethyl-4-acetoxy-40-hydroxy-8,30-neolign-7,70-dien-
Yield 89%, mp = 65–68 °C; 1H NMR (CDCl3,
300 MHz): d 1.31 (t, 6H, 2ꢁ CH3), 2.24 (s, 3H, OAc) 4.24 (m, 4H,
2ꢁ OCH2), 6.12 (d, 1H, CH, aromatic, J = 7.5 Hz), 6.23–6.28 (d, 1H,
@CH, J = 16.2 Hz), 6.86 (d, 1H, CH, aromatic, J = 8.4 Hz), 7.05 (d,
2H, CH, aromatic, J = 8.4 Hz), 7.44 (m, 3H, 3ꢁ CH, aromatic), 7.52
(s, 1H, CH, olefinic), 7.55–7.61 (d, 1H, CH@, J = 16 Hz). 13C NMR
(CDCl3, 75 MHz): d 14.70, 14.76, 21.50, 60.80, 62.40, 110.72,
116.22, 122.43, 122.43, 126.79, 126.79, 127.34, 127.34, 128.48,
130.20, 131.07, 132.03, 138.16, 144.66, 151.11, 161.41, 167.69,
169.80, 170.57. Electrospray mass: 463 [M+K]+; ESI-HRMS:
425.1591 (calcd for C24H25O7, 425.1600), 447.1410 (calcd for C24-
H24O7Na, 447.1420), 463.1156 (calcd for C24H24O7K, 463.1159),
calcd: 491.24099; IR (cmꢀ1): 3449, 2928, 1737, 1719, 1702, 1654,
1603, 1233.
5.2.3.5.
7,70-dien-9,90-dionate (11).
Diethyl-4,40-dihydroxy-5,50-dimethoxy-8,30-neolign-
Yield 26%, mp = 127–29 °C; 1H
NMR (CDCl3, 300 MHz): d 1.34 (t, 6H, 2ꢁ CH3), 3.87 (s, 3H,
OCH3), 3.91 (s, 3H, OCH3), 4.29 (q, 4H, 2ꢁ OCH2), 5.27 (s, 2H, 2ꢁ
OH, both phenolic OH), 6.10 (d, 1H, CH aromatic, J = 6.9 Hz),
6.27–6.33 (d,1H, CH@, olefinic, J = 15.9 Hz), 6.90 (s, 3H, 3ꢁ CH, aro-
matic), 7.02 (s, 1H, CH, aromatic), 7.18 (s, 1H, CH@, olefinic), 7.61–
7.66 (d, 1H, olefinic, J = 15.9 Hz). 13C NMR (CDCl3, 75 MHz): d 14.69,
14.75, 55.97, 56.42, 60.84, 62.30, 87.91, 109.16, 112.29, 114.92,
116.34, 118.30, 119.90, 126.26, 129.03, 131.87, 144.97, 145.12,
146.45, 146.45, 147.12, 150.32, 167.67, 170.66. Electrospray mass
(MeOH): 465 [M+Na]+; Negative mode: 441 [MꢀH]ꢀ; IR (cmꢀ1):
3422, 2934, 1737, 1718, 1606, 1520, 1270, 1172.
5.2.5.3. Diethyl-4-acetoxy-40-hydroxy-5,50-dimethoxy-8,30-neo-
lign-7,70-dien-9,90-dionate (20).
Yield 92%, mp = 106–
5.2.4. General procedure for the synthesis of lignans 17 and 18
through Stobbe’s condensation
108 °C; 1H NMR (CDCl3, 300 MHz): d 1.31 (t, 6H, 2ꢁ CH3), 2.26 (s,
3H, O–CO–CH3), 3.80 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.26 (q,
4H, 2ꢁ OCH2), 6.16 (d, 1H, CH, aromatic, J = 7.8 Hz), 6.25–6.31 (d,
1H, CH@, olefinic, J = 15.9 Hz), 6.88–7.01 (m, 4H, aromatic proton),
7.31 (s, 1H, CH@, olefinic), 7.59–7.64 (d, 1H, @CH, olefinic,
J = 15.9 Hz). 13C NMR (CDCl3, 75 MHz): d 14.69, 14.69, 21.03,
56.39, 56.58, 60.82, 62.39, 110.54, 110.54, 112.34, 116.53, 118.32,
118.66, 123.48, 126.03, 129.27, 139.03, 140.20, 144.86, 145.15,
147.20, 148.10, 151.73, 168, 169, 170.51; Electrospray mass
(MeOH): 507 [M+Na]+. IR (cmꢀ1): 3449, 2983, 2930, 1768, 1734,
1702, 1609, 1275.
5.2.4.1.
Diethyl-3,30,4,40,5,50-hexamethoxy-lign-7,70-dien-9,
90-dionate (18).
Pre-washed sodium hydride (138 mg, 5.75
mmol) was stirred in dry DMF (5 mL) at room temperature under
inert atmosphere (N2). To this diethyl succinate (200 mg,
1.15 mmol)
and
3,4,5-trimethoxybenzaldehyde
(271 mg,
1.38 mmol) were added as solution in DMF (5 mL) through syringe.
The reaction mixture was refluxed at 100 °C for 6 h. On completion,
the reaction mixture was cooled and quenched with dil HCl (5%,
10 mL). It was extracted with ethyl acetate (3 ꢁ 25 mL), washed
with water and dried over anhydrous sodium sulfate and evapo-
rated to dryness. The residue thus obtained was a complex mixture
and was difficult to purify. So, the crude mass thus obtained was as
such processed for esterification with diethyl sulfate in potassium
carbonate-DMSO/acetone (1:3) system. After routine work-up, it
was purified through column chromatography over silica gel
(100–200 mesh) to get 18 as viscous oil.
5.2.5.4.
9,90-diacetate (24).
Diethyl-4,40,5,50-tetramethoxy-8,30-neolign-7,70-dien-
Yield 91%, oil, 1H NMR (CDCl3,
300 MHz): d 2.00, 2.02 (s, 6H, 2ꢁ CH3), 3.51 (s, 3H, OCH3), 3.66
(s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 4.61–4.64
(m, 2H, CH2), 4.80 (s, 2H CH2), 6.51–6.74 (m, 8H, aromatic, and ole-
finic). 13C NMR (CDCl3, 75 MHz) 21.36, 21.36, 55.52, 56.04, 56.23,
61.08, 64.16, 65.47, 108.61, 111.38, 112.76, 119.01, 120.19,
121.42, 122.78, 132.37, 132.42, 133.15, 134.55, 135.52, 147.09,
147.72, 148.93, 154.01, 170.08, 171.44. Electrospray mass (MeOH):
Compound 18: Yield 49%; oil, 1H NMR (CDCl3, 300 MHz): d 1.25
(t, 6H, 2ꢁ CH3), 3.81 (s, 12H, 4ꢁ OCH3), 3.92 (s, 6H, 2ꢁ OCH3), 4.13–
4.20 (m, 4H, OCH2), 6.77 (s, 4H, CH, aromatic), 7.82 (s, 2H, olefinic,

1352
B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
493 [M+Na]+, 495.5 [M+Na+2]+, 497 [M+Na+4]+. IR (cmꢀ1): 2931,
2839, 1735, 1720, 1704, 1687, 1595, 1513, 1461, 1252, 1159, 1024.
and stirred for an hour. After this it was further stirred at room
temperature for 3 h. On completion, it was quenched by adding
dil HCl (5%, 10 mL), extracted with ethyl acetate, washed with
water and dried over anhydrous sodium sulfate. The organic layer
was dried in vacuo to get a residue. It was recrystallised with
CHCl3–Pet ether (1:4) to get a gummy solid.
5.2.6. General procedure of synthesis of 21 and 22
The procedure followed is same as described at Section 5.2.1.
5.2.6.1. Diethyl-4,40-dimethoxy-8,30-neolign-7,70-dien-9,90-dio-
Compound 27: Yield 87%, gummy, 1H NMR (CDCl3, 300 MHz): d
3.54 (s, 3H, OCH3), 3.61 (s, 3H, OCH3), 3.80 (s, 6H, 2ꢁ OCH3), 4.17–
4.23 (m, 2H, OCH2), 4.32 (s, 2H, OCH2), 6.17–6.87 (m, 8H, aromatic
and olefinic). 13C NMR (CDCl3, 75 MHz): d 55.49, 56.00, 56.08,
56.23, 62.19, 62.34, 108.4, 111.08, 111.42, 111.75, 112.86, 118.74,
119.82, 122.52, 127.77, 130.44, 135.63, 147.55, 148.42, 148.88,
152.94, 153.19. Electrospray mass (MeOH): 409 [M+Na]+, 425
[M+K]+. IR (cmꢀ1): 3426, 2926, 1654, 1636, 1628, 1460, 1438,
1144, 1024.
nate (21).
Yield 91%, gummy, 1H NMR (CDCl3, 300 MHz): d
1.26 (t, 6H, 2ꢁ CH3), 3.78 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.22
(m, 4H, 2ꢁ OCH2), 6.21–6.26 (d, 1H, @CH, J = 15.9 Hz), 6.69 (m,
2H, CH, aromatic, J = 8.7 Hz), 7.02 (m, 4H, 4ꢁ CH, aromatic), 7.52
(s, 1H, CH, aromatic), 7.56–7.61 (m, 1H, CH@, J = 16.5 Hz), 7.81 (s,
1H, CH@). 13C NMR (CDCl3, 75 MHz): d 14.71, 14.71, 55.61, 56.18,
60.71, 61.27, 111.70, 114.20, 114.20, 116.39, 126.74, 127.64,
127.87, 130.13, 131.29, 132.34, 132.34, 140.84, 144.49, 159.79,
160.71, 160.71, 167.68, 168.14. Electrospray mass: 411.3 [M+H]+,
433.2 [M+Na]+, 449.2 [M+K]+, 843.5 [2M+Na]+, 1253.6 [3M+Na]+;
IR (cmꢀ1): 2978, 1706, 1602, 1510, 1254, 1174.
5.2.9. 4,40,5,50-Tetramethoxy-8,30-neolign-7,70-dien-9,90-dioic
acid (25)
Diester 22 (42 mg, 0.09 mmol) was stirred in 5% aqueous meth-
anolic (1:4) KOH (10 mL). The reaction mixture was heated to 50 °C
for 2 h. Reaction was neutralized with dil HCl (5%, 10 mL),
extracted with ethyl acetate, washed with water and dried over
anhydrous sodium sulfate. Solvent was evaporated to dryness
and the residue thus obtained was crystallized from methanol to
get 25.
5.2.6.2.
9,90-dionate (22).
Diethyl-4,40,5,50-tetramethoxy-8,30-neolign-7,70-dien-
Yield 87%, gummy, 1H NMR (CDCl3, 300
MHz): d 1.28 (t, 6H, 2ꢁ CH3), 3.45 (s, 3H, OCH3), 3.74 (s, 3H, OCH3),
3.83 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.26 (q, 4H, 2ꢁ OCH2), 6.26–
6.32 (d, 1H, @CH, olefinic, J = 15.9 Hz), 6.53 (s, 1H, CH, aromatic),
6.72 (d, 1H, CH, aromatic, J = 8.1 Hz), 6.84 (d, 1H, CH, aromatic,
J = 8.4 Hz), 6.94 (s, 1H, CH, aromatic), 7.09 (s, 1H, aromatic), 7.54–
Yield 86%, oil, 1H NMR (CDCl3, 300 MHz): d 3.47 (s, 3H, OCH3),
3.84 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.28–
6.33 (d, 1H, @CH, olefinic, J = 15.9 Hz), 6.56 (s, 1H, CH, aromatic),
6.74 (d, 1H, CH, aromatic, J = 7.4 Hz), 6.86 (d, 1H, CH, aromatic,
J = 8.1 Hz), 6.99 (s, 1H, CH, aromatic), 7.13 (s, 1H, CH, aromatic),
7.64–7.69 (d, 1H, CH@, J = 15.9 Hz), 7.92 (s, 1H, CH, olefinic). 13C
NMR (CDCl3, 75 MHz): 55.56, 56.23, 56.33, 61.14, 62.00, 111.09,
111.20, 111.69, 117.05, 119.01, 125.20, 127.24, 130.78, 148.80,
150.03, 172.02, 173.01. Electrospray mass (MeOH): 437.2
[M+Na]+, 453.1 [M+K]+, Negative mode: 413.3 [MꢀH]ꢀ; IR
(cmꢀ1): 3448, 2934, 1686, 1514, 1263, 1147.
7.59 (d, 1H, olefinic, CH@, J = 15.9 Hz), 7.80 (s, 1H, CH, olefinic); 13
C
NMR (CDCl3, 75 MHz): d 14.73, 14.73, 55.51, 56.18, 56.30, 60.89,
61.01, 61.50, 111.05, 111.31, 112.47, 117.92, 124.08, 125.64,
126.65, 127.63, 131.09, 131.86, 141.06, 144.30, 148.71, 149.59,
150.52, 153.62, 167.31, 168. Electrospray mass (MeOH): 493.2
[M+Na]+, 509.2 [M+K]+; IR (cmꢀ1): 2937, 1706, 1635, 1599, 1513.
5.2.7. General procedure of synthesis of amides 23 and 26
5.2.7.1. 4,40-Dihydroxy-5,50-dimethoxy-8,30-neolign-7,70-dien-
9,90-diamide (23).
Neolignan 11 (110 mg, 0.25 mmol) was
stirred in 30% aqueous ammonia (2 mL) with cooling at 0–10 °C
for an hour. The reaction mixture was stirred overnight (16–
18 h) at room temperature. The reaction mixture was quenched
with dil HCl (5%, 10 mL) and extracted with ethyl acetate, washed
with water and dried over anhydrous sodium sulfate. The organic
layer was dried in vacuo to get a residue. The residue was purified
through column chromatography over silica gel (60–120 mesh).
The desired amide 23 was obtained as viscous liquid.
5.3. Bioevaluation
5.3.1. Cell culture
Human breast cancer cell lines, MCF-7 and MDA-MB-231 were
originally obtained from American type of cell culture collection
(ATCC), USA and stocks are maintained in laboratory. HEK-293
cells were obtained from institutional cell repository of animal tis-
sue culture facility (CSIR-CDRI). Cells were grown in tissue culture
flasks in DMEM (Dulbecco modified Eagle medium, Sigma) supple-
mented with 10% fetal bovine serum with 1ꢁ stabilized antibiotic–
antimycotic solution (Sigma) in a CO2 incubator (Sanyo, Japan) at
37 °C with 5% CO2 and 90% relative humidity. The cells at subcon-
fluent stage were harvested with 1ꢁ porcine pancreatic trypsin
(Sigma) and seeded in required density in tissue culture plates
for assay.
Compound 23: Yield 58%, oil, 1H NMR (CDCl3, 300 MHz): d 3.47
(s, 3H, OCH3), 3.89 (s, 3H, OCH3), 5.80 (bs, NH), 6.02 (bs, NH), 6.23–
6.28 (d, 1H, @CH, olefinic, J = 15.9 Hz), 6.56 (s, 1H, CH, aromatic),
6.74–6.76 (m, 2H, CH, aromatic), 6.96 (s, 1H, CH, aromatic), 7.04
(s, 1H, CH, aromatic), 7.54–7.59 (d, 1H, CH@, olefinic, J = 15.9 Hz),
7.83 (s, 1H, olefinic, @CH). Electrospray mass (MeOH): 385
[M+H]+, 397 [M+Na]+, Negative mode: 383 [MꢀH]ꢀ; IR: 3369,
2924, 1702, 1655, 1595, 1494, 1271.
5.2.7.2. 4,40,5,50-Tetramethoxy-8,30-neolign-7,70-dien-9,90-dia-
mide (26).
5.3.2. In vitro cell inhibition assay
Yield 62%, oil, 1H NMR (CDCl3, 300 MHz): d 3.38
The cell inhibiting activity of the compounds of the series were
determined using MTT assay as described earlier.38 In brief, cells
were seeded @ 1 ꢁ 104 cells/well in each well of 96-well microcul-
(s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 6.19–6.33 (bp, 4H, 2ꢁ NH2), 6.46 (s, 1H), 6.63–6.65 (d,
1H), 6.74–6.77 (d, 1H), 6.87–6.89 (s, 2H), 7.12–7.18 (d, 1H), 7.58–
7.60 (d, 1H), 7.73 (s, 1H, aromatic). Electrospray mass (MeOH):
413 [M+H]+, 451 [M+K]+; IR (cmꢀ1): 3425, 3400, 2927, 1703,
1686, 1595, 1582, 1512, 1253, 1147.
ture plates in 200 lL DMEM (Sigma), supplemented with 10% FBS
and 1ꢁ stabilized antibiotic–antimycotic solution (Sigma) and
incubated for 24 h at 37 °C in a CO2 incubator. Compounds, diluted
to the desired concentrations in culture medium DMEM without
phenol red, supplemented with 0.5% FBS, were added to the wells
5.2.8. 4,40,5,50-Tetramethoxy-8,30-neolign-7,70-dien-9,90-diol
(27)
Diester 22 (100 mg, 0.21 mmol) was stirred in 10 mL THF at
0 °C. To this, lithium borohydride (120 mg, 5.71 mmol) was added
with respective vehicle control. After 24 h of incubation, 20 lL of
5 mg/mL MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazo-
lium bromide) (Sigma) was added to each well and the plates were
further incubated for 3 h. Then the supernatant from each well was

B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
1353
carefully removed without disturbing the formazan crystals. The
formazan crystals were dissolved in 200 L of dimethyl sulfoxide
These ADMET descriptors were calculated and checked for com-
pliance with their standard ranges. The octanol–water partition
coefficient (LogP) has been implicated in LogBB penetration and
permeability studies. The descriptor values of 90% orally active
compounds follows Lipinski’s rule. This descriptor has been
shown to correlate well with passive molecular transport through
membranes. Calculations of other important ADME properties of
10 and 19 were performed through QikProp, version 3.2, Schrö-
dinger, LLC, USA (2010). Compounds 10 and 19 were also
screened for LogS, Caco-2 and number of primary metabolites
(Schrödinger, USA, 2010).
l
(DMSO) using plate shaker (Biosan) and absorbance at 570 nm
wavelength was recorded in a microplate reader (Microquant;
BioTek). The cell inhibition of analogues 10 and 19 was also evalu-
ated at 48 and 72 h incubation. Analogues 10 and 19 were also
evaluated for toxicity against human embryonic kidney cells, that
is, HEK-293. Podophyllotoxin (PDT) and tamoxifen (TAM) were
used as positive controls.
5.3.3. Cell cycle analysis
Cell cycle analysis using PI-staining of cells was carried out as
per earlier reported method.39 MCF-7 and MDAMB-231 cells were
5.3.6. In vivo acute oral toxicity
plated and treated with 10
l
M of compound 10 for 24 h, and har-
In view of potent anti-cancer activity of lignan 10 in in vitro
model, acute oral toxicity of the same was carried out in Swiss al-
bino mice. Experiment was conducted in accordance with the
Organization for Economic Co-operation and Development (OECD)
test guideline No. 423 (1987).
vested. For flowcytometry analysis, collected cells were washed
with cold PBS, re-suspended at the rate of 2 ꢁ 106 cells/ml, fixed
in absolute ethanol, treated with RNase A (10 mg/mL), and then
stained with propidium iodide (50 lg/mL; Sigma, St. Louis, MO,
USA) for 30 min at room temperature. The DNA content of the cells
was measured using a FACS Calibur flow cytometer (Becton–Dick-
inson, San Jose, CA, USA) and CellQuest software.
For this study 30 mice (15 male and 15 female) were taken and
divided into four groups comprising 3 male and 3 female mice in
each group weighing between 20 and 25 g. The animals were
maintained at 22 5 °C with humidity control and also on an auto-
matic dark and light cycle of 12 h. The animals were fed with the
standard mice feed and provided ad libitum drinking water. Mice
of group 1 were kept as control and animals of groups 2, 3, 4 and
5 were kept as experimental. The animals were acclimatized for
7 days in the experimental environment prior to the actual exper-
imentation. Neolignan 10 was solubilized in dimethyl sulfoxide
and then suspended in caboxymethyl cellulose (0.7%) and was gi-
ven at 5, 50, 300 and 1000 mg/kg body weight to animals of groups
2, 3, 4 and 5, respectively, once orally. Control animals received
only vehicle.
The animals were checked for mortality and any signs of ill
health at hourly interval on the day of administration of drug
and there after a daily general case side clinical examination was
carried out including changes in skin, mucous membrane, eyes,
occurrence of secretion and excretion and also responses like lach-
rymation, pilo-erection, respiratory patterns etc. Also changes in
gait, posture and response to handling were also recorded.42 In
addition to observational study, body weights were recorded and
blood and serum samples were collected from all the animals on
7th day of the experiment in acute oral toxicity. The samples were
analysed for total RBC, WBC, differential leucocytes count, haemo-
globin percentage and biochemical parameters like ALKP, SGPT,
SGOT, total cholesterol, triglycerides, creatinine, bilirubin, serum
protein, tissue protein, malonaldehyde and reduced GSH activity.
The animals were then sacrificed and were necropsed for any gross
pathological changes. Weights of vital organs like liver, heart, kid-
ney, etc. were recorded.43
5.3.4. Tubulin polymerisation assay
Tubulin polymerization experiment was done as per reported
method using ‘assay kit’ from Cytoskeleton, USA.40 In brief, tubulin
protein (3 mg/mL) in tubulin polymerization buffer (80 mM PIPES,
pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP and 15% glycerol)
was placed in pre-warmed 96-well microtiter plates at 37 °C in
the presence of test compounds with variable concentrations. All
samples were mixed well and polymerization was monitored
kinetically at 340 nm every min for 1 h using Spectramax plate
reader. Podophyllotoxin (PDT) was used as standard inhibitor of
tubulin polymerisation, while paclitaxel (taxol) was used as stan-
dard stabilizer of tubulin polymerisation. DMSO was used as neg-
ative control.
5.3.5. In silico studies
5.3.5.1. Molecular docking studies.
To find the possible bio-
active conformations of 10 and 19, the Sybyl X 1.3 interfaced with
Surflex–Dock module was used for molecular docking. Program
automatically docks ligand into binding pocket of a target protein
by using protomol-based algorithm and empirically produced
scoring function. The X-ray crystallographic structures of tubulin
complex with ligand (taxol) [PDB: 1TUB]41 was taken from the
protein data bank (PDB) and modified for docking calculations.
Co-crystallized ligand was removed from the structure, water
molecules were removed, H-atoms were added and side chains
were fixed during protein preparation. Protein structure minimi-
zation was performed by applying Tripos force field and partial
atomic charges were calculated by Gasteiger–Huckel method. In
reasonable binding pocket, all the compounds were docked into
the binding pocket and 20 possible active docking conformations
with different scores were obtained for each compound. During
the docking process, all of the other parameters were assigned
their default values.31,32
5.3.7. Statistical analysis
Data were expressed as means. Statistical analysis was done by
ANOVA and Newman Keul’s test. A value of P values <0.05 were
considered a statistically significant difference.
Acknowledgements
5.3.5.2. Screening for pharmacokinetic properties.
As we
Authors are thankful to Directors (CSIR-CIMAP and CSIR-CDRI)
for constant support and co-operation. Financial support from
BSC203 (SIP) is duly acknowledged.
know that most of drugs in discovery process fail to cross clinical
trials because of poor pharmacokinetic profile (PK). The proper-
ties of PK such as absorption, distribution, metabolism, excretion,
and toxicity (ADMET) are important descriptors for human ther-
apeutic use of any compound. The compound distribution in hu-
man depends on factors, for example, blood–brain barrier
(LogBB), permeability (apparent Caco-2 and MDCK permeability,
LogKp for skin permeability), the volume of distribution and plas-
ma protein binding refer by LogKhsa for serum protein binding.31
Supplementary data
Supplementary data (1H NMR, 13C NMR, and Mass of com-
pounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2013.12.067.

1354
B. Sathish Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1342–1354
22. Haq, A.; Malik, A.; Khan, M. T. H.; Haq, A.; Khan, S. B.; Ahmad, A.; Choudhary, M.
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