Phenolate based metallomacrocyclic xanthate complexes of CoII/CuII and their exclusive deployment in [2 : 2] binuclear N,O-Schiff base macrocycle formation and in vitro anticancer studies

Article abstract of DOI:10.1039/c5dt03407h

Potassium salts of phenolate based polydentate xanthate ligands 4,4′-bis(2-dithiocarbonatobenzylideneamino)diphenyl ether (K₂xan¹) and 4,4′-bis(2-dithiocarbonatonaphthylmethylideneamino)diphenyl ether (K₂xan²) h

Full text of DOI:10.1039/c5dt03407h

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Phenolate Based Metallomacrocyclic Xanthate Complexes of CoII/ CuII and Their
Exclusive Deployment in [2:2] Binuclear N, OꢀSchiff Base Macrocycle Formation and in
vitro Anticancer Studies†
Vinay K. Singh,*a Rahul Kadu,a Hetal Roy,b Pallepogu Raghavaiahc and Shaikh M. Mobind
† Dedicated to Professor Pradeep Mathur on the occasion of his 60th Birthday
aDepartment of Chemistry, Faculty of Science, The M. S. University of Baroda, Vadodaraꢀ
390 002, India
bDepartment of Zoology, Faculty of Science, The M. S. University of Baroda, Vadodaraꢀ390
002, India
cDepartment of Chemistry, Dr. Harisingh Gour University, Sagarꢀ470003, India
dDepartment of Chemistry, Indian Institute of Technology Indoreꢀ452 017, India
*Email: vinay.singhꢀchem@msu.ac.in
Table of contents entry
Crystallographic and in vitro anticancer studies of phenolate based CoII/ CuII xanthate
metallomacrocycles and their N, Oꢀ Schiff base derivatives.
Graphics:
___________________________________________________________________________
Abstract: Potassium salts of phenolate based polydentate xanthate ligands 4,4’ꢀbis(2ꢀ
dithiocarbonatobenzylideneamino)diphenyl
ether
(K2xan1)
and
4,4’ꢀbis(2ꢀ
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dithiocarbonatonaphthylmethylideneamino)diphenyl ether (K2xan2) have been synthesized
and characterized, prior to use. Reaction of K2xan1 or K2xan2 with M(OAc)2 in Et3N affords
access to a rare series of binuclear metallomacrocyclic xanthate complexes of the type [M2ꢀ
µ2ꢀbisꢀ(κ2S,Sꢀxan1/xan2)] (1ꢀ4) which quickly forms [2:2] binuclear N, Oꢀ bidentate Schiff
base macrocyclic complexes of the type [M2ꢀµ2ꢀbisꢀ(κ2N,OꢀL1/ L2)] (L1 = 4,4’ꢀbis(2ꢀ
hydroxybenzylideneamino)diphenyl ether, L2 = 4,4’ꢀbis(2ꢀhydroxynaphthylmethylideneꢀ
amino)diphenyl ether) 5ꢀ8 via evolution of CS2 in solution. Compounds were characterized
by microanalysis, relevant spectroscopy (FTꢀIR, UVꢀvisible), mass spectrometry (ESIꢀMS),
powder and single crystal XRD techniques. In vitro anticancer activity of all the compounds
was evaluated against HEP 3B (Hepatoma) and IMR 32 (Neuroblastoma) by the MTT assay.
Remarkably, the binuclear copper(II) xanthate complexes were found extremely active
against both the cell lines (IC50: 8.1±0.8 ꢁM (3), 8.8±1.7 ꢁM (4) against HEP 3B and 1.9±0.3
ꢁM (3) and 7.3±0.6 ꢁM (4) against IMR 32) and this projects them in the vein of good
candidates as potent antitumor agent and the IC50 values confirm their better potency than the
reference drug cisplatin. Flowꢀcytometric density plot illustrates the induction of apoptosis in
HEP 3B and IMR 32 cells after the treatment with K2xan1, 1, 3, 6 and 7.
Keywords: Xanthate, Schiff base, metallomacrocycle, XRD, anticancer activity.
___________________________________________________________________________
Introduction
The interest in the chemistry of discrete metallomacrocyclic structures with diverse
cavity size and shape1 has been continued due to their potential applications in catalysis,2
hostꢀguest chemistry,3 molecular and ion sensing,4 separation, transport and storage,5 in drug
delivery, twoꢀphase transport and biosensing.6 In particular, transition metal complexes offer
additional opportunities for development of new therapeutic agents not accessible to organic
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compounds,7 by means of varied coordination numbers, geometries, redox states,
thermodynamicꢀkinetic characteristics, and intrinsic properties of both metal ions and ligands.
The platinum based anticancer drug, cisplatin and its analogues have already proved to be
indispensable in cancer chemotherapy. The efforts in the evaluation of anticancer drugs have
been shifted to nonꢀplatinum metalꢀbased agents that have improved pharmacological
properties and aimed at different targets,8 to avoid doseꢀlimiting side effects9 and due to the
emergence of drug resistance associated with platinum chemotherapeutics.10 In this context,
copper and cobalt complexes have shown encouraging results, 11, 12 mainly due to their redox
activity and accessibility for transmetallation.
A careful literature search revealed that a wide range of natural product bearing
macrocyclic motif and their synthetic derivatives have long been clinically used as they offer
structural preꢀorganization with sufficient flexibility for better binding with the target
molecules and exhibit high degree of potency as well as selectivity.13 However, a class of
inorganic compounds “metallomacrocyclic structures” have scarcely been investigated14,15
from a medical perspective especially for antitumor activity. Very recently, we have
developed a series of binuclear diphenyltinIVdithiocabamate macrocyclic scaffolds
i
s
n
[(Ph2SnIV)2ꢀµ2ꢀbisꢀ{(κ2S,SꢀS2CN(R)CH2CONHC6H4)2O}] {R = Pr (1), Bu (2), Bu (3), Cy
(4), 2ꢀfurfuryl (5) or benzyl (6) that have shown exceptional anticancer activity against
Neuroblastoma and Hepatoma human cancer cell lines.15 Sulfur being an important
constituent of biomolecules, plays a crucial role in transporting and addressing the molecule
to the targets as well as in the protection of the pharmacophore against untimely exchanges
with biomolecules.9,16 Sulfurꢀrich compounds exhibit good DNA/protein binding/cleaving
and catalytic activity toward glutathione.17 Besides, Schiff bases have also been used in the
preparation of many potential drugs that are known to possess a broad spectrum of biological
activities.18 Literature suggests that when administered as their metal complexes, the
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anticancer activity of these complexes is enhanced in comparison to the free Schiff base
ligands.19 Moreover, metal complexes derived from salicylaldehyde is known to cleave
DNA.20
Tanaka et al.,21 have reported primary investigations on inꢀvivo mutagenicity of 4,4’ꢀ
diaminodiphenyl ether and its Nꢀacetyl derivative towards Salmonella typhimurium TA98 and
TA100 in 1985. However there are very few reports15,22 on further investigations of
biological properties of 4,4’ꢀdiaminodiphenyl ether and its derivatives. In this contribution,
we have synthesized potassium salts of 4,4’ꢀbis(2ꢀdithiocarbonatobenzylideneamino)diphenyl
ether (K2xan1) and 4,4’ꢀbis(2ꢀdithiocarbonatonaphthylmethylideneamino)diphenyl ether
(K2xan2). Phenolate based xanthate K2xan1 and K2xan2 ligands have been used to derive
binuclear metallomacrocyclic CoII/ CuII xanthate complexes 1ꢀ4. Interestingly 1ꢀ4 undergo a
facile transformation into their corresponding (2:2) binuclear metallomacrocyclic N,OꢀSchiff
base complexes 5ꢀ8, selectively. In a conventional approach for the synthesis of (2:2)
binuclear metallomacrocyclic N,OꢀSchiff base complexes, lack of selectivity appears to be
the main barrier. To the best of our knowledge, this is the first report on binuclear
metallomacrocyclic xanthate complexes derived from phenolate based polydentate xanthate
ligands and their selective transformations into (2:2) binuclear N,OꢀSchiff base macrocycles.
All the compounds were investigated for their potential in vitro cytotoxic and apoptosis
inducing properties against HEP 3B and IMR 32 human cancer cells. With anticipation, the
incorporation of the xanthate moiety in the molecular framework of the ensuing complexes
would enhance the possibility of stacking and intercalation through a number of noncovalent
interactions with DNA/proteins and may lead to the superior anticancer activity.
Results and discussion
Synthesis and characterization
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Ligand precursors 4,4’ꢀbis(2ꢀhydroxybenzylideneamino) ether (L1) and 4,4’ꢀbis(2ꢀ
hydroxynaphthylmethylideneamino)diphenyl ether (L2) have been synthesized by using a
literature procedure23 (Scheme S1). The potassium salts of polydentate xanthate ligands
(K2xan1 or K2xan2) were synthesized by the reaction of these phenolates L1 or L2 with CS2
in the aqueous KOH solution shown in Scheme 1. Though numerous reports support the easy
accessibility of xanthate ligands derived from alcohols and their use in synthetic organic,24
coordination25 chemistry, the use of phenolates based xanthates in the development of
metallomacrocyclic coordination compounds have not yet been investigated. The lack of
stability of transition metal xanthate complexes,31 primarily limit their use in coordination
chemistry. However, we could use phenolate based K2xan1 and K2xan2 ligands successfully
to derive binuclear metallomacrocyclic CoII/ CuII xanthate complexes 1ꢀ4 (Scheme 2) from a
metathetical reaction of K2xan1 or K2xan2 with corresponding metal acetates in Et3N.
O
O
N
O
N
O
H2O, KOH
CS2
N
N
HO
OH
SK
KS
O
S
S
N
O
N
N
O
N
(L1)
(L2)
:
(K2xan1) OR
(K2xan2)
N
OR
:
N
O
O
O
Scheme 1. General scheme for the synthesis of K2xan1 and K2xan2.
O
S
S
S
S
M
O
O
N
N
N
O
N
O
K2xan1
OR
K2xan2
CH2Cl2
ꢀCS2
M(OAc)2
Et3N
M
M
O
O
O
N
O
N
S
S
S
S
M
O
O
N
N
O
M = Co(II); xan1 (1), xan2 (2)
M = Cu(II); xan1 (3), xan2 (4)
M = Co(II); L1 (5), L2 (6)
M = Cu(II); L1 (7), L2 (8)
Scheme 2. General synthetic methodology used for binuclear CoII/CuII xanthate
metallomacrocycles and their respective binuclear N,Oꢀbidentate Schiffꢀbase analogues.
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These complexes 1ꢀ4 are stable in the solid state but are found to be unstable,
especially in chlorinated solvents. The instability associated with the binuclear
metallomacrocyclic CoII/ CuII xanthate complexes 1ꢀ4 helped us to use them further in
selective transformation reactions leading to the corresponding (2:2) binuclear
metallomacrocyclic N,OꢀSchiff base complexes 5ꢀ8.
Literature suggests that a direct approach to obtain (2:2) binuclear Schiff base
metallamacrocycles suffer the disadvantage of giving a mixture of products viz. 1:1, 2:2, 3:3,
4:4 and the (1:1)∞ polymeric species which are insoluble in most organic solvents.27 Further
reports suggest that the conformational modes of the ligand, nuclearity and ultimately the
structures of the resulting complexes, greatly depend upon the selection of metal ion. For
instance, J. Zhang et. al. have isolated a dinuclear mesocate structure for CuII and a pseudo
C3ꢀsymmetrical torusꢀlike molecular structure for CoII ions with the same ligand system.28
Hence, in the development of efficient synthetic methodology for the formation of binuclear
metallomacrocyclic complexes, lack of selectivity is one of the main barriers. Thus, the
present synthetic methodology to obtain (2:2) binuclear metallomacrocyclic N, OꢀSchiff base
complexes selectively from their respective binuclear metallamacrocylic CoII/CuII xanthate
complexes is of great interest. The presence of Schiff base (ꢀN=C) moiety in close proximity
to xanthate (ꢀOCS2) moiety in 1ꢀ4, causes instability in solution, enforcing these molecules to
undergo a facile transformation into their corresponding (2:2) binuclear metallomacrocyclic
N, OꢀSchiff base complexes 5ꢀ8 selectively, via CS2 evolution which was easily detected by
GC. Earlier, CS2 evolution from 1,1ꢀdithiolato complexes in chlorinated solvents has been
observed.29 Moreover, the isolation of binuclear metallamacrocylic CoII xanthate complexes
became possible due to the poor electron density at CoII centers, apparently revealed by DFT
study (Table S13), thereby preventing them from ready oxidation, unlike earlier reports.30
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The newly synthesized compounds were characterized by elemental, ESꢀMS,
thermogravimetric analysis and relevant spectroscopic techniques. The microanalysis and
spectroscopic data were sufficient to unequivocally assign the structure of potassium salts of
xanthate lignads K2xan1 and K2xan2. The IR spectra of these ligands display characteristic IR
bands due to ν(C=N), ν(CꢀO) and ν(CS2) in the region of 1619ꢀ1624, 1084ꢀ1147 and 982ꢀ
1038 cmꢀ1 respectively. In the 1H NMR spectrum, the disappearance of phenolic ꢀOH signals
and significant downfield shift of imine (ꢀN=CH) signals to the 8.97 and 9.688 ppm are
indicative of the formation of K2xan1 and K2xan2, respectively. In the 13C NMR spectra,
most characteristic signals appeared at 190.86 and 190.24 ppm due to the xanthate (–OCS2)
moiety, apart from the signals that appeared in the regions 161.83ꢀ168.69 ppm and 108ꢀ162
ppm due to the imine (ꢀN=CH) and aromatic groups respectively. The IR spectra of binuclear
xanthate complexes 1ꢀ4 exhibit most characteristic bands in the region 1041ꢀ967 cmꢀ1,
diagnostic of S,S coordination of the xanthate ligands.31 The appearance of shoulder or split
bands in the region of 1041ꢀ967 cmꢀ1, is indicative of anisobidentate coordination mode of
the xanthate moiety in 1ꢀ4. The absence of –OCS2 bands and subsequent splitting of ν(C=N)
stretching bands with significant shifting (compared to ν(C=N) bands in L1 or L2) in the IR
spectra of 5ꢀ8 suggests the formation of corresponding binuclear metallomacrocyclic N,Oꢀ
Schiff base complexes. The structural variations in K2xan1, its binuclear xanthate complex 1
and the corresponding binuclear N,OꢀSchiff base complex 5 can easily be seen in their
overlapped IR spectra. (Supporting Information)
Table 1 UVꢀvisible and fluorescence bands for L1, L2, K2xan1, K2xan2 and 1ꢀ8.
Entry UVꢀVisible data (10−4 M DMSO)
Fluorescence data (10−4 M DMSO)
λmax nm (ε, L mol−1 cm−1)
λem nm (Intensity)
λex (nm)
L1
L2
320 (15633) π→π*, 364 (13555) n→π*,
395 CT
319 (26530) π→π*, 365 (21589) n→π*,
480 (17534) CT
432 (16.0497),
513 (6.1781)
519 (25.7266)
360
318
366
497
K2xan1 319 (26530) π→π*, 367 (21589) n→π*,
444 (7920) CT
520 (9.5477)
532 (57.9323)
K2xan2 321 (28560) π→π*, 366 (24090) n→π*,
490 (19778) CT
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1
2
3
318 (32593) π→π*, 366 (27634) n→π*,
405 (15852) CT, 531 dꢀd
469 (11.1489),
519(12.6580)
634 (21.6423)
364
316 (53760) π→π*, 367 (39718) n→π*,
433 (34477) CT, 556 dꢀd
314 (4903) π→π*, 348 (4032) n→π*,
380 (4534) n→π*, 452 (4156) CT,
683 dꢀd
317
314
628(32.09)
4
5
6
7
317 (6527) π→π*, 366 (5346) n→π*,
461(4959) CT, 674 dꢀd
314 (35817) π→π*, 349 (29221) n→π*,
390 CT, 538 dꢀd
317 (36314) π→π*, 365 (30300) n→π*,
458 (28921) CT, 565 dꢀd
318 (5726) π→π*, 354 (4696) n→π*,
464(4419) CT, 677 dꢀd
634 (14.013)
410 (178.54)
317
314
317
317
545 (21.5474)
634 (40.7225)
634(12.84)
8
317 (5411) π→π*, 352 (4252) n→π*,
423(4379) , 469 (4342) CT, 685 dꢀd
No fluorescence
317, 352
Metallomacrocyclic xanthate complex 2 and metallomacrocyclic N,Oꢀ Schiff base
complexes 6ꢀ7 clearly displayed (M+H) molecular ion peaks along with the other
fragmentation peaks. (Supporting Information) The instability of 1ꢀ4 in solution limited our
attempts to obtain single crystals suitable for single crystal XRD study. The powder Xꢀray
diffraction study was performed on representative compounds (K2xan1, 1, 3, 5 and 7) to
understand features like crystal lattice and lattice parameters. The powder XRD data have
been indexed and refined applying least square refinement by commonly used32 program
‘POWDERX’33 (Supporting Information). Notably, the results of PXRD analysis for xanthate
ligand K2xan1 and its CoII/CuII complexes 1/3 as well as their corresponding Schiff base
complexes 5/7 suggest their different packing patterns in the solid state. The indexing of
powder XRD data for 5 and 7 gave the lattice system similar to that obtained from single
crystal data with the differences in cell parameters probably arising due to the involvement of
solvent of crystallization.
Further, geometry optimization for representative compounds L1, K2xan1, binuclear
xanthate macrocycles 1, 3 and their corresponding binuclear Schiff base macrocyclic
derivatives 5, 7 was performed using DFT study. (Supporting Information) Optimized
geometry of K2xan1 gives the required orientation of xanthate moieties essential for the
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formation of metallomacrocyclic xanthate complexes 1ꢀ4. The calculated structural
parameters for complex 5 and 7 are comparable to their structural parameters obtained from
Xꢀray crystallography (Supporting Information). The calculated HOMOꢀLUMO gaps are
comparable to the experimental UVꢀvisible absorption data (Table S6) and further validate
the computed data as well as optimized structures.
The UV–visible absorption (10˗4 M DMSO solution) and emission (10˗4 M DMSO
solution) properties of L1ꢀL2, K2xan1ꢀK2xan2 and 1–8 were investigated at room temperature
(Table 1 and Figure S21ꢀS22). The electronic spectra of 1ꢀ8 clearly revealed the structural
changes in xanthate and Schiff base complexes. The shorter absorption band ~315 nm is
assigned to π→π* (phenyl) transitions and the longer absorption band ~360 nm is assigned to
n→π* (imine) transitions whereas the absorption bands appeared in 400ꢀ490 nm regions are
attributable to the intraligand charge transfer transitions in respective compounds.
Evidently, imine n→π* transition bands of L1, L2 remain unaffected in corresponding
xanthate ligands K2xan1, K2xan2 and binuclear xanthate complexes 1ꢀ4. However, a
distinguishable blue shift of this transition band by 10ꢀ15 nm in the binuclear N,OꢀSchiff base
complexes 5ꢀ8 was observed which clearly underlines the participation of imine moiety in the
complex formation. The fluorescence spectra of L1 and K2xan1 display weak emission bands
at 432, 513 and 520 nm upon excitation at λex 360 and 318 nm, respectively. However, L2 and
K2xan2 display strong emission bands at 519 and 532 nm upon excitation at λex 318 and 497
nm respectively with a significant Stokes shift. Among 1ꢀ8, complex 5 fluoresced with
maximum emission at 410 nm upon excitation at λex 314 with a concomitant Stokes shift of ≈
138 nm. Expectedly, all the CuII complexes display no significant fluorescence properties, as
CuII is a well known fluorescence quencher.34
Crystallographic Study
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Complexes 5ꢀ8 have crystallized in orthorhombic Pbca (5), monoclinic P21/c (6),
orthorhombic Pbca (7.DMSO), monoclinic C2/c (7.2CH2Cl2) and monoclinic P21/c
(8.CHCl3.8H2O) centrosymmetric space groups. The Xꢀray crystal structures show one
complete molecule in their asymmetric units, except 7.2CH2Cl2 and 8.CHCl3.8H2O which
contains half and two complete molecules in their asymmetric units, respectively. All the five
compounds contain voids which are occupied by disordered solvent molecules in their crystal
lattice, compound 5 and compound 6 have voids space which can be accounted for four water
molecules each per unit cell respectively. A satisfactory disorder model for the solvent was
not found in both of these cases, therefore the OLEX2 Solvent Mask routine (similar to
PLATON/SQUEEZE) was used to mask out the disordered density. However, each
asymmetric units of Compound 7.DMSO has one DMSO molecule, compound 7.2CH2Cl2 has
one CH2Cl2 molecule and compound 8.CHCl3.8H2O has one CHCl3 molecule and eight water
molecules respectively. The crystal structure of compound 8.CHCl3.8H2O is obtained from
poor quality data ought to weekly diffracting crystals and our efforts to get best diffraction
quality crystals have been failed and we report this structure only for connectivity purpose.
The reported structure is refined isotopically except Cu and its four connecting atoms and
chlorine atoms of CHCl3 solvent. The ORTEP view at 40 % probability for these complexes
is shown in Fig. 1aꢀe. Details about data collection, refinement, and structure solution are
recorded in Table S2, and the selected geometrical parameters are provided in Table S3.
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(a) 5
(b) 6
(c) 7. DMSO
(d) 7. 2CH2Cl2
(e) 8. CHCl3.9H2O
Fig. 1 ORTEP diagram with atoms labelled showing 40% probability ellipsoids for (a) 5, (b)
6, (c) 7.DMSO, (d) 7.2CH2Cl2 and (e) 8.CHCl3.8H2O. Hydrogens and disordered solvent
molecules are removed for clarity.
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In 5ꢀ8, each of the CoII/ CuII centers are bonded to the available coordination sites of
L1 and L2 through two imine nitrogen and two phenolic oxygen atoms. While the angles
between the two O−M−N planes deviate largely from coplanarity, the O−M−O, and N−M−N
angles differ significantly from 90° in all the molecules. The geometry around the metal ions
in these complexes is thus essentially distorted tetrahedral. It appears that M–O and M–N
bond lengths around one metal center in the binuclear CoII complexes 5, 6 is comparable with
the similar distances around other metal center whereas these distances deviate significantly
in the binuclear CuII complex 7 (Table S3). It is noteworthy that the Co–O (1.884ꢀ1.908 Å)
and CoꢀN (1.954ꢀ1.988 Å) bond distances in binuclear CoII complexes 5ꢀ6 are significantly
longer than the similar distances observed in the planar Schiff base CoII complex (1.835ꢀ
1.847 Å for Co–O and 1.861ꢀ1.864 Å for Co–N).35 However, longer Cu–O (1.888ꢀ1.976 Å)
and shorter CuꢀN (1.949ꢀ1.982 Å) distances, compared to planar Schiff base CuII complex
(1.874 Å for Cu–O and 2.009 Å for Cu–N) were observed.36 However, these distances are
indeed consistent with the similar measurements observed in analogous tetrahedral CuII
complex reported earlier.27a In all the binuclear complexes, the ethereal bond angle of one
linker appeared in the normal range23 (117.5ꢀ118.0º) whereas significant shrinking of a
similar angle (115.4ꢀ116.4º) associated with the other linker was observed. This inward
bending of the diphenyl ether linkers suggests flexibility of the coordinated ligands, essential
for the formation of macrocyclic structures. In addition, the unique coordination geometry
adopted by CoII or CuII ions and the presence of two arc shaped ligands form double helical
molecular unit of 5ꢀ8 as exemplified in Fig. 2.
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Fig. 2 General representation of double helical molecular unit formed in complexes 5ꢀ8.
Further, singleꢀcrystal Xꢀray diffraction data is useful in revealing the supramolecular
structures of 5ꢀ8 and also in gauging the crucial role of aromatic moieties present in the
molecular framework and that of solvent molecules on the association of molecules in the
solid state. The study revealed that the presence of phenyl/naphthyl rings and solvent
molecules induces electronic and conformational changes (dihedral angles) that apparently
modify the nature and number of donorꢀacceptor sites for noncovalent interactions such as
π...π, CH...π, CH...O, Cl...O and/ or O...S, leading to diverse crystal packing patterns.
Relevant discussion on significant nonꢀconventional intermolecular interactions observed in
5ꢀ8 is summarized in the supporting information.
In vitro anticancer activity
All the compounds L1, L2, K2xan1, K2xan2, 1ꢀ8 were screened against the malignant
cell lines HEP 3B and IMR 32 for their in vitro cytotoxicity by the MTT assay.37 The
cytotoxicity of these compounds were compared with the clinically used antineoplastic drug
cisplatin and lead compound 4,4’ꢀdiaminodiphenyl ether (L). The 50% inhibition
concentration (IC50) values obtained after incubation for all the compounds against HEP 3B
(6 h ) and IMR 32 (14 h) are summarized in Table 2, Fig. 3.
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Table 2 In vitro cytotoxicity MTT assay IC50 (ꢁM) for entry 1ꢀ16 against HEP 3B and IMR
32 cancer cell lines.
Entry
Compound
Antitumor activity (IC50 values)b
HEP 3B ꢁM (1mL) ±SE
IMR 32 ꢁM (1mL) ±SE
272.4±2.4
104.0±3.3
166.9±11.1
23.2±1.4
1a
2
3
4
5
6
7
8
L
415.4±2.2
137.8±13.9
36.7±5.0
17.8±2.9
79.5±6.1
52.9±1.3
54.4±0.3
8.1±0.8
L1
L2
K2xan1
K2xan2
26.2±1.3
51.8±2.9
64.4±3.8
1.8±0.3
1
2
3
9
4
8.8±1.7
7.3±0.6
10
11
12
13
14
15
16
5
6
7
69.7±3.6
43.4±4.7
19.2±2.1
14.3±1.3
290.9±4.1
42.9±4.3
74.6±1.3
99.7±6.6
50.17±2.5
13.27±1.1
28.8±1.4
343.2±17.1
19.0±2.9
8
CoII
CuII
Cisplatin
107.9±0.3
(a: IC50 value retrieved from ref22; : The data are expressed as ꢁM concentration and value
b
represents the average of three sets of independent experiments.)
Fig. 3 In vitro cytotoxicity ( IC50) for entries 1ꢀ16 against malignant HEP 3B and IMR 32
cells.
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Fig. 4 Effect of functionality changes on in vitro cytotoxicity against HEP 3B and IMR 32
cells.
The condensation of salicyladehyde with lead compound L resulted into L1 with a ≈
3 fold increase in the cytotoxicity (IC50: 137.8±13.9 ꢁM against HEP 3B and 104.0±3.3 ꢁM
against IMR 32). However, condensation of 2ꢀhydroxy naphthaldehyde with L resulted into
L2 with
>10 and a two fold increase in the cytotoxicity against HEP 3B (36.7±5.0 ꢁM) and
IMR 32 (166.9±11.0 ꢁM) cell lines, respectively. The cytotoxicity of L1 and L2 further
augmented on the formation of their respective xanthate salts and ensuing complexes,
particularly copper xanthate complexes. All binuclear xanthate complexes displayed
comparable anticancer activity, except 3 which exhibits differential activity against both the
cell lines. Interestingly, binuclear CuIIxanthate complexes were found extremely active
against both the cell lines (IC50: 8.1±0.8 ꢁM (3), 8.8±1.7 ꢁM (4) against HEP 3B and 1.9±0.3
ꢁM (3) and 7.3±0.6 ꢁM (4) against IMR 32) and this projects them as good candidates for
being employed as potent antitumor agents. Unlike binuclear xanthate analogues, binuclear
N, OꢀSchiff base complexes could not preserve the anticancer activity. Binuclear CuII N, Oꢀ
Schiff base complexes 7 and 8 exhibit 2ꢀ7 fold better potency than that of cobalt analogues 5ꢀ
6. The overall data suggests that K2xan1, K2xan2, and complex 1ꢀ8 are more cytotoxic
against both the cell lines than the clinically used antineoplastic drug cisplatin (Table 2). In
general, all the complexes bearing copper center are more potent compared to their cobalt
bearing congeners probably due to the predominant favouring of the CuII ion in biological
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systems than that of CoII. The probable mode of action leading to cell death at molecular
level can be explained on the basis of several previously observed effects of isostructural
dithiocarbamate complexes and complexes derived from N, Oꢀ donor ligands (Supporting
Information). Among the investigated compounds, L1, L2, K2xan1 and K2xan2 can be
classified as moderate cytotoxic agents, binuclear N,OꢀSchiff base analogues as good
whereas binuclear xanthate complexes can be excellent cytotoxic agents; especially 3ꢀ4 with
optimum cytotoxic ability (Fig. 4) against both the cell lines.
In analogy with the recent investigations,38 we have calculated the stereoꢀelectronic
parameters such as highest occupied molecular orbital (HOMO), lowest unoccupied
molecular orbital (LUMO) energies, dipole moment and charges on the N/O/S/M atoms using
density functional theory (DFT). Calculated stereoelectronic parameters and experimental
IC50 values against HEP 3B and IMR 32 cells for model compounds are summarized in Table
S13. Among the model complexes 1, 3 and 5, 7 bearing similar ligand framework, complexes
3 and 7 with lower HOMO energy and lower dipole moment (greater lipophilicity)
(Supporting Information) exhibit higher cytotoxicity, suggestive of higher bioavailability at
the site of action and reactivity in the biological conditions. Notably, complex 3 bearing
negative charge on copper and positive charges on sulfur atoms exhibits higher reactivity
towards biological receptors, which could underline the extraordinary ability of 3 to arrest the
cell growth against both the cell lines. These theoretical parameters correlate well with the
experimental results since complex 3 is the most active against both the cell lines.
Induction of Apoptosis
In higher organism, apoptosis is a key process for boy haemostasis during
development, embryonic development, the immune system, and ageing. It is also known to
play an important role in many pathological conditions like cancer and neurodegeneration.39
Apoptotic cell death is postulated to be the crucial mechanism in natural tumor suppression
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and cancer treatment, which abolish abnormal, malignant cells and reduce the tumor size.40
Thus, it is important to develop novel chemical agents to specifically induce apoptosis for
therapeutic purposes. In addition to the MTT assay, ‘Flow Cytometry’ is considered as the
analytical tool for investigation of potency, not only for cell viability, but also to evaluate
membrane and chromosomal damage, cellꢀcycle analysis and morphological changes. Hence,
for quantification of the extent of apoptotic and necrotic cell death, flow cytometry studies
were performed upon treatment of some representative compounds on HEP 3B and IMR 32
cells. The flowꢀcytometric density plot (Figure S30) illustrates the induction of apoptosis in
HEP 3B and IMR 32 cells after the treatment with K2xan1 and binuclear metallamacrocyclic
complexes 1, 3, 6 and 7.
With reference to Table 3, less than 1% live cell population was observed for both the
cell lines after treatment which reinforced the extreme cytotoxicity of the compounds
investigated by the MTT assay. Interestingly, the apoptotic cell populations viz. 32.1%,
70.2%, 13.5%, 62.3% 10.9% were observed for HEP 3B cells whereas 76.3%, 19.5%, 51.9%,
54.3% 1.2% were observed for the IMR 32 cells, after treating with K2xan1, 1, 3, 6 and 7,
respectively. It may be noted that 64.1% of proꢀapoptotic HEP 3B cells were observed upon
treatment with K2xan1 whereas 77.3% and 43.2% of proꢀapoptotic IMR 32 cell population
was observed upon treatment with 1 and 3, respectively.
Table 3 Apoptotic/ necrotic population of HEP 3B and IMR 32 cells upon treatment with
K2xan1, binuclear metallamacrocyclic complexes 1, 3, 6 and 7.
Sample
HEP 3B (%)
IMR 32(%)
Necrotic Apoptotic Live
Proꢀ
apoptotic
64.1
3.8
Necrotic Apoptotic Live
Proꢀ
apoptotic
2.5
cells
0.0
0.2
0.0
0.1
0.2
99.8
0.0
cells
0.0
0.2
0.1
0.0
1.0
99.7
...
K2xan1
3.8
25.8
85.9
30.8
88.3
0.1
32.1
70.2
13.5
62.5
10.9
0.0
21.2
3.0
4.8
43.3
97.6
0.1
76.3
19.5
51.9
54.3
1.2
77.3
43.2
2.3
0.3
0.2
1
3
6
7
0.5
6.7
0.5
0.1
Control
Cisplatin
0.0
...
10.3
30.4
59.3
...
...
In particular, HEP 3B and IMR 32 cells treated with 7 and HEP 3B cells treated with
3, predominantly dyed with propidium iodide and evident for the induction of necrosis to a
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great extent, whereas IMR 32 cells treated with 6 showed simultaneous binding of annexin V
and propidium iodide, indicative of the transition of apoptosis to necrosis (late apoptosis and
secondary necrosis).41 Apoptosis inducing ability of compounds K2xan1, 1 and 6 is clearly
reflected by the sum of apoptotic and proꢀapoptotic cell populations (96.2%, 74% and 69.2%
for HEP 3B; 78.8%, 96.8% and 56.6% for IMR 32) of treated cells. The distinct behavior of
the binuclear metallomacrocyclic xanthate complex 3 towards both the cell lines is also
reflected by the sum of apoptotic and proꢀapoptotic cell populations of 95.1% (IMR 32) and
14% (Hep 32). The differential apoptotic, proꢀapoptotic and necrotic cell populations in
treated HEP 3B and IMR 32 cells is indicative of the involvement of different receptors in the
cytotoxicity.
Conclusion
A metathetical reaction of 4,4’ꢀbis(2ꢀdithiocarbonatobenzylideneamino)diphenyl ether (xan1)
or 4,4’ꢀbis(2ꢀdithiocarbonatonaphthylmethylideneamino)diphenyl ether (xan2) with CoII or
CuII acetates in Et3N affords access to novel series of phenolate based binuclear CoII or CuII
xanthate metallomacrocyclic complexes of the type [M2ꢀµ2ꢀbisꢀ(κ2S,Sꢀxan1/xan2)] (1ꢀ4).
These metastable products undergo a facile transformation into [2:2] binuclear N, OꢀSchiff
base macrocycles of the type [M2ꢀµ2ꢀbisꢀ(κ2N,OꢀL1/ L2)] (L1 = 4,4’ꢀbis(2ꢀhydroxybenzꢀ
ylideneamino)diphenyl ether, L2 = 4,4’ꢀbis(2ꢀhydroxynaphthylmethylideneamino)diphenyl
ether) 5ꢀ8. The unique coordination geometry of metal ions together with the arc shaped
ligands form double helical molecular unit of 5ꢀ8. Interestingly, the binuclear
metallamacrocycle 3 was found to be the most active against the human cancer cells and the
cytotoxicity data confirms better potency of all the investigated compounds than the reference
drug “cisplatin”. These scaffolds, with excellent IC50 values were explored as a novel class of
active chemical agents to induce apoptosis, required for a major therapeutic implication in
cancer therapy. Owing to the unique characteristics of macrocyclic scaffolds and considering
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the advantage of their bioavailability, versatile coordination ability and the ability to change
their oxidation state (in case of copper/ cobalr centers) these binuclear macrocyclic
complexes project themselves as efficacious anticancer agents.
Experimental Section
Materials
All the synthetic manipulations were performed under nitrogen atmosphere. The solvents
were of laboratory grade available at various commercial sources and distilled prior to use by
following the standard procedures. 4,4’ꢀdiaminodiphenyl ether (99%) (National Chemicals),
potassium hydroxide, carbon disulfide (99%), metal acetates such as Co(OAc)2.4H2O and
Cu(OAc)2.H2O (all Merck), 2ꢀhydroxybenzaldehyde (98%), 2ꢀhydroxynaphthaldehyde (98%)
(Chemlabs) were used as received. Ligand precursors 4,4’ꢀbis(2ꢀhydroxybenzylideneamino)
diphenyl ether (L1) and 4,4’ꢀbis(2ꢀhydroxynaphthylmethylideneamino)diphenyl ether (L2)
were synthesized following literature procedure.23
Instrumentation
IR (KBr pellets) spectra were recorded in the 4000ꢀ400 cm–1 range using a PerkinꢀElmer
FTIR Spectrometer. The NMR spectra were obtained on a Bruker 400 MHz spectrometer in
DMSOꢀd6 unless otherwise noted. Elemental analyses were performed on a PerkinꢀElmer
Series II CHNS/O Analyzer 2400. Mass spectra were obtained on a Thermo Scientific DSQꢀ
II and AB SCIEX 3200 Q TRAP LC/MS/MS system. Single crystal Xꢀray analysis was
carried out on Agilent's Gemini diffractometer equipped with a Eos CCD detector. UV–
visible spectra were recorded on a Perkin Elmer Lambda 35 UV–vis spectrophotometer.
TGA/DTA plots were obtained using SII TG/DTA 6300 in flowing N2 with a heating rate of
10 °C min–1. GC analysis was carried out on CLARUS500, PE AutoSystem type GC
equipped with FID detector. Powder Xꢀray diffraction studies were performed on a ‘X
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Calibur, Eos, Gemini’ Xꢀray diffractometer using Cu source, CrysAlisPro data reduction:
Agilent Technologies Version 1.171.37.33 program(s) was used to process the data and the
‘POWDERX’ program for indexing the powder XRD data. Flow cytometry studies were
performed on Fluorescent Activated Cell Sorter (FACS) analyzer with high speed sorter
model BD FACS Aria III.
General procedure for synthesis of xanthate salts K2xan1 and K2xan2
To a solution of KOH (2.5 mmol, 141 mg) in 25 ml distilled water, 4,4’ꢀbis(2ꢀ
hydroxyarylmethylideneamino)diphenyl ether precursors L1 (1 mmol, 408 mg) or L2 (1
mmol, 508 mg) was added with stirring. After 15 minutes, an excess amount of CS2 (2 ml)
was added and the reaction was allowed to continue at room temperature for 12 h. The
solvent was evaporated under vacuum and solid was washed with diethyl ether to yield the
product K2xan1 or K2xan2. The compound was dried under vacuum, stored under a nitrogen
atmosphere and samples were taken for analysis. This reaction is outlined in Scheme 1.
K2[4,4’ꢀbis(2ꢀdithiocarbonatobenzylideneamino)diphenyl ether] (K2xan1). Yellow solid;
Yield: ca 624 mg, 98%; Melting/Decomposition point: 205ꢀ210 °C. Anal. Calcd for
[C28H18K2N2O3S4]: C, 52.80; H, 2.85; N, 4.40. Found: C, 52.87; H, 2.95; N, 4.49. IR (KBr
1
pellet, cm–1): 1620s ν(C=N), 1148s, 1109m ν(CꢀO), 1031w, 996s, 982m ν(CS2). H NMR
(400 MHz, DMSOꢀd6): (δ from TMS) 6.880(m, 4HꢀPh), 7.110(d, 4HꢀPh), 7.354(m, 2HꢀPh),
7.44(d, 4HꢀPh), 7.645(dd, 2HꢀPh), 8.97(s, 2HꢀN=CH). 13C NMR (400 MHz, DMSOꢀd6): (δ
from TMS) 115.24, 115.34, 117.57, 119.38, 119.93, 121.42, 123.19, 123.50, 124.36, 127.61,
133.38, 135.63, 144.54, 146.12, 148.93, 158.45 (all corresponds to Ph), 162.02 (–N=CH),
190.86(–CS2).
K2[4,4’ꢀbis(2ꢀdithiocarbonatonaphthylmethylideneamino)diphenyl ether] (K2xan2). Dark
yellow solid; Yield: ca 722 mg, 98%; Melting/Decomposition point: 180ꢀ185 °C
decomposes. Anal. Calcd for [C36H22K2N2O3S4]: C, 58.67; H, 3.01; N, 3.80. Found: C, 58.08;
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H, 3.12; N, 3.85. IR (KBr pellet, cm–1): 1624s ν(C=N), 1140s, 1084m ν(CꢀO), 1038w, 998s,
970m ν(CS2). 1H NMR (400 MHz, DMSOꢀd6): (δ from TMS) 7.05(dd, 2HꢀPh), 7.18(dd, 4Hꢀ
Ph), 7.36(t, 2HꢀPh), 7.545(m, 2HꢀPh), 7.71(dd, 4HꢀPh), 7.8(d, 2HꢀPh), 7.93(d, 2HꢀPh),
8.525(d, 2HꢀPh), 9.688(s, 2HꢀN=CH). 13C NMR (400 MHz, DMSOꢀd6): (δ from TMS)
109.14, 118.85, 118.91, 120.10, 120.92, 122.21, 122.72, 122.93, 123.90, 123.95, 127.19,
128.49, 129.44, 133.51, 136.88, 140.63, 155.69, 156.29 (all corresponds to Ph), 169.62 (–
N=CH), 190.24 (–CS2).
General procedure for synthesis of 1ꢀ4
To a solution of xanthate ligand K2xan1 (0.5 mmol, 318.45 mg) or K2xan2 (0.5 mmol,
368.51mg) in 20 ml of distilled Et3N, Co(OAc)2.4H2O / Cu(OAc)2.H2O (0.6 mmol, 149.45
mg) / (0.6 mmol, 119.79 mg) was added with rigorous stirring (Scheme 2). The reaction was
allowed to continue for 12h. The solid residue was filtered and washed with Et3N followed by
distilled water and hexane to yield the products 1ꢀ4. These products were dried under high
vacuum, stored under a nitrogen atmosphere and samples were taken for analysis.
[Co2ꢀµ2ꢀbisꢀ(κ2S,Sꢀxan1)] (1). Buff solid; Yield: ca 305 mg, 99%; Melting/Decomposition
point: 180ꢀ185 °C. Anal. Calcd for [C56H36Co2N4O6S8]: C, 54.45; H, 2.94; N, 4.54; S, 20.77.
Found: C, 54.12; H, 3.05; N, 4.50; S, 20.75. IR (KBr pellet, cm–1):, 1620s ν(C=N), 1152m,
1112m, 1104m ν(CꢀO), 1031w, 1012w, 983m ν(CS2).
[Co2ꢀµ2ꢀbisꢀ(κ2S,Sꢀxan2)] (2). Orange solid; Yield: ca 344 mg, 96%; Melting/Decomposition
point: 200ꢀ205 °C. Anal. Calcd for [C72H44Co2N4O6S8]: C, 60.24; H, 3.09; N, 3.90; S, 17.87.
Found: C, 60.25; H, 3.21; N, 3.85; S, 17.70. Mass (MS ES+): 1436.1 (M+H). IR (KBr pellet,
cm–1): 1616s, 1615s, 1601s ν(C=N), 1145m, 1110m ν(CꢀO), 1041w, 1012m, 980m ν(CS2).
[Cu2ꢀµ2ꢀbisꢀ(κ2S,Sꢀxan1)] (3). Brown solid; Yield: ca 305 mg, 98%; Melting/Decomposition
point: 160ꢀ165 °C. Anal. Calcd for [C56H36Cu2N4O6S8]: C, 54.04; H, 2.92; N, 4.50; S, 20.61.
Found: C, 54.10; H, 2.99; N, 4.55; S, 20.91. IR (KBr pellet, cm–1): 1614s ν(C=N), 1149s,
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1127m, 1104m ν(CꢀO), 1029w, 1010m, 982w ν(CS2).
[Cu2ꢀµ2ꢀbisꢀ(κ2S,Sꢀxan2)] (4). Brown solid; Yield: ca 336 mg; 93% Melting/Decomposition
point: 100ꢀ105 °C. Anal. Calcd for [C72H44Cu2N4O6S8]: C, 59.86; H, 3.07; N, 3.88; S, 17.76.
Found: C, 59.23; H, 3.35; N, 3.72; S, 17.91. IR (KBr pellet, cm–1): 1617s, 1602s ν(C=N),
1142m, 1103m ν(CꢀO), 1041w, 1011w, 982w, 967w ν(CS2).
General procedure for synthesis of 5ꢀ8. Binuclear metallomacrocyclic xanthate complexes
(0.5 mmol) 1 (617.65 mg), 2 (717.67 mg), 3 (622.26 mg) or 4 (722.38 mg) were dissolved in
dichloromethane and volatiles were allowed to evaporate at room temperature (Scheme 2).
The brownish 5 and 6 (in case of 1 and 2) and blackish brown 7 and 8 (in case of 3 and 4)
crystalline solids obtained, were washed with diethyl ether for three times. Solids were dried
under vacuum to yield corresponding binuclear metallomacrocyclic bisꢀN,OꢀSchiff base
complexes 5ꢀ8; products were stored under a nitrogen atmosphere and samples were taken for
analysis.
[Co2ꢀµ2ꢀbisꢀ(κ2N,OꢀL1)] (5). Pale brown solid, Yield: ca 377 mg, 81%;
Melting/Decomposition point: 170ꢀ175 °C. Anal. Calcd for [C52H36Co2N4O6]: C, 67.10; H,
3.90; N, 6.02. Found: C, 67.20; H, 3.95; N, 5.96. IR (KBr pellet, cm–1): 1608s, 1584s
ν(C=N).
[Co2ꢀµ2ꢀbisꢀ(κ2N,OꢀL2)] (6). Dark brown solid, Yield: ca 526 mg, 93%;
Melting/Decomposition point: 200ꢀ205 °C. Anal. Calcd for [C68H44Co2N4O6]: C, 72.22; H,
3.92; N, 4.95. Found: C, 72.18; H, 4.05; N, 4.91. Mass (MS ES+): 1131.0 (M+H). IR (KBr
pellet, cm–1): 1616s, 1601s ν(C=N).
[Cu2ꢀµ2ꢀbisꢀ(κ2N,OꢀL1)] (7). Blackish brown solid; Yield: ca 423 mg, 90 %;
Melting/Decomposition point: 200ꢀ205 °C. Anal. Calcd for [C52H36Cu2N4O6.CH2Cl2]: C,
62.11; H, 3.74; N, 5.47. Found: C, 62.13; H, 3.75; N, 5.45. Mass (MS ES+): 931 (M+H). IR
(KBr pellet, cm–1): 1617s, 1602s ν(C=N).
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[Cu2ꢀµ2ꢀbisꢀ(κ2N,OꢀL2)] (8). Blackish brown solid; Yield: ca 542 mg, 95 %;
Melting/Decomposition point: 150ꢀ155 °C. Anal. Calcd for [C68H44Cu2N4O6]: C, 71.63; H,
3.89; N, 4.91. Found: C, 71.60; H, 3.95; N, 4.89. IR (KBr pellet, cm–1): 1616s, 1603s
ν(C=N).
Xꢀray structure determinations
Single crystals suitable for Xꢀray crystallographic study were obtained by slow evaporation
of dichloromethane solution of 5ꢀ7 at 4 °C and a chloroform solution of 8. The crystals of 7
were also grown by the slow diffusion of hexane in a DMSO solution in order to explore the
solvent effect on crystallization and thus its impact on supramolecular packing in the solid
state. The intensity data for 5 [C52H36Co2N4O6], 7.DMSO [C54H40Cu2N4O7S], 7.2CH2Cl2
[C54H40Cl4Cu2N4O6] and 8.CHCl3.9H2O [C137H105Cl3Cu4N8O22] was collected on Oxford X
Calibur and Gemini diffractometer respectively equipped with Eos CCD detector at 298 K.
Monochromatic CuꢀKα (λ = 1.54184) (in case of 5) and MoꢀKα Xꢀray (λ=0.71073 Å) (in case
of 7ꢀ8) radiations were used for the measurements. Data was collected and reduced by using
the ‘‘CrysAlispro’’ program (Version 1.171.37.33 Agilent Technologies, 2014).42a Intensity
data for 6 [C68H44Co2N4O6] were collected on a Bruker Smart Apex CCD diffractometer
using graphiteꢀmonochromated MoꢀKαradiation (λ= 0⋅71073 Å). The data integration and
reduction were processed with SAINT42b software. An empirical absorption correction was
applied to the collected reflections with SADABS.42c All the crystal structure were solved by
the direct methods, and the refinement was carried out by fullꢀmatrix least squares against F2
using SHELXLꢀ97 program package43 and Olex2 (version 1.2.5) program package.44 MASK
procedure of Olex2 is used to treat the disordered solvent molecules in each structure. All
nonꢀhydrogen atoms were refined anisotropically.
In vitro cytotoxic study
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Cell line and culture. Hepatoma (HEP 3B) and Neuroblastoma (IMR 32) cell lines were
procured from the National Center for Cell Science, Pune. Dubecoos Modified Essential
Medium (DMEM) and Foetus Bovine Serum (FBS) were procured from HiMedia whereas
cisplatin from Sigma Aldrich. The human cell lines HEP 3B and IMR 32 were established in
DMEM with 10% FBS in humidified atmosphere supplied with 5% CO2 at 37 °C. The IMR
32 cell line was differentiated as a neuron by using sodium butyrate for 9 days of incubation
at 37°C at a concentration of 5% CO2. The compounds reported in this article were screened
for their antitumor activity against both the cell lines at varying concentration.
MTT assay for cell viability/ proliferation. The cell growth inhibition was determined by
MTT assay with some modifications.37 Schiff base precursors (L1, L2), xanthate ligands
(K2xan1, K2xan2), binuclear xanthate complexes (1ꢀ4) and binuclear Schiff base complexes
(5ꢀ8) were dissolved in DMSO and then diluted with water. The content of DMSO in each
sample was 1%. Cells were seeded in 96ꢀwell plates at a density of 1 × 103 cells per well and
incubated for 24 h. These cells were treated with different concentrations of compounds
reported in this paper for 6 h (against HEP 3B) and 14 h (against IMR 32). Cisplatin was also
screened against both the cell lines under similar experimental conditions. After removal of
the media, the culture was incubated with 20 ꢁL of media containing 5 mg/ml stock solution
of MTT in PBS and 60 ꢁL of DMEM for 6 h at 37 °C in 5% CO2 incubator. Formazan crystal
formed by metabolically viable cells were dissolved in DMSO and the optical density was
measured at 570 nm by ELISA reader (METERTECHꢀ∑960). The number of viable cells
was proportional to the extent of formazan production.
Statistical Analysis for Determination of IC50. Six sets of each sample were screened and
the data obtained, was analyzed in Prism/OriginPro 8 for standard error and probit analysis.
The percent cytotoxicity index (% CI) was as follows:
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% CI= [1− (OD of treated cells/OD of control cells)] × 100 % (where, CI= cytotoxicity index,
OD= optical density.)
A plot of % CI versus concentration was obtained from the experimental data for each set of
experiments. IC50 values (50% growth inhibition of cell) were determined from the graph.
Each test was repeated thrice and the results were expressed as mean IC50 ± SD.
Annexin V/PI Double Staining for Cell Death Analysis. The cell death assessment was
performed using annexin V alexa Fluro 4888 and propidium iodide apoptosis kit, procured
from Himedia. For apoptosis measurement, HEP 3B and IMR 32 cells (5 × 106 cells/mL)
were treated with selective compounds (1/3 of the IC50 concentration) for 16 hours. After
washing in PBS, 1×106 cells were resuspended in 100µl of annexin binding buffer. FITCꢀ
Annexin V and propidium iodide were added and then incubated for 15 minutes at room
temperature in the dark. After the incubation period, 400 µL of annexinꢀbinding buffer was
added then kept in an ice bath for 5 minutes. Cells were centrifuged and fixed in 1%
formalin. Cells were resuspended in 1% FBS and 0.5% in BSA after centrifugation and then
these were analyzed by flow cytometry (BD FACSAria III).
Acknowledgements
VKS acknowledges CSIR, New Delhi, India for the financial support (Project No.
01/2733/13/EMRꢀII). One of the authors, R. Kadu acknowledges UGC, New Delhi, India for
the financial support in the form of fellowship. We also thank the Department of Science and
Technology (DST), New Delhi for the PURSE project under which the single crystal Xꢀray
diffractometer was acquired in the Faculty of Science. We would like to thank Dr. Sanjio
Zade for his help in computational study.
25

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DOI: 10.1039/C5DT03407H
Supporting Information Available: Synthesis, Characterization, Crystallographic (CIF)
(CCDC No. 986523 for 6, 986524 for 5, 986525 for 7.DMSO, 986526 for 7.2CH2Cl2 and
1426720 for 8.CHCl3.8H2O), Computational and TGꢀDTA data.
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