Pharmacophore hybrid approach of new modulated bis-diimine Cu II /ZnII complexes based on 5-chloro isatin Schiff base derivatives: Synthesis, spectral studies and comparative biological assessment Mohammad. Shakir, Summaiya Hanif, Mohd. Asif Sherwani, Owais Mohammad, Mohammad. Azam, Saud I. Al-Resayes PII: DOI: Reference:
S1011-1344(15)30007-5 doi: 10.1016/j.jphotobiol.2016.01.019 JPB 10237
To appear in: Received date: Accepted date:
18 August 2015 29 January 2016
Please cite this article as: Mohammad. Shakir, Summaiya Hanif, Mohd. Asif Sherwani, Owais Mohammad, Mohammad. Azam, Saud I. Al-Resayes, Pharmacophore hybrid approach of new modulated bis-diimine CuII /ZnII complexes based on 5-chloro isatin Schiff base derivatives: Synthesis, spectral studies and comparative biological assessment, (2016), doi: 10.1016/j.jphotobiol.2016.01.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Pharmacophore hybrid approach of new modulated bis-diimine CuII/ ZnII complexes based on 5-chloro Isatin Schiff base derivatives: Synthesis, spectral studies and
T
comparative biological assessment.
a*
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India.
c
NU
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India. Department of Chemistry, Science College, King Saud University, PO Box 2455, Riyadh
MA
b
SC R
Mohammad. Azamc and Saud I. Al-Resayesc
IP
Mohammad. Shakira*, Summaiya Hanifa, Mohd. Asif Sherwanib, Owais Mohammadb,
D
11451 Saudi Arabia.
TE
.*To whom correspondence should be addressed
CE P
Email: [email protected]
AC
Phone No. +91-9837430035
1
ACCEPTED MANUSCRIPT Abstract Novel bioactive 5-chloro isatin based Schiff base ligands, (N,N'E,N,N'Z)-N,N'-(5-
T
chloroindoline-2,3-diylidene)bis(5-nitrobenzo[d]thiazol-2-amine), L1 and (N,N'E,N,N'Z)-
IP
N,N'-(5-chloroindoline-2,3-diylidene)bis(5-nitrothiazol-2-amine), L2 derived from 2-amino 5-
SC R
nitrobenzothiazole and 2-amino 5-nitrothiazole and their metal complexes, [Cu(L1)2]Cl2; 1, [Zn(L1)2(H2O)2]Cl2; 2, [Cu(L2)2]Cl2; 3 and [Zn(L2)2(H2O)2]Cl2; 4 have been synthesized. The
NU
composition, stoichiometry and geometry of the proposed ligands and their complexes have been envisaged by the results of elemental analyses and spectroscopic data (FT-IR, 1H NMR 13
C NMR, Mass and EPR). The molar conductivity values of the metal complexes
MA
and
revealed their ionic nature. The thermal stability of metal complexes was demonstrated by
D
TGA/DTA studies while the crystalline nature of the complexes has been ascertained by
TE
XRD. Furthermore, a comparative account of in vitro antibacterial study against different bacterial strains with respect to standard antibiotic and scavenging activity against standard
CE P
control at different concenterations unfolded pronounced antibacterial and radical scavenging potencies of the metal complexes as compared to free ligands. In addition, in vitro
AC
cytotoxicity of ligands and its metal complexes was also screened on MCF7 (Human breast adenocarcinoma), HeLa (Human cervical carcinoma) and HepG2 (Human Hepatocellular carcinoma), cell lines and normal cells (PBMC). The antiproliferative outcomes revealed that metal complexes exhibit superior activity in general as compared to free ligands (L1 and L2) where metal complexes (1 and 2) of 5-chloro isatin linked benzothiazole motif (L1) are found to have better prospect of acting as chemotherapeutic agents which can be explained in terms of greater biopotency, planarity and conjugation against all the tested cancer cell lines with IC50 g┴> 2.0023) in both the complexes envisage a square planar geometry around Cu(II)
NU
ions. The geometric parameter, G is calculated by using Kneubuhl’s method, G= g|| -2/ g┴ -2
MA
gives an account of measure of extent of exchange interaction [47]. The calculated G values of 2.81 and 2.48 indicate that the exchange coupling do take place in the complexes as G is
3.7. Thermal Studies
TE
D
lesser than 4 [48].
CE P
In order to determine the thermal stability of metal complexes, thermogravimetric analysis (TGA/DTA) was carried out under N2 atmosphere at a heating rate of 20◦C in the temperature
AC
range 25-800◦C (Fig. 7). It is noticeable that TGA curves of complexes 1 and 3 show two step weight losses whereas complexes 2 and 4 indicated weight loss in three steps. Thermograms of complexes 1 and 3 were found to be stable upto 200-230◦C which indicates the absence of coordinated water molecules in the complexes [49]. The first step revealed the simultaneous degradation of chlorine atoms and benzothiazole/thiazole moieties in the temperature range 240-420◦C accompanied by weight loss (Obs = 65.49% and 58.58%; Calc = 65.31% and 58.41%). The endothermic DTA peak in the range 310-390 ◦C supported this decomposition. Further elevation in temperature lead to second stage of decomposition from 550-650 ◦C alongwith an exothermic peak in the temperature range 570-640 ◦C corresponding to the loss of remaining organic moieties and which is in agreement with weight loss (Obs = 29.58%
14
ACCEPTED MANUSCRIPT and
35.63%; Calc = 29.44% and 35.31%). The TGA curves of complexes 2 and 4
correspond to weight loss (Obs = 2.95% and 3.51%; Calc = 2.89% and 3.44%) within the
T
temperature range of 100-220 ◦C corresponding to the loss of coordinated water molecules
IP
[47] alongwith an endothermic peak in the temperature range of 120-140◦C. In the second
SC R
step, a weight loss (Obs = 63.43% and 56.70%; Calc = 63.30% and 56.23%) in the temperature range of 240-510◦C has been observed, attributable to the decomposition of chlorine atoms and benzothiazole/thiazole moieties alongwith an endothermic peak in the
NU
temperature range of 330-390◦C. The final decomposition step appeared in the range 500-
MA
690◦C with weight loss (Obs = 28.70% and 33.87%; Calc = 28.44% and 33.99%) which may reasonably be accounted for the degradation of remaining organic moieties of ligand framework. The DTA peak in the temperature range of 600-650 ◦C further confirming this
TE
D
decomposition step. However, horizontal lines in the TGA curves beyond 650◦C have been observed in all the metal complexes indicated no further weight loss, implying that a metal
CE P
residue may be the final product [50]. The thermal analysis data agree well with the proposed stoichiometry derived from the results of elemental analysis findings
AC
.
3.8. X- Ray diffraction analysis All the inordinate efforts have failed to grow single crystals of metal complexes suitable for single X- ray crystallography. Therefore, X- ray powder diffraction studies of the metal complexes have been carried out to obtain further evidence about their structure. The X- ray powder diffraction studies of metal complex crystallites, 1-4 (Fig. 8a-d) displayed well defined patterns confirming the crystalline nature of the complexes. The X- ray diffraction of complex 1 exhibited reflecting peaks at 2Ɵ scattering angles of 19.85, 22.86, 32.55 and 45.67 assigned to (111), (200), (220) and (400) crystal planes, respectively characteristic of well ordered cubic arrangement of Cu atoms (Space group: Fm3m, JCPDS: 01-1241) while
15
ACCEPTED MANUSCRIPT complex 2 depicted reflecting peaks at 2Ɵ scattering angles of 24.99, 30.55, 30.67, 35.29, 35.99, 36.90, 37.92, 43.61, 45.01, 48.93, 55.72 and 58.39 assigned to (102), (110), (004),
T
(112), (200), (201), (104), (203), (114), (211), (301) and (214) crystal planes, respectively
IP
characteristic of well ordered hexagonal arrangement of Zn atoms (Space group: P63/mmc,
SC R
JCPDS: 01-1244). The complex 3 demonstrated reflecting peaks at 2Ɵ scattering angles of 22.76, 32.07, 37.95 and 39.33 assigned to (200), (220), (311) and (222) crystal planes, respectively characteristic of well ordered cubic arrangement of Cu atoms (Space group:
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Fm3m, JCPDS: 02-1225) whereas complex 4 displayed fine indexed reflecting peaks at 2Ɵ
MA
scattering angles of 20.62, 24.23, 30.43, 35.12, 36.05, 39.92 and 44.37 assigned to (101), (102), (110), (112), (201), (202), (203) and (105) crystal planes, respectively characteristic of
D
well ordered hexagonal arrangement of Zn atoms (Space group: P63/mmc, JCPDS: 01-1238).
TE
The different lattice parameters observed for 1-4 metal complexes (Table 4) revealed that complex particles possess similar crystal structure as that of bulk [51]. The detailed XRD
CE P
parameters of these complexes are listed in Table 4. In addition, the average crystallite size for the above mentioned complexes was estimated using the Debye Scherrer’s formula [52]:
AC
D = 0.9λ/β.cos Ɵ
Where constant 0.9 is the shape factor, D is the particle size, λ is the X- ray wavelength of CuKα radiation (1.5406 A◦), Ɵ is the Bragg diffraction angle and β is the full width at half maximum (FWHM) value of Ɵ . The average crystallite size of the complexes, 1-4 were found to be 26.58 nm, 33.89 nm, 29.48 nm and 22.27 nm, respectively.
4. Biological studies 4.1. In vitro antibacterial activity It is well documented fact that metal complexes based on Schiff base ligand acted as superficial chemical scaffolds against different bacterial pathogens because of presence of
16
ACCEPTED MANUSCRIPT imine group which imports in elucidating the mechanism of transamination and resamination reaction in biological systems [53]. Hence, the comparative screening of antibacterial activity
T
of synthesized Schiff base ligands, L1 and L2 and their metal complexes, 1-4 have been
IP
carried out on gram negative and gram positive bacteria using Ciprofloxacin as positive
SC R
control reference drug. The diameter of the zone of inhibition (mm) was used to compare the antibacterial activity with the Ciprofloxacin as presented in Table 5 and Fig. 9. In vitro antibacterial studies revealed that all the complexes demonstrated varying degree of
NU
inhibition. Among all the metal complexes, complex 1 exhibited sound activity against E.coli
MA
and V. Cholerae with zone diameter 34.23 mm and 32.89 mm, respectively which is almost equivalent to inhibition zone of standard control while complex 2 having zone diameter 27.34
D
mm showed better activity as compared to complexes 3 and 4 against E. coli. In case of S.
TE
aureus complexes 1 and 2 showed potential inhibition zone diameter 30.12 mm and 26.36 mm respectively, as compared to complexes 3 and 4. On the other hand, all metal complexes
CE P
showed influential zone of inhibition against L. monocytogenes and S. typhimurium as compared to P. Aeruginosa. However, complexes 2, 3 and 4 were found to exhibit substantial
standard.
AC
activity against V. Cholera with zone diameter 27.12, 19.21 and 18.23 mm with respect to the
The MIC’s of ligands L1 and L2 and their complexes (1-4) against different bacterial strains are given in Table 6 and it is clear from the data that complexes 1 and 2 (MIC : 6.25 µg/ml and 12.50 µg/ml, respectively) have shown best bacterial activity which is comparable to the control (Ciprofloxacin) against E. coli in comparison to complexes 3 and 4 (MIC : 25 µg/ml and 50 µg/ml, respectively) whereas complexes 1, 2 and 3 (MIC: 12.5 µg/ml) have shown good activity than complex 4 (MIC : 50 µg/ml ) in comparison to standard against S. aureus. Further, complexes 1 and 2 (MIC : 6.25 µg/ml and 6.25 µg/ml, respectively) exhibit better activity against L.monocytogenes as compared to complexes 3 and 4 (MIC : 25 µg/ml and 50
17
ACCEPTED MANUSCRIPT µg/ml, respectively). However, metal complexes were found to exhibit significant MIC’s results against S. typhimurium and V.cholerae as compared to P.aeruginosa in comparison to
T
standard control. From the data presented in Table 5 and 6, it is evident that the complexes
IP
which possess lower MIC’s showing effectual zone of inhibition and hence active bacterial
SC R
agents.
A comparative evaluation of antibacterial results suggested that the metal complexes, 1-4 demonstrated enhanced antibacterial activity as compared to free ligands, L1 and L2 as
NU
previously accounted [4]. The enhancement in the antibacterial activity of the complexes as
MA
compared to free ligands can be supported by Tweedy’s chelation theory [54]. It is known that chelation enhances the ligand to act as more powerful and potent bactericidal agents by
D
inhibiting the bacterial growth, thus zones of inhibition of metal complexes were found to be
TE
higher compared to its free ligands. The chelation reduces the polarity of metal ion considerably because of the partial sharing of its positive charge with the hetero donor atoms
CE P
of the ligand and also due to π-electron delocalization over the whole chelate ring system. The lipids and polysaccharides are the vital constituents of the cell wall and membranes
AC
which are ideal for metal ion interaction. Apart from this, the cell walls also possess phosphates, carbonyl and cystenyl ligands which assist in sustaining the integrity of the membrane by acting as a diffusion barrier and also provide suitable sites for binding [55]. Furthermore, the diminution in polarity boost the lipophilic character of the chelates and thus an interface between the lipid and metal ion is preferred which may subsequently breakdown the permeability barrier of the cell resulting in involvement with the normal cell processes and lead to the blockage of metal binding sites in the enzymes of the microorganisms. These metal chelators may also held in perturbing the respiration process of the cell and thus obstruct the synthesis of proteins that consequently inhibits further growth of the organism. Further, it has been reported that metal complexes containing Schiff base ligand with nitrogen
18
ACCEPTED MANUSCRIPT and oxygen donor atoms might hinder enzyme production and that result in cell death [56]. The variation in the activity of the metal complexes against different microbes depend either
T
on the difference in the ribosomes of the microbial cells or on the impermeability of the cells
IP
of the microbes. The lower activity of complexes as compared to others may be attributed due
SC R
to low lipid solubility, so that the metal ion may not be able to reach the favourable site of action of the cell wall to get interfere with the normal cell activity. Although chelation itself play a significant role in determining antibacterial behaviour of the complexes but
NU
concurrently different factors such as solubility, size, dipole moment, coordinating sites,
MA
redox potential of metal ion, solubility, bond length between metal and the ligand, geometry of complexes, steric, pharmacokinetic, concenteration and hydrophobicity have substantial
D
influence on the antibacterial activity. Thus, it is apparent from the results that enhanced
TE
antibacterial activity of the metal complexes may not be only due to chelation but also
CE P
intricate combinations of several other factors as well [57-60].
4.2. In vitro antioxidant activity
AC
It is well established earlier that derivatives of isatin and benzothiazole/thiazole exhibit significant antioxidant activity which manifested them as potentially effective drugs [20-22, 61] hence, we carried out a methodical analysis to investigate the antioxidant efficacy of free ligands and metal complexes by DPPH method using L-ascorbic acid (standard). The antioxidant data of the compounds at variable concenterations (5-20 µM) and concenteration dependent curves have been shown (Table 7 and Fig. 10) which revealed that chelation effect exhibits metal complexes (1-4) as effective antioxidants in comparison to the respective free ligands, L1 and L2 [62]. While the comparative assessment of antioxidant data of metal complexes (1 and 2) of L1 have shown pronounced scavenging activity whereas complexes (3 and 4) of L2 have resulted moderate activity in comparison to L- ascorbic acid which may be
19
ACCEPTED MANUSCRIPT explained in terms of coordination environment and the redox properties of metal ions. It has been reported that the redox properties of the complex depend on various factors such as
T
chelate ring size, axial ligation, degree of unsaturation in the chelate ring [63]. Therefore, it
IP
may be concluded that these complexes could be of therapeutic importance for further in vivo
SC R
studies in the field of pharmacy.
4.3. In vitro cytotoxic activity
NU
On the basis of considerable outcomes obtained after antibacterial and antioxidant screening,
MA
it was considered sensible to explore the cytotoxic potential of newly synthesized 5-chloro isatin derived Schiff base compounds against MCF7, HeLa and HepG2 cancer cell lines of
D
different histological origins by determining the number of viable cells surviving after
TE
incubation with the Schiff base compounds for the specified time period using MTT method [34]. The cytotoxicity assay implies a variable cytotoxic trend of synthesized compounds
CE P
against above mentioned cell lines which can be ascribed due to the intrinsic cytotoxic potential of these compounds. The various curves of dose dependent effects of all the
AC
examined compounds on cell viability of different cancer cell lines and normal cells (PBMC) are showed in Fig. 11. The results from experimental data demonstrated an increment in cytotoxicity in the cell lines with increasing the exposure to drug concenteration. The absorption values were articulated as the cell viability in percent, according to the control group as 100%. The assays were carried out in triplicate on three independent experiments. The concenteration entailed for 50% inhibition of cell viability (IC50) was calculated using the software “Prism 3.0”. The cytotoxic potential of all the examined compounds against cancer cell lines with IC50’s < 3.60µM (Table 8). In view of structure activity relationships, numerous correlations can be concluded from the cytotoxic data of tested compounds. On evaluating the cytotoxic potential against the MCF7,
20
ACCEPTED MANUSCRIPT HeLa and HepG2 cancer cell lines, ligands L1 and L2 were found to be least active in comparison to metal complexes under similar conditions. The experimental IC50 data
T
revealed that complex 1 demonstrates relatively greater cytotoxic effect than other metal
IP
complexes against all cancerous cell lines with substantial IC50 values of 1.06±0.18 µM
SC R
(MCF7), 1.86±1.30 µM (HeLa) and 2.44±0.61µM (HepG2). The complex 2 also displayed significant activity against MCF7 and HeLa with IC50
S1011-1344(15)30007-5 doi: 10.1016/j.jphotobiol.2016.01.019 JPB 10237
To appear in: Received date: Accepted date:
18 August 2015 29 January 2016
Please cite this article as: Mohammad. Shakir, Summaiya Hanif, Mohd. Asif Sherwani, Owais Mohammad, Mohammad. Azam, Saud I. Al-Resayes, Pharmacophore hybrid approach of new modulated bis-diimine CuII /ZnII complexes based on 5-chloro isatin Schiff base derivatives: Synthesis, spectral studies and comparative biological assessment, (2016), doi: 10.1016/j.jphotobiol.2016.01.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Pharmacophore hybrid approach of new modulated bis-diimine CuII/ ZnII complexes based on 5-chloro Isatin Schiff base derivatives: Synthesis, spectral studies and
T
comparative biological assessment.
a*
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India.
c
NU
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India. Department of Chemistry, Science College, King Saud University, PO Box 2455, Riyadh
MA
b
SC R
Mohammad. Azamc and Saud I. Al-Resayesc
IP
Mohammad. Shakira*, Summaiya Hanifa, Mohd. Asif Sherwanib, Owais Mohammadb,
D
11451 Saudi Arabia.
TE
.*To whom correspondence should be addressed
CE P
Email: [email protected]
AC
Phone No. +91-9837430035
1
ACCEPTED MANUSCRIPT Abstract Novel bioactive 5-chloro isatin based Schiff base ligands, (N,N'E,N,N'Z)-N,N'-(5-
T
chloroindoline-2,3-diylidene)bis(5-nitrobenzo[d]thiazol-2-amine), L1 and (N,N'E,N,N'Z)-
IP
N,N'-(5-chloroindoline-2,3-diylidene)bis(5-nitrothiazol-2-amine), L2 derived from 2-amino 5-
SC R
nitrobenzothiazole and 2-amino 5-nitrothiazole and their metal complexes, [Cu(L1)2]Cl2; 1, [Zn(L1)2(H2O)2]Cl2; 2, [Cu(L2)2]Cl2; 3 and [Zn(L2)2(H2O)2]Cl2; 4 have been synthesized. The
NU
composition, stoichiometry and geometry of the proposed ligands and their complexes have been envisaged by the results of elemental analyses and spectroscopic data (FT-IR, 1H NMR 13
C NMR, Mass and EPR). The molar conductivity values of the metal complexes
MA
and
revealed their ionic nature. The thermal stability of metal complexes was demonstrated by
D
TGA/DTA studies while the crystalline nature of the complexes has been ascertained by
TE
XRD. Furthermore, a comparative account of in vitro antibacterial study against different bacterial strains with respect to standard antibiotic and scavenging activity against standard
CE P
control at different concenterations unfolded pronounced antibacterial and radical scavenging potencies of the metal complexes as compared to free ligands. In addition, in vitro
AC
cytotoxicity of ligands and its metal complexes was also screened on MCF7 (Human breast adenocarcinoma), HeLa (Human cervical carcinoma) and HepG2 (Human Hepatocellular carcinoma), cell lines and normal cells (PBMC). The antiproliferative outcomes revealed that metal complexes exhibit superior activity in general as compared to free ligands (L1 and L2) where metal complexes (1 and 2) of 5-chloro isatin linked benzothiazole motif (L1) are found to have better prospect of acting as chemotherapeutic agents which can be explained in terms of greater biopotency, planarity and conjugation against all the tested cancer cell lines with IC50 g┴> 2.0023) in both the complexes envisage a square planar geometry around Cu(II)
NU
ions. The geometric parameter, G is calculated by using Kneubuhl’s method, G= g|| -2/ g┴ -2
MA
gives an account of measure of extent of exchange interaction [47]. The calculated G values of 2.81 and 2.48 indicate that the exchange coupling do take place in the complexes as G is
3.7. Thermal Studies
TE
D
lesser than 4 [48].
CE P
In order to determine the thermal stability of metal complexes, thermogravimetric analysis (TGA/DTA) was carried out under N2 atmosphere at a heating rate of 20◦C in the temperature
AC
range 25-800◦C (Fig. 7). It is noticeable that TGA curves of complexes 1 and 3 show two step weight losses whereas complexes 2 and 4 indicated weight loss in three steps. Thermograms of complexes 1 and 3 were found to be stable upto 200-230◦C which indicates the absence of coordinated water molecules in the complexes [49]. The first step revealed the simultaneous degradation of chlorine atoms and benzothiazole/thiazole moieties in the temperature range 240-420◦C accompanied by weight loss (Obs = 65.49% and 58.58%; Calc = 65.31% and 58.41%). The endothermic DTA peak in the range 310-390 ◦C supported this decomposition. Further elevation in temperature lead to second stage of decomposition from 550-650 ◦C alongwith an exothermic peak in the temperature range 570-640 ◦C corresponding to the loss of remaining organic moieties and which is in agreement with weight loss (Obs = 29.58%
14
ACCEPTED MANUSCRIPT and
35.63%; Calc = 29.44% and 35.31%). The TGA curves of complexes 2 and 4
correspond to weight loss (Obs = 2.95% and 3.51%; Calc = 2.89% and 3.44%) within the
T
temperature range of 100-220 ◦C corresponding to the loss of coordinated water molecules
IP
[47] alongwith an endothermic peak in the temperature range of 120-140◦C. In the second
SC R
step, a weight loss (Obs = 63.43% and 56.70%; Calc = 63.30% and 56.23%) in the temperature range of 240-510◦C has been observed, attributable to the decomposition of chlorine atoms and benzothiazole/thiazole moieties alongwith an endothermic peak in the
NU
temperature range of 330-390◦C. The final decomposition step appeared in the range 500-
MA
690◦C with weight loss (Obs = 28.70% and 33.87%; Calc = 28.44% and 33.99%) which may reasonably be accounted for the degradation of remaining organic moieties of ligand framework. The DTA peak in the temperature range of 600-650 ◦C further confirming this
TE
D
decomposition step. However, horizontal lines in the TGA curves beyond 650◦C have been observed in all the metal complexes indicated no further weight loss, implying that a metal
CE P
residue may be the final product [50]. The thermal analysis data agree well with the proposed stoichiometry derived from the results of elemental analysis findings
AC
.
3.8. X- Ray diffraction analysis All the inordinate efforts have failed to grow single crystals of metal complexes suitable for single X- ray crystallography. Therefore, X- ray powder diffraction studies of the metal complexes have been carried out to obtain further evidence about their structure. The X- ray powder diffraction studies of metal complex crystallites, 1-4 (Fig. 8a-d) displayed well defined patterns confirming the crystalline nature of the complexes. The X- ray diffraction of complex 1 exhibited reflecting peaks at 2Ɵ scattering angles of 19.85, 22.86, 32.55 and 45.67 assigned to (111), (200), (220) and (400) crystal planes, respectively characteristic of well ordered cubic arrangement of Cu atoms (Space group: Fm3m, JCPDS: 01-1241) while
15
ACCEPTED MANUSCRIPT complex 2 depicted reflecting peaks at 2Ɵ scattering angles of 24.99, 30.55, 30.67, 35.29, 35.99, 36.90, 37.92, 43.61, 45.01, 48.93, 55.72 and 58.39 assigned to (102), (110), (004),
T
(112), (200), (201), (104), (203), (114), (211), (301) and (214) crystal planes, respectively
IP
characteristic of well ordered hexagonal arrangement of Zn atoms (Space group: P63/mmc,
SC R
JCPDS: 01-1244). The complex 3 demonstrated reflecting peaks at 2Ɵ scattering angles of 22.76, 32.07, 37.95 and 39.33 assigned to (200), (220), (311) and (222) crystal planes, respectively characteristic of well ordered cubic arrangement of Cu atoms (Space group:
NU
Fm3m, JCPDS: 02-1225) whereas complex 4 displayed fine indexed reflecting peaks at 2Ɵ
MA
scattering angles of 20.62, 24.23, 30.43, 35.12, 36.05, 39.92 and 44.37 assigned to (101), (102), (110), (112), (201), (202), (203) and (105) crystal planes, respectively characteristic of
D
well ordered hexagonal arrangement of Zn atoms (Space group: P63/mmc, JCPDS: 01-1238).
TE
The different lattice parameters observed for 1-4 metal complexes (Table 4) revealed that complex particles possess similar crystal structure as that of bulk [51]. The detailed XRD
CE P
parameters of these complexes are listed in Table 4. In addition, the average crystallite size for the above mentioned complexes was estimated using the Debye Scherrer’s formula [52]:
AC
D = 0.9λ/β.cos Ɵ
Where constant 0.9 is the shape factor, D is the particle size, λ is the X- ray wavelength of CuKα radiation (1.5406 A◦), Ɵ is the Bragg diffraction angle and β is the full width at half maximum (FWHM) value of Ɵ . The average crystallite size of the complexes, 1-4 were found to be 26.58 nm, 33.89 nm, 29.48 nm and 22.27 nm, respectively.
4. Biological studies 4.1. In vitro antibacterial activity It is well documented fact that metal complexes based on Schiff base ligand acted as superficial chemical scaffolds against different bacterial pathogens because of presence of
16
ACCEPTED MANUSCRIPT imine group which imports in elucidating the mechanism of transamination and resamination reaction in biological systems [53]. Hence, the comparative screening of antibacterial activity
T
of synthesized Schiff base ligands, L1 and L2 and their metal complexes, 1-4 have been
IP
carried out on gram negative and gram positive bacteria using Ciprofloxacin as positive
SC R
control reference drug. The diameter of the zone of inhibition (mm) was used to compare the antibacterial activity with the Ciprofloxacin as presented in Table 5 and Fig. 9. In vitro antibacterial studies revealed that all the complexes demonstrated varying degree of
NU
inhibition. Among all the metal complexes, complex 1 exhibited sound activity against E.coli
MA
and V. Cholerae with zone diameter 34.23 mm and 32.89 mm, respectively which is almost equivalent to inhibition zone of standard control while complex 2 having zone diameter 27.34
D
mm showed better activity as compared to complexes 3 and 4 against E. coli. In case of S.
TE
aureus complexes 1 and 2 showed potential inhibition zone diameter 30.12 mm and 26.36 mm respectively, as compared to complexes 3 and 4. On the other hand, all metal complexes
CE P
showed influential zone of inhibition against L. monocytogenes and S. typhimurium as compared to P. Aeruginosa. However, complexes 2, 3 and 4 were found to exhibit substantial
standard.
AC
activity against V. Cholera with zone diameter 27.12, 19.21 and 18.23 mm with respect to the
The MIC’s of ligands L1 and L2 and their complexes (1-4) against different bacterial strains are given in Table 6 and it is clear from the data that complexes 1 and 2 (MIC : 6.25 µg/ml and 12.50 µg/ml, respectively) have shown best bacterial activity which is comparable to the control (Ciprofloxacin) against E. coli in comparison to complexes 3 and 4 (MIC : 25 µg/ml and 50 µg/ml, respectively) whereas complexes 1, 2 and 3 (MIC: 12.5 µg/ml) have shown good activity than complex 4 (MIC : 50 µg/ml ) in comparison to standard against S. aureus. Further, complexes 1 and 2 (MIC : 6.25 µg/ml and 6.25 µg/ml, respectively) exhibit better activity against L.monocytogenes as compared to complexes 3 and 4 (MIC : 25 µg/ml and 50
17
ACCEPTED MANUSCRIPT µg/ml, respectively). However, metal complexes were found to exhibit significant MIC’s results against S. typhimurium and V.cholerae as compared to P.aeruginosa in comparison to
T
standard control. From the data presented in Table 5 and 6, it is evident that the complexes
IP
which possess lower MIC’s showing effectual zone of inhibition and hence active bacterial
SC R
agents.
A comparative evaluation of antibacterial results suggested that the metal complexes, 1-4 demonstrated enhanced antibacterial activity as compared to free ligands, L1 and L2 as
NU
previously accounted [4]. The enhancement in the antibacterial activity of the complexes as
MA
compared to free ligands can be supported by Tweedy’s chelation theory [54]. It is known that chelation enhances the ligand to act as more powerful and potent bactericidal agents by
D
inhibiting the bacterial growth, thus zones of inhibition of metal complexes were found to be
TE
higher compared to its free ligands. The chelation reduces the polarity of metal ion considerably because of the partial sharing of its positive charge with the hetero donor atoms
CE P
of the ligand and also due to π-electron delocalization over the whole chelate ring system. The lipids and polysaccharides are the vital constituents of the cell wall and membranes
AC
which are ideal for metal ion interaction. Apart from this, the cell walls also possess phosphates, carbonyl and cystenyl ligands which assist in sustaining the integrity of the membrane by acting as a diffusion barrier and also provide suitable sites for binding [55]. Furthermore, the diminution in polarity boost the lipophilic character of the chelates and thus an interface between the lipid and metal ion is preferred which may subsequently breakdown the permeability barrier of the cell resulting in involvement with the normal cell processes and lead to the blockage of metal binding sites in the enzymes of the microorganisms. These metal chelators may also held in perturbing the respiration process of the cell and thus obstruct the synthesis of proteins that consequently inhibits further growth of the organism. Further, it has been reported that metal complexes containing Schiff base ligand with nitrogen
18
ACCEPTED MANUSCRIPT and oxygen donor atoms might hinder enzyme production and that result in cell death [56]. The variation in the activity of the metal complexes against different microbes depend either
T
on the difference in the ribosomes of the microbial cells or on the impermeability of the cells
IP
of the microbes. The lower activity of complexes as compared to others may be attributed due
SC R
to low lipid solubility, so that the metal ion may not be able to reach the favourable site of action of the cell wall to get interfere with the normal cell activity. Although chelation itself play a significant role in determining antibacterial behaviour of the complexes but
NU
concurrently different factors such as solubility, size, dipole moment, coordinating sites,
MA
redox potential of metal ion, solubility, bond length between metal and the ligand, geometry of complexes, steric, pharmacokinetic, concenteration and hydrophobicity have substantial
D
influence on the antibacterial activity. Thus, it is apparent from the results that enhanced
TE
antibacterial activity of the metal complexes may not be only due to chelation but also
CE P
intricate combinations of several other factors as well [57-60].
4.2. In vitro antioxidant activity
AC
It is well established earlier that derivatives of isatin and benzothiazole/thiazole exhibit significant antioxidant activity which manifested them as potentially effective drugs [20-22, 61] hence, we carried out a methodical analysis to investigate the antioxidant efficacy of free ligands and metal complexes by DPPH method using L-ascorbic acid (standard). The antioxidant data of the compounds at variable concenterations (5-20 µM) and concenteration dependent curves have been shown (Table 7 and Fig. 10) which revealed that chelation effect exhibits metal complexes (1-4) as effective antioxidants in comparison to the respective free ligands, L1 and L2 [62]. While the comparative assessment of antioxidant data of metal complexes (1 and 2) of L1 have shown pronounced scavenging activity whereas complexes (3 and 4) of L2 have resulted moderate activity in comparison to L- ascorbic acid which may be
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ACCEPTED MANUSCRIPT explained in terms of coordination environment and the redox properties of metal ions. It has been reported that the redox properties of the complex depend on various factors such as
T
chelate ring size, axial ligation, degree of unsaturation in the chelate ring [63]. Therefore, it
IP
may be concluded that these complexes could be of therapeutic importance for further in vivo
SC R
studies in the field of pharmacy.
4.3. In vitro cytotoxic activity
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On the basis of considerable outcomes obtained after antibacterial and antioxidant screening,
MA
it was considered sensible to explore the cytotoxic potential of newly synthesized 5-chloro isatin derived Schiff base compounds against MCF7, HeLa and HepG2 cancer cell lines of
D
different histological origins by determining the number of viable cells surviving after
TE
incubation with the Schiff base compounds for the specified time period using MTT method [34]. The cytotoxicity assay implies a variable cytotoxic trend of synthesized compounds
CE P
against above mentioned cell lines which can be ascribed due to the intrinsic cytotoxic potential of these compounds. The various curves of dose dependent effects of all the
AC
examined compounds on cell viability of different cancer cell lines and normal cells (PBMC) are showed in Fig. 11. The results from experimental data demonstrated an increment in cytotoxicity in the cell lines with increasing the exposure to drug concenteration. The absorption values were articulated as the cell viability in percent, according to the control group as 100%. The assays were carried out in triplicate on three independent experiments. The concenteration entailed for 50% inhibition of cell viability (IC50) was calculated using the software “Prism 3.0”. The cytotoxic potential of all the examined compounds against cancer cell lines with IC50’s < 3.60µM (Table 8). In view of structure activity relationships, numerous correlations can be concluded from the cytotoxic data of tested compounds. On evaluating the cytotoxic potential against the MCF7,
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ACCEPTED MANUSCRIPT HeLa and HepG2 cancer cell lines, ligands L1 and L2 were found to be least active in comparison to metal complexes under similar conditions. The experimental IC50 data
T
revealed that complex 1 demonstrates relatively greater cytotoxic effect than other metal
IP
complexes against all cancerous cell lines with substantial IC50 values of 1.06±0.18 µM
SC R
(MCF7), 1.86±1.30 µM (HeLa) and 2.44±0.61µM (HepG2). The complex 2 also displayed significant activity against MCF7 and HeLa with IC50
