Four new copper complexes have been synthesized and isolated quantitatively. These have been obtained in crystallized form. These have been structurally characterized and the spectroscopic results are in harmony with structural elucidation results. Moreover, the biological relevance has been ascertained through their DNA-binding affinity (experimental and theoretical) as well as their anti-oxidant and anti-bacterial activity.
3.1 FTIR study
FTIR spectroscopy of the pure samples of the complexes revealed all the peaks which were expected in the structures of the synthesized complexes. The most prominent peaks were those of the stretching vibrations of the carboxylate moiety. The asymmetric stretch of the carboxylate moieties of all the complexes were observed in the range 1621-1544 cm-1 while the symmetric stretching vibrations were in the range 1465-1428 cm-1. The difference between the two peaks ∆ν for complexes 1-4 was 200, 189, 200 and 216 cm-1, respectively. These ∆ν values were in the range (150-250 cm-1) typical of bridging bidentate coordination mode of the carboxylate moiety [19]. When the ∆ν values are below 150 cm-1, it shows a chelate bidenatate coordination mode while ∆ν values above 250 cm-1 has been found to indicate monodentate or uncoordinated carboxylate moiety [19]. Thus for metal carboxylate complexes, ∆ν value can give valuable clue regarding the nature of carboxylate moiety.
Aromatic C-H was observed on 3058, 3078, 3074 and 3040 cm-1, respectively for complexes 1-4. A closely lying peak was that of methylene CH2 which was observed just below 3000 cm-1 for all the complexes. Similarly, ligand to copper bond was indicated by Cu-O bond stretching observed at 482, 493, 503 and 508 cm-1, respectively for complexes 1-4 as observed for the same functionality in other structurally characterized copper(II) complexes [20,21]. The aromatic C=C bond was also indicated by its respective peaks. The FTIR spectra were in agreement to the structural data of the complexes.
3.2 Structural description
Molecular structures of complexes 1-4, drawn in mercury have been shown in Fig. 1 (A-D) while their crystallographic parameters have been listed in Tables 1 and 2. The complexes crystallized in triclinic crystal system with space group P-1. Each complex is dinuclear with two copper ions bonded by four carboxylate ligands in syn-syn fashion. These four Cu-O bonds around each copper ion constitute the square base of the square pyramidal geometry around each copper. The apical position of the square pyramid is formed by the oxygen atom of the DMSO molecule in complexes 1 and 2 and by the nitrogen atom of 3-methylpyridine and 3-bromopyridine in 3 and 4, respectively.
The paddlewheel complexes of copper(II) have both copper ions drawn closer enough by the four OCO bridges so that the inter-nuclear distance between the two ions is comparable to a covalent bond. However, a bond cannot be formed between the two copper ions along that axis because there is no suitably oriented orbital on each copper to overlap and form a bond along that axis. The central Cu-Cu bond in these molecules is not a genuine bond, rather a bond drawn by the software owing to the shorter distance between two copper ions. A crystallography software/program usually draws a bond between such ions owing to the short distance but that bond is not meaningful and metal ions are actually 5-coordinated square pyramidal in these complexes.
The metal ion is bulging out of the square base towards the apical position in such complexes. It means that the Cu···Cu ̶ O angle is less than 90o and the O ̶ Cu ̶ O/Napical is greater than 90o. The Cu···Cu distance in paddlewheel complexes is sensitive to the basic strength of the carboxylate ligand. Thus varying the basic strength of the donor atoms, this distance can be increased or decreased and the properties dependent on this parameter can be tuned. Changing the carboxylate ligand from trifloroacetato (2.766(1) Å) to trimethylacetato (2.630(18) Å) the Cu···Cu distance is shortened as the basicity of the ligand is increased [22]. The corresponding Cu···Cu distances in the synthesized complexes lie in the range 2.619(5) - 2.671(13) Å. The basic strength of the carboxylate ligands is moderate so Cu···Cu separation is less than that of the trifloroacetate based complexes [22].
Table 1: Structural refinement parameters of complexes 1-4.
Complex
|
1
|
2
|
3
|
4
|
Empirical formula
|
C46H44Cu2O12N2Br2
|
C42H58Cu2O12N4
|
C42H54Cu2O15S3
|
C42H50N4Cu2O19S3
|
Formula weight (g mol-1)
|
1103.73
|
938
|
1100.24
|
608.12
|
Temperature (K)
|
296 (2)
|
296 (2)
|
296(2)
|
296(2)
|
Space group
|
P -1
|
P -1
|
P -1
|
P -1
|
a (Ǻ)
|
10.1938(18)
|
10.2587(5)
|
10.2769(3)
|
8.3318(3)
|
b (Ǻ)
|
11.1038(19)
|
11.0604(7)
|
10.5506(3)
|
9.4496(5)
|
c (Ǻ)
|
11.3014(17)
|
11.2080(6)
|
13.1292(4)
|
16.8061(7)
|
α (°)
|
86.680(6)
|
87.823(3)
|
77.156(2)
|
95.045(4)
|
β(°)
|
66.562(6)
|
66.689(2)
|
70.7940(10)
|
100.412(3)
|
γ (°)
|
79.318(6)
|
78.992(3)
|
69.967(2)
|
92.032(4)
|
Volume (A3)
|
1153.1(3)
|
1145.42(11)
|
1253.32(7)
|
1294.57(10)
|
Z
|
1
|
1
|
1
|
2
|
ρ(calc.) (g cm-3)
|
1.589
|
1.360
|
1.458
|
1.560
|
Absorption coeff. (mm-1)
|
2.718
|
0.991
|
1.081
|
1.063
|
F(000)
|
558
|
492
|
574
|
630
|
Reflections collected
|
3101
|
3367
|
5419
|
4840
|
Goodness-of-fit on F2
|
1.047
|
1.072
|
1.049
|
1.042
|
Final R index [I > 2σ(I)]
|
0.0502
|
0.103
|
0.0427
|
0.0791
|
Table 2: Selected bond lengths and angles of complexes 1-4.
Bond Lengths Å
|
|
1
|
2
|
|
3
|
4
|
Cu(1)-O(1)
|
1.957(3)
|
1.980(4)
|
Cu(1)-O(1)
|
1.971(18)
|
1.964(3)
|
Cu(1)-O(2)
|
1.967(3)
|
1.973(4)
|
Cu(1)-O(2)
|
1.980(18)
|
1.958(3)
|
Cu(1)-O(3)
|
1.973(3)
|
1.959(4)
|
Cu(1)-O(3)
|
1.958(18)
|
1.965(4)
|
Cu(1)-O(4)
|
1.966(4)
|
1.974(4)
|
Cu(1)-O(4)
|
1.976(18)
|
1.965(4)
|
Cu(1)-N(1)
|
2.187(3)
|
2.171(5)
|
Cu(1)-O(5)
|
2.162(17)
|
2.138(3)
|
Cu(1)-Cu(1)
|
2.639(9)
|
2.671(13)
|
Cu(1)-Cu(1)
|
2.619(5)
|
2.639(10)
|
Bond Angles (o)
|
O(2)-Cu(1)-O(3)
|
89.41(12)
|
89.20(2)
|
O(2)-Cu(1)-O(3)
|
89.37(9)
|
88.43(19)
|
O(1)-Cu(1)-O(2)
|
168.02(11)
|
166.84(17)
|
O(1)-Cu(1)-O(2)
|
168.79(7)
|
167.77(15)
|
O(4)-Cu(1)-O(3)
|
168.12(11)
|
166.76(17)
|
O(4)-Cu(1)-O(3)
|
168.66(7)
|
167.75(16)
|
O(1)-Cu(1)-O(3)
|
90.01(12)
|
89.70(2)
|
O(1)-Cu(1)-O(3)
|
89.62(9)
|
89.58(19)
|
O(4)-Cu(1)-O(2)
|
88.82(12)
|
88.30(2)
|
O(4)-Cu(1)-O(2)
|
89.50(9)
|
89.52(18)
|
O(1)-Cu(1)-O(4)
|
89.29(12)
|
89.80(2)
|
O(1)-Cu(1)-O(4)
|
89.31(8)
|
89.87(19)
|
O(2)-Cu(1)-N(1)
|
98.40(12)
|
95.92(18)
|
O(2)-Cu(1)-O(5)
|
96.34(8)
|
95.35(14)
|
O(3)-Cu(1)-N(1)
|
96.40(12)
|
93.87(19)
|
O(3)-Cu(1)-O(5)
|
97.57(7)
|
96.88(14)
|
O(1)-Cu(1)-N(1)
|
93.50(12)
|
97.24(19)
|
O(1)-Cu(1)-O(5)
|
94.86(8)
|
96.33(15)
|
O(4)-Cu(1)-N(1)
|
95.48(12)
|
99.32(19)
|
O(4)-Cu(1)-O(5)
|
93.76(7)
|
95.88(15)
|
Supra-molecular chemistry: Since there is no O/F/N─H∙∙∙O/F/N interactions in the lattices of all the four complexes, there is no intermolecular H-bonding interactions. However, there are some C─H∙∙∙O interactions in complex 4. A further insight into these interactions was obtained by performing the Hirshfeld surface analysis. These intermolecular interactions have been indicated by bright red spots on the Hirshfeld surfaces mapped over dnorm for complexes 1-4 as shown in Fig. 2 A-D. These contacts have been expanded to a neighboring molecule as well across the Hirshfeld surface as evident in these figures for each molecule. The fewer C─H∙∙∙O interactions in complex 4 have been shown in Fig. 2E as dotted lines.
The contributions of H∙∙∙C, H∙∙∙N, H∙∙∙O and H∙∙∙H interactions to the overall packing of complexes 1-4 have been indicated in the 2D fingerprint plots shown in Fig. 3. Amongst H∙∙∙C, H∙∙∙N, H∙∙∙O and H∙∙∙H interactions, the highest percentage is that of H∙∙∙H linkage indicating a major contribution of the van der Waal’s forces in the crystal lattice of the complexes 1-4. A considerable contribution of H∙∙∙C interactions indicates contribution of C─H∙∙∙π interactions as well. There is very small percentage of C∙∙∙C interactions indicating small overlap of aromatic rings and less possibility of π ─π staking interactions.
3.3 Stability studies in solution form
In order to ascertain whether the structure and geometry of the synthesized complexes remains intact in solution form, their solutions were subjected to absorption spectroscopic studies at various intervals. Since the absorption spectrum of a copper(II) complex is a function of geometry (octahedral, square planar, square pyramidal, trigonal bipyramidal and tetrahedral) around copper ion, the spectra for solution studies were taken in visible region of spectrum as shown in Fig. 4. These spectra show that the complexes remain unchanged for at least 24 hr in common solvents.
3.4 DNA-binding studies of the complexes
DNA-binding potential of the complexes was explored using four different techniques. These are discussed below in detail one by one.
UV-Visible spectrophotometry
The complexes gave rise to ligand based well-defined absorption peaks in the UV-region following the Beer-Lambert’s Law. It means that reduction in concentration of the complexes will result in reduction in their absorbance value as per the Beer-Lambert’s Law. Adding incremental quantity of DNA will result the reduction in concentration of the complex owing to the complex-DNA adduct formation. This has been shown for 1-4 in Fig. 5, where the absorbance value of the pure complex represented by the uppermost plot has suffered proportionate diminution on DNA-addition. This indicates that each complex effectively binds with DNA majorly through intercalative mode of binding [5,6]. Since there is pronounced reduction in absorbance of each complex on addition of DNA and the wave length of complex suffers no appreciable change along the successive addition, such binding mode is called intercalative binding where the complex molecules simply insert between the DNA base pairs [5,6]. This binding mode is not unexpected because the complexes have plenty planar aromatic moieties in the structure through which these molecules get inserted into the DNA double strand. The binding ability has been quantitatively ascertained by calculating the binding constant Kb using a simplified form of the Benesi-Hildebrand equation [23]. This was done by plotting the reciprocal molar concentration of DNA vs. the relative absorbance value as shown in Fig. 6 and the Kb value was calculated from the slope to intercept ratio of the plot. The Kb values calculated were 1.85, 2.05 and 1.88 and 1.92 × 104 M-1, respectively for complexes 1-4. Similar DNA binding potency was observed for other copper complexes as well [24].
DNA-binding study using viscometry
The viscosity of DNA is sensitive to its strand length; longer strains have higher viscosity than shorter strands. Planar molecules are able to intercalate between base pairs of DNA double strands which will lengthen the DNA strand resulting in higher viscosity [5,6]. When the complexes 1-4 were added gradually to the DNA solution, the viscosity of DNA solution increased steadily as seen in the Fig. 7. This also indicated the intercalative mode of complexes with DNA. The same mode was indicated by UV-Visible spectroscopy as well.
DNA-binding study using spectrofluorimetry
Ethidium bromide (EtBr) is a potent intercalator into the DNA-base pairs. It has high emission intensity in DNA-bound form while in free state its emission intensity is quenched by the buffered solvent medium. If another molecule competing for intercalation into the DNA-base pairs is added to the solution containing DNA-EtBr adduct, it will compete with EtBr and will tend to replace the later [25]. If the added molecule successfully replaces the EtBr and gets inserted into the DNA strand, the emission intensity of the EtBr will be reduced since the quantity of the DNA-bound and emissive EtBr has been reduced. On successive addition of the synthesized complexes 1-4 to the solution containing DNA-EtBr adduct, its emission intensity was reduced on each addition. This showed that the complexes are able to compete successfully with EtBr for intercalation into the DNA base pairs as shown in Fig. 8 for complexes 1-4. This technique indicated that the complexes are more potent intercalators than EtBr and that the mode of binding of the complexes with DNA is intercalation as deduced from the previous techniques.
DNA-binding study using cyclic voltammetry
Change in the concentration of an electro-active compound can be followed by cyclic voltammetry where the resulting current is changed with change in concentration. In this context, cyclic voltammograms of the complexes were recorded before and after adding variuos quantities of DNA. The voltammograms did suffer deminution in current on addition of DNA which indicated binding with the complexes as shown in Fig. 9. The diminution in current of the complexes on addition of DNA was used to calculate the binding constant of the complexes with DNA using plots shown in Fig. 10. The results of the four techniques on DNA-binding study are in harmony with each other.
3.5 Molecular Docking Studies
The binding interactions of the compounds 1-4 with DNA were assessed through molecular docking analysis, which allowed us to examine how these complexes interact with their target. Fig. 11 shows the best-docked conformations of complexes 1-4. In all instances, the ligands bind to DNA's major groove, with binding scores falling within the range of -6.5 to -7.4 kcal/mol. In all cases multiple conventional intermolecular hydrogen bonds are developed between the ligands and both strands of DNA base pairs showing a dual binding mode. The most effective among the docked complexes is compound 4, with a docking score of -7.3 kcal/mol. It acts as four hydrogen bond acceptor through the oxygen of -OS(CH3)2 and -NO2 groups. One hydrogen bond is formed with A:DA5 and three hydrogen bonds with B:DA17 and B:DA18 residues. It is also involved in π-donor hydrogen bonding through the phenyl ring and A:DG4 residue, π-sulphur interaction with A:DA6 and π-π T-shaped interaction with B:DA18 and DG10, indicating its strong binding affinity. Compounds 1-2 show equal binding affinity with a binding score of -6.9 kcal/mol. Compound 1 acts as two H-bond acceptor and interacts with A:DG2 and B:DC21. Further interactions of 1 with DNA include π-anion interaction with B:DT19, π-π T-shaped with B:DT20 and π-alkyl interaction with B:DG22. Compound 2 forms two conventional hydrogen bonds with A:DA6 and B:DA18, as well as a carbon-hydrogen bond with A:DT7, π-anion interaction with A:DG4, π-π T-shaped interaction with A:DC3 and B:DA17, and π-alkyl interaction with B:DT20. Compound 3 shows the least binding score of -6.5 kcal/mol and forms three hydrogen bonds with A:DC3 and B:DC21. The other binding interactions of 3 include carbon-hydrogen interactions, π-anion, π-π T-shaped, π-donor hydrogen bonds, and π-alkyl interactions. In summary, the molecular docking analysis reveals a robust binding relationship between these complexes and DNA, indicating their potential as promising anticancer agents.
3.6 Anti-oxidant activity of complexes
Another activity of biological relevance is the ability of a complex to quench free radicals which are produced continuously in bio-systems. In order to ascertain this activity, the synthesized complexes have been treated with a free radical DPPH and the decrease in the concentration of DPPH indicated reaction between the two and quenching of the later. Plots in Fig. 12 show that with increase in concentration of complexes 1-4, their scavenging activity also increased. The activity of each of complexes 2 and 3 was comparable to that of the ascorbic acid which was used as standard. The relative inhibition of the complexes showed that these complexes can be used to quench the free radicals as well.
3.7 Anti-bacterial studies
The in vitro antibacterial activity of the synthesized complexes against three Gram-positive strains (Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus) and a Gram-negative strain (Escherichia coli). The activity was measured by calculating the area of zone of inhibition by the complexes and was classified accordingly as in the literature [12]. Table 3 lists the observed activity of the complexes where the highest activity was observed for complexes 1 and 4 (zone of inhibition 20 and 21) against Micrococcus luteus and Staphylococcus aureus. However, their activity against Bacillus subtilis and Escherichia coli was low (zone of inhibition = 17 mm for both complexes). Complexes 1 and 4 exhibited good activity against the rest of the bacterial species. Only complex 2 showed significant activity against E. coli while moderate and low activities were exhibited against the rest of the species. The significant activity of complexes 1 and 2 might be due to the presence of bromo- and nitro-groups, respectively which are able to promote covalent and ionic interactions with cellular membranes and tissues of target bacteria. Anti-bacterial activity indicates potential biological relevance of the synthesized complexes.
Table 3: Antibacterial data of the synthesized complexes
Bacterial strain
Parameter
|
Bacillus
subtilis
|
Micrococcus
luteus
|
Staphylococcus
aureus
|
Escherichia
Coli
|
Average zone of inhibition (mm)
|
Complex 1
|
17
|
20
|
20
|
17
|
Complex 2
|
18
|
17
|
14
|
19
|
Complex 3
|
16
|
16
|
14
|
13
|
Complex 4
|
17
|
21
|
21
|
17
|
Cefixime
|
27
|
30
|
32
|
31
|
Minimum Inhibitory Concentration (mg/mL)
|
Complex 1
|
0.25
|
0.25
|
0.25
|
0.25
|
Complex 2
|
0.23
|
0.23
|
0.23
|
0.23
|
Complex 3
|
0.20
|
0.20
|
0.20
|
0.20
|
Complex 4
|
0.25
|
0.25
|
0.25
|
0.25
|
Concentration of each complex: 1 mg/mL in DMSO. Cefixime: 1 mg/mL.