Introduction

Cancer, one of the most staggering and death causing diseases in the world involves abnormal cell proliferation in the body (Mahmoud et al. 2018; Thirunavukkarasu et al. 2018; Devi et al. 2018a, b). On one hand cisplatin and its derivatives, on the other hand, a large number of metal complexes are introduced in the treatment of a variety of cancers. In 2015 about 90.5 million individuals were accounted for to have cancer (Srividya et al. 2019). Along these lines, there have been instances of various sorts of tumors which reach up to an incredible number of 14.1 million every year and the sickness has nearly asserted about 8.8 million lives (15.7%) till date (Global et al. 2016). In this regard, metal complexes having sulphur and nitrogen atoms in the core moiety of ligands are one of the growing interest fields of coordination chemistry (Mohammadtabar et al. 2016), owing to activity binding and cleaving the DNA under physiological conditions (Muralisankar et al. 2016). For the past few decades, metal complexes like platinum and copper complexes occupy an eminent position in cancer diagnosis and treatment (Wernyj et al. 2004; Ramadan et al. 2018). Some of the metal complexes having anticancer activity were shown in Fig. 1 (Muralisankar et al. 2016; Kelland et al. 2007). However, it was observed that there are several drawbacks regarding the usage of complexes like dose limiting efficiency, limited clinical usage to several tumors, a wide range of side effects (Wernyj et al. 2004). Hence, the chemists make serious efforts to design, develop, synthesis new, safe drug-like molecules with high selectivity, maximum efficacy and minimum toxicity. One among them Schiff base metal complexes have a broad spectrum of pharmacological properties (Abdel-Rahman et al. 2015, 2016; Abdel Aziz et al. 2017a, b). These complexes also remarkable biological activities like antioxidant, antitumor antimicrobial, anti-inflammatory, antimalarial (Khedr et al. 2012; Mesbah et al. 2018; Miri et al. 2013; Mumtaz et al. 2016; El-Boraey and El-Salamony 2019).

Fig. 1
figure 1

Some representative examples for the metal complexes having the anticancer activity

Full size image

In view of the aforementioned biological importance of the metal complexes and Schiff bases, herewith we report the novel metal complexes of Pd(II), Zn(II), Cu(II), Ni(II) and Co(II) with Schiff base in a single molecular framework and their potential in vitro anticancer, antioxidant, antimicrobial activities and in silico studies (DFT and molecular docking).

Results and discussion

Synthesis

The new ligand (L) was prepared by treating 4-aminobenzoic acid with 2-aminonicotinaldehyde under stirring conditions (Scheme 1). The metal(II) complexes (1–5) were synthesized by mixing the different metal chlorides with ligand in methanol. The resulting mixtures were refluxed for 4 h at 70 °C (Scheme 2). The complexes are stable at room temperature and are non-hygroscopic. Analytical data of the compounds are presented in Table 1. All the complexes are soluble in DMSO and dimethylformamide but insoluble in common organic solvents. The authenticities of the synthesized compounds were ascertained using various spectroscopic methods and elemental analysis.

Scheme 1
scheme 1

Synthetic route of ligand (L)

Full size image
Scheme 2
scheme 2

Synthetic route of the mental(II) complexes

Full size image
Table 1 Analytical and physical parameters for the ligand (L) and its metal complexes (1–5)
Full size table

Spectroscopy

The rationale positions of the selected characteristic IR bands unambiguously unveiled the formation of ligand 4-(((2-aminopyridin-3-yl)methylene)amino)benzoic acid and its metal complexes. The important absorption frequencies are presented in Table 2. The ligand showed the strong band at 1629 cm−1 due to ν(C=N), on the other hand, this band is lowered by 20–30 cm−1 in the complexes pointing to the fact that the nitrogen of this group is involved in binding with the metal ion (Ebrahimi et al. 2014; Abu Al‐Nasr and Ramadan 2013; Ramadan et al. 2014; Abdel Aziz et al. 2017a, b). The amine group nitrogen of the ligand showed the band at 3420 cm−1 due to ν(ΝH2) has been found lower frequencies by (3401–3371 cm−1) in the complexes, this kind of shift suggested that amine nitrogen is bonded to the metal (Burns 1968; Jyothi et al. 2015). The carboxylic acid (C=O) and (C=N)py stretching frequencies of the ligand occurring at 1701 and 1567 cm−1. These bands do not undergo any perceptible shifts in the metal complexes suggesting that there are no interactions between oxygen and nitrogen of these groups. The aforementioned results proved that the ligand coordinating with metal ions through the amine nitrogen atom and the azomethine nitrogen. In complexes (1–5) the peaks observed in the range of 478–520 cm−1 and 275–321 cm−1 corresponds to υ(M–N) and υ(M–Cl), respectively. The conductance measurements of the metal complexes (1–5) in DMF solution were made at 10−3 mol dm−3 concentrations. All the complexes showed only residual molar conductance values (11–19 Ω−1 cm2 mol−1) and hence they may be considered as non-electrolytes (Saif et al. 2011; Firdaus et al. 2009). This implies that both the two chloride anions associated with these complexes are present inside the coordination sphere. Thus these compounds may be formulated as [ML2Cl2]. The results are given in Table 1. The UV–Visible spectra of the ligand exhibited the bands at 30,390 cm−1 and 33,245 cm−1 corresponding to n → π* and π → π* transitions, respectively. The electronic spectra of Co(II) complex (1) exhibits three bands around 9000 cm−1 (4T1g(F) → 4T2g(F) (\(\vartheta\)1)), 18,500 cm−1 (4T1g(F) → 4A2g(F) (\(\vartheta\)2)) and 20,400 cm−1 attributable to 4T1g(F) → 4T1g(P), respectively, characteristic of octahedral geometry (Shukla et al. 2008; Chandra 2004). The octahedral geometry is also more supports the \(\vartheta\)2/\(\vartheta\)1 value, which fall in the range (2.02) observed for the octahedral complex (Devi et al. 2018a, b). The Racah inter electronic repulsion parameter (B) value for the Co(II) complex was found to be 756 cm−1 which is lower than the free ion value (B1 = 971 cm−1) indicates a good overlap of the orbitals involved (Konakanchi et al. 2018a, b). Further, the nephelauxetic effect parameter (β = B/B1) value (0.78) is less than one suggesting that M–L bonds are covalent in character. The results are given in Table 3. The Ni(II) complex exhibits three peaks in the region of 9800, 15,900 and 25,800 cm−1. These peaks have been attributed, respectively to the transitions 3A2g(F) → 3T2g(F), 3A2g(F) → 3T1g(F) and 3A2g(F) → 3T1g(P) of octahedral geometry (Nigam et al. 2000; Saleh 2005; Saif et al. 2012). And also the value \(\vartheta\)2/\(\vartheta\)1 (1.61) further supports the octahedral geometry. The values of B (820 cm−1) and β (0.80) observed for Ni(II) complex indicate that the orbital overlap and M–L bonds are covalent in character (Devi et al. 2018a, b). The Cu(II) complex reveals a peak at 18,351 cm−1 and a shoulder at 13,825 cm−1, respectively as is usually expected for square planar geometry (Raman et al. 2004). Zn(II) complex shows no bands in the visible region as is expected for d10 system and also showed the peak around 28,500 cm−1 due to charge transfer. Octahedral geometry has been proposed based on the empirical formulae (Ganesan et al. 2019; Devi et al. 2012), Pd(II) complex show three peaks at 18,700 (1A1g → 1A2g), 21,500 (1A1g → 1B1g) and 24,850 cm–1 (1A1g → 1Eg) these transitions characteristic of square-planar geometry (Goggin et al. 1972). Further the band at 28,000 cm−1 is due to the charge transfer. Further, the \(\vartheta\)2/\(\vartheta\)1 value (1.14) also supports the square planar geometry (Gajendragad and Aggarwala 1975). The Co(II), Ni(II) complexes observed magnetic moment (µeff) values are 4.95, 3.22 BM, respectively suggests the octahedral arrangement (Omar and Mohamed 2005; Patil and Kulkami 1984; Ramadan 2012; Mohamed et al. 2001; Cotton et al. 1999; Kavitha , 2012). From the literature, the square planar copper(II) complexes, µeff values are observed in the range of 1.82–1.86 B.M. (Sabastiyan and Venkappayya 1990). In the present investigation, the µeff value is 1.83 B.M. which indicates a square planar arrangement. The Zn(II) and Pd(II) complexes are diamagnetic. The results are given in Table 3. In the 1H NMR spectra of the ligand, the carboxylic acid attached O–H proton showed the signals at 12.93 ppm. In the complexes, this O–H proton observed the signals around 12.82–12.07 ppm. In the ligand, the azomethine CH proton and NH2 attached pyridine ring was observed at 8.72 ppm and 7.38 ppm, respectively. In the spectra of complexes, the azomethine CH and NH2 protons appeared in the range of 8.74–8.61 ppm (CH=N) and 7.33–7.28 ppm (NH2), respectively. 13C NMR spectra of the ligand showed the chemical shift values at 167.49, 163.88 and 158.44 ppm indicate the carboxylic acid (C=O), (C=N) and (C–NH2) groups, respectively. The aromatic carbons were observed in the range of 158.44–112.34 ppm. The 1H and 13C NMR spectra of the ligand and its metal complexes were shown in Figs. 2, 3 and 4, respectively.

Table 2 Infrared absorption frequencies (cm−1) of ligand (L) and its metal complexes (1–5)
Full size table
Table 3 Electronic spectral data, ligand field parameters and magnetic data of the complexes (1–5)
Full size table
Fig. 2
figure 2

a 1H NMR and b 13C NMR spectrum of the ligand (L)

Full size image
Fig. 3
figure 3

a 1H NMR and b 13C NMR spectrum of zinc(II) complex (4)

Full size image
Fig. 4
figure 4

a 1H NMR and b 13C NMR spectrum of palladium(II) complex (5)

Full size image

DFT computations

Required calculations were made using the Gaussian 09/DFT program package (Frisch et al. 2010). Beck’s three parameter hybrid exchange functional B3 (Becke 1993) in conjunction with Lee-Yang-Parr (LYP) correlation functional (Lee et al. 1988) using enlarged basis set 6-311+ +G(d,p) /SDD was employed for the purpose. These basic functionals are more accurate and reduce the computational rate and include few relativistic effects in the calculations (Bergner et al. 1993; Kaupp et al. 1991; Dolg et al. 1993). The optimized molecular geometry for the ligand and its metal complexes (1–5) were shown in Fig. 5. The results of bond angles, bond lengths and dihedral angles were presented in Tables 4 and 5. On the optimization of geometry, the observed global minimum energy of the compounds were observed at − 816.95688 Hartree (ligand), − 2698.19907 Hartree (1), − 2723.30339 Hartree (2), − 2749.76705 Hartree (3), − 2779.56640 Hartree (4) and − 1865.00019 Hartree (5), respectively.

Fig. 5
figure 5

Optimized structures along with atomic numbering of a ligand, b complex 1, c complex 2, d complex 3, e complex 4 and f complex 5

Full size image
Table 4 Optimized geometry parameters of ligand (L)
Full size table
Table 5 Selected bond lengths and bond angles of the complexes (1–5)
Full size table

Molecular electronic properties i.e. electron affinity (A), ionization potential (I), chemical potential (µ), global hardness (\(\eta\)) and global electrophilicity power (\(\omega\)) of ligand and its metal complexes obtained from frontier molecular orbital energies comprising of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) using the following expressions (Gece 2008; Fukui 1982; Koopmans 1933; Parr et al. 1999).

$${\text{I }}\, = \, - \,{\text{E}}_{{{\text{HOMO}}}} ;\,{\text{A}}\, = \, - \,{\text{E}}_{{{\text{LUMO}}}} ; = \,\left( { - \,{\text{E}}_{{{\text{HOMO}}}} \, + \,{\text{E}}_{{{\text{LUMO}}}} } \right)/{2};\,\mu \, = \,\left( {{\text{E}}_{{{\text{HOMO}}}} \, + \,{\text{E}}_{{{\text{LUMO}}}} } \right)/{2};\,{\text{and}}\,\omega \, = \,\mu^{{2}} /{2}$$

HOMO and LUMO are known as frontier molecular orbitals. They are important in quantum chemistry, as they determine the molecular reactivity of conjugated systems (Choi and Kertez 1997) and the ability of a molecule to absorb electromagnetic radiation. HOMO plays the role of an electron donor, whereas LUMO act as an electron acceptor (Gece 2008; Fukui 1982). The results are presented in Table 6 and illustrated in Fig. 6. The molecules under consideration, the FMO energy gap was found to be L > 3 > 2 > 5 > 1 > 4. Generally, molecules with a small frontier orbital gap are readily polarizable and normally exhibit high chemical reactivity and low kinetic stability (Sinha et al. 2011; Lewis et al. 1994; Kosar and Albayrak 2011). The FMO gap of metal complexes (1–5) are small compared to the ligand, hence the metal complexes are more polarizable than the ligand (Table 6). As the chemical potential (μ) for the compounds being investigated are negative (Table 6) it is stable.

Table 6 Frontier molecular orbital parameters of ligand (L) and its metal complexes (1–5)
Full size table
Fig. 6
figure 6

Frontier molecular orbitals of a ligand, b complex 1, c complex 2, d complex 3, e complex 4 and f complex 5

Full size image

Non-linear optical (NLO) behavior

DFT has been widely used as a powerful method to examine the NLO materials (Prasad and Wiliams 1991). To understand NLO behavior of the ligand and its metal complexes, computation of the total molecular dipole moment (μt) and its components, total molecular polarizability (αt) and its components, anisotropy of polarizability (Δα), and first order static hyperpolarizability (βt) was attempted according to Buckingham’s definition (Buckingham 1967) using density functional theory based on finite field approach. Generally, polarizabilities (αt) and first order hyperpolarizabilities (βt) were estimated to the response of compounds in presence of an applied electric field to predict cross-section of various scattering processes, molecular interactions and NLO properties of the system (Meyers et al. 1994; Hinchliffe and Munn 1985). βt is a third rank tensor. Therefore, it was measured as a 3 × 3 × 3 matrix. From the basis of Kleinman symmetry (Kleinman 1962), all the 27 components were condensed to ten components indicated as βxxx, βxxy, βxyy, βyyy, βxxz, βxyz, βyyz, βxzz, βyzz, βzzz. So, by using these components X, Y, Z we can calculate the μt, αt and βtot by the following equations.

$$\mu = \mu^{2}_{x} + \mu^{2}_{y} + \mu^{2}_{z}$$
(1)
$$\alpha_{o} = \frac{{\alpha_{xx} + \alpha_{yy} + \alpha_{zz} }}{3}$$
(2)
$$\Delta \alpha = 2^{ - 1/2} [(\alpha_{xx} - \alpha_{yy} )^{2} + (\alpha_{yy} - \alpha_{xx} )^{2} + 6\alpha^{2}_{xx} ]^{1/2}$$
(3)
$$\beta = (\beta^{2}_{x} + \beta^{2}_{y} + \beta^{2}_{z} )^{1/2}$$
(4)
$$\beta_{x} = \beta_{xxx} + \beta_{xyy} + \beta_{xzz}$$
(5)
$$\beta_{y} = \beta_{yyy} + \beta_{xxy} + \beta_{yzz}$$
(6)
$$\beta_{z} = \beta_{zzz} + \beta_{xxz} + \beta_{yyz}$$
(7)

In this work, αt, βt and µt were calculated using the DFT approach and the results are given in Table 7. It is usual practice to determine the NLO behavior of compounds by comparing its μt and βt with corresponding values of urea, which are 1.3732 Debye and 0.3728 × 10−30 cm5/ e.s.u, respectively. The calculated βt value of the ligand is 16.962 × 10−30 cm5/e.s.u, which was 45 times greater than urea. Hence, the ligand exhibits NLO properties. The complexes 2, 3 and 5 showed more first order hyperpolarizability values compared to the free ligand. The remaining complexes exhibited lesser first order hyperpolarizability values than the ligand. The non-linear optical activity was associated with the intra-molecular charge transfer, obtaining from the electron cloud movement through resonance (Arivazhagan and Jeyavijayan 2011). Hence, the ligand and its metal complexes 2, 3 and 5 are strong candidates for the expansion of NLO materials. The components of first order hyperpolarizability were also helpful to understand charge delocalization of the compounds.

Table 7 The dipole moment (in Debye), polarizability and first order hyperpolarizability (in 10−30 cm5/e.s.u ) values of ligand and its metal complexes (1–5)
Full size table

EPR spectral studies

EPR studies are performed to identify the number of unpaired electrons and type of the bonds between the ligand and its Cu(II) complexes. The EPR parameters are listed in Table 8 and Fig. 7. The EPR spectrum of the Cu(II) complex was recorded at room temperature and shows two bands, one of small intensity towards the low field and another one large intensity towards the high field. Based on these bands the values of \({g}_{\parallel }\) and \({g}_{\perp }\) have been calculated (Anees et al. 2019; Kneubuhl 1960). From the results, \({g}_{\parallel }\) = 2.22 and the other to \({g}_{\perp }=\) 2.05. i.e. > \({g}_{\parallel }{g}_{\perp }\), pointing out that metal ion contains its unpaired electron in its dx2− y2 orbital, and suggests a square-planar geometry (Konakanchi et al. 2018a, b). Kivelson and Neimann 1961) showed that \({g}_{\parallel }>\) 2.3 for ionic environment and < 2.3 for covalent compounds. From Table 8 that \({g}_{\parallel }\) obtained for the present complex is less than 2.3 indicating the covalent character of M–L band. According to Hathaway and Billing 1970), if the G value is larger than 4, the exchange interaction is negligible, whereas if its value is less than 4 indicates considerable interaction in solid complexes. In the present case G is found to be greater than 4, thus ruling out solid state exchange interactions between copper centers. Further comparison of \({{K}^{2}}_{\parallel }\) and \({{K}^{2}}_{\perp }\) values (Table 8) obtained points out that complex 3, \({{K}^{2}}_{\parallel }\)> \({{K}^{2}}_{\perp }\), suggest that out of plane pi-bonded (Konakanchi et al. 2018a, b). The spin–orbit coupling constant (λ) calculated using the relation.

$$g_{\parallel } = [2 - (8 \lambda /10 \, Dq)$$
Table 8 ESR spectral data of Cu(II) complex (3)
Full size table
Fig. 7
figure 7

ESR spectrum of copper(II) complex (3)

Full size image

The λ value for the complex 3 (− 442 cm−1) is found to be much lower than the free ion value λ0 (− 828 cm−1) which supports covalent character (Kavitha et al. 2013).

Biological evaluation

Antimicrobial screening

Microorganisms used in this work were three gram negative bacteria like Proteus vulgaris, Klebsiella pneumoniae and Pseudomonas aeruginosa, two gram positive bacteria like Staphylococcus aureus and Bacillus subtilis and few fungal strains like Aspergillus niger, Aspergillus flavus, Curvularia lunata, Rhizoctonia bataticola and Candida albicans by using disc diffusion method (Mallela et al. 2018; Arun et al. 2015). Streptomycin and Ketoconazole were chosen as reference drugs for bacteria and fungi, respectively. The MIC values of the tested compounds and reference drugs were expressed in µM. The results are present in Table 9 (antibacterial) and Table 10 (antifungal), respectively. From the results, the complex 3 exhibited excellent activity against S. aureus and P. vulgaris strains with MIC values of 4.21 and 8.18 µM, respectively and also these compunds showed significant activity against B. subtilis (MIC: 5.13 µM) and P. aeruginosa (MIC: 6.01 µM). The complex 5 exhibited broad spectrum activity against the strains with MIC values of 5.17 µM (S. aureus), 7.69 µg/mL (B. subtilis), 10.54 µM (P. vulgaris), 8.24 µM (K. Pneumoniae) and 7.25 µM (P.aeruginosa), respectively. Complex 4 against B. Subtilis (MIC: 11.50 µM), complex 2 against P. vulgaris (MIC: 21.01 µM) and complex 1 against P.aeruginosa (MIC: 10.91 µM) exhibited moderate activity compared to reference drug. The antifungal activity results are observed that the ligand is ineffective and it shows only marginal activity against the microorganisms tested. While complexes 2 and 3 have shown potent activity against A. niger (MIC = 3.94 µM) and R. bataticola (MIC = 6.24 µM), respectively compared to standard drug Ketoconazole. The complexes 3 and 5 exhibited significant activity against A. niger with MIC values of 4.17 and 40.3 µM, respectively. Complexes 1, 2 and 3 against C. albicans showed good activity with MIC values of 7.12, 7.99 and 8.18 µM, respectively. Complex 2 against A. flavus (MIC = 10.77 µM) and complex 4 against C. lunata (MIC = 6.41 µM) registered moderate activity. In few of the complexes showed the same level of activity as the ligand. This difference might be due to their difference in Gram status.

Table 9 MIC of the synthesized compounds against the growth of bacteria
Full size table
Table 10 MIC of the synthesized compounds against the growth of fungi
Full size table

Antioxidant activity

Antioxidant activity of the synthesized compounds has attracted a lot of interest and has been evaluated mostly in the in vitro systems (Sathyadevi et al. 2012; Mohanraj et al. 2016). The ligand and its metal complexes were investigated for their antioxidant ability and were carried out by using DPPH (1,1-diphenyl-2-picrylhydrazyl) radical as a reagent in the spectrophotometric test (Rohini et al. 2018a, b). Ascorbic acid is used as a reference antioxidant. The compounds were performed in triplicate and calculated the standard deviation (Gul et al. 2013). The IC50 (µM) values were given in Table 11 and Fig. 8. From the results, the complex 4 exhibited significant antioxidant activity with an IC50 value of 6.11 ± 0.54 µM compared to the reference drug showed their IC50 value of 4.61 ± 0.93 µM. Complex 1 exhibited good activity with IC50 value of 7.63 ± 1.57 µM. All the other complexes IC50 values in between 13.16 ± 0.85 µM and 29.47 ± 0.18 µM. According to the IC50 values the order of the ligand and its complexes are 4 > 1 > 2 > 3 > 5 > L.

Table 11 Antioxidant activity of the ligand and its metal complexes
Full size table
Fig. 8
figure 8

Antioxidant activity of the ligand and its metal complexes (1–5)

Full size image

In vitro antiproliferative evaluation

The successful evaluations of the antimicrobial and scavenging activity, we have also examined the anticancer evaluation against different cancer cell lines, IMR-32 (neuroblastoma), HeLa (cervical), MCF-7 (breast), A549 (lung), HepG-2 (liver) and HEK293 (embryonic kidney) by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Mosmann 1983; Rohini et al. 2018a, b). The MTT assay results were shown in IC50, expressed in micromolar units and summarized in Table 12. The percentage of cell viability versus concentration graphs is shown in Fig. 9. Cisplatin is used as a reference drug. The results clearly indicate that complex 4 showed excellent anti-proliferative activity against IMR-32, A549 and HepG-2 with an IC50 value of 7.81 ± 0.52 µM, 6.18 ± 1.15 µM and 15.28 ± 1.26 µM, respectively. Which are close to standard drug cisplatin (IC50 = 5.78 ± 0.12 µM, 4.90 ± 0.31 and 10.52 ± 0.40, respectively) and also the complex 4 showed good activity against MCF-7 (IC50 = 7.41 ± 0.32 µM) and HeLa (IC50 = 5.99 ± 0.23 µM), respectively. Similarly complex 1 exhibited potent with broad spectrum activity with IC50 values against HeLa (5.94 ± 1.13 µM), IMR-32 (8.51 ± 0.12 µM), HepG-2 (16.51 ± 1.18 µM), A549 (8.27 ± 0.38 µM) and MCF-7 (13.26 ± 0.19 µM). The observed higher efficiency of complexes 1 and 4 are may be due to the presence of ligand coordinated to central Cobalt and Zinc metal ions. Also complex 2 against HeLa (IC50 = 12.72 ± 0.27 µM) and L against A549 (IC50 = 19.38 ± 1.07 µM) exhibited moderate activity. The other compounds exhibited the least activity against the cell lines. In addition, we have also tested the cytotoxicity of the potent compounds 1 & 4 against normal cancer cell line HEK293 with IC50 values of 96.57 ± 0.42 µM and 81.37 ± 0.10 µM, respectively. None of the potent complexes (1 and 4) interrupted the viability of the normal cell line, suggesting that the potent compounds are not toxic.

Table 12 Cytotoxic activity of the newly synthesized compounds on human cancer cell lines IMR-32, HeLa, MCF-7, A549 and HepG-2 [in vitro (IC50 μM)]
Full size table
Fig. 9
figure 9

Survival curves of cell lines: a IMR-32, b HeLa, c MCF-7 d A549 and e HepG-2

Full size image

Molecular docking

The in silico molecular docking analysis of the ligand and its metal complexes against human epidermal growth receptor (HER2) and epidermal growth factor (EGFR) was carried out to verify the relation between the in vitro antiproliferative activity results and binding affinities of the inhibitors by using auto dock program. It plays a major role in malignant growth from various origins (Jost et al. 2013). It is expressed on the surfaces of most of the cells of human body (Jost et al. 2013). The overexpression of the HER2 involved in several cancers like breast, adenocarcinoma of lungs, stomach (Buza et al. 2014), ovarian (Santin et al. 2008), uterine cancers (Buza et al. 2014; Santin et al. 2008) etc., It is a suitable target for kinase inhibitors (Jost et al. 2013). On the other hand, EGFR is the prominent cell-surface receptor and belongs to the EGFR family (Sebastian et al. 1998). The TKD (tyrosine kinase domain) and extracellular mutations of EGFR causes non small cell lung cancer and glioblastoma respectively (Foloppe and MacKerell 2000; Breneman and Wiberg 1990; Walker et al. 2009). Its overexpression also leads to epithelial tumors of the head, neck and anal cancer (Lynch et al. 2004; Davis and Teague 1999). By considering the aforementioned reasons, we have chosen the target protein receptors HER2 and EGFR for the docking studies. Herein, the main aim is to explore the binding behaviour (in terms of Binding energy) of the target compounds against HER2 and EGFR and their results were compared with well established inhibitors (in vitro and in vivo) such as Canertinib (HER2), Afatinib (HER2), Lapatinib (EGFR) and Gefitinib (EFGR) (Schroeder et al. 2014). The comparative docking studies of the compounds [ligand and its metal complexes (1–5)] and the reported inhibitors (Canertinib, Afatinib, Lapatinib and Gefitinib) against the proteins HER2 and EGFR along with their corresponding binding energies, which were listed in Table 13. As shown in Table 13, docking results revealed that complexes 1 and 4 showed the least binding energies compared to other metal complexes against receptors HER2 and EGFR with their binding energies − 7.31, − 6.46 kcal/mol (for complex 1); − 8.02, − 7.35 kcal mol−1 (for complex 4), respectively. Hence HER2 and EGFR were taken as the target protein receptors for the insightful deep discussion for complexes 1 and 4. The best docking poses of the complexes 1 and 4 were shown in Figs. 10 and 11 the molecular docking results for the complex 1 against HER2 exhibited four hydrogen bonds; one strong hydrogen bond in between the OH group of carboxylic acid moiety and the amino acid residue LYS875 with bond length 1.80 Å, one hydrogen bond in between an amino group of 2-aminopyridyl ring and amino acid residue SER720 with bond length 2.28 Å, one hydrogen bond in between OH group of benzoic acid moiety and the amino acid residue ALA722 with bond length 2.90 Å and one carbon–hydrogen bond in between –CH group of pyridyl ring and amino acid residue GLY724 with bond length 3.03 Å. The pyridyl ring interacts with the amino acid residues ARG841 and CYS797 and the phenyl ring of the benzoic acid moiety interacts with amino acid residue ALA722 through hydrophobic interactions. In relation the complex 1 against EGFR exhibited eight hydrogen bonds; one strong hydrogen bond in between -O-atom of carboxylic acid moiety and the amino acid residues ARG817 and GLY697 with bond length 1.87 Å and 3.28 Å respectively, one hydrogen bond in between H-atom of the carboxylic acid moiety and the amino acid residue ASP813 with bond length 2.14 Å, one hydrogen bond in between carbonyl oxygen atom of the carboxylic acid group and the amino acid residue LYS721 with hydrogen bond length 3.04 Å, one hydrogen bond in between amino group of 2-aminopyridyl ring and amino acid residue ARG817 with bond length 2.08 Å, one hydrogen bond in between N-atom of pyridyl ring and the amino acid residue CYS773 with bond length 2.87 Å and one carbon-hydrogen bond in between –CH group of pyridyl ring and amino acid residue ASP831 with bond length 3.16 Å, one hydrogen bond in between N-atom of the imine group and the amino acid residue ARG817. The pyridyl ring interacts with the amino acid residues LEU820, ARG817 and VAL702 and the phenyl ring of the benzoic acid moiety interact with amino acid residue ARG817 through hydrophobic interactions. The hydrogen bonding interactions and hydrophobic interactions of the complex 1 was shown in Figs. 10 and 11.

Table 13 Binding energies of ligand and its metal complexes (1–5) against protein receptors HER 2 and EGFR
Full size table
Fig. 10
figure 10

The best docking pose of complex 1 with HER2. a Hydrogen bond interactions, b hydrophobic interactions

Full size image
Fig. 11
figure 11

The best docking pose of complex 1 with EGFR. a Hydrogen bond interactions, b hydrophobic interactions

Full size image

In case of complex 4 against HER2 exhibited five hydrogen bonds; one strong hydrogen bond in between the –OH group of benzoic acid moiety and amino acid residue PRO877 with bond length 1.69 Å, one strong hydrogen bond in between the –OH group of benzoic acid moiety and amino acid residue ASP837 with bond length 1.85 Å, one hydrogen bond in between –CH group of imine moiety with amino acid residue ALA722 with bond length 3.56 Å, one hydrogen bond in between –CH group of pyridyl moiety with amino acid residue GLY719 with bond length 3.69 Å and one hydrogen bond in between carbonyl group of benzoic moiety with amino acid residue PRO877 with bond length 3.77 Å. The pyridyl ring interacts with the amino acid residues ARG841, LYS745 and VAL726 through hydrophobic interactions. In addition to this, there was an electrostatic interaction between the pyridyl moiety with the amino acid residues ASP855 and CYS797. In relation the complex 4 against EGFR exhibited eight hydrogen bonds; three hydrogen bonds in between carbonyl oxygen atom of the carboxylic acid group and the amino acid residue ARG779, LYS851 and LYS889 with hydrogen bond lengths 1.93 Å, 1.95 Å and 3.37 Å respectively, one strong hydrogen bond in between the –OH group of benzoic acid moiety and amino acid residue LYS889 with bond length 2.98 Å, two hydrogen bonds in between an amino group of 2-aminopyridyl ring and amino acid residue SER696 with bond length 1.61 Å, and 1.92 Å, one hydrogen bond in between N-atom of pyridyl ring with amino acid residue GLY695 with bond length 3.57 Å and One Pi-donor hydrogen bond in between pyridyl ring and amino acid residue SER696 with bond length 2.97 Å. The pyridyl ring interacts with the amino acid residues ARG817 through hydrophobic interactions. The hydrophobic interactions also played a vital role in increasing the affinity in between the synthesized compounds and targeted proteins. The hydrogen bonding interactions and hydrophobic interactions of the complex 4 was shown in Figs. 12 and 13. Finally, the docking results clearly suggest the affinity of the synthesized compounds (1–5 and ligand) towards the protein receptor HER2 is better than that of the protein receptor EGFR. The docking results were quite consistent with experimental anticancer activity. The best docking poses of the standard compounds against the proteins HER2 and EGFR were included in the supplementary file (Figs. S1, S2).

Fig. 12
figure 12

The best docking pose of complex 4 with HER2. a Hydrogen bond interactions, b hydrophobic interactions

Full size image
Fig. 13
figure 13

The best docking pose of complex 4 with EGFR. a Hydrogen bond interactions, b hydrophobic interactions

Full size image

Conclusion

In summary, the complexes (1–5) with Schiff base ligand has been designed, synthesized and characterized, further with an aim to evaluated for their antioxidant, antimicrobial, cytotoxic activity, DFT calculations and HER2, EGFR target based in silico docking studies. The metal–ligand stoichiometry in the complexes (1–4) corresponds to 1:2, in the case of Pd 1:1 stoichiometry, wherein ligand behaves in a bidentate manner towards metals coordinating through azomethine nitrogen atom and nitrogen of amine group. Based on different experimental evidences, the complexes ensured that the octahedral geometry have been proposed for complexes 1, 2 and 4, square planar for complexes 3 and 5. All the complexes show non-electrolytic nature in DMF. The evaluation of ESR parameters from the ESR spectra confirms the M–L bonds are covalent and out of plane π-bonded. From the antimicrobial activity results, it is known that the metal complexes exert higher effectiveness compared to the ligand indicating that the metals are actually in action. Especially, complex 3 has shown excellent antimicrobial activity, complex 5 exhibited broad spectrum antibacterial activity, and complex 2 has shown superior antifungal activity compared to the standards streptomycin and ketoconazole. Antioxidant properties of the compounds, the compound 4 has shown very good activity compared to reference drug ascorbic acid. In Addition, complexes 4 and 1 showed potent anti-proliferative activity against IMR-32 (IC50 = 7.81 ± 0.52, 8.51 ± 0.12 µM), A549 (IC50 = 6.18 ± 1.15, 8.27 ± 0.38 µM) and HepG-2 (IC50 = 15.28 ± 1.26, 16.51 ± 1.18 µM), respectively. The docking results revealed that complexes 1 and 4 showed least binding energies against receptors HER2 and EGFR with their binding energies − 7.31, − 6.46 kcal/mol (for complex 1); − 8.02, − 7.35 kcal mol−1 (for complex 4), respectively compared to Canertinib (HER2), Afatinib (HER2), Lapatinib (EGFR) and Gefitinib (EFGR). From the results, it is clear that the complexes are strongly bound to HER2 and EGFR protein receptors. Interestingly, these compounds showed the most potent anticancer activity and minimum binding energies obtained by the docking study. The results indicate that in silico molecular docking studies were well correlated with the experimental anti-proliferative activity results.

General considerations

All chemicals and solvents used in these investigations were purchased from Sigma Aldrich and Spectrochem as high purity materials. The infrared spectra of the samples were recorded using a PerkinElmer 100S FTIR spectrometer. The absorption spectra of compounds in DMF were performed from Perkin-Elmer UV–Visible Spectrophotometer. The molar conductivity measurements of the complexes in DMF were made at 10−3 M concentration. NMR spectra were recorded in DMSO solvent using TMS as an internal standard on a Brukner 400 MHz spectrometer. Analytical data for the compounds were obtained from the Vario EL − III CHNS analyzer. The Electron paramagnetic spectrum of the copper complex is recorded by using JOEL X-Band ESR spectrometer.

Synthesis of 4-(((2-aminopyridin-3-yl)methylene)amino)benzoic acid (L)

A mixture of 2-aminonicotinaldehyde (1.22 g, 10 mmol) and 4-aminobenzoic acid (1.37 g, 10 mmol) in methanol was stirred for 1 h, to obtain the yellow color precipitate. The product was collected by filtration, washed with methanol and dried in a desiccator over calcium chloride. Yield: 95%. Yellow color solid. M.p.: 270–272 °C. Anal. Calcd. for C13H11N3O2 (%): C, 64.72; H, 4.60; N, 17.42. Found: C, 64.65; H, 4.56; N, 17.48. FT-IR (KBr): υ, cm−1 3420 (N − H), 1629 (C=N), 1567 (C=N)py, 1701 (C=O)carbaxilic acid. 1H NMR (400 MHz, DMSO) δ: 12.93 (s, 1H, OH), 8.72 (s, 1H, CH=N), 8.14 (d, J = 6.0 Hz, 1H, aromatic-H), 8.00–7.87 (m, 5H, aromatic-H), 7.38 (s, 2H, NH2), 6.72–6.69 (m, 1H, aromatic-H). 13C NMR (100 MHz, DMSO) δ: 167.49, 163.88, 158.44, 155.10, 152.18, 143.40, 131.11, 128.37, 121.77, 112.46, 112.34.

Synthesis of metal complexes (1–5)

An appropriate amount of methanolic solution of ligand (2 mmol, 0.482 g) and methanolic solution containing the chlorides of the Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) (1 mmol) were mixed with constant stirring for 4 h under reflux, and then the precipitate formed collected by filtration, washed with methanol and dried in a desiccator over calcium chloride.

[Co(II)(L)2Cl2] (1)

CoCl2 6H2O (0.237 g, 1 mmol) was used. Brown, Yield: 71%. M.p.: 350–352 °C. Anal. Calcd. for C26H22Cl2CoN6O4 (%): C, 51.00; H, 3.62; N, 13.72; Co, 9.62. Found: C, 50.93; H, 3.65; N, 13.66; Co, 9.69. ∧m = 12. UV–Vis (DMF): λmax, nm (cm−1) 1111 (9000), 540 (18,500), 490 (20,400). FT-IR (KBr): υ, cm−1 3371 (N–H), 1604 (C=N), 1564 (C=N)py, 1699 (C=O)carbaxilic acid. µ = 4.95 BM.

[Ni(II)(L)2Cl2] (2)

NiCl2 6H2O (0.237 g, 1 mmol) was used. Yield: 79%. Light green. M.p.: 310–312 °C. Anal. Calcd. for C26H22Cl2N6NiO4 (%): C, 51.02; H, 3.62; N, 13.73; Ni, 9.59. Found: C, 50.97; H, 3.58; N, 13.67; Ni, 9.52. ∧m = 16. UV–Vis (DMF): λmax, nm (cm−1) 1020 (9800), 628 (15,900), 387 (25,800). FT-IR (KBr): υ, cm−1 3389 (N–H), 1663 (C=N), 1569 (C=N)py, 1704 (C=O). µ = 3.22 BM.

[Cu(II)(L)2Cl2] (3)

CuCl2 (0.134 g, 1 mmol) was used. Yield: 84%. green. M.p.: 328–330 °C. Anal. Calcd. for C26H22Cl2CuN6O4 (%): C, 50.62; H, 3.59; N, 13.62; Cu, 10.30. Found: C, 50.58; H, 3.55; N, 13.59; Cu, 10.25. ∧m = 19. UV–Vis (DMF): λmax, nm (cm−1) 544 (18,351), 723 (13,825). FT-IR (KBr): υ, cm−1 3390 (N–H), 1592 (C=N), 1562 (C=N)py, 1703 (C=O). µ = 1.83 BM. EPR ‘g’ values 2.22, 2.05.

[Zn(II)(L)2Cl2] (4)

ZnCl2 (0.136 g, 1 mmol) was used. Light Yellow. Yield: 75%. M.p.: 378–380 °C. Anal. Calcd. for C26H22Cl2N6O4Zn (%): C, 50.47; H, 3.58; N, 13.58; Zn, 10.57. Found: C, 50.41; H, 3.55; N, 13.54; Zn, 10.50. ∧m = 15. FT-IR (KBr): υ, cm−1 3384 (N–H), 1592 (C=N), 1571 (C=N)py, 1697(C=O). 1H NMR (400 MHz, DMSO): δ: 12.07 (s, 1H, OH), 8.74 (s, 1H, CH=N), 8.13–7.91 (m, 6H, aromatic-H), 7.33 (s, 2H, NH2), 6.72 (s, 1H, aromatic-H). 13C NMR (100 MHz, DMSO) δ: 166.98, 163.78, 157.30 154.88, 152.08, 142.96, 131.01, 129.01, 121.67, 112.42, 112.31.

[Pd(II)(L)Cl2] (5)

Pd(II) chloride (0.177 g, 1 mmol) was used. Yield: 88%. Orange. M.p.: 336–338 °C. Anal. Calcd. for C13H11Cl2N3O2Pd (%): C, 37.30; H, 2.65; N, 10.04; Pd, 25.42. Found: C, 37.37; H, 2.59; N, 10.09; Pd, 25.31. ∧m = 11. UV–Vis (DMF): λmax, nm (cm−1) 534 (18,700), 465 (21,500), 402 (24,855), 357 (28,000). FT-IR (KBr): υ, cm−1 3401 (N–H), 1608(C=N), 1570 (C=N)py, 1705 (C=O). 1H NMR (400 MHz, DMSO) δ: 12.82 (s, 1H, OH), 8.61 (s, 1H, CH=N), 8.03 (d, J = 6.0 Hz, 1H, aromatic-H), 7.89–7.77 (m, 5H, aromatic-H), 7.28 (s, 2H, NH2), 6.62–6.59 (m, 1H, aromatic-H). 13C NMR (100 MHz, DMSO) δ: 167.39, 163.79, 158.44, 155.00, 152.09, 143.30, 131.01, 128.27, 121.68, 112.36, 112.25.

Biological evaluations

The methods used for antibacterial, antifungal, radical scavenging, and in vitro anti-proliferative activity studies were explained in the supplementary information file.