Original Article

Table 1: Chemical analyses and some physical data of the solid complexes.

Physical measurements

Elemental analyses contents (C, H and N) were determined at the Microanalytical Unit, Cairo University. Molar conductivities measurements were carried out using Jenco-3173. The IR spectra in the 400-4000 cm^-1 range were recorded in KBr using 4100 Jasco Spectrophotometer. The electronic spectra of metal complexes were recorded in DMSO in the range (200-900 nm) using UV-1601 spectrophotometer. ¹H-NMR and ¹³C-NMR spectra of L and some of its metal complexes were recorded on Varian Gemini (200 MHz) Spectrometer in d6-DMSO. Magnetic moments were determined using a Sherwood balance at room temperature (25°C) with Hg[Co(NSC)₄] as a calibrate. The diamagnetic corrections for L and the metal atoms were computed using Pascal's constants [9]. Thermal analysis measurements (TGA and DTG) were recorded on a Schimadzu model 50 instrument using 20 mg. The nitrogen flow rate and heating rates were 20 cm³/min and 10°C/min, respectively. The mass spectra were carried out on a Shimadzu of the type (GC/MS-QP5050) at the regional center for mycology and biotechnology, Al-Azhar University, Egypt. The metal contents were determined using the standard methods [10].

Results and Discussion

The chemical analyses and some physical data of the isolated solid complexes are recorded in Table 1. All the isolated metal complexes are colored, stable against light and insoluble in most common organic solvents but easily soluble in DMF and DMSO. The molar conductivities of the complexes in DMSO at 25°C lie in the range 1.5-13 Ω^-1 cm² mol^-1 indicating the non-electrolyte nature [11] of the complexes. The high melting points (?300°C) of the isolated complexes suggest the stability of these compounds as indicated also from the results of TGA.

IR spectra

The IR spectrum of lamotrigine (L) in KBr shows several bands at 3450 (vs), 3319 (s), 3211(s), 1620 (vs) and 1554 (s) cm^-1 (Figure. 2). These bands are assigned to ν_as(NH2, free), νs(NH2, free), ν_as(NH₂, hydrogen-bonded) [12], ν(C=N) and ν(C=C) vibrations, respectively. The observation of weak broad bands in the 1948-1800 cm^-1 region suggests the existence of inter and/or intra-hydrogen bonding of the type (N-H….N) as shown in Figure. 1.

Figure 2: IR spectrum of lamotrigine (L) in KBr.

The ¹H-NMR spectrum of L in d₇-DMF (Figure. 3) displays five signals at 9.03, 7.52, 7.46, 6.95 and 6.56 ppm, relative to TMS. The first three signals are assigned to the protons of CH (C3-H19), (C2-H18) and (C1-H17), respectively. The latter two signals at 6.95 and 6.56 ppm are attributed to the protons of NH₂ attached to (C8-N13) and (C10-N14), respectively. The signal at 6.59 ppm is assigned to the first NH₂ signal (C8-N13) and observed downfield of TMS in comparison to the second NH₂ signal (C10-N14) since the former group is hydrogen-bonded. All the results are taken an evidence of the existence of inter-or intra-hydrogen bonded within the molecule.

Figure 3: ¹H-NMR spectrum of L in d₆-DMSO.

The ¹³C-NMR spectrum of L in d₇-DMF (Figure. 4) displays seven signals at 155, 139.7, 137.6, 132.7, 132.3, 130.9 and 128.7, relative to TMS. The first three signals are assigned to the carbons of C8-N13 (hydrogen-bonded), C10-N14 (free) and C7, respectively. Also, the results are taken as strong evidence for the existence of hydrogen bonding (inter-or intra hydrogen-bonded) either from the data of IR and ¹H-NMR. Finally, the latter four signals at 132.7, 132.3, 130.9 and 128.7 ppm are attributed to the carbons of C5, C6, C3, C2 and C1, respectively.

Figure 4: ¹³C-NMR spectrum of L in d₇-DMF.

A comparison of the IR spectra of the free L with its complexes allows us to determine the mode of bonding. The IR spectra indicate that the L behaves in a monodentate manner in all metal complexes and coordinates via the NH₂ group. The negative shift of the NH₂ band to lower wavenumbers shows that this group is participate in coordination. Also, the other functional groups remain more or less at the same positions indicating that the other functional didn’t participate in bonding. Moreover, in comparing the IR spectra of L with its metal acetate complexes (Ni²⁺ and Hg²⁺) we observed that the difference between υ_as and υ_s of the acetate group is 199 and 212 cm^-1 for Ni²⁺ and Hg²⁺ complexes, respectively indicating that this group behaves in a monodentate manner [13]. The band observed in the 410-490 cm^-1 range is assigned to the υ (M-N) vibration [14].

The UV spectra of lamoterigine in DMSO shows two bands at 32850 and 38460 cm^-1 attributed to n → π* transitions for C=N and N=N groups, respectively [15] as shown in Figure. 1. The spectra of all complexes were carried out in DMSO. The electronic spectrum of the Cu²⁺ complex, [Cu₂(L)₂Cl₄].½H₂O, shows a broad band centered at 10870 cm^-1 attributed to ²T₂ → ²E₂transition in a tetrahedral geometry around the Cu²⁺ ion [15] as shown in Figure. 5. The value of magnetic moment is 1.55 BM. is taken as additional evidence for the existence of small Cu-Cu interaction.

Figure 5: Structure of [Cu₂(L)₂Cl₄].½H₂O.

The electronic spectrum of [Co(L)₂Cl₂].1½EtOH shows multiple bands at 14,903 and 16290 cm^-1 attributed to the 4A1 → 4T1 (P) transitions in a pseudo-tetrahedral geometry around the Co²⁺ ion [16,17]. The value of magnetic moment. (4.2 BM) is taken as additional evidence for the pseudo-tetrahedral geometry around Co²⁺ ion as shown in Figure 6.

Figure 6: Structure of [Co(L)₂Cl₂].½H₂O.

The determination of energies of the HOMO (π-donor) and LUMO (π-acceptor) are important parameters in quantum chemical calculations. The HOMO (Highest Occupied Molecular Orbitals) is the orbital that primarily acts as an electron donor and the LUMO (Lowest Unoccupied Molecular Orbital) is the orbital that largely act as the electron acceptor. These molecular orbitals are also called the frontier molecular orbitals (FMOs) as shown in Figure. 7.

Figure 7: HOMO and LUMO of the Co²⁺ complex.

The electronic spectrum of the Fe³⁺ complex, [Fe(L)₂Cl₃H₂O], shows a band at 17860 cm^-1 assigned to ⁶A_1g → ⁴T_2g, ⁴T_1g transitions in an octahedral geometry around the Fe³⁺ ion [14,16]. The value of magnetic moment. (5.6 BM) supports a high-spin octahedral geometry as shown in Figure. 8. The band observed at 37740 cm^-1 is assigned to charge-transfer of the type L → M. The low value of the molar conductance (3.6 ohm^-1.cm² mol^-1) indicates that the three chloride ions reside inside the coordination sphere [11] as shown in Figure. 8.

Figure 8: Structure of [Fe(L)₂Cl₃H₂O].

The magnetic moment value of the Ni²⁺ complex (3.3 BM) as well as the observation of multiplet bands centered at 15060 cm^-1 in the electronic spectrum of the Ni²⁺ complex, [Ni₂(L)(Ac)₄(H₂O)₂].4H₂O, assigned to ³A₂ → ³T₁ (P) transition suggests a tetrahedral geometry around the two Ni²⁺ ions [16] as shown in Figure. 9. Also, the molar conductance value (13 ohm^-1. cm². mol^-1) indicates that the acetate ions are non-conducting [11].

Figure 9: Structure of [Ni₂(L)(Ac)₄(H₂O)₂].4H₂O.

Finally, the Cr³⁺ complex, [Cr(L)₃Cl₃], shows two bands at 16500 and 22222 cm^-1 assigned to ⁴A₂g → ²T_2g, ²E_g and ⁴A_2g → ⁴T_2g transitions in an octahedral geometry around the Cr³⁺ ion [16]. The value of magnetic moment (3.6 BM) is taken as additional evidence for octahedral geometry around the Cr³⁺ ion [18,19] as shown in Figure. 10. The low molar conductance value (7 ohm^-1.cm² mol^-1) suggests that the chloride ions are non-conducting [11,20].

Figure 10: Structure of [Cr(L)₃Cl₃].

DFT method concepts can indicate the chemical reactivity and site selectivity of the molecular systems. The energies of frontier molecular orbitals (E_HOMO+E_LUMO) and energy band gap (E_HOMO-E_LUMO) explains the eventual charge transfer interaction within the molecule [21,22].

Thermal studies

The steps of decomposition, temperature extent and decomposition products and the percentages of weight loss for the metal complexes are reported in Table 2. The results are in good agreement with the proposed structures. Also, the data suggest that the complexes are stable up to 800°C since the complexes didn’t decomposed completely to get the metal oxides and/or the nitride.

Table 2: Decomposition steps and weight loss of the metal complexes.

In vitro anticancer activity

In vitro cytotoxicity tests were conducted utilizing five complexes derived from lamotrigine (L) against human tumor cell lines of the types MCF-7 and Hela normal cell line using a colorimetric assay (MTT assay) that is a measurement for mitochondrial dehydrogenase activity as an indication of cell viability [21,22]. The activities correspond to the viability of cancer cell growth. In parallel, the impact of widely utilized anticancer drug, 5-fluorouracil, has been also assayed as a standard. The IC50 values were estimated from the plot between the percent cell viability and concentration. The IC₅₀ of both cell lines for the five complexes covered a large range of activity ranging from 8.2 up to 46.3 μg/ml as shown inFigure. 11(A, B, C, D, E, F). The results suggest that the five complexes monitored antitumor activity against the cell lines without damaging the normal cells. There is obviously direct correlation between strong interaction of metal ion complex with DNA and ant proliferative activity. The results reveal that [Fe(L)₂Cl₃H₂O] and [Cr(L)₃Cl₃] complexes have high potential activity than [Cu₂(L)₂Cl₄].½H₂O, [Ni₂(L)(Ac)₄(H₂O)₂].4H₂O and [Hg₃(L)(Ac)₆] complexes.

Figure 11: IC₅₀ of metal complexes (B, C, D, E, F) with reference to 5-fluorouracil against to MCF-7 and Hela (A).

The results are explained on the basis of three factors. Firstly, the ionic radii of the Cr³⁺ and Fe³⁺ ions are smaller than the other divalent ions (Cu²⁺, Ni²⁺and Hg²⁺) which permit the penetration of the trivalent ions (Cr³⁺ and Fe³⁺) to the membrane of the cells more than the divalent ions and coordinated to DNA. Secondly, both the Cr³⁺ and Fe³⁺complexes contain only one metal ion while the rest involve two or three ions as shown from the chemical formulae. The existence of more than one metal ion may cause a damage of the cell membrane. Finally, the stereochemistry of the metal complexes plays role on targeting cells in which the octahedral structure is more effective than the other geometries. All these observations suggest that the DNA may be the targeting molecules of the metal complexes anti-carcinogenic action and the metal complexes bind significantly enhances the drugs anticancer activity. This means that the complex is essential in designing and synthesizing novel anticancer drugs. So, it is unmistakably watched that coordination with Cr³⁺ and Fe³⁺ ions has a synergistic impact on the cytotoxicity. Also, it means that the chemical structure of compounds is very important to explain the complex biological activity and it can be essential in designing and synthesizing novel anticancer drugs.

Conclusion

New mono, di and tri-metallic metal complexes derived from lamotrigine (L) were synthesized and characterized by elemental analyses, conductance, spectral (IR, UV-Vis., ¹H-NMR and ¹³C-NMR), magnetic and thermal measurements. A distorted-octahedral geometry is proposed for the Cu²⁺ complex, tetrahedral for Ni²⁺ and Co²⁺ and octahedral structures around the Cr³⁺ and the Fe³⁺. TGA data suggest the mechanism of decomposition for the metal complexes and indicates also the stability of these complexes at high temperature. The cytotoxic activity assay of the Cr³⁺ and Fe³⁺ complexes have the highest cytotoxic activity against human tumor cell lines of the types MCF-7 and Hela normal cell line, while other complexes showed low cytotoxic activity. The role of oxidation state of the metal ions, nature and geometry of complex is proposed.

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