Structural elucidation of a Mn(III) derivative anchored with a tetradentate Schiff base precursor: in vitro cytotoxicity study

A new mononuclear Mn(III) derivative, [Mn(L)(H2O)2]ClO4 (1) was obtained by reacting manganese perchlorate with the tetradentate Schiff base precursor, H2L, [where H2L=C6H3(OMe)(OH)CH=N(CH2)3N=CH(OH)(OMe)C6H3] and characterized by various spectral techniques. Single crystal X-ray diffraction analysis revealed that in 1, the central Mn atom is hexa-coordinated via the NNOO donor sites of the Schiff base precursor and two water molecules, yielding a distorted octahedral geometry. Also, the EPR spectra of 1 were recorded at the solid state by varying the temperature (from 90 to 300 K) as well as in different organic solvents; DMSO, MeOH and a mixture CH2Cl2/toluene; the results obtained agree with the geometrical environment around the metal. The cytotoxic activity of the Mn(III) derivative was evaluated against normal and cancer cell lines. The derivative exhibited comparatively better cytotoxic activity against MCF-7 cells compared to SiHa and fibroblast cells.


Introduction
Because of their structural diversification, Schiff basemediated metal derivatives are the indispensable stereochemical frameworks for coordination and organometallic chemistry (Rezaeivala and Keypour 2014;Low et al. 2016). The design of new multidentate precursors containing oxygen and nitrogen donors and of their corresponding metal derivatives has long attracted chemists due to their wide use in the discovery of new drugs, as well as catalytic and biological applications (Mahmoudabadi et al. 2019;Habibi et al. 2018;Keypour et al. 2017). Their ease of synthesis and reactivity with almost all metal ions present in the periodic table make them suitable synthons for the development of coordination chemistry. Furthermore, they form stabilized metal complexes with various oxidation states (Cozzi 2004;Köse et al. 2015;Ourari et al. 2014). Schiff base manganese complexes are efficient catalysts in biological chemistry because of their binding modes with O and N donor atoms (Luaces et al. 1996); they can be sterically and electronically modified to enhance their catalytic activity (Bagherzadeh et al. 2008;Ray et al. 2014;Ceyhan et al. 2012) by modifying the substituents on the aromatic ring. Due to the importance of Mn(III) Kuheli Das and Manasa G. Shetty have contributed equally to this work.
in diverse chemical and biological fields in recent years, research in connection with the organometallic chemistry of Mn(III) has been very active (Kirby et al. 1981;Christou 1989;Brudvig and Crabtree 1989;Vincent and Christou 1989;Wieghardt 1989;Que and True 1990). To obtain high-valent manganese(III) complexes, a high number of different ligating systems were probed to see if they could mimic the structural components of the active site; thus, the Schiff base precursors can participate as effective biometric ligands. Manganese(III) salen complexes [salen = N, N%-bis(salicylideneaminato)ethylene] have been previously reported (Chidara and Du 2013;Noritake et al. 2013;Rouco et al. 2020) and have been exploited in distinct areas such as catalysis (Routier et al. 1997;Muller et al. 1994;Puglisi et al. 2004), as simple models for active sites of metalloenzymes and proteins (Pecoraro 1992;Rousselot-Pailley et al. 2009), for mimicking the function of cytochrome p-450 and as models as well as for the treatment of ROS (reactive oxygen species)-associated disease including ischemic tissue damage (Park and Lim 2009;Bruce et al. 1996;Doctrow et al. 2002) and as molecular magnets (Bhargavi et al. 2009;Clérac et al. 2002). Fluorescence spectroscopy is a key tool for the qualitative and quantitative measurements of the fluorophores present in the compound. It helps in the identification of specific additives present in the sample. Moreover, fluorescence decay measurements provide information about the stability of the fluorophore in the excited state. Its intensity depends on the excitation wavelength, self-absorption by the sample and concentration of fluorophore (Lakowicz 2006 (Thakurta et al. 2010) prepared in an aqueous methanolic medium. To characterize the complex, spectral studies (IR, UV-Vis, Emission, Fluorescence and EPR) were performed. The derivative was biologically evaluated against fibroblast (normal), SiHa (human cervical cancer) and MCF-7 (human breast cancer) cell lines.

Materials
Mn(ClO 4 ) 2 ·6H 2 O was acquired from Merck, India, whereas o-vanillin and 1,3-diaminopropane were purchased from Sigma-Aldrich. The reagent-grade solvents were used without further purification. The Schiff base precursor was synthesized according to the literature (Mani et al. 1994). All the reactions were carried out in aerobic conditions.

Physical measurements
A Perkin-Elmer 2400 CHNS/O elemental analyzer was used for recording the elemental analysis data (C, H, and N). The FTIR spectra were collected as KBr pellets on a Perkin-Elmer RX-1 spectrophotometer in the range 3550-450 cm −1 . UV-vis absorption spectra were acquired using a standard laboratory grade spectrophotometer (Vari-oskanTM LUX multimode microplate reader, Thermo Scientific, India). The fluorescence spectroscopy was performed using the same spectrophotometer, and the sample fluorescence was measured over a spectral range of 238-650 nm. The sample was prepared by dissolving 1 mg of the compound in 1 ml of dimethylsulfoxide (DMSO) and loaded in a corning flat bottom 96 well plate in triplicates for the fluorescence acquisition. The fluorescence spectra were analyzed and plotted using the OriginPro software. EPR spectra were recorded from 0 to 8000 Gauss in the temperature range 90-300 K with an X-band (9.4 GHz) Bruker EMX spectrometer equipped with an HP 53150A microwave frequency counter and a variable temperature unit. The microwave frequency was 9.40-9.41 GHz, microwave power was 20 mW (which is, with the ER4119 HS resonator, below the saturation limit), time constant was 81.92 ms, modulation frequency 100 kHz, modulation amplitude 0.4 mT, and resolution 8192 points.

Synthesis of the Schiff base ligand (H 2 L)
1,3-Diaminopropane (10 mmol, 0.835 ml) and o-vanillin (20 mmol, 3.04 g) were refluxed in 50 ml of MeOH. The resulting mixture was cooled down and the excess solvent was removed under vacuum. A yellow-colored crystalline solid was obtained with a 73% yield.

Synthesis of compound 1
A 10 ml methanolic solution of the ligand (1 mmol) was added dropwise to a 1 ml methanolic solution of Mn(ClO 4 ) 2 ·6H 2 O (0.361 g, 1 mmol) under constant stirring. The resulting mixture was boiled for ten minutes. After that, the brown-colored solution was kept undisturbed at room temperature. Dark brown, rectangular-shaped single crystals of 1 were generated after 1 week. The crystals were isolated very carefully by filtration and dried in air before carrying out the physical experiments. Yield: 0.58 g Anal. Calc

X-ray crystallography
The crystal structure of compound 1 was determined by X-ray diffraction methods. Intensity data and cell parameters were recorded at 190(2) K on a Bruker ApexII diffractometer (MoKα radiation λ = 0.71073 Å) equipped with a CCD area detector and a graphite monochromator. The raw frame data were processed using the programs SAINT and SADABS to yield the reflection data files (AXS, SADABS Bruker, and Wisconsin Madison 1999). The structure was solved by direct methods using the SIR97 program (Altomare et al. 1999) and refined on F o 2 by full-matrix least-squares procedures, using the SHELXL-2014/7 program (Sheldrick 2008) in the WinGX suite v.2014.1 (Farrugia 2012). The structure of the title compound was refined as a two-component inversion twin. All non-hydrogen atoms were refined with anisotropic atomic displacements, except for two disordered oxygen atoms of a perchlorate anion. The carbon-bound H atoms were placed in calculated positions and refined isotopically using a riding model with C-H ranging from 0.95 to 0.99 Å and Uiso(H) set to 1.2/1.5Ueq(C). The H atoms of the water molecules were found in the difference Fourier map and were subsequently refines setting the O-H distance to 0.87 Å with Uiso(H) = 1.5Ueq(O). The weighting scheme used in the last cycle of refinement was . Crystal data and experimental details for data collection and structure refinement are reported in Table 2.

Cell culture
Fibroblast (normal), human cervical cancer (SiHa) and human breast cancer (MCF-7) cell lines were obtained from ATCC, and cells were grown in Dulbecco's Modified Eagle Medium (DMEM), DMEM F12 (Himedia) supplemented with 10% FBS (Himedia). The stock solutions of 1 were prepared in dimethyl sulfoxide (DMSO) (Merck) and added at desired concentrations to the cell culture. All the cell lines were cultured at humidified atmosphere containing 5% CO 2 at 37 °C. The DMSO concentration did not exceed 0.1% in the final culture.

Cell viability assessment
Cancer and normal human cells were seeded at a density of 1.0 × 10 4 (SiHa and MCF-7) and 5.0 × 10 3 (fibroblast) cells per well, respectively, in a 96-well cell tissue culture plates with the medium supplemented with 10% FBS and the cells were allowed to attach for 24 h. The cells were then treated with various concentrations (0.1-100 µg/ml) of the compound. After 48 h of treatment, the cells were treated with 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) at 37 °C for 4 h. The media were removed after incubation, and DMSO (Merck) was added to each well to dissolve the formazan crystals. The absorbance at 570 nm and 630 nm of each well was measured using a microplate reader (VarioskanTM Thermo Scientific). The IC50 values of the compound in all the three cell lines were calculated using the Prism 8 software (GraphPad Software, CA). Experiments were conducted in triplicate (Mosmann 1983).

Synthesis and characterization
The tetradentate Schiff base precursor, H 2 L [where H 2 L=C 6 H 3 (OMe)(OH)CH=N(CH 2 ) 3 N=CH(OH)(OMe) C 6 H 3 ] is a condensed product of o-vanillin and 1,3-diaminopropane in aerobic condition. The stoichiometric reaction of H 2 L and Mn(ClO 4 ) 2 ·6H 2 O in methanol afforded compound 1 with a 65% yield (see Scheme 1). The crystals were collected via vacuum filtration and washed with cold methanol.
The FTIR spectrum of 1 was carried out using KBr pallets in the 4000-400 cm -1 range and showed the key stretches diagnostic of the formation of the metal derivative. The vibration ν C-O (phenolic) is responsible for the band observed at 1254 cm -1 for the free ligand (Nakamoto 2009); after complexation this band shifts at 1250 cm -1 . A band at 3450 cm -1 is observed for the free ligand due to the ν (O-H) stretching frequency, but it disappears after complex formation. The stretching frequency ν C=N for 1 appears in the lower region at 1611 cm -1 , indicating that the metal center is coordinated with the nitrogen atom (You and Zhu 2004), while the same band appears at 1643 cm -1 for the free ligand. The appearance of a corresponding band at 459 cm -1 , which is primarily due to the ν M-N stretching frequency, supports ligand coordination to the metal center.

UV-Visible spectroscopy
In the range from ultraviolet to visible (200-800 nm), the absorption spectra were measured via synchronize scanning. The sample was dissolved in methanol. The free ligand shows maximum absorbance at 224, 264 and 415 nm with a weak shoulder at 292 nm. These are basically π → π* and n → π* transition bands. For the Mn-complex, it exhibits the electronic bands at 238 nm (ε = 1.0 × 10 4 M −1 cm −1 ) and 390 nm (ε = 1.8 × 10 3 M −1 cm −1 ) with a weak shoulder at 292 nm (ε = 4.3 × 10 3 M −1 cm −1 ). Absorbance values give similar extinction coefficients along with the λ max found in previous experiments (Fronko et al. 1988;Lever 1984). Complex 1 exhibits a d-d band at 570 nm which reveals that Mn is in the oxidation state + 3 in a hexa-coordinated 1 3 environment; indeed, Mn(II) does not show any d-d transition bands as high spin octahedral d 5 systems are spin forbidden.

Crystal structure
The crystal structure of 1 was elucidated through X-ray diffraction analysis on single crystals. It consists of a manganese complex of general formula [Mn(L)(H 2 O) 2 ]ClO 4 , where H 2 L is the Schiff base CH 2 [CH 2 N=CH(C 6 H 3 OCH 3 OH)] 2 comprising two OH groups. The complex crystallizes in the orthorhombic space group Pca21, with two independent complexes labeled A and B in the asymmetric unit. The two complexes show very similar geometrical parameters (see Fig. S1); the molecular structure of complex A is shown in Fig. 1.
The main feature of the crystal structure is the formation of supramolecular chains along the b-axis direction in which molecules A and B alternate, connected by means of H-bonds involving the coordinated water molecules as donors and the Mn-bound oxygen anions as acceptors (as shown in Fig. 2 and Table 2). The perchlorate anions are placed between the parallel chains, forming C-H•••O interactions with the ligand.

Fluorescence spectroscopy
The sample fluorescence was measured at four different excitation wavelengths, specifically at 238 nm, 260 nm, 360 nm and 390 nm as shown in Fig. 3. The emission wavelength was set at a difference of 30 nm from the excitation wavelength, and the sample was scanned up to 650 nm with a step size of 2 nm. SkanIt Software 2.4.3 RE was used for the spectral acquisition with a measurement time of 100 ms. From the fluorescence spectrum, we observed multiple emission peaks as marked in Fig. 3.

Scheme 1. The synthetic procedure of the Schiff base ligand
Moreover, the fluorescence decay of the sample was measured at the same excitation wavelengths with the emission set at their respective fluorescence maxima. The sample excited at 238 nm showed a longer lifetime, as shown in Fig. 3e.
The decay curve was fitted with a bi exponential curve using the OriginPro software as represented in Fig. 4. The equation underlying (Monisha et al. 2021;Das et al. 2017) the fit is given as follows.
where τ 1 and τ 2 are lifetime values, A 1 and A 2 represent amplitudes obtained from fitted curve, and t1 and t2 are time Fig. 1 View of the molecular structure of [Mn(L)(H 2 O) 2 ]ClO 4 with atom labeling scheme. The disorder from the perchlorate anion has been omitted for clarity  Labeling scheme as in Table 2 1 3 constants. The overall lifetime of the compound is measured using the following relation, and it is found to be 69.34 µs.
The fluorescence emission of Mn-doped compound is observed at 385 nm, which supports the current study's findings (Tan et al. 2014). Furthermore, Randall's research demonstrates the fluorescence property of various Mn-based complexes (Randall 1939).

EPR spectroscopy
The EPR spectra on polycrystalline 1 were recorded in the solid state as a function of the temperature from 300 to 90 K (Fig. 5) and on the powder dissolved in an organic solvent like DMSO, MeOH and a mixture CH 2 Cl 2 /toluene 60/40 v/v (Fig. 6). All the spectra are EPR-silent and do not change with varying the temperature and the solvent, suggesting that the structure and oxidation state of 1 are maintained when varying the temperature and the organic solvent.
The lack of signals can be ascribed to the even number of electrons, to the large zero field splitting and fast relaxation time (Drago 1977;Mabbs and Collison 1992). These features indicate an oxidation state + 3 for Mn, which is generally inactive at the X-band frequency (Carlin 1966);  (Mani et al. 1994;Campbell et al., 2001;Syiemlieh et al. 2018). The very weak signal around g = 2 is assigned to Mn(II) (S = 5/2), often present as impurity in Mn(III) compounds (Bryliakov et al. 1999;Talsi and Bryliakov 2004). In DMSO an EPR signal at g value of ca 7.8 is detected. This resonance does not appear in MeOH or in the mixture CH 2 Cl 2 /toluene probably for the different solubility of the complex 1 in these solvents (Fig. 6). The absorption can be ascribed to the forbidden transition in the non-Kramers doublet, observed in Mn(III) and not in Mn(II) species (Bryliakov et al. 1999;Talsi and Bryliakov 2004;Dexheimer et al. 1989).

Magnetic susceptibility
To get an insight about the oxidation number and stereochemistry of the central metal ions in coordination frameworks, magnetic moments are often used in conjunction with electronic spectra. The spin and orbital angular momentum of its electrons are accountable for the magnetic properties of a complex. Nevertheless, in all cases, the spin-only magnetic moment persists, and the total numbers of unpaired electrons are correlated with it. The magnetic moment of complex 1 was calculated at room temperature, using the solid sample. The observed magnetic data (µ eff = 4.92 µ B ) are in good agreement with other Mn(III) derivatives in the literature (Dutta and Syamal 1982). Mn(III) is d 4 (S = 2) and given that both configurations exhibit unpaired electrons, it would be paramagnetic. Indeed, the magnetic studies evidence that complex 1 exhibits a high spin state, indicating that the energy gap between successive orbitals is low enough to allow electrons to occupy them; this results in a high spin atom with 4 unpaired electrons.

Cytotoxicity measurement
To assess the cytotoxic effects of the Mn(III) derivative on the survival of fibroblast, SiHA, and MCF-7 cell lines with concentration ranging from 0.1 to 100 µg/ml, a MTT assay was performed (Fig. 7) applications where the compound is used as the chemical sensor. The complex was evaluated for its cytotoxic activity against three different human cell lines (2 cancer cell lines and one normal cell line). Among the three cell lines, the derivative exhibited a better anti-proliferative activity against MCF-7 in comparison to SiHa. Also, it was least cytotoxic toward fibroblast cells. Further studies must be performed to synthesize new metal derivatives which could be good candidates for the biological process that involves the enzymatic breakdown of a cell's cytoplasm or cytoplasmic components.
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