Synthesis, Characterization, Single Crystal Structural Studies, Antibacterial Activity and DFT Investigations of 2-Chloro-5-Ethoxy-3,6-Bis(Methylamino)-1,4-Benzoquinone

A new compound 2-chloro-5-ethoxy-3,6-bis(methylamino)-1,4-benzoquinone (cemb) has been synthesized and characterized by various physicochemical techniques. The compound crystallizes in the triclinic system with the space group P-1 and contains extended hydrogen bonding interaction providing supramolecular architecture. The compound contains CH∙∙∙π and oxygen∙∙∙π interaction which stabilizes the crystal structure and is also responsible for the semiconducting behavior. The DFT calculations in the gaseous phase of cemb with B3LYP hybrid functional show good correlation with experimental results (single crystal XRD data) and are used to predict the molecular properties. Excluding the side ethyl group, the structure possesses Cs symmetry. The ring breathing mode of vibration is found to have frequency (551 cm-1) which is much lower as compared to the corresponding mode of benzene (991 cm-1). The bioefficacy of the compound has been examined against the growth of bacteria in vitro to evaluate antimicrobial potential. The pphotoluminescence properties indicate that the compound has characteristic fluorescence emissions. The HOMO and LUMO energy gap reveals that the energy gap reflects the chemical activity of the molecule. Synthesis, Characterization, Single Crystal Structural Studies, Antibacterial Activity and DFT Investigations of 2-Chloro-5-Ethoxy-3,6Bis(Methylamino)-1,4-Benzoquinone


Introduction
Naturally occurring compounds containing a quinone group are involved in various bioenergetic processes as electron-transport agents. Some quinones play important roles in the biochemistry of energy production and serve as vital links during electron transport. These compounds have attracted considerable attention because of their biological activity and chemotherapeutic values [1]. Various quinone containing oxygenated aromatic rings show biological activities [2]. Quinones are found in all respiring animal and plant cells where they function primarily as components of the electron transport chains involved in cellular respiration [3] and photosynthesis [4]. These compounds occur in other natural contexts and as pollutants [4][5][6][7][8]. Quinones form an important class of toxic metabolites and paradoxically can be either mutagenic and, therefore, are potentially carcinogenic, or effective anticancer agents [9]. Some quinones possibly play roles in the cellular defence since they effectively inhibit growth of bacteria, fungi and/or parasites [10][11][12][13].
Quinones are a large class of compounds endowed with rich and fascinating chemistry [14]. They are widely distributed in the natural world [15], being found in bacteria, plants and arthropods and hence, quinones are ubiquitous to living systems. Their role as electron transfer agents in primary metabolic processes like photosynthesis and respiration is vital to human life. A large number of chemical derivatives with 1,4-benzoquinone as the basic subunit exhibit prominent pharmacological applications such as antibiotic, [16,17] antitumor, [18][19][20][21] antimalarial, [22] antineoplastic, [23] anticoagulant [24] and herbicidal activities [25]. Coordination chemistry of quinones is also quite rich from the perspective of designing magnetic materials [26] and understanding photo physical properties [27]. The chemistry of quinones and hydroquinones is of interest because of their involvement in the electron and hydrogen transfer reactions in various processes

Crystal structure determination
Crystals suitable for X-ray analysis of cemb were grown at the room temperature. The crystal structure data were collected on an Oxford Gemini diffractometer equipped with a CrysAlis CCD software using a graphite mono-chromated Mo Kα ( λ=0.71073 Å) radiation source at 293 K for cemb. Multiscan absorption correction was applied to the X-ray data collection and the structure was solved by direct methods (SHELXS-08) and refined against all data by full matrix leastsquare on F 2 using anisotropic displacement parameters for all nonhydrogen atoms. All hydrogen atoms were included in the refinement at geometrically ideal position and refined with a riding model [34]. The MERCURY package and ORTEP-3 for Windows program were used for generating structures [35,36]. The details of data collection, structure solution and refinement are listed in Table 1. Selected bond lengths and angles are given in Table 2. ORTEP diagram of cemb with atom numbering schemes is shown in Figure 1A.The single crystal X-ray diffraction studies indicate that the starting compound chloranil is converted to cemb during the course of reaction. The grown single crystals of cemb were analyzed by single crystal X-ray data. The bond lengths and angles in the ring are normal [37,38]. The structure is stabilized by intermolecular N-H···O and C-H···O hydrogen bonding occurring between hydrogen of substituted N-methyl amine group to oxygen of nearby molecule and CH•••π interaction occurring between hydrogen of substituted ethoxy group to centroid of ring of a nearby molecule at a distance of 3.561 Ǻ and Oxygen···π at a distance of 3.557 Ǻ producing linear chain structure. The structure is also stabilized by Carbon, hydrogen, nitrogen contents were estimated on a CHN Model CE-440 Analyser and on an Elementar Vario EL III Carlo Erba 1108. IR spectra were recorded in the 4000-400cm -1 region in KBr pellets on a Perkin Elmer/Varian Excalibur 3100 FT-IR spectrophotometer. 1 H and 13 C NMR spectra were recorded in DMSO-d 6 on a JEOL AL300 FT-NMR spectrophotometer. The electrochemical experiments were performed with a CHI-660C (CH Instruments, USA), using threeelectrode system, platinum wire as counter electrode (area ~ 8.0 cm 2 ) and Hg/HgO was used as the reference electrode. Electrical conductance of pressed pellets was measured by a conventional two-probe method in the 313-443 K range with contacts made on the pellet surfaces by means of silver paint. A Keithley 236 SMU was used to measure the sample resistance. The fluorescent data were collected at room temperature with a Varian CARYECLIPSE spectrophotometer. Six bacterial pathogens Salmonella typhi, Shigella flexneri, Staphylococcus aureus, Aeromonas hydrophila, E. coli and Enterococcus faecalis were used to test the antibacterial activity of the compound. Commercial antibacterial drugs, streptomycin sulphate and chloramphenicol (Himedia) in same concentration of 10-100 µg/disc are used to compare effectiveness of the test compounds.

Synthesis of cemb
For the synthesis of cemb, sodium acetate (1.148 g, 14 mmol) was added pinch by pinch within 20 min to a reaction mixture of chloranil (0.492 g, 2 mmol), which was added drop wise to methylamine hydrochloride (0.270 g, 4 mmol) in 15 ml of absolute ethanol with constant stirring for 1 h and refluxed 4h at 65ºC. The solid separated was filtered off, washed out successively with ethanol-water mixture (50:50 v/v) and air dried. The precipitates were suspended in ethanol (20 mL) to which pyridine (1 mL) was added and refluxed for 1 h at 65ºC. The resulting clear solution was filtered and kept for crystallization. Single crystals of cemb suitable for X-ray analysis were obtained by slow evaporation of the solutions over a period of 25 days (Scheme 1). Grey color crystals were obtained with: Yield: 72%; m.p>290ºC; Anal. Found: C, 49

Antibacterial assay
The antibacterial assay was done according to the Bayer method [33]. The test compounds were dissolved in DMSO to a final concentration of 5 mg/mL. Sterilized Whatman no. 1 filter paper discs (5 mm) were impregnated with different volume (2,4,8,12,16 and 20 µL) of compounds to get a final concentration of 10, 20, 40, 60, 80 and 100 µg per disc. Sterilized paper disc loaded with the 10 µL of DMSO was taken as a control. The bacterial test pathogens were spread on fresh Müller Hinton Agar (MHA) plates with the help of cotton swabs to form an even lawn of the test bacteria. The filter paper disc impregnated with the test compounds were placed on the surface of the MHA plates seeded with test bacteria and the plates were incubated in a BOD Incubator (Caltan-152, Narang Scientific Works, New Delhi, India) for 24 h at 37 ± 2ºC. The inhibition zones around each disc were  Table 1: Crystallographic data for cemb molecule.

UV-Vis spectra
The experimental UV-vis spectrum of cemb in DMSO is shown in Figure 2. It shows a transition at 336.50 nm.

Photoluminescence emission spectra
The photoluminescence emission spectrum of cemb was recorded in DMSO at room temperature ( Figure 3). On excitation with a 335 nm laser source at room temperature, a strong fluorescence emission peaked at 426 nm is observed. The photoluminescence property shows that the compound has characteristic fluorescence emissions. The main chromospheres in the compound are the carbonyl and halogen groups and its conjugation degree is further enhanced by the amine group. The solution state photoluminescence property of compound in DMSO indicates that increase in the dilution of solution resulted in a blue shift and decrease in intensity at various concentrations due to weakening of the CH•••π and CH•••Cl interactions, but the luminescence property persists even in dilute solution due to existence of the intermolecular interactions [40].

Computational Details
All calculations were performed using the Gaussian 03 and Gauss View 4.1 software [41]. The structure optimization of cemb was done using DFT method with the functional B3LYP and basis set 6-311 ++ G(d,p) [42]. The input geometry of the molecule for the DFT calculations was generated from the single crystal X-ray investigation carried out in the present study. The HOMO-LUMO energies, MEP, UV-vis spectra, linear polarizability and first hyperpolarizability were also calculated by the DFT method [43,44]. To facilitate the vibrational analysis potential energy distributions (PEDs) were also computed using the GAR2PED software.

Results and Discussion
Chloranil react with methylamine hydrochloride in presence of sodium acetate and pyridine to yield compound cemb. There is substitution of two methylamine and one ethoxy group with replacement of three chlorine atoms through a condensation reaction, which is confirmed by X-ray diffraction studies. The Scheme 1 depicts the formation of cemb. The compound is air stable, non-hygroscopic crystalline solids, which is insoluble in common organic solvents but soluble in DMF, DMSO and can be kept in desiccators over a prolonged period without any sign of decomposition with melting point at >290ºC.

Geometry optimization
The optimized geometry (optimized energy -2037.988 a.u) of cemb are shown in Figure 4(A). There is good agreement between the geometrical parameters obtained by X-ray crystallography to those generated by DFT method as shown in Table 2. The slight disagreement in the bond lengths and angles are due to the fact that the experimental results are for the solid phase and the theoretical calculations are for the gas phase [45]. In the solid state, the existence of a crystal field along with the intermolecular interactions connect the molecules together, which results in the differences in geometrical parameters between the calculated and experimental values.

UV-Vis spectra
The DFT calculations were performed for the gas phase and DMSO solvent. Calculations predict an intense electronic transition at 328.24 nm.
Out of the rest four modes of the CH 2 group the scissoring mode is calculated to have magnitude 1476 (ν 62 ) cm -1 , the wagging mode is found at 1384 ( 53 ) cm -1 while the twisting and the rocking modes have the magnitudes 1268 (ν 49 ) and 1141 (ν 44 ) cm -1 .

C=O modes (6 modes):
The two carbonyl stretching modes couple strongly with the ring stretching modes to give rise to the computed frequencies 1661, 1639, 1595 and 1584 cm -1 ; the former two frequencies being assigned to the @ opc ν (C=O) and @ ipc ν (C=O) modes whereas the latter two frequencies are assigned to ring stretching modes. The two γ (C=O) modes are assigned at 757 and 745 cm -1 whereas the 2 β (C=O) modes are assigned as the frequencies 412 and 321 cm -1 in

Vibrational assignments
The cemb molecule is a 29 atomic molecule with 81 normal modes of vibration. The observed and calculated IR and Raman spectra of cemb are reproduced in Figures 5 and 6. The distribution of the normal modes could be divided under the following sections:-(i)

IR
Vibrational assignment (%PED) Theoretical wave number    accordance with the assignment reported in literature [52].

Ring modes (12 modes):
In addition to the two ring stretching modes mentioned above (section 5.1.2) the frequencies 1321 (ν 51 ), 1300 ( 50 ), 974 (ν 36 ) and 551 (ν 25 ) cm -1 have been identified as the ring stretching modes. The lowest frequency mode 551 (ν 25 ) cm -1 corresponds to the ring breathing mode. Out of the three planar ring deformation frequencies 641 (ν 27 ), 507 ( 23 ) and 457 (ν 22 ) cm -1 the first frequency appears to be due to the trigonal phenyl ring bending mode whereas the latter two correspond to the modes 6 (a,b) of the benzene molecule. The three normal modes of vibration corresponding to the non-planar ring deformations are calculated to have magnitudes 608 (ν 26 ), 146 ( 8 ) and 128 (ν 7 ) cm -1 . All the 12 normal modes of the ring are strongly coupled with the other modes.
*With the scale factor of 0.9906 for calculated wave numbers lower than 1100 cm -1 and the scale factor 0.9728 for larger wave numbers.

Molecular electrostatic potential and contour maps
The molecular electrostatic potential (MEP) mapped surfaces illustrate the charge distributions of molecules three dimensionally which give clear signature of the interactions of the molecules. The electrostatic potential is used to find the reactive site of a molecule. The MEP mapped surface of the molecules are calculated by DFT/ B3LYP/6-311G (d,p) method at the 0.02 isovalues and 0.004 density values. In color scheme of MEP plot, the red region show atoms with lone pairs or negative electrostatic potentials; the intensity of the color is proportional to the absolute value of the potential energy. The positive electrostatic potentials are shown in blue/yellow color region and characterize the polar hydrogen in O-H bonds. The Green areas cover parts of the molecule where electrostatic potentials are close to zero (C-C, C-N and C-S bonds). The MEP mapped surface of the cemb is shown in Figure 4(B).
Molecular electrostatic potential and electrostatic potential are the useful quantities to illustrate the charge distributions of molecules are used to visualize variably charged regions of a molecule. Therefore, the charge distributions can give the information about how the molecules interact with another molecule. At any given point r (x, y, z) in the vicinity of a molecule, the molecular electrostatic potential (MEP), V(r) is defined in terms of the interaction energy between the electrical charge generated from the molecule electrons and nuclei and a positive test charge (a proton) located at r. The molecular electrostatic potential is related to the electronic density and a very useful descriptor for determining sites for electrophilic attack and nucleophilic reactions as well as hydrogen-bonding interactions [54]. In Figure 4b and 4c, whereas electrophilic reactivity is presented by the negative (red) regions, nucleophilic reactivity is shown by the positive (blue) regions of MEP. The red region is localized on the vicinity of oxygen and reflects the most electronegative region (excess negative charge). On the other hand, the nucleophilic reactivity of the molecules is localized on the hydrogen atoms. In this respect, the compound (cemb) is found to be useful to both bonds metallically and interact intermolecularly. Based on these results, it can be ascertained that how the change of the chlorine group affects the molecular properties of the compound. One can see from the Figure 4b that the blue color on methyl amine is found to be the predominant because of the positive charge distribution on the molecule. Moreover, according to the ESP Figure 4c, the most negative ESP is spread over the oxygen and chlorine atoms, indicating the delocalization of electrons over the oxygen and chlorine atoms of the molecule which also reveal extended conjugation of the benzene ring with these groups [55]. Furthermore, the total charge density contour map is given in the Figure 4d. A contour plot is a two-dimensional XY plot of a three-dimensional XYZ surface showing lines where the surface intersects planes of constant elevation (Z). The contour maps are used to show lines of constant density or brightness, such as electrostatic potentials and are drawn in the molecular plane. The electron rich red lines are around oxygen and nitrogen whereas electron deficient region are shown by greenish-yellow lines. The contour maps are also calculated by same basis sets at 0.004 density values at same level of calculations of the MEP mapped surface of the molecules [56].

Frontier molecular orbital analysis
The analysis of the wave function indicates that the electron absorption corresponds to the transition from the ground to the first excited state and is mainly described by one-electron excitation from the HOMO to LUMO. Both the HOMO and LUMO are the main orbitals taking part in the chemical reaction. The HOMO energy characterizes the capability of electron donating; The LUMO characterizes the capability of electron accepting [57]. The HOMO and LUMO, calculated at the B3LYP/6-311G (d, p) level can be seen in Figure 7 for the gas phase. The positive phases are marked as red and the negative ones as green. Figure 7 shows distribution and energy levels of the HOMO and LUMO orbitals. For the HOMO of cemb, electron density are localized on all atom except on methyl of ethoxy group with E HOMO =-0.21294 a.u. For the LUMO, electron density are localized on the entire atom except on ethoxy group with E LUMO =-0.10412. The value of the energy separation between the HOMO and LUMO is 0.10882 a.u (2.961068 eV). In the contour map, the lines with red colors are regarded as the negative phase and the lines with green colors are the positive phase. The contour HOMO-LUMO diagram is in accordance with the frontier orbital. The frontier orbital gap helps to characterize the chemical reactivity, optical polarizability and chemical hardnesssoftness of a molecule [58].The small HOMO-LUMO energy gap means low excitation energy for the compound, a good stability and a low chemical hardness. The molecules having a large energy gap are known as hard, and molecules having a small energy gap are known as soft molecules. The soft molecules are more polarizable than the hard ones, because they need small energy for excitation. The hardness value of a molecule can be determined by the formula [59]

Global reactivity descriptors
On the basis of Koopman's theorem, global reactivity descriptors [60] are calculated using the energies of Frontier molecular orbitals E HOMO , E LUMO and given by Equations (a)-(e). where A and I are the ionization potential and electron affinity of the compounds respectively. Electron affinity refers to the capability of a ligand to accept precisely one electron from a donor. However, in many kinds of bonding viz., covalent hydrogen bonding, and partial charge transfer occurs. Softness (S) is the reciprocal of hardness and is a property of compound that measures the extent of chemical reactivity.
Recently Parr et al. [61] have defined a new descriptor to quantity the global electrophilic power of the compound as electrophilicity index (ω), which defines a quantitative classification of the global electrophilic nature of a compound. Parr et al. have proposed electrophilicity index (ω x) as a measure of energy lowering due to maximal electron flow between donor and acceptor. They defined electrophilicity index (ω) as follows The energies of frontier molecular orbitals (E HOMO , E LUMO ), energy band gap (E HOMO ,E LUMO ), electro negativity (χ), ionization potential (A), electron affinity (I), chemical potential (μ), global hardness (η), global softness (S) and global electrophilicity index (ω) for cemb are listed in Table 5.

In-vitro antibacterial activity
Six bacterial pathogens Salmonella typhi, Escherichia coli, Shigella flexneri, Staphylococcus aureus, Aeromonas hydrophila and Enterococcus faecalis were used to test the antibacterial activity of the cemb. The activity of cemb and commercial antibacterial drugs streptomycin sulphate and chloramphenicol were tested against the bacterial pathogens and the inhibition zones (cm) of the compounds were presented in the Table 6. The test results of the compound have also been compared with those of the standard drugs and results show significant activity of the compound against S. aureus than all other test pathogens. The cemb displays higher zone of inhibition (3.5 cm) against S. aureus at 100 μg/disc and does not display any zone of inhibition against Aeromonas hydrophila and E.coli at all concentration. A clearing zone around the disc indicates the inhibitory activity of the compound on the organism (Figure 8a-8d). The results are given in Table 6 which clearly indicate that the inhibitions by the compound is lower as compared to the standard drugs and activity increases with increasing concentration of the compound (since concentration plays a vital role in increasing the degree of inhibition) which is almost comparable to the size of the reference antibiotics. 1,4-Benzoquinones is a crossconjugated enediones, have ability to accept one electron initially, forming the semiquinone anion radical, followed by a further electron to give the dianion, is the dominant feature of quinone chemistry. It is this reversible reduction process that accounts for the biological activity [62]. Apart from this, other factors such as solubility, conductivity and intermolecular interaction may also be the possible reasons for activity. Furthermore, free HNCCO moiety increases the delocalization of π-electrons over the molecule of cemb and enhances the lipophilicity Electrophilicity Index (ω) -0.184 Table 5: Reactivity parameters of cemb.  No zone was observed *inhibition zone is the average of the diameter of zone from two sides in cm. (A. Hydrophila, E. coli does not show activity). which leads to the breakdown of the permeability barrier of the cell and thus retards the normal cell processes. The mode of action may involve the formation of a hydrogen bond through the nitrogen /oxygen atom in the compound with the active centers of the cell constituents, resulting in interference with the normal cell process, i.e., prolonging the lag phase of the growth cycle and increasing the generation time of the organisms so that it takes each organism more time to complete cell division [63].

Conclusions
This paper reports the synthesis, spectral and crystal structure investigations of a new compound: 1,4-benzoquinone derivative, 2-chloro-5-ethoxy-3,6-bis (methylamino)-1,4-benzoquinone (cemb), formed via condensation reaction. It was found that during the reaction, chlorine group of the compound is replaced with ethoxy group generating a new molecule which is stabilized via weak intermolecular interactions. The results of antibacterial activity shows that the new compound cemb inhibit growth of Gram-positive bacteria. Keeping in view the single molecule approximation, the X-ray crystallographic results are well reproduced by DFT method. From the vibrational analysis it turns out that the ring breathing mode (551 cm -1 ) and the trigonal ring bending mode (641 cm -1 ) are considerably reduced as compared to the benzene modes. In the present case one γ(C=O) is found in the range 700-800 cm -1 while the other γ(C=O) and two β(C=O) are found to be in the range 300-400 cm -1 while for 2,5-diamino-3,6-dichloro-1,4-benzoquinone [46] the two γ (C=O) and one β (C=O) were found in the range 700-800 cm -1 and the other β (C=O) in the range 300-400 cm -1 . Inspite of the low symmetry (C 1 ) of the molecule the modes of the identical groups couple strongly. According to the MEP, the most negative ESP is spread over the oxygen and chlorine atoms, indicating the delocalization of electrons over the oxygen and chlorine atoms of the molecule which also reveal extended conjugation of the benzene ring.