Spectroscopic investigations, DFT calculations, molecular docking and MD simulations of 3-[(4-Carboxyphenyl) carbamoyl]-4-hydroxy-2-oxo-1, 2- dihydroxy quinoline-6-carboxylic acid

By FT-IR, FT-Raman and DFT computations spectral characterisation of 3-[(4Carboxyphenyl) carbamoyl]-4-hydroxy-2-oxo-1, 2-dihydroxy quinoline-6carboxylic acid was performed. Computational calculations were done using B3LYP/6-31G(d’) basis set. Vibrational assignments of wavenumbers were performed on the basis of potential energy distribution. Donor acceptor interactions were evaluated using NBO analysis. To foresee the important reactive sites of the title compound we combined DFT calculations and molecular dynamics (MD) and visualized the ALIE and Fukui functions. Sensitive nature of the compound towards autoxidation and degradation in the presence of water was investigated by the calculation of BDE and RDF. By molecular docking the compound forms a stable complex with ubiquinol-cytochrome–c reductase inhibitor.

The FT-IR spectrum ( Fig.1) was recorded using KBr pellets on a DR/Jasco FT-IR 6300 spectrometer. The FT-Raman spectrum (Fig.2) was obtained on a Bruker RFS 100/s, Germany. For excitation of the spectrum the emission of Nd: YAG laser was used, excitation wavelength 1064 nm, maximal power 150mW, measurement on solid sample.

Computational Details
Calculations of the wavenumbers, molecular geometry, polarizability values, frontier molecular orbital analysis were carried out with Gaussian 09 program [21] using the B3LYP/ 6-31G(d') quantum chemical calculation method. A scaling factor of 0.9613 is used to scale the theoretically obtained wavenumbers [22] and the assignments of the vibrational wavenumbers are done by using Gauss View [23] and GAR2PED software [24]. Parameters corresponding to optimized geometry of the title compound (Fig. 3) are given in Table 1. Jaguar 9.0 and Schrodinger materials science suite 2015-4 was used for the investigation of the reactivity of the compound [25]. DFT calculations with the Jaguar were carried out using B3LYP exchange correlation functional, with 6-311++G(d,p), 6-31+G(d,p) and 6-311G(d,p) basis sets for the calculations of ALIE, Fukui functions and BDEs, respectively. Desmond program was used for MD simulations which was performed by OPLS 2005 force field [26], with simulation time set to 10 ns. The pressure was set at 1.0325 bar while temperature was set to 300 K. Cutoff radius was set to 12 Å, while the modelled system was of isothermal-isobaric (NPT) ensemble class.
For the solvent of SPC model [27] was used here. For the modelling of system CPCHODQ6C molecule was placed alone into the cubic box with ~3000 water molecules. For the preparation of input files and output analysis Schrodinger materials science suite 2015-4 was used [28]. (125.8°), C3-C15-C18 (121.3°) and N14-C20-C18 (114.2°) respectively [30]. The presence of higher electro negative group C=O would be the reason for the lesser bond angle of N10-C12-C14 (116.0°).

IR and Raman Spectra
The observed IR and Raman bands and calculated (scaled) wavenumbers and assignments are given in Table 2. The C12=O16 and C13=C14 stretching vibrations are assigned at 1678 cm -1 (DFT), 1670 cm -1 (IR) and at 1562 cm -1 (DFT), 1551 cm -1 (IR), 1548 cm -1 (Raman) respectively. The C=O stretching mode has high IR intensity and PED of 37%. The C=O stretching vibration in the spectra of carboxylic acid give rise to strong bands in the region 1600-1700 cm -1 [31]. The bands observed at 1746, 1741 cm -1 theoretically with IR intensities 362.66, 401.29, Raman activities 269.81, 169.61 and with PEDs of 73, 72% are assigned as C35=O36, C31=C32 stretching modes of the title compound. The stretching band of C13-O15 is expected in the region 1220 ± 40 cm -1 [32][33][34] and the band at 1292 cm -1 (DFT) is assigned as C-O stretching vibration with IR intensity of 165.43, Raman activity of 5.51 and a PED of 17% of the title compound while the reported value is 1206 cm -1 (DFT) [30]. The O-H stretching vibration gives rise to a band at 3050 ± 150 cm -1 [31]. The band observed at 2666 cm -1 experimentally and 2793 cm -1 in DFT calculation is assigned as the O-H stretching vibration. The downshift of the OH stretching mode is due to the strong hydrogen bonded system present in the title compound as reported in literature [35,36]. The O-H in-plane and out-of-plane deformation modes are expected at 1395 ± 55 cm -1 and at 905 ± 70 cm -1 respectively [32]. For the title compound the band at 1352 cm -1 (DFT) is assigned as the inplane O-H deformation band. Similarly, the band at 922 cm -1 (DFT) is assigned as the O-H out-of-plane deformation band of the title compound. The in-plane O-H bending mode has a low IR intensity 68.74 and high Raman activity of 955.36. Rajeev et al. [30] reported a band at 1412 cm -1 as the in-plane O-H deformation. The N-H stretching vibrations are expected [37] in the range 3500-3300 cm -1 . In the present study the bands observed at 3440, 3392 cm -1 in the IR spectrum and 3454 cm -1 theoretically are assigned as N-H stretching vibrational mode which has a PED of 100%, IR intensity 50.89 and Raman activity 108.89. In the present case the N-H stretching mode splits into a doublet and downshifted from the computed value which indicates the weakening of the N-H bond [38,39]. N-H group shows bands at 1510-1500, 1350-1250 and 740-730 cm -1 [39]. According to literature if N-H is a part of a closed ring [40] the N-H deformation band is absent in the region 1510-1500 cm -1 .
In the present case the N-H in-plane deformation band is observed at 1439 cm -1 theoretically and this mode has IR intensity 35.79, Raman activity 131.12 with a PED 22%. The out-ofplane deformation bands of N-H are expected in the range 650 ± 50 cm -1 and the bands observed at 612 cm -1 (DFT) are assigned as γN-H mode of the title compound. This mode has 73% PED with 60.95 as IR intensity and a low Raman activity less than 10.00. In the present case, the quinoline CC stretching ring modes are observed at 1413 cm -1 in the IR spectrum, 1414 cm -1 in the Raman spectrum, 1413 cm -1 theoretically with high Raman activity and the C-N stretching modes are at 1114 cm -1 in the IR spectrum, 1233, 1106,1082 cm -1 theoretically. Both the modes possess moderate IR intensities. Rajeev et al. reported the quinoline stretching modes at 1610, 1445, 1020 cm -1 (C-C), 1262 cm -1 (C-N) in the IR spectrum, 1609, 1051, 1022 cm -1 (C-C), 1202 cm -1 (C-N) in the Raman spectrum, 1607, 1433, 1045, 1035 cm -1 (C-C), 1270, 1230 cm -1 (C-N) theoretically [30]. The DFT calculations give the C-H stretching modes of the phenyl ringI and phenyl ringII of the title compound at 3128, 3101, 3067 cm -1 and 3151, 3099, 3098, 3061 cm -1 . Similarly, the bands observed at 3132, 3103, 3078 cm -1 (Raman) and 3157, 2990 cm -1 (IR) are assigned as C-HI and C-HII stretching modes of the phenyl rings of parent molecule [31] [31]. In asymmetric trisubstituted benzene, when all the three substituents are heavy, the ring breathing mode appears above 1100 cm -1 [32]. For the tri-substituted phenyl ring PhI, the ring breathing mode is assigned at 1066 cm -1 theoretically with moderate IR intensity and PED 18%.
Madhavan et al. [41] reported the ring breathing mode for a compound having two trisubstituted benzene rings at 1110 and 1083 cm -1 respectively. In the present case, the band observed at 1070 cm -1 in Raman spectrum and 1072 cm -1 theoretically with a PED contribution of 36% and high IR intensity is assigned as the ring breathing mode of the phenyl ring II which is expected in region 1020-1070 cm -1 [32]. Panicker et al. [42] reported the ring breathing mode of di-substituted benzene at 1018 cm -1 (IR), 1034 cm -1 (Raman) and

Frontier Molecular Orbitals
Frontier molecular orbital study is used to explain the chemical behaviour and stability of the molecular system. The atomic orbital components of the frontier molecular orbitals are shown in Fig. 4. The delocalization of HOMO and LUMO over the molecular system shows the charge transfer within the molecular system. The HOMO-LUMO gap is found to be 3.157 eV. The chemical descriptors can be evaluated by using HOMO and LUMO orbital energies, EHOMO, and ELUMO as ionization energy I= −EHOMO, electron affinity A= −ELUMO, hardness η = (I−A)/2, chemical potential µ = −(I+A)/2 and electrophilicity index (ꞷ = µ 2 /2η [43]. For the title compound CPCHODQ6C, I = 8.482, A = 5.325, η = 1.579, µ = −6.904 and ꞷ = 15.093 eV (Table 3). For the title molecule, HOMO is delocalized over the phenyl group (PhII), amide group and partially over the quinoline ring while the LUMO is delocalized strongly over the entire molecule except carboxyl group of quinoline ring. For 7Cl, HOMO is delocalized strongly over the quinoline ring and substituted chlorine atom while LUMO is delocalized strongly over the entire molecule except NH groups. For 8Cl and 9Cl HOMO is over the phenyl ring PhI and partially over the pyridine ring and LUMO is over the entire molecule except carboxyl group of PhI and carbonyl group of pyridine ring. For 7Br HOMO is over the entire molecule except carboxyl group of PhI and LUMO is over the entire molecule. For 8Br and 9Br HOMO is over the entire molecule except carboxyl group of PhI and carbonyl group of pyridine ring and LUMO is over the entire molecule except carboxyl group of PhI and NH group of pyridine ring. For 7F, HOMO is over the entire molecule except carboxyl group of PhI and NH of pyridine while LUMO is over the entire molecule except NH group of amide group. For 8F, HOMO is over the entire quinoline ring while for 9F, HOMO is over the entire molecule. LUMO is delocalized over the entire molecule except carboxyl group of PhI, carbonyl group of pyridine and NH group of amide for 8F and 9F.
The chemical potential decreases for the halogen substitution in the order 7Cl, 8Cl, 9Cl < 7F, 8F, 9F < 7Br, 8Br, 9Br < CPCHODQ6C. Chemical potential value of 8Cl is deviated maximum from the parent molecule while all other halogen substitution shows minimum deviation. Halogen substitution results in reduction in the µ value in comparison with the parent molecule and for 8Cl it is minimum. Halogen substitution also results a decrease in electrophilicity index and is minimum for 8Cl. Global hardness is higher for 8Cl because of its large HOMO-LUMO gap which results a decrease in polarizability.

Molecular Electrostatic Potential
Molecular electrostatic potential and electron density are related to each other to find the reactive sites for electrophilic and nucleophilic sites [44,45]. The negative (red and yellow) regions of MEP map (Fig.5) were related to electrophilic reactivity while the positive (blue) regions to nucleophilic reactivity. For the parent molecule, most electrophilic (red and Yellow) regions are C=O group of both carboxyl group, slightly over PhII and the nucleophilic regions (blue) are deeply over the NH bond of quinoline ring, slightly over the hydrogen atom of the OH groups. For 7Cl, 8Cl and 9Cl, electrophilic regions are strongly over the carbonyl group of both carboxyl group and slightly over the phenyl ring while the nucleophilic regions are over the NH group of quinoline ring and slightly over the hydrogen atoms of the OH groups and more intense in the case of 8Cl. For fluorine substitution the electrophilic regions are similar to that of chlorine substitution while the nucleophilic regions are same that of chlorine substitution but blue region of NH bond of quinoline in 8F is more pronounced. For bromine substitution also the electrophilic and nucleophilic behaviour is identical to that of chlorine and fluorine substitution while blue region around bromine is higher than that in fluorine substitution. The nucleophilic region of fluorine substitution is less than that in chlorine and bromine substitution.

NBO Analysis
The natural bond orbitals (NBO) calculations were performed using NBO 3.1 program [46] and the important interactions are presented in tables 4 and 5. The strong interactions are

Nonlinear Optical Properties
The calculated first hyper polarizability of the title compound is 15.827×10 −30 esu which is 121.75 times that of standard NLO material urea (0.13 ×10 −30 esu) ( Table 6) [47]. The reported value of first hyper polarizability of similar derivative is 2.24 ×10 −30 esu [48]. The phenyl ring stretching vibrations at 1593, 1505 cm -1 in the IR spectrum have their counterparts in the Raman spectrum at 1602, 1503 cm -1 respectively with IR and Raman intensities are comparable. These types of organic molecules have conjugated π-electron system and large hyper polarizability which leads to nonlinear optical properties [49]. The C−N distances in the calculated molecular structure vary from 1.3745 to 1.4047 Å which are in between those of a CN single and double bond and this suggest an extended π-electron delocalization over the molecular system which is also responsible for the nonlinearity of the molecule [50]. We conclude that the title compound is an attractive object for future studies of non-linear optical properties.

ALIE surfaces and Fukui functions
Average local ionization energy (ALIE) is a quantum molecular descriptor which indicates the energy required to remove an electron from the molecule. So, we can say that the sites with least values of ALIE are more open for an electrophilic attack [51,52]. According to the equation given below ALIE is the sum of orbital energies weighted by the orbital density.
where N stands for the number of electrons in reference state of the molecule, while  stands for the fraction of electron which default value is set to be 0.01 [55] . By plotting Fukui functions to electron density surfaces we get a lot of information about the important molecular sites acting as a reactive centres [51,52]. The Fukui function plot is represented in Fig. 7. The colour coding in the plot is as follows, purple (positive) colour in Fukui function f + means the electron density has been increased by the addition of charges to the system while red (negative) colour in Fukui function f -means the electron density has been diminished by the addition of charges. Electron density is increased in the near vicinity of carbon atom C21 and electron density is decreased near the O16, O36, O37 atoms.

Reactive and degradation properties based on autoxidation and hydrolysis
Degradation properties based on autoxidation and hydrolysis mechanisms are explained using RDFs and BDEs. Calculations of BDE for hydrogen abstraction allow the possibility to predict molecular sites where autoxidation process could start. It provides details about upto what extent some molecule are sensitive to presence of oxygen in open air, a parameter that is of very much importance in pharmaceutical industry. Forced degradation studies can also be studied using BDE, since they can be used for confirmation and determination of degradation path of some organic pharmaceutical molecule [56][57][58][59]. Wright et al. says that the target molecule is most vulnerable to autoxidation if the BDE for hydrogen abstraction ranges from 70 to 85 kcal/mol [60]. BDE values for hydrogen abstraction lower than 70 kcal/mol, are not suitable for the autoxidation mechanism since formed radicals are resistant for O2 insertion [60][61][62]. kcal/mol so we can say that the molecule is stable in the presence of oxygen. To find the extend of hydrolysis we have also calculated the RDF for the molecule. In Fig.9 RDFs of atoms with the most pronounced interactions with water molecules are presented. In RDF plot, g(r) represents the probability of finding a particle in the distance r from another particle [63]. Results provided in Fig.9 indicate that only four atoms of CPCHODQ6C molecule have relatively significant interactions with water molecules. These atoms are C12, C24, O19, O33, O37 , H11, H17, H34, H38, H39 which shows similar g(r) profile. According to the maximal g(r) values the most important RDF is certainly for H34 atom. Here the presence of hydrogen atoms shows the low stability of the molecule in the surroundings of water, so the role of this title compound in the pharmaceutical industry is irrelevant.

Solubility parameter
One of the demanding fields in pharmaceutical is the development of new products and finding the active component. To be considered for the pharmaceutical drug production, the active ingredient has to fit in to certain physical properties such as stability, solubility etc. If the active component doesn't meet the required parameters, then these properties must be modified. One way to modify the properties without structural modification is to find a suitable excipient and mixing them up. Suitable excipient can be identified using experimental methods but its time consuming, while computational methods can be effectively used to narrow down the possibilities. Active component and excipient must be mutually compatible, one of the parameters for compatibility is the solubility parameter.
Which means, the solubility parameter of the active component must have a value similar to the one of the excipient compounds [17][18][19]. Solubility parameter can be computationally predicted by applying the MD simulations and the following equation: In this work, the solubility parameter has been calculated for the CPCHODQ6C molecule and it has been compared with three compounds frequently used as excipients polyvinylpyrrolidone polymer (PVP), maltose, and sorbitol). MD systems used to calculate this quantity consisted of 32 molecules placed in a cubic simulation box. Solubility parameters of all mentioned compounds have been summarized in Table 7.
As indicated by the results presented in Table 7, the CPCHODQ6C molecule has the highest compatibility with the Maltose compound. In this case, the difference between corresponding values of solubility parameter is less than 0.2 MPa 1/2 , indicating very high compatibility.
Solubility parameter of sorbitol is higher than the solubility parameter of the CPCHODQ6C molecule, while PVP has lesser value than our title molecule. Therefore, the MD calculations suggest that it is reasonable to consider Maltose as an excipient for CPCHODQ6C molecule.

Molecular Docking
Antimalarial drugs constitute a major part of antiprotozoal drugs. Malaria remains a major health problem, mainly in sub-Saharan Africa and parts of Asia and South America [64] with over 200 million clinical infections and nearly half a million deaths annually. Malaria is caused by protozoan parasites belonging to the genus Plasmodium and is transmitted via the bite of a female Anopheles mosquito. There are four major species of the parasite that cause malaria in humans, namely, Plasmodium falciparum, P. vivax, P. ovale and P. malaria, while a fifth parasite, P. knowlesi, is now recognized [65]. Historically, a range of drugs has been used to treat or prevent malaria, including several derived from the quinoline ring system such as quinine, chloroquine (CQ), amodiaquine, piperaquine, mefloquine, and primaquine [66]. Quinoline and its related derivative comprise a class of heterocycles, which has been exploited immensely than any other nucleus for the development of potent antimalarial agents. Various chemical modifications of quinoline have been attempted to achieve analogs with potent antimalarial properties against sensitive as well as resistant strains of Plasmodium sp., together with minimal potential undesirable side effects [67]. From PASS (Prediction of Activity Spectra) [68] analysis we have to choose the favorable target for docking study and different types of activities predicted as in Table 8. We choose the activity ubiquinolcytochrome-c reductase inhibitor with Pa value 0.858 and high-resolution crystal structure of corresponding receptor atovaquone-inhibited cytochrome BC1 complex with (PDB ID: 4PD4) was downloaded from the RCSB protein data bank website. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding [69]. cyt bc1 inhibitors are generally classified as slow-onset anti-malarials, we found that a single dose of endochinlike quinolone-400 (ELQ-400) rapidly induced stasis in blood-stage parasites, which was associated with a rapid reduction in parasitemia in vivo. ELQ-400 also exhibited a low propensity for drug resistance and was active against atovaquone-resistant P. falciparum strains with point mutations in cyt bc1. ELQ-400 shows that cyt bc1 inhibitors can function as single-dose, blood-stage anti-malarials and is the first compound to provide combined treatment, prophylaxis, and transmission blocking activity for malaria after a single oral administration [70]. This remarkable efficacy suggests that metabolic therapies, including cyt bc1 inhibitors, may be valuable additions to the collection of single-dose anti-malarials in current development. All docking calculations were performed on AutoDock4.2 [71], Auto Dock-Vina software [72] and as in literature [73]. The amino acids of the receptor Tyr275, Asn96, Asn271 forms H-bond with OH group and other electrostatic interactions are detailed in Fig.10. The docked ligand forms a stable complex with the receptors atovaquone−inhibited cytochrome BC1 complex as depicted in Fig.11 and the binding free energy value is -9.1 kcal/mol ( Table 9). The docked ligand is embedded with the catalytic site of cytochrome BC1 complex as shown in Fig.12. These preliminary results suggest that the compound having               Table 8 Pass Analysis Table 9 Docking

Graphical Abstract
Tables Table 1 Optimized    Table 7.
Values of solubility parameters [MPa 1/2 ] for studied molecules and selected frequently used excipients Table 8 PASS prediction for the activity spectrum of CPCHODQ6C compound. Pa represents probability to be active and Pi represents probability to be inactive.