Pharmaceutica Analytica Acta

The physical properties of the solid state seen in crystals and powders of both drugs and pharmaceutical excipients are of interest because they can affect both the production of dosage forms and the performance of the finished product. The nature of the crystalline form of a drug may affect its stability in the solid state, its solution properties and its absorption. In order to study the stability and bioactivity of the title pharmaceutical compound, it is chosen for crystal growth and its properties are studied theoretically in detail.

The use of arsenic and its compounds are very popular in the production of pesticides, herbicides and insecticides. They are used as food additives in the poultry and swine industries in developing countries [1-3]. As a food additive, they control disease, simulate growth and improve both feed efficiency and conversion in animals. Majority of the organoarsenic compounds are not metabolized in the poultry and are excreted chemically unchanged and the manure is turned into fertilizer pellets for commercial use.

After the application of the fertilizer to soil, microbial activity and the presence of ultraviolet light lead to the formation of other organic arsenic species [4-6]. This in turn poses a number of health and environmental concerns. Since arsenic, in its various forms, is a known Carcinogen [7-11] it has been correlated with hypertension as well as other cardio metabolic diseases [12]. Arsanilic acid is used in the laboratory, for instance in recent modification of nanoparticles. It is also launched in chemotherapeutic approach for treating infectious diseases of humanbeings.

Density Functional Theory (DFT) is an effective cum economical tool for studying the structural properties of the molecule. Spectroscopic techniques eminently help in determining the dynamic behaviour of the electronic and molecular structures of natural products at microscopic level [13,14]. In this work, DFT technique is employed to study the complete vibrational spectra of p-Arsanilic acid.

The pure sample of p-Arsanilic acid is purchased from Spectro. Chem Ltd, Mumbai, India and used as such for crystal growth. From the solubility test, sodium carbonate solution is found to be the good solvent. To the 50ml of sodium carbonate solution, the titled substance is dissolved up to the supersaturated state. Then the solution was filtered and kept undisturbed for slow evaporation. The nucleation began after 30 days. A single crystal of 20 x 10 x 5 mm³ size was harvested at the seventh week. The transparency of the crystal is shown in Figure 1.

Figure 1: Grown crystal of p-Arsanilic acid.

The grown crystal is subjected to powder x-ray diffraction studies to reveal the crystallinity of the substance. The powdered sample is scanned in the range 10-90° C at a scan rate of 2^o/min using the JEOL JDX services instrument with CuKα (λ = 1.5406A°) radiation. The room temperature FTIR spectrum of the title compound is measured in the region 4000- 400 cm^-1 with the scanning speed of 10 cm^-1 min^-1 and the spectral resolution of 4.0 cm^-1 by employing Perking-Elmer spectrometer. The FT-Raman spectrum of the compound is recorded using Bruker FRA 106/S instrument equipped with Nd: YAG laser source operating at 1064 nm line widths with 100mW power. The spectrum is recorded in the range of 4000-10 cm^-1. ¹H and ¹³C NMR (400 MHz; DMSO) spectra are recorded using BRUKER TPX-400 FT-NMR spectrometer. The optical absorption spectrum is recorded using Perkin-Elmer Lamda 935 UVVIS- NIR spectrometer. The non- linear optical activity of the compound is determined using Kurtz powder technique.

Density functional theory (DFT) is extensively used due to their accuracy and low computational cost to calculate a wide variety of molecular properties and provided reliable results which were in accordance with experimental data. The molecular structure of pAsA and corresponding vibrational harmonic frequencies are calculated using Becke3-Lee-Yang-Parr (B3LYP) with 6-31+G^** basis set using GAUSSIAN 09W [15] program package. No constraints are imposed on the structure during the geometry optimizations. The vibrational analyses, calculated at the same level of theory, indicate that the optimized structures are at the stationary points corresponding to local minima without any imaginary frequency.

Electronic properties: HOMO-LUMO energies, absorption wavelengths and oscillator strengths are calculated using B3LYP method, based on the optimized structure in gas phase. Thermodynamic properties of the title compound at different temperatures are calculated in gas phase. Moreover, the dipole moment, linear polarizabilities, hyperpolarizabilities and Mulliken atomic charge are also studied. The natural bonding orbital (NBO) calculations are performed using Gaussian 09 package at the same level in order to understand various second order interactions between the filled orbitals of one subsystem and vacant orbitals of another subsystem, which quantify the intermolecular delocalization or hyper conjugation. The second order perturbation theory analysis of Fock matrix in NBO basis of pAsA is carried out to evaluate the donor-acceptor interactions. The interactions result is a loss of occupancy from the localized NBO of the idealized Lewis structure into an empty non- Lewis orbital. ¹H and ¹³C chemical shifts were calculated with GIAO approach [16] by applying B3LYP/6-31+G^** method.

Powder X-Ray diffraction studies

The powder x-ray diffraction analysis helps in determining the crystalline nature of the grown crystal. Using the JEOL JDX services instrument with CuKα (λ = 1.5406A°) radiation the sample is scanned with the range 10–90° C at a scan rate of 2^o /min. The crystal belongs to monoclinic type and the lattice parameters obtained from the Powder XRD data are given below.

a = 7.241 (2)

b = 6.214(1)

c = 8.643 (1)

β = 101.19 (1)°

V= 381.5 (1) A^o3

The crystallinity of pAsA is well defined by the prominent peaks at specific 2θ values which can be seen in Figure 2. The d-spacing and their relative intensities of the diffraction peaks are tabulated in Table 1.

Figure 2: Powder X-ray diffraction pattern of pAsA crystals.

Table 1: X-ray powder diffraction data of pAsA crystals.

Conformational stability and molecular geometry

In order to determine the most stable structure of p-AsA, the energies of various conformers were calculated. The optimization was performed for the most stable conformer with global minimum energy. The conformers with their energy values are shown in Figure 3.

Figure 3: Various Conformers of p-Arsanilic acid.

The molecular geometry of the title compound is described on the basis of bond lengths, bond angles and dihedral angles. The optimized parameters are tabulated in Table 2. The compound under investigation belong to C₁ point group symmetry with the global minimum energy E=-2747.732 Hartree. The optimized structure reveals that pAsA to be zwitter ionic form and it is shown in Figure 3a. The C-C bond length for pAsA calculated at B3LYP/6-31+G^** level lies in the range 1.3883 to 1.4106 A0. This coincides with the analogous molecule whose C-C bond length varies from 1.358 to 1.491 A0 [17]. The presence of electronegative arsenic atom has reduced the calculated C-N bond length and it slightly varies from the experimental value (1.47 A0) [18]. The substitution of arsenic in the ring exerts a valence electron cloud of nitrogen atom resulting in an increase force constant and decrease in bond length. The bond length between carbon and arsenic is probably large due to the electronegative nature of the arsenic. The As14-O15 and As14-O17 bond lengths, have the values 1.7702 and 1.7825 A0, which coincides with the analogous molecule respectively [19]. The As14-O19 bond distance is reduced to 1.63 A0 because of the inductive effect of the amino group. The O15-H16 bond and O17-H18 bond takes the value 0.9699 and 0.9705 A0 respectively, which coincide with the analogous molecule [20].

Table 2: Optimized geometrical parameters of p-Arsanilic acid.

Figure 3a: Optimised structure of p-AsA with atom numbering.

The distortion in the ring is mainly due to the substituents and its consideration is very important. Among the six angles of ring, the angle C6-C1-C2 and C5-C4-C3 have a slight variation because of the presence of amine and arsenic group. The geometry optimization performed on the title compound indicates that it exhibits intramolecular hydrogen bond interaction. The magnitude of the bond angles O15-As14-O17 and O19-As14-O17 are 98.72o and 111.24o respectively, which indicates that As14-O17 is not symmetrically disposed on As14 and tilted towards O19 atom to form H-bonding between O19 and H18 atoms. The magnitude of the bond angles C1-N11-H12 and C1-N11-H13 is 117.6 and 117.70o respectively, which indicates that C1-N11 bond, is symmetrically disposed on C₁.

Vibrational analysis

A complete vibrational assignment for the title compound is obtained using Gauss view program. The potential energy distribution (PED) illustrates the frequency distribution for the molecule under study. Most of the calculated frequencies are in coincidence with the available data. The optimized structure of pAsA comes under C₁ symmetry and has 51 normal modes of vibrations. The observed and calculated frequencies are summarised in the Table 3. The experimental and calculated FTIR spectra and FT-RAMAN spectra are shown in Figure 4 and 5.

Table 3: Observed and B3LYP/6-31+G^**level calculated vibrational frequencies (in cm^-1 ) of p-Arsanilic acid.

Figure 4: Comparison of observed and B3LYP/6-31+G** calculated FTIR spectra of p-Arsanilic acid.

Figure 5: Comparison of Observed and B3LYP/6 -31+G** Calculated FTRAMAN spectra of p-Arsanilic acid.

C-C vibrations: Generally, the C-C stretching vibration occurs in the region 1625-1430 cm^-1. The actual position of these modes is determined not so much by the form of substituents but by the form of substitution around the ring [21].The bands at 1571 cm^-1, 1414 cm^-1, 1289 cm^-1, 1096 cm^-1 in the IR and 1289 cm^-1, 1094 cm^-1 in Raman are assigned for CC stretching. The corresponding calculated frequencies take the values 1575, 1542, 1400, 1282, 1066 cm^-1. In aromatic ring, some bands are below 700 cm^-1. These bands are quite sensitive to change in nature and position of the substituents [22-25]. The in-plane vibration is at higher frequency than the out-of-plane vibration, which is due to the substituent group. The ring vibrations are observed at 516, 610, 616 cm^-1 in IR, 614 cm^-1 in Raman and their corresponding calculated frequencies are 514, 592 and 620 cm^-1. The ring torsional deformation vibrations are predicted at 726, 415 and 72 cm^-1, which are active in Raman.

C-H vibrations: The aromatic C-H stretching vibrations are normally found between 3100 and 3000 cm^-1. In this region, the bands are not affected appreciably by the nature of the substituents. The CH stretching mode is assigned to the peak at 2802, 2905 and 3033 cm^-1 in the IR and the Raman active modes are at 2993, 3061 and 3150 cm^-1.The calculated frequencies at 3069,3065 and 3042 cm^-1 also depict the CH stretching vibrations.

The region 1300-1000 cm^-1 is expected for CH in-plane bending vibrations. Similarly for the title molecule the bands at 1326 and 1140 cm^-1 are allotted for in-plane bending in IR spectrum. The CH out - of - plane vibrations is expected in the range 1000-700 cm^-1. For this molecule, the peak at 976 cm^-1 is assigned for out –of –plane bending vibration and the calculated frequencies are 970 and 990 cm^-1.

As-O vibrations: The As-O stretching and bending vibrations are expected to appear in the region 1000-300 cm^-1 [26]. However, the calculated band located at 928 cm^-1 is assigned to the stretching mode of As-O band. The bond distance of As-OH types are 1.78 and 1.77Ao. The greatest distance corresponds to the lowest wavenumbers at 670 and 651 cm^-1. The bands due to symmetric and asymmetric bending vibrations are identified in the 550-400 cm^-1 frequency region in IR spectra. Two bands located at 319 and 282 cm^-1 are assigned to asymmetric bending mode, whereas the symmetric mode appears at 136 cm^-1.

O-H and N-H vibrations: In dilute solutions, O-H stretching appears as a sharp band at higher frequency around 3600 cm^-1 due to free O-H group. In spectra of undiluted liquids or solids, intermolecular hydrogen bonding broadens the band and shifts its position to lower frequency (3200–3500 cm^-1)[27]. The peak at 3603 cm^-1 in Raman is assigned to O-H stretching and the calculated frequency also falls at 3633 cm^-1 with 100% PED contribution. The OH bending vibrations are observed in the region 965 and 948 cm^-1 which coincides with the experimental data.

It is stated that in amines, the N-H stretching vibrations occur in the range 3400-3300 cm^-1 [28]. With reference to this, the vibrational frequencies observed at 3482 in Raman and 3405 in IR are assigned to NH stretching modes. The peak observed at 1234 cm^-1 is allotted for C-NH₂ stretching mode. The in-plane –NH₂ bending vibration falls from 1650-1580 cm^-1. The IR peak at 1602 cm^-1 and Raman peak at 1594cm^-1 is allotted for NH₂ bending vibration. Likewise, the out- ofplane bending -NH₂ band at 826 cm^-1 in the IR 830 cm^-1 in Raman is assigned to the amino group deformation mode.

NMR analysis

NMR serves as a great resource in determining the structure of an organic compound by revealing the hydrogen and carbon skeleton. Chemical shifts of pAsA are determined experimentally and the theoretical chemical shifts are predicted using Gauge – Invariant Atomic Orbitals (GIAO). The ¹H atom is mostly localized on periphery of the molecules and their chemical shifts would be more susceptible to inter molecular interactions in the aqueous solutions as compared to that of other heavier atoms. The chemical shift (δ) value provides information on the magnetic/chemical environment of the protons. Protons next to electron withdrawing groups are deshielded, whereas protons next to electron-donating groups are shielded [29].The experimental and calculated¹³C and ¹H NMR chemical shifts of the title molecule are gathered in Table 4. The hydrogen atoms attached to the electron withdrawing oxygen atom in the hydroxyl group decrease the shielding. This results in the low chemical shift for the hydroxyl protons. Whereas, the protons attached to the ring are in the range 6.87 - 8.04 ppm and the experimental values also fall in the same range coinciding with the theoretical data. Usually aromatic carbons possess the chemical shift values from 100 -150 ppm. Due to the influence of electronegative nitrogen atom, the chemical shift value of C₁ of pAsA is significantly differing in the shift position and the corresponding value is 151.08 ppm. The experimental and calculated NMR spectra of ¹H and ¹³C are shown in Figures 6 and 7.

Table 4: Experimental and Theoretical isotropic chemical shifts of p-Arsanilic acid.

Figure 6: Experimental and Calculted 1H NMR Spectrum of pAsA.

Figure 7: Experimental and Calculted ¹³C NMR Spectrum of pAsA.

Frontier molecular orbitals

The analysis of the wave function indicates that the electron absorption corresponds to a transition from the ground state to the excited state and is mainly described by one electron excitation from the HOMO to LUMO. Both HOMO and LUMO are the main orbital taking part in chemical reaction. HOMO energy characterizes the capability of electron giving; LUMO characterizes the capability of electron accepting [30]. The frontier orbital gap helps to characterize the chemical reactivity, optical polarizability, chemical hardness and softness of a molecule [31]. The surfaces for the frontier orbital are drawn to understand the bonding scheme of the title compound. Two important molecular orbital (MO) were examined for the title compound, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are given in Figure 8. The calculated HOMO and LUMO energies are -0.23498eV and -0.03750eV and the resulting band gap energy is 0.19748eV. The chemical stability of a molecule is determined by the hard and soft nature of it. HOMO-LUMO energy gap helps to find whether the molecule is hard or soft. Hard molecules have large energy gap and soft molecules have small energy gap. 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 as η = (-HOMO+LUMO)/2. The value of η of the title molecule is 0.13624eV. Hence, it shows that the title compound belongs to soft material.

Figure 8: The atomic orbital compositions of the frontier molecular orbital for p- Arsanilic acid.

NBO analysis

The NBO analysis is already proved to be an effective tool for the chemical interpretation of hyperconjugative interaction and electron density transfer from the filled lone pair electron [32]. In order to investigate the various second - order interaction between the filled orbitals of one subsystem and vacant orbitals of another subsystem the DFT/B3LYP level has been used, and it predicts the delocalization or hyperconjugation[33]. The hyperconjugative interaction energy can be deduced from the second-order perturbation approach [34]:

E⁽²⁾ =ΔE_ij =q_j F(i,j)²/ε_j - ε_i

Where qj is the ith donor orbital occupancy, εj , εi are diagonal elements(orbital energies) and F(i,j) is the off-diagonal NBO Fock matrix elements.

The intramolecular interactions are formed by the orbital overlap between bonding (C-C), (C-As) and (As-O) antibonding orbital which results in the intra molecular charge transfer (ICT) causing stabilization of the system. These interactions are observed as increase in electron density(ED) in C-As, As-O antibonding orbital that weakens the respective bonds. A large number of stabilizing orbital interactions are calculated in pAsA and they are listed in Table 5.

Table 5: The second-order perturbation energies E (2) (kcal/mol) corresponding to the most important charge transfer interactions (donor-acceptor) in pAsA by B3LYP/6- 31+G^** method.

The strong intra-molecular hyper-conjugative interaction of LP(2) O15→ σ^*(As14-O19) increases the electron density ED(0.051e) that weakens the bonds leading to stabilization of 6.13 kcal/mol. Also the strong intra-molecular hyper-conjugative interaction of LP (2) O15→ σ^*(As14-O17) weakens the respective bonds leading to stabilization 7.68 kcal/mol. Another strong intra – molecular hyper- conjugative interactions of C1-C6 from LP (1) N11 → π^*C1-C6 increases ED (0.095e) that weakens the respective bonds leading to stabilization of 32.32 kcal/mol. These interactions are observed as an increase in electron density(ED) in C-C antibonding orbital that weakens the respective bonds. The increased electron density at the carbon atom leads to the elongation of respective bond length and a lowering of the corresponding stretching wave number.

UV-Vis spectra analysis

The grown crystal is subjected to UV-Vis-NIR spectral analysis and the lower cut-off wavelength is found to be 240nm. The wide transparency region in the visible and NIR region proves the molecule to be a good one for optical applications. The absorbance peak of the UV-Vis-NIR spectra is shown in Figure 9. Energy gap of pAsA is calculated by using the formula given below.

Figure 9: Theoretically calculated and experimental UV-Vis spectrum of p-Arsanilic acid.

E = 1.243 X 10³ [35]

λ_max

Where, λ is the lower cutoff wavelength and the energy gap value is found as 5.1375 eV

Time-dependent density functional theory (TD-DFT) calculation is performed for pAsA on the basis of fully optimized ground state structure to investigate the electronic absorption properties. The λ_max values which are the function of electron availability, electronic excitation energies and oscillator strength are obtained from the UVVisible spectra, simulated theoretically with B3LYP/6-31+G^** basis set. The experimental and calculated visible absorption maxima are tabulated in Table 6. The theoretically predicted UV-Vis spectra are visualized in Figure 9, as can be seen from Table 6, the calculated absorption maxima values for pAsA have been found to be 267.96, 263.03 and 260.36nm. The oscillator strength for 260.36 nm is of higher in magnitude compared to other transitions. The absorption band of pAsA at the longer wave length region 267.96nm is caused by the n – π^* transition.

Table 6: Experimental and Theoretical electronic absorption spectra values of p-Arsanilic acid using TD-DFT/B3LYP/6-31+G^**.

Molecular electrostatic potential

The molecular electrostatic potential (MEP) is used primarily for predicting sites and relative reactivities towards electrophilic attack, in studies of biological recognition and hydrogen bonding interactions [36]. To predict the reactive sites for electrophilic and nucleophilic attack for pAsA, the MEP at the B3LYP/6-31+G^** method is calculated as shown in Figure 10. Different colours on the MEP represent the different values of the electrostatic surface. The electrostatic potential increases in the order red

Figure 10: The total electron density isosurface mapped with molecular electrostatic potential of p-Arsanilic acid.

As seen from the Figure 10, in the pAsA the amine group region has negative potential and As-OH region has positive potential. The predominance of the light green region of MEPs surface corresponds to a potential halfway between the two extremes red and dark blue colour. The positive (blue) region of MEP is related to electrophilic reactivity and the negative (red) regions to nucleophilic reactivity.

Mulliken population analysis

The calculated Mulliken charges and natural charges of pAsA are listed in Table 7. The Mulliken analysis is the most common population analysis method. Mulliken atomic charge calculation has a significant role in the application of quantum chemical calculations to molecular systems because the atomic charges affect some properties of molecular system including dipole moment and molecular polarizability.

Table 7: Mulliken and Atomic charges of pAsA.

The obtained atomic charges for H7, H8, and H9 are smaller than the charges for H12 and H13, which is due to the presence of electronegative oxygen atom. In addition, the results illustrate that the charge of the oxygen atoms exhibits a negative charge, which are donor atoms. The results also show that the hydrogen atoms H16 and H17 have more positive atomic charge than the other hydrogen atoms. This is due to the presence of electronegative oxygen atom O17 and O19; the hydrogen atoms attract the positive charge from the oxygen atom.

NLO studies

In order to confirm the enhancement of nonlinearity of pAsA, second harmonic efficiency test is performed by the modified version of powder technique developed by Kurtz and Perry [37,38]. The powdered sample of pAsA crystal is illuminated using the fundamental beam of 1064nm from Q-switched ND:YAG laser. The input pulse energy of 1.9 mJ/pulse and pulse width 8 ns and repetition rate of 10 Hz are used.

The second harmonic signal generated by the crystal was confirmed from the emission of green radiation of wavelength 532nm. The output voltage was 41mV and it was 0.5 times greater than the KDP value (76 mV).

In order to investigate the relationships between molecular structures and non-linear optical properties(NLO) , the polarizibilities and first order hyperpolarizabilities of the pAsA compound are calculated using DFT/B3LYP method with 6-31+G^** basis set, based on finite field approach.

The polarizability and hyperpolarizability tensors can be obtained by a frequency job output file of Gaussian. The mean polarizability(α_tot), anisotropy of polarizability(Δα) and the average value of the first order hyperpolarizaility(β_tot) can be calculated using the equations

α_tot = α_xx. + α_yy + α_zz / 3

The polarizability and hyperpolarizability are reported in atomic units(a.u), the calculated values have been converted into electrostatic units (esu) (for α : 1 a.u = 0.1482 x 10^-24)esu, for β:1 a.u = 8.6393 x 10^-33) esu.

The total dipole moment (μ) for the title compound can be calculated using the following equation.

Theoretically calculated values of polarizability, first order hyperpolarizability and dipole moment are shown in Table 8. It is well-known that the higher value of dipole moment, molecular polarizability and first order hyperpolarizability are important for more active NLO properties. The large value of hyperpolarizability, β which is a function of the non-linear optical activity of the molecular system is associated with the intra molecular charge transfer. The physical properties of these conjugated molecules are governed by the high degree of electronic charge delocalization along with the charge transfer axis and by the low band gaps. The calculated first order hyperpolarizability of the title compound is 6.258 X 10^-30esu, which is 48 times greater than that of urea (0.13 x 10^-30 esu) [39]. So, it is revealed that the title molecule is an attractive object for future studies of non-linear optical properties.

Table 8: The average polarizability (β_tot), first order hyperpolarizability(β_tot) and dipolemoment (µ) value of pAsA.

Thermodynamic properties

The zero point energies, thermal correction to internal energy, enthalpy, Gibbs free energy and entropy and heat capacity for a molecular system are computed from the frequency calculations. The computed thermodynamic parameters are listed in Table 9. The correlation of heat capacity at constant pressure (C_p), entropy(S) and enthalpy change (ΔH_o→T) with temperature are delineated in Figure 11. As the temperature is increased from 100 to 1000K, the thermodynamic parameters also increase linearly. Here, all the mentioned thermodynamic calculations are done in gas phase. As per the second law of thermodynamics in thermochemical field [40], these calculations can be used to compute the other thermodynamic energies and help to estimate the directions of chemical reactions.

Table 9: The temperature dependence of thermodynamic parameters of p-Arsanilic acid.

Figure 11: Temperature dependence of heat capacity, entropy and enthalpy change at constant pressure of p-AsA.

Single crystals of p-Arsanilic acid have been grown using the slow evaporation technique. The crystallinity and the cell parameters have been revealed by the powder XRD results. The detailed vibrational analysis has been studied using DFT/B3LYP method and most of the calculated frequencies coincide with the experimental FTIR and FT-RAMAN data. The UV studies show a wide transparency region above the lower cut-off region and large band gap energy. This proves the optical quality of the crystal. The emission of green radiation from the SHG studies is yet another proof for title compound to be a good NLO material. The stability, chemical reactivity, intramolecular interaction of the molecule are analysed with the help of theoretical calculations in detail. . The molecular electrostatic potential surface (ESP) provides information regarding the size, shape, charge density distribution and sites of chemical reactivity of the title molecule .The intermolecular interactions in the compound is found out with the help of the reactive sites. A deep insight into the charge transfer is elucidated by NBO analysis. The correlations between the thermodynamics and temperature are also obtained.

The authors are sincerely thankful to the SHG measurement facility extended by Prof.P.K.Das, Department of Inorganic and physical chemistry, Indian Institute of Science, Bangalore. The authors are also thankful to Sophisticated Analytical Instrumentation Facility (SAIF), IIT, Chennai, and St. Joseph’s College, Trichirappalli, India for providing spectral measurements.