Accepted Manuscript Spectroscopic investigation (FT-IR, FT-Raman and SERS), vibrational assignments, HOMO-LUMO analysis and molecular docking study of Opipramol Y. Sheena Mary, C. Yohannan Panicker, C.N. Kavitha, H.S. Yathirajan, M.S. Siddegowda, Sandra M.A. Cruz, Helena I.S. Nogueira, Abdulaziz A. Al-Saadi, Christian Van Alsenoy, Javeed Ahmad War PII: DOI: Reference:

S1386-1425(14)01297-9 http://dx.doi.org/10.1016/j.saa.2014.08.106 SAA 12627

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

3 July 2014 10 August 2014 24 August 2014

Please cite this article as: Y. Sheena Mary, C. Yohannan Panicker, C.N. Kavitha, H.S. Yathirajan, M.S. Siddegowda, S.M.A. Cruz, H.I.S. Nogueira, A.A. Al-Saadi, C. Van Alsenoy, J.A. War, Spectroscopic investigation (FT-IR, FTRaman and SERS), vibrational assignments, HOMO-LUMO analysis and molecular docking study of Opipramol, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa. 2014.08.106

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Spectroscopic investigation (FT-IR, FT-Raman and SERS), vibrational assignments, HOMOLUMO analysis and molecular docking study of Opipramol Y.Sheena Marya, C.Yohannan Panickerb*, C. N. Kavithac, H. S. Yathirajanc, M. S. Siddegowdad, Sandra M. A. Cruze, Helena I. S. Nogueirae, Abdulaziz A.Al-Saadif, Christian Van Alsenoyg, Javeed Ahmad Warh a

Department of Physics, Fatima Mata National College, Kollam, Kerala, India.

b c

Department of Physics, TKM college of Arts and Science, Kollam, Kerala, India.

Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570006,

India d

R. L. Fine Chem., Bangalore-560 064, India

e

Department of Chemistry, CICECO and TEMA-NRD, University of Aveiro, 3810-193 Aveiro,

Portugal f

Department of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran 31261,

Saudi Arabia. g

University of Antwerp, Chemistry Department, Universiteitsplein 1, B2610, Antwerp, Belgium.

h

INSPIRE Fellow, Department of Chemistry, HSG University, Sagar, M.P. India.

*author for correspondence : email: [email protected] Phone: 91 9895370968 Abstract FT-IR and FT-Raman spectra of Opipramol were recorded and analyzed. SERS spectrum was recorded in silver colloid. The vibrational wave numbers were computed using DFT quantum chemical calculations. The data obtained from wave number calculations are used to assign vibrational bands obtained in infrared and Raman spectra as well as in SERS of the studied molecule. Potential energy distribution was done using GAR2PED program. The geometrical parameters (DFT) of the title compound are in agreement with the XRD results. The presence of CH2 stretching modes in the SERS spectrum indicates the close of piperazine ring with the metal surface and the interaction of the silver surface with this moiety. NBO analysis, HOMO-LUMO, first hyperpolarizability and molecular electrostatic potential results are also reported. The inhibitor Opipramol forms a stable complex with P4502C9 as is evident from the ligandreceptor interactions and a -9.0 kcal/mol docking score and may be an effective P4502C9 inhibitor if further biological explorations are carried out. 1

Keywords: Opipramol; SERS; molecular docking; piperazine. 1.

Introduction Piperazines are a broad class of chemical compounds, with many important

pharmacological properties. It has been reported that some piperazine-based compounds are capable of inducing apoptosis in some cancer cells [1]. For instance, the effect of 1,4-bis-(4-(1Hbenzo[d]imidazol-2-yl-phenyl)piperazine on U937 leukemia cell viability was investigated by Sampson et al. [2]. Sharma and Ravani [3] reported the synthesis and screening of piperazine derivative as anticancer agents. Hatnapure et al. [4] reported the synthesis and biological evaluation of novel piperazine derivatives of flavones as potent anti-inflammatory and antimicrobial agents. Additionally, some piperazine derivatives were proven to possess a high biological activity for multidrug resistance in cancer and malaria [5]. The title compound opipramol is piperazine-based compound. Opipramol (systematic IUPAC name: 4-[3-(5HDibenz[b,f] azepin-5-yl)propyl]-1-piperazinethanol] is an antidepressant and anxiolytic agent typically used in the treatment of generalized anxiety disorder [6]. Opipramol, which is a tricyclic compound with no reuptake-inhibiting properties, is also considered as an iminostilbene derivative and belongs to the dibenzazepine group developed by Schindler and Blattner in 1961 [7]. However, it has pronounced D2-, 5-HT2-, and H1-blocking potentials and exhibits a high affinity to sigma receptors (α-1 and α-2). The crystal structure studies of opipramol [8] and some of its important derivatives such as opipramoldipicrate [9], opipramol dihydrochloride [10] and opipramoliumfumarate [11] have been reported. The synthesis, characterization and pharmacological screening of various impurities present in opipramol, pargeverine and propiverine bulk drugs are also reported [12]. For more information regarding the drug activity of opipramol, a recent review on that subject has recently been published [13]. In the present work, the vibrational spectroscopic study along with surface enhanced Raman scattering of the title compound are reported. To the best of our knowledge, a detailed description of the spectroscopic behavior of the title compound with the help of vibrational spectral techniques and quantum chemical calculations along with NLO properties has not been given to date. Due to the different potential biological activity of the title compound, molecular docking of the title compound is also reported. 2.

Experimental

2

The title compound was obtained as a gift sample from R.L.Fine Chem. Ltd., Bangalore, India. The compound was re-crystallized from acetone. In the title compound, the seven membered, 5H-dibenz[b,f]azepine ring (N2-C5-C14-C15-C17-C19-C28) adopts a boat conformation [14] and the overall molecular shape is that of a butterfly [15] whereas the piperazine ring (N3C38-C41-N4-C44-C47) adopts a chair conformation [14]. In the crystal, O-H…N and C-H…O hydrogen bond link the molecules into a layer parallel to the bc plane. The FT-IR spectrum (Fig.1) was recorded using KBr pellets on a DR/Jasco FT-IR 6300 spectrometer. The FT-Raman spectrum (Figs. 2 and 3) was obtained on a Bruker RFS100/s FT-Raman spectrometer (Nd:YAG laser, 1064 nm excitation). SERS spectrum in the range 2500-3200 cm-1 is given in Fig. S1 (supporting material). An aqueous silver colloid was used in the SERS experiments, prepared by reduction of silver nitrate by sodium citrate using the Lee-Meisel method [16]. Solutions of the samples were made in methanol (0.05 mmol in 1 mL of solvent) and transferred using a micropipette into the silver colloid (20 μl in 0.5 mL of colloid) such that the overall concentration of the sample was 2×10-3 mol L-1. Colloid aggregation was induced by addition of an aqueous solution of MgCl2 (1drop of a 2 mol L-1 solution). Polyvinylpyrrolidone was then used to stabilize the colloid (1drop of a 1% aqueous solution). The final colloid mixture was placed in a glass tube and the Raman spectrum registered. 3.

Computational details Calculations of the title compound are carried out with Gaussian09 program [17] using

the B3LYP functional methods to predict the molecular structure and vibrational wave numbers. The 6-311++G(d,p)(5d,7f) basis set was employed in this study for the molecule optimization and harmonic frequency calculation. The DFT hybrid B3LYP functional method tends to overestimate the fundamental modes; therefore scaling factor of 0.9613 has to be used for obtaining a considerably better agreement with experimental data [18]. Structural parameters corresponding to the optimized geometry of the title compound (Fig.4) are given in Table 1 and are compared with the XRD data [8]. The absence of imaginary wave numbers on the calculated vibrational spectrum confirms that the structure deduced corresponds to a minimum energy. The assignments of the calculated wave numbers are aided by the animation option of GAUSSVIEW program, which gives a visual presentation of the vibrational modes [19]. The potential energy distribution is calculated with the help of GAR2PED software package [20]. 4.

Results and discussion 3

4.1.

IR and Raman spectra In the following discussion, the C5-C6-C8-C10-C12-C14, C20-C22-C24-C26-C28-C19, N2-C5-

C14-C15-C17-C19-C28 and piperazine rings are designated as RingI, RingII, RingIII and Pz respectively and the experimental results are compared with the B3LYP/6-311++G(d,p)(5D, 7F) theoretically obtained results. The observed IR, Raman and SERS bands and calculated wave numbers (scaled) are given in Table 2. 4.1.1. Phenyl ring vibrations The phenyl CH stretching vibrations occur above 3000 cm-1 and are typically exhibited as multiplicity of weak to moderate bands compared with the aliphatic CH stretching [21]. For the title compound, the DFT calculations give CH stretching vibrations of the phenyl rings at 3070, 3062, 3046, 3038 cm-1 and 3072, 3063, 3046, 3037 cm-1 for RingI and RingII, respectively. The bands observed at 3059 in the IR spectrum and 3058 cm-1 in the Raman spectrum are assigned as CH stretching modes of the phenyl rings. The benzene ring possesses six ring stretching modes of which the four with the highest wave numbers occurring near 1600, 1580, 1490 and 1440 cm-1 are good group vibrations [22]. With heavy substituent, the bands tend to shift to somewhat lower wave numbers and the greater the number of substituent on the ring, the broader the absorption regions [22]. In the case of C=O substitution, the band near 1490 cm-1 can be very weak [22]. The fifth ring-stretching mode is active near 1315 ± 65 cm-1, a region that overlaps strongly with that of the in-plane CH deformation [22]. The sixth ring-stretching mode, which is the ring breathing vibration, appears as a weak band near 1000 cm-1 in mono, 1,3-di and 1,3,5trisubstituted benzenes. In the otherwise substituted benzenes, however, this mode is substituent sensitive and difficult to distinguish from the other modes. The C-C ring stretching modes are expected in the range 1260-1615 cm-1 for Rings I and II (ortho substituted) [22, 23]. The DFT calculations give the ring stretching modes at 1290, 1409, 1446, 1542, 1567 and 1306, 1419, 1454, 1542, 1569 cm-1 for Ring I and II, respectively. These modes are observed at 1406, 1569 cm-1 in the IR spectrum and at 1310, 1418, 1445, 1571 cm-1 in the Raman spectrum. In ortho disubstitution the ring breathing mode has three frequency intervals according to whether both substituent are heavy or one of them is heavy while the other is light or both of them are light. In the first case the interval is 1100-1130 cm-1, in the second case 1020-1070 cm-1 while in the third case 630-790 cm-1 [23]. The bands calculated at 1024 and 1028 cm-1 are assigned as the ring breathing modes of ortho-substituted phenyl rings I and II. Kaur et al. [24] reported the ring 4

breathing mode of ortho-substituted benzene rings at 1026 and 1023 cm-1 theoretically. The inplane CH bending modes are expected above 1000 cm-1 [22] and in the present case the in-plane CH deformation bands are assigned at 1261, 1139, 1101, 1032 cm-1 for Ring I and at 1226, 1148, 1095, 1028 cm-1 for Ring II theoretically. Experimentally these modes are observed at 1149, 1139, 1103 cm-1 in the IR spectrum. The out-of-plane CH deformation bands of the phenyl ring are assigned at 947, 918, 850, 756 cm-1 for RingI and 949, 920, 858, 750 for Ring II theoretically and bands are observed in the IR spectrum at 861,757 and at 862 cm-1 in the Raman spectrum. In the case of 1,2-disubstituted benzenes only one strong absorption in the region 755 ± 35 cm -1 is observed and is due to γCH [22]. The strong band observed at 757 cm-1 in the IR spectrum is assigned as this mode. Most of the modes are not pure according to PED calculations, but contain significant contributions from other modes also. The substituent sensitive modes of the phenyl rings and other modes are also identified and assigned. 4.1.2. Piperaizne ring vibrations For 1-phenylpiperazine, the CH2 stretching vibrations have been reported at 2944, 2910, 2881 and 2884 cm-1 [25]. Krishnakumar and Seshadri have reported the CH2 stretching modes of 2-methylpiperazine at 3078 and 2532 cm-1 [26]. For the title compound, the asymmetric CH2 stretching modes are observed at 2947, 2922 cm-1 in the IR spectrum, 2953, 2935, 2915 cm-1 in the Raman spectrum and 2954, 2946, 2939, 2918 cm-1 theoretically. The symmetric CH2 stretching modes are assigned at 2810 cm-1 in the IR spectrum, 2792 cm-1 in the Raman spectrum and in the range 2780-2806 cm-1 theoretically. The CH2 scissoring vibrations of piperazine molecule were reported at 1455 and 1446 cm-1 [27] while these modes are reported at 1452 cm-1 for 1-phenylpiperazine [25] and at 1406 and 1293 cm-1 for 2-methylpiperazine [26]. The CH2 scissoring vibrations observed for piperazine and 1-phenylpiperazine are found to be consistent with the results of the title compound. For the title compound the scissoring modes of the CH2 are observed at 1435 cm-1 in IR, 1445 cm-1 in Raman and in the range 1430-1446 cm-1 theoretically. The wagging and twisting modes of the CH2 groups are assigned in the range 13791317 and 1107-1278 cm-1 theoretically for the title compound. The piperazine ring stretching modes are highly characteristic and in a study on the determination of piperazine rings in ethyleneamines, poly(ethyleneamine) and polyethylenimine by Infrared spectroscopy, Spell reported that the piperazine ring was found to be associated with sharp, well define absorptions at 1380-1345 cm-1, 1125-1170 cm-1 and 1010-1025 cm-1 regions 5

of IR spectrum [28]. In accordance with Spell [28] we have also observed a very strong peak in the IR spectrum at 1342 cm-1 corresponding to CH2 wagging mode of the piperazine ring. The theoretically calculated corresponding wave number for the mode is 1346 cm-1 with a PED of 65% and a calculated IR intensity of 54.48. For the title compound, the rocking modes of the CH2 are observed at 1041, 965, 834 cm1

in the IR spectrum and at 1052, 1039, 961, 831 cm-1 theoretically. A very sharp and intense

band was observed at 1037 cm-1 by da Silva et al. [29] and was assigned to the ring CH2 rocking motions. As stated by Spell, [28] this is one of the most useful bands for detecting the presence of di-substituted piperazines. In the present case, we have also observed a band at 1041 cm-1 in the IR spectrum with calculated value 1039 cm-1. For the title compound, the piperazine ring stretching modes are observed at 1149, 1122, 1005, 912, 720 cm-1 in the IR spectrum and at 1124, 1112, 724 cm-1 in the Raman spectrum. The calculated values corresponding to these modes are 1148, 1125, 1116, 1052, 1001, 915 and 726 cm-1. El-Emam et al. [30] reported the CN stretching vibrations of the piperazine ring in the region 1154-756 cm-1. The C-C stretching vibrations in the piperazine ring were reported at 972, 903 cm-1 [30]. Two absorption characteristic for the piperazine ring at 1130 and 1168 cm-1 and assigned for the CN stretching modes were observed by da Silva et al. [29]. In the present case, we have observed bands in the IR spectrum at 1149 and 1122 cm-1 corresponding to the piperazine ring stretching modes and the shift in the wavenumber may be attributed to the bulky groups attached to the piperazine ring. Piperazine ring modes are reported at 1240, 1155, 1134, 1043, 1032, 1018, 1002, 974, 880 cm-1 theoretically and at 1238, 1143, 1045, 1004, 874 cm-1 experimentally [31]. Gunasekaran and Anita reported the piperazine ring stretching modes at 1055, 1173, 1199, 1218, 1268, 1323 cm-1 in the IR spectrum and at 1049, 1120, 1186, 1294 cm-1 in the Raman spectrum [32]. The inplane and out-of-plane piperazine ring modes are also identified and assigned (Table 2). 4.1.3. CH2 vibrations The vibrations of the CH2 group, the asymmetric stretch υasCH2, symmetric stretch υsCH2, scissoring vibration δCH2, appear in the region 2945 ± 45, 2885 ± 45 and 1445 ± 35 cm-1, respectively [22, 33]. The DFT calculations give υasCH2 in the range 2941-2969 cm−1 and υsCH2 in the range 2774-2902 cm-1. The bands observed at 2971, 2844 cm-1 (Raman) and 2972, 2947, 2870, 2842, 2810 cm-1 (IR) are assigned as the stretching modes of CH2 group. In the present case, the band observed at 1470, 1458 cm-1 in the IR, 1465 cm-1 in the Raman spectrum and in 6

the range 1467-1426 cm-1 (DFT) are assigned as the scissoring mode of CH2. Bands of hydrocarbons due to CH2 twisting and wagging vibrations are observed in the region 1180–1390 cm-1 [33, 34] The CH2 wagging and twisting modes are assigned in the range 1346-1383 cm-1 (DFT) and bands are observed at 1385, 1360, 1342 cm-1 in the IR spectrum. The bands observed at 1270, 1248, 1062, 788 cm-1 in the IR spectrum and 1284, 1267, 1248, 909, 744 cm-1 in the Raman spectrum are assigned as the twisting and rocking modes of the CH2 groups. Theoretically these modes are assigned in the range 1137-1293 and 740-1065 cm-1 and these assignments are in agreement with literature [22, 33, 34]. 4.1.4. OH, CN and other modes The OH group provides three normal vibrations υOH, δOH and γOH. The DFT calculations give the υOH band at 3403 cm-1 and a broad band is observed in the IR spectrum at 3421 cm-1. The in-plane OH deformation[22] is expected in the region 1400 ± 40 cm-1 and the band at 1370 cm-1 (DFT) are assigned as this mode, which is not pure but contains significant contribution from other modes. The stretching of the hydroxyl group υC-O appears at 1287 cm-1 theoretically. This band is expected [23, 33] in the region 1220 ± 40 cm-1. The out-of-plane OH deformation is observed 556 cm-1, theoretically, which is expected [22] in the region 650 ± 80 cm-1. The C-O stretching and OH deformations modes are reported at 1240 and 620 cm-1, respectively [35]. The identification of C-N vibration is a very difficult task, since the mixing of several bands are possible in this region. However, with the help of theoretical PED analysis, the C-N stretching modes are calculated. The CN stretching modes are active in the region 1275 ± 55 cm1

[22]. In the present investigation the CN stretching modes (C50-N4, C35-N3, C2-N29, C5-N2, C28-

N2) are assigned at 1208, 1201, 1125, 1101, 1095 cm-1 theoretically and observed at 1122, 1103 cm-1 in the IR spectrum and at 1206, 1124 cm-1 in the Raman spectrum. For the title compound the C-C stretching modes [33, 34] are observed at 1062, 1041, 940 cm-1 in the IR spectrum, 1044, 1015, 942 cm-1 in the Raman spectrum and at 1138, 1065, 1041, 1018 cm-1 theoretically and these modes contains contributions from other modes also. The C=C stretching mode is assigned at 1620 cm-1 in the IR spectrum, 1622 cm-1 in the Raman spectrum and at 1611 cm-1 theoretically [33, 34]. The CH modes associated with the ring III are assigned at 3029, 3010 (stretching), 1393, 1218 (in-plane deformation) and 989, 795 cm-1 (out-of-plane bending)

7

theoretically and corresponding bands are observed at 3012 cm-1 in the IR spectrum and 3012, 991, 794 cm-1 in the Raman spectrum. In order to investigate the performance of vibrational wave numbers of the title compound, the wave numbers are also calculated at the B3LYP/SDD method and the root mean square (RMS) value between the calculated and observed wave numbers were calculated. The RMS values of wave numbers were calculated using the following expression [36].

RMS 



1 n calc i  iexp  n 1 i



2

.

The RMS errors of the observed IR and Raman bands are found to be 20.06, 20.85 for B3LYP/SDD and 4.27, 3.96 for B3LYP/6-311++G(d,p)(5D, 7F) methods, respectively. The small differences between experimental and calculated vibrational modes are observed. This is due to the fact that experimental results belong to solid phase and theoretical calculations belong to gaseous phase. 4.2.

SERS spectrum Surface-Enhanced Raman Scattering (SERS) technique is already regarded as a valuable

method because of its high sensitivity, which enables the detection of even a single molecule [37]. The vibrational information contained in the Raman spectrum provide the molecular specificity required to characterize the adsorbate surface interactions, specifically, the orientation of the adsorbed species on the metal surface. The relative intensities from the SERS spectra are expected to differ significantly from normal Raman spectra owing to specific surface selection rules [38]. The surface selection rules suggest that for a molecule adsorbed flat on the metal surface, its out-of-plane vibrational modes will be more enhanced when compared with its inplane vibrational modes and vice versa when it is adsorbed perpendicular to the surface [39, 40]. It is further seen that vibrations involving atoms that are close to the silver surface will be enhanced. When the wave number difference between Raman bands in the normal and SERS spectra is not more than 5 cm-1, the molecular plane will be perpendicular to the silver surface [41]. The CH stretching mode of the phenyl ring has shown to be an unambiguous probe in the determination of surface orientation of substituted aromatics [42]. Since the CH stretching modes do not mix significantly with other vibrational modes of the aromatic ring, their SERS intensities are known to provide the most specific evidence of the orientation of the adsorbate with respect to the surface [43, 44]. The presence or absence of the phenyl ring CH stretching mode is a 8

reliable probe for the perpendicular or parallel orientation, respectively, of the phenyl ring with respect to the metal surface [42, 45]. In the present case, CH stretching mode of the rings PhII and RingIII are present at 3049, 3011 cm-1 in the SERS spectrum, while for the ring PhI this mode is not seen in the SERS spectrum. The in-plane CH deformation band is present in the SERS spectrum at 1224 cm-1 for PhII and out-of-plane CH deformation band is present at 797 cm-1 for Ring III. The presence of the in-plane deformation mode in the SERS spectrum suggests a perpendicular orientation for PhII. The phenyl ring stretching vibrations are present in the SERS spectrum at 1592, 1309 cm-1 for PhII and at 1570, 1407 cm-1 for PhI with substantial band broadening as in literature [46]. This indicates a somewhat flat orientation on the silver surface. For the ring PhII, the phenyl ring stretching mode at 1571 cm-1 in the normal Raman spectrum is shifted to 1592 cm-1 in the SERS, while for PhI, corresponding to the bands at 1570 and 1407 cm-1, no bands are seen in the normal Raman spectrum. These shifts observed for the ring modes could also be attributed to the interaction of the π-electrons with the silver surface. When a molecule is adsorbed flat on the silver surface, the ring breathing mode is known to undergo a red shift of more than 10 cm-1 with substantial band broadening [47]. In the present case, the ring breathing modes of the phenyl rings are absent in the SERS spectrum and the probability of a flat orientation is rare. For the ring III, bands at observed in the SERS spectrum at 1628 cm-1 (C=C stretching mode), 685, 160 cm-1 (deformation modes) and the presence of these modes suggest a titled orientation for RingIII. The presence of CH2 modes associated with the piperazine ring at 2929, 2911, 2815 cm-1 in the SERS spectrum indicates the closeness of piperzine ring with metal surface and interaction of the silver surface with the CH2 groups. The deformation modes of the piperazine rings are observed at 475, 387, 330, 240 cm-1 in the SERS spectrum. The presence of these modes suggests that the piperazine ring is oriented tilted to the silver surface [41]. Arenas et al [48] reported strong SERS bands at 1080, 728, 507, 349 cm-1 for a pyrazine derivative adsorbed on silver electrode. Li et al [49] reported the SERS of pyrazine, pyridine and benzene adsorbed on nano particles on smooth electrode and reported bands at 1246, 1021, 744, 701, 637 and 438 cm-1 in the SERS spectrum for pyrazine. Also the piperazine ring stretching modes are observed at 1127 and 715 cm-1 in the SERS spectrum support this fact. The presence of CH2 stretching modes at 2972, 2883, 2848, 2815 cm-1 in the SERS spectrum indicates the close of piperazine ring with the metal surface and the interaction of the silver surface with this moiety. This 9

supported by the presence of CH2 deformation modes at 1475, 1165, 965, 495 cm-1 and CC stretching modes at 1041 and CN deformation band at 416 cm-1 in the SERS spectrum. Similar observations are reported by Mary et al for CH2 modes [50]. The charge transfer mechanism of SERS can be explained by the resonant Raman mechanism in which charge transfer excitation from the metal to the adsorbed molecule or vice versa occurs at the energy of the incident laser wave number [51, 52]. The frontier orbital theory plays a significant role in the understanding of the charge transfer mechanism of SERS [41, 53]. Two types of charge transfer mechanisms are predicted. One is molecule-to-metal and the other is metal-to-molecule. Molecule-to-metal charge transfer excitation occurs when an electron is transferred from the highest occupied molecular orbital (HOMO) of the adsorbate to the Fermi level of the metal. Conversely, transfer of an electron from the Fermi level of the metal to the lowest unoccupied molecular orbital (LUMO) results in metal-to-molecule charge transfer [41, 54]. The theoretical results show that the HOMO+1, HOMO, LUMO and LUMO +1 energies of the molecule are -8.269, -8.018, 5.530 and -4.447 eV, respectively , which are energetically much lower than the Fermi level of silver ( +5.48 eV) [55]. Hence, we conclude that metal to molecule charge transfer interaction is more preferred here. The electron is probably transferred from metal to the LUMO of the molecule. 4.3.

Natural Bond Orbital analysis The Natural Bond Orbital (NBO) calculations [56] were performed using NBO3.1

program implemented in the Gaussian09 package [17] at the B3LYP/6-311++G(d,p) method. It offers a handy basis for exploring charge transfer or conjugative interaction in molecular systems and is an efficient method for studying intra- and intermolecular bonding and interaction among bonds. The larger the stabilization energy value, the more intensive is the interaction between electron donors and electron acceptors, i.e. the more donating tendency from electron donors to electron acceptors and the greater the extent of conjugation of the whole system. The NBO analysis is already proved to be an effective tool for chemical interpretation of hyper-conjugative interaction and electron density transfer from the filled lone pair electron. Interaction between both filled and virtual orbital spaces was correctly explained by the NBO analysis and it could enhance the analysis of intra- and intermolecular interactions. The second order Fock matrix was carried out to evaluate donor (i)-acceptor (j) i.e. interaction between donor level bonds and acceptor level bonds in the NBO analysis [57]. The result of interaction is a loss of occupancy 10

from the concentration of electron NBO of the idealized Lewis structure into an empty nonLewis orbital. For each donor (i) and acceptor (j) the stabilization energy E(2) associated with the delocalization i→j is as follows: E(2)= Eij = qi

( Fi , j ) 2 ( E j  Ei )

where qi is the donor orbital occupancy, Ei and Ej are the diagonal elements and Fi,j is the off diagonal NBO Fock matrix element. In NBO analysis large E(2) value shows the intensive interaction between electron-donors and electron- acceptors, and greater the extent of conjugation of the whole system, the possible intensive interaction are given in NBO Table S1( supporting material). The second-order perturbation theory analysis of Fock-matrix in NBO basis shows strong intermolecular hyper conjugative interactions are formed by orbital overlap between n(O) and σ*(C-C) and that between n(N) and π*(C-C),σ*(C-C) bond orbital which result in ICT causing stabilization of the system. These interactions are observed as an increase in electron density (ED) in C-C anti-bonding orbital that weakens the respective bonds. There occurs a strong inter molecular hyper-conjugative interaction of C50-C53 from O1 of n2(O1)→σ*(C50-C53) which increases ED(0.02746e) that weakens the respective bonds leading to stabilization of 2.59 kJ/mol. Also there occurs a strong inter molecular hyper-conjugative interaction of C5-C6 from N2 of n1(N2)→π*(C5-C6) which increases ED(0.36779) that weakens the respective bonds leading to stabilization of 14.86 kJ/mol and also the hyper-conjugative interaction of C32-C35 from N3 of

n1(N3)→σ*(C32-C35) which increases ED(0.01853) that

weakens the respective bonds leading to stabilization of 1.69 kJ/mol. Also a strong hyperconjugative interaction of C50-C53 from N4 of n1(N4)→σ*(C50-C53) which increases ED(0.02746) that weakens the respective bonds leading to stabilization of 2.30 kJ/mol. The increased electron density at the oxygen atoms leads to the elongation of respective bond length and a lowering of the corresponding stretching wave number. The electron density (ED) is transferred from the n(O) to the anti-bonding σ* orbital of the C-C and n (N) to the anti-bonding π* orbital of the C–C bond, explaining both the elongation and the red shift [58]. The hyper conjugative interaction energy was deduced from the second-order perturbation approach. Delocalization of electron density between occupied Lewis-type (bond or lone pair) NBO orbital and formally unoccupied (anti bond or Rydberg) non-Lewis NBO orbital corresponds to a stabilizing donor–acceptor interaction. 11

The NBO analysis also describes the bonding in terms of the natural hybrid orbital n2(O1), which occupy a higher energy orbital(-0.26720a.u.) with considerable p-character (99.82%) and low occupation number (1.96408) and the other n1(O1) occupy a lower energy orbital(-0.55434) with p-character (49.07%) and high occupation number (1.98240). Thus, a very close to pure p-type lone pair orbital participates in the electron donation to the σ*(C–C) orbital for n2(O1)→σ*(C-C), π*(C-C) orbital for n1(N2)→π*(O-C), σ*(C-C) orbital for n1(N3)→σ*(CC), and σ*(C-C) orbital for n1(N4)→σ*(C-C) interaction in the compound. The results are tabulated in Table S2 (supporting material). 4.4

Non-linear optical effects Non-Linear Optical (NLO) effect is the forefront of current research because of its

importance in providing the key functions of frequency shifting, optical modulation, optical switching, optical logic and optic memory for the emerging technologies in areas such as telecommunications, signal processing and optical interconnections [59-61]. The first hyperpolarizability of the title compound is calculated using the B3LYP/6-311++G(d,p) basis set based on the finite field approach. In the presence of an applied electric field, the energy of a system is a function of electric field. The first hyperpolarizbility is a third rank tensor that can be described by a 3×3×3 matrix. The 27 components of the matrix can be reduced to 10 components due to Kleinman symmetry [62]. The components of β are defined as the coefficients in the Taylor series expansion of the energy in the external electric field. When the electric field is weak and homogeneous, this expansion becomes E  E0    i F i  i

1 1 1  ij F i F j    ijk F i F j F k    ijkl F i F j F k F l  ...  2 ij 6 ijk 24 ijkl

where E0 is the energy of the unperturbed molecule, Fi is the field at the origin, µi, αij, βijk and γijkl are the components of dipole moment, polarizability, the first hyperpolarizabilities, and second hyperpolarizabilities, respectively. The calculated first hyperpolarizability of the title compound is 2.47 × 10-30 esu, which is comparable with the reported values of piperizine derivatives [63]. The calculated hyperpolarizability of the title compound is 19 times that of the standard NLO material urea (0.13 × 10-30 esu) [64]. We conclude that the title compound is an attractive object for future studies of nonlinear optical properties. 4.5.

Frontier Molecular orbital analysis

12

Investigation of molecular orbital and the spatial distribution of other molecular properties are useful for many purposes. Molecular orbital can provide important insight into bonding and other chemical properties. The analysis of 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 highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). HOMO and LUMO are very important parameters for quantum chemistry. The HOMO-LUMO energy gap for the title compound was calculated at the DFT level of theory. The frontier orbital gap helps to characterize the chemical reactivity and kinetic stability of the molecule. A molecule with a small orbital gap is more polarizable and is generally associated with a high chemical reactivity, low kinetic stability and is also termed as soft molecule [65]. The HOMO and LUMO orbital of the title compound are illustrated in Fig.S2 (supporting material). It is clear from the figure that , while the HOMO is occupied over the rings I, II and III, and methylene groups between the pyrazine rings and the nitrogen atom N2, LUMO is especially localized fully on rings I, II and III. The calculated energy values of the HOMO and LUMO are –8.018 and –5.530 e V, respectively. The energy gap between HOMO and LUMO indicates the molecular chemical stability. In this molecule, the value of energy separation between HOMO and LUMO is 2.488eV. The electronic properties of the molecules are calculated from the total energies and the Koopmans’ theorem [66]. The ionization energy I= -EHOMO = 8.018, electron affinity A= -ELUMO= 5.530. The other important quantities such as hardness (η = (I-A)/2 =1.244 and chemical potential μ = -(I+A)/2= 6.774 and electrophilicity index (ω= μ2/2η)= 18.443 were deduced from ionization energy and electron affinity values. 4.6.

Molecular electrostatic potential Molecular electrostatic potential (MEP) generally present in the space around the

molecule by the charge distribution is very useful in understanding he sites of electrophilic attacks and nucleophilic reaction for the study of biological recognition process [67] and hydrogen bonding interaction actions [68-70]. The electrostatic potential is also well suited for analyzing processes based on the “recognition” of one molecule by another as in drug-receptor, and enzyme-substrate interactions, because it is through their potentials that the two species first “see” each other [71, 72]. To predict reactive sites of electrophilic and nucleophilic attacks for the title compound, MEP at the B3LYP optimized geometry was calculated. The negative (red 13

and yellow) regions of MEP were related to electrophilic reactivity and the positive (blue) regions to nucleopilic reactivity (Fig.5). From the MEP it is evident that the negative charge covers the CH2OH group and the positive regions are over the hydrogen atoms. The more electro negativity in the CH2OH group makes it the most reactive part in the molecule. 4.7.

Geometrical parameters The piperazine ring in the title compound adopts an equatorial-equatorial configuration

with the lone pairs of electrons on the two nitrogen atoms being in the axial positions. The bond lengths (DFT/XRD) of the piperazine ring are N4-C44 = 1.4601/1.4641Å, N4-C41= 1.4611/1.4633Å,

C41-C38

=

1.5250/1.5168Å,

N3-C47=

1.4618/1.47667Å,

N3-C38

=

1.4634/1.4719Å, C44-C47 = 1.5218/1.5160Å. The reported values of corresponding bond lengths are 1.4796, 1.4927, 1.4997, 1.4807, 1.4837, 1.4937Å [73] and 1.4887, 1.4811, 1.5364, 1.4721, 1.4746, 1.5456Å [63]. The DFT calculations give the bond angles (DFT/XRD) within the piperazine ring as N4-C41-C38 = 110.6/111.0˚, N4-C44-C47 = 111.0/110.5˚, N3-C47-C44 = 110.8/110.5˚, N3-C38-C41 = 110.9/111.6˚, C47-N3-C38 =109.8/107.9˚ whereas the corresponding reported values are 111.2, 113.6, 110.1, 110.4, 116.7˚ [63] and 110.0, 109.7, 109.7, 110.0, 110.0˚ [30]. Gao et al. [73] reported the dihedral angles C44-N4-C41-C38 = 56.9˚, C47-N3-C38-C41 = 58.8˚, N4-C41-C38-N3 = -56.7˚, C38-N3-C47-C44 = -58.9˚ which are in agreement with our values, 57.1, 57.5, -57.9, -57.0 (DFT), 58.7, 56.5, -58.7, -57.8˚ (XRD) and the piperazine ring (N3-C38C41-N4-C44-C47) adopts a chair conformation [14]. The C-C bond lengths in the ring I lie between 1.3899-1.4132Å (DFT), 1.3900-1.4109Å (XRD) and in ring II lie between 1.3894-1.4129Å (DFT), 1.3870-1.4072Å (XRD). Here for the title compound, benzene rings are a regular hexagon with bond lengths somewhere in between the normal values for a single (1.54Å) and a double (1.33Å) bond [74]. For the title compound, the C-N bond lengths (DFT/XRD) are C50-N4 = 1.4588/ 1.4644Å, C35-N3 = 1.4750/ 1.4621Å, C29-N2 = 1.4681/ 1.4669Å, C28-N2 =1.4237/1.4286Å, C5-N2=1.4263/ 1.4284Å. The C15=C17 bond length (DFT/XRD) of the title compound is 1.3461/ 1.3446Å and this is in agreement with the reported values [44]. The bond angles at C5, C14, C28 and C19 positions, reveal the strain in the rings I, II and III. In the title compound, the seven membered, 5H-dibenz[b,f]azepine ring (N2C5-C14-C15-C17-C19-C28) adopts a boat conformation [14], and the overall molecular shape is that of a butterfly [15] as is evident from the torsion angles (DFT/XRD), C26-C28-C19-C17 =178.0/175.3˚, C20-C19-C28-N2 = 175.1/174.4˚, C20-C19-C17-C15 =148.7/144.6˚ and C26-C28-N2-C5 14

= -115.5/-111.9˚. Major torsion angles show good agreement between experimental and DFT values. The C-O bond length (DFT/XRD) of the hydroxyl group in the present case is 1.4276/1.4222Å which is greater than the average distance of 1.3620Å found among phenols [75]. The increase in the C-O distance is due to the intra molecular hydrogen bonding experienced by the molecule [76]. 4.8.

Molecular docking studies PASS (Prediction of Activity Spectra for Substances) [77] is an online server to predict

the activity of a compound. Among the various activities predicted for the compound we explored the molecule for its inhibitor activity against human cytochrome P450 2C9(P4502C9). Molecular docking is an efficient tool to get an insight into ligand-receptor interactions and screen molecules for the binding abilities against a particular receptor. All molecular docking calculations were performed on AutoDock-Vina software [78]. The 3D crystal structure of P4502C9 was obtained from Protein Data Bank (PDB ID: 1R9O) [79]. The ligand was prepared for docking by minimizing its energy at B3LYP/6-311++g(d,p)(5D, 7F) level of theory. The AutoDockTools graphical user interface was used to calculate Geisteger charges, add polar hydrogen and partial charges using Kollman united charges. The active site of the enzyme was defined to include residues of the active site within the grid size of 40Å× 40Å× 40Å. The most popular algorithm, Lamarckian Genetic Algorithm (LGA) available in Autodock was employed for docking [80, 81]. The docking protocol was tested by docking the co-crystallized inhibitor onto the enzyme catalytic site which showed perfect synergy with the co crystallised ligand with RMSD [82] well within the allowed range of 2Å [83]. Amongst the docked conformations the best scored conformation predicted by AutoDock scoring function was visualized for detailed interactions.

The ligand binds at the catalytic site of the substrate by weak non-covalent

interactions. The terminal oxygen forms two hydrogen bonds one with Arg433 and other with Cys435. The other nearest neighbor amino acids interacting with the molecule are Leu366, Leu362, Leu208, Ile205, Ala297 and Ile205 which interact mostly by alkyl-π and π-π interactions. The inhibitor Opipramol forms a stable complex with P4502C9 as is evident from the ligand-receptor interactions and a -9.0 kcal/mol docking score. We conclude that the compound in consideration may be an effective P4502C9 inhibitor if further biological explorations are carried out. Figs. 6 and 7 show the ligand-substrate interactions and Table S3 15

(supplementary data) gives the binding affinity values of different poses of the title compound predicted by Autodock Vina. PASS prediction of activity spectrum of the title compound is given in Table 3. 5.

Conclusion The spectroscopic techniques such as FT-Raman, FT-IR and SERS supported by the

development of computational tool such as DFT method allow the structural analysis of the title compound to be conducted in a seamless way. FT-IR, FT-Raman and surface enhanced Raman scattering spectra of the title compound were recorded and analyzed. The vibrational wave numbers were examined theoretically using the Gaussian09 set of quantum chemistry codes, and the normal modes were assigned by potential energy distribution calculations. The geometrical parameters of the title compound are in agreement with XRD results. A computation of the first hyperpolarizability indicates that the compound may be a good candidate as a NLO material. Stability of the molecule arising from hyper-conjugative interaction leading to its bioactivity, charge delocalization has been analyzed using NBO analysis. The presence of CH2 modes associated with the piperazine ring in the SERS spectrum indicates the closeness of piperzine ring with metal surface and interaction of the silver surface with the CH2 groups. The deformation modes of the piperazine rings are also observed in the SERS spectrum and the presence of these modes suggest that the piperazine ring is oriented tilted to the silver surface. The inhibitor Opipramol forms a stable complex with P4502C9 as is evident from the ligandreceptor interactions and we conclude that the compound in consideration may be an effective P4502C9 inhibitor if further biological explorations are carried out. Acknowledgments One of the authors Javeed would like to thank DST, New Delhi, for a INSPIRE fellowship (IF120399). A.A.Al-Saadi thanks King Fahd University for Petroleum and Minerals (KFUPM) for providing the computing facility to support this work. The authors are thankful to University of Antwerp for access to the university’s CalcUA supercomputer cluster. References [1]

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D.J. Kempf, K.C. Marsh, J.F. Denissen, E. McDonald, S. Vasavanonda, C.A. Flentge, B.E. Green, L. Fino, C.H. Park, X.P. Kong, N.E. Wideburg, A. Saldivar, L. Ruiz, W.M. Kati, H.L. Sham, T. Robins, K.D. Stewart, A. Hsu, J.J. Plattner, J.M. Leonard, D.W. Norbeck, Proceedings of the National Academy of Sciences of the United States of America 92 (1995) 2484-2488.

Figure Captions Fig.1 FT-IR spectrum of Opipramol Fig.2 FT-Raman spectrum of Opipramol Fig.3 SERS spectrum of Opipramol Fig.4 Optimized geometry (B3LYP/6-311++G(d,p)(5D,7F) of Opipramol Fig.5 MEP plot of Opipramol Fig.6 The ligand and the co-crystallized inhibitor embedded into the catalytic site of P4502C9

21

Fig.7 Ligand-protein interations, showing the amino acids interacting with the ligand, hydrogen bonds are shown by green dotted line and other interactions by pink lines. The distance between the interacting atoms are shown over the dotted line.

22

Table1 Optimized geometrical parameters (B3LYP/6-311++g(d,p)(5D, 7F)) of Opipramol with XRD data Bond lengths (Å) Label

DFT

XRD

Label

DFT

XRD

C53-O1

1.4276

1.4222

H56-O1

0.9612

0.8962

C5-N2

1.4263

1.4284

C28-N2

1.4327

1.4286

C29-N2

1.4681

1.4669

C35-N3

1.4750

1.4621

C38-N3

1.4634

1.4719

C47-N3

1.4618

1.4766

C41-N4

1.4611

1.4633

C44-N4

1.4601

1.4641

C50-N4

1.4588

1.4644

C5-C6

1.3998

1.3988

C5-C14

1.4132

1.4109

C6-H7

1.083

0.9500

C6-C8

1.3930

1.3932

C8-H9

1.0843

0.9500

C8-C10

1.3927

1.3908

C10-H11

1.0838

0.9500

C10-C12

1.3899

1.3900

C12-H13

1.0852

0.9500

C12-C14

1.4037

1.4057

C14-C15

1.462

1.4641

C15-H16

1.0869

0.9500

C15-C17

1.3461

1.3446

C17-H18

1.0870

0.9500

C17-C19

1.4624

1.4644

C19-C20

1.4041

1.4029

C19-C28

1.4129

1.4072

C20-H21

1.0852

0.9500

C20-C22

1.3894

1.3870

C22-H23

1.0838

0.9500

C22-C24

1.3929

1.3879

C24-H25

1.0843

0.9500

C24-C26

1.3925

1.3941

C26-H27

1.0827

0.9500

C26-C28

1.3993

1.4002

1

C29-H30

1.0925

0.9900

C29-H31

1.0925

0.9900

C29-C32

1.5342

1.5295

C32-H33

1.095

0.9900

C32-H34

1.0931

0.9900

C32-C35

1.532

1.5275

C35-H36

1.0921

0.9900

C35-H37

1.1081

0.9900

C38-H39

1.106

0.9900

C38-H40

1.0936

0.9900

C38-C41

1.5250

1.5168

C41-H42

1.1068

0.9900

C41-H43

1.0934

0.9900

C44-H45

1.0934

0.9900

C44-H46

1.1074

0.9900

C44-C47

1.5258

1.5160

C47-H48

1.0923

0.9900

C47-H49

1.1069

0.9900

C50-H51

1.0936

0.9900

C50-H52

1.1069

0.9900

C50-C53

1.523

1.5163

C53-H54

1.0971

0.9900

C53-H55

1.0991

0.9900

Bond angles(˚) Label

DFT

XRD

Label

DFT

XRD

C53-O1-C56

109.0

105.3

C5-N2-C28

116.5

114.7

C5-N2-C29

117.2

116.4

C28-N2-C29

118.2

116.8

C35-N3-C38

112.0

110.9

C35-N3-C47

113.3

109.6

C38-N3-C47

109.8

107.9

C41-N4-C44

110.2

107.7

C41-N4-C50

112.9

111.2

C44-N4-C50

113.0

110.2

N2-C5-C6

121.0

121.1

N2-C5-C14

119.7

119.5

C6-C5-C14

119.2

119.4

C5-C6-H7

119.5

119.5

C5-C6-C8

121.1

121.1

H7-C6-C8

119.3

119.5

2

C6-C8-H9

119.6

120.1

C6-C8-C10)

120.0

119.8

H9-C8-C10

120.4

120.1

C8-C10-H11

120.5

120.2

C8-C10-C12

119.3

119.7

H11-C10-C12

120.2

120.2

C10-C12-H13

119.7

119.3

C10-C12-C14

121.8

121.3

H13-C12-C14

118.5

119.3

C5-C14-C12

118.5

118.7

C5-C14-C15

122.7

123.1

C12-C14-C15

118.8

118.1

C14-C15-H16

115.2

116.4

C14-C15-C17

127.3

127.2

H16-C15-C17

117.2

116.4

C15-C17-H18

117.2

117.1

C15-C17-C19

127.3

125.7

H18-C17-C19

115.1

117.1

C17-C19-C20

118.7

118.0

C17-C19-C28

122.7

123.3

C20-C19-C28

118.6

118.7

C19-C20-H21

118.5

119.2

C19-C20-C22

121.8

121.6

H21-C20-C22

119.8

119.2

C20-C22-H23

120.2

120.3

C20-C22-C24

119.2

119.4

H23-C22-C24

120.5

120.3

C22-C24-H25

120.4

119.9

C22-C24-C26

120.0

120.1

H25-C24-C26

119.6

119.9

C24-C26-H27

119.2

119.6

C24-C26-C28

121.2

120.7

H27-C26-C28

119.6

119.6

N2-C28-C19

119.5

118.9

N2-C28-C26

121.2

121.7

C19-C28-C26

119.2

119.4

N2-C29-H30

112.8

109.1

N2-C29-H31

106.3

109.1

N2-C29-C32

113.1

112.5

H30-C29-H31

106.2

107.8

H30-C29-C32

109.2

109.1

H31-C29-C32

108.9

109.1

C29-C32-H33

106.8

109.1

C29-C32-H34

110.9

109.1

3

C29-C32-C35

113.0

112.3

H33-C32-H34

106.6

107.9

H33-C32-C35

110.7

109.1

H34-C32-C35

108.7

109.1

N3-C35-C32

113.3

113.4

N3-C35-H36

107.7

108.9

N3-C35-H37

111.2

108.9

C32-C35-H36

108.4

108.9

C32-C35-H37

109.6

108.9

H36-C35-H37

106.5

107.7

N3-C38-H39

111.5

109.3

N3-C38-H40

108.6

109.3

N3-C38-C41

110.9

111.6

H39-C38-H40

107.8

108.0

H39-C38-C41

108.8

109.3

H40-C38-C41

109.3

109.3

N4-C41-C38

110.6

111.0

N4-C41-H42

111.6

109.4

N4-C41-H43

108.6

109.4

C38-C41-H42

108.8

109.4

C38-C41-H43

109.2

109.4

H42-C41-H43

108.0

108.0

N4-C44-H45

109.4

109.5

N4-C44-H46

111.1

109.5

N4-C44-C47

111.0

110.5

H45-C44-H46

107.5

108.1

H45-C44-C47

108.8

109.5

H46-C44-C47

109.0

109.5

N3-C47-C44

110.8

110.5

N3-C47-H48

109.4

109.5

N3-C47-H49

111.1

109.5

C44-C47-H48

108.6

109.5

C44-C47-H49

109.0

109.5

H48-C47-H49

107.8

108.1

N4-C50-H51

108.5

108.7

N4-C50-H52

111.5

108.7

N4-C50-C53

114.0

114.2

H51-C50-H52

106.9

107.6

H51-C50-C53

107.1

108.7

H52-C50-C53

108.6

108.7

O1-C53-C50

108.7

109.6

O1-C53-H54

110.8

109.8

O1-C53-H55

110.4

109.8

C50-C53-H54

110.6

109.8

4

C50-C53-H55

108.6

109.8

H54-C53-H55

107.8

108.2

Label

DFT

XRD

Label

DFT

XRD

N2- C5-C6-C8

174.7

175.5

N2-C5-C14-C12

-174.4

-175.9

N2-C5-C14-C15

5.2

1.8

N2-C29-C32-C35

62.7

61.7

N3-C47-C44-N4

57.3

61.6

N4-C50-C53-O1

-74.4

-69.4

N4-C41-C38-N3

-57.9

-58.7

C5-N2-C28-C19

67.5

71.0

C5-C14-C15-C17

31.0

31.8

C5-N2-C28-C26

-115.5

-111.9

C5-N2-C29-C32

-151.1

-158.5

C5-C6-C8-C10

0.6

0.7

C6-C8-C10-C12

0.4

0.0

C6-C5-C14-C15

-178.3

178.5

C6-C5-C14-C12

2.1

0.9

C8-C10-C12-C14

-0.0

-0.3

C10-C12-C14-C5

-1.2

-0.1

C10-C12-C14-C15

179.2

177.9

C12-C14-C15-C17

-149.4

-150.5

C14-C5-C6-C8

-1.8

-1.2

C14-C15-C17-C19

0.1

3.7

C15-C17-C19-C28

-31.2

-33.5

C15-C17-C19-C20

148.7

144.6

C17-C19-C28-C26

178.0

175.3

C17-C19-C20-C22

-178.7

-175.6

C17-C19-C28-N2

-4.9

-7.5

C19-C20-C22-C24

0.0

-0.9

C20-C19-C28-N2

175.1

174.4

C20-C22-C24-C26

-0.4

-0.7

C20-C19-C28-C26

-1.9

-2.8

C22-C24-C26-C28

-0.4

0.6

C24-C26-C28-C19

1.6

1.2

C24-C26-C28-N2

-175.5

-175.8

C28-C19-C20-C22

1.2

2.6

C28-N2-C5-C6

116.0

116.9

C28-N2-C5-C14

-67.6

66.5

C28-N2-C29-C32

61.3

61.0

C29-N2-C28-C19

-144.7

-147.8

Dihedral angles (˚)

5

C29-N2-C28-C26

32.3

29.3

C29-C32-C35-N3

-176.0

-176.0

C29-N2-C5-C6

-32.2

24.5

C29-N2-C5-C14

144.2

152.2

C35-N3-C38-C41

-175.7

-176.6

C35-N3-C47-C44

176.9

178.6

C38-N3-C35-C32

162.6

173.8

C38-N3-C47-C44

-57.0

-57.8

C41- N4-C44-C47

-56.9

-60.3

C41-N4-C50-C53

153.7

165.8

C44-N4-C41-C38

57.1

58.7

C44-N4-C50-C53

-80.5

-74.9

C47-N3-C35-C32

-72.4

-67.1

C47-N3-C38-C41

57.5

56.5

C50-N4-C41-C38

-175.6

-179.6

C50-N4-C44-C47

175.8

178.2

6

Table 2. Calculated vibrational wave numbers (scaled), measured IR and Raman bands and assignments of Opipramol B3LYP/SDD υ(cm-1) IRI

B3LYP/6-311++g(d,p)(5D,7F) RA

υ(cm-1) IRI

RA

IR

Raman

SERS Assignmentsa

υ(cm-1) υ(cm-1)

υ(cm-1)

-

3416

62.85 96.75

3403

36.14 130.96

3421

-

-

υOH(100)

3107

16.17 402.76

3072

11.57 228.14

-

3075

-

υCHII(80)

3106

44.99 143.42

3070

13.55 222.24

-

-

-

υCHI(80)

3093

35.03 74.87

3063

21.82 191.67

-

-

-

υCHII(95)

3092

27.20 54.20

3062

25.06 131.88

3059

3058

-

υCHI(71)

3074

20.26 51.05

3046

11.80 95.01

-

-

3049

υCHII(73), υCHI(17)

3074

4.36

123.57

3046

8.77

131.49

-

-

-

υCHI(74), υCHII(13)

3065

26.22 140.52

3038

12.20 150.68

-

-

-

υCHI(47), υCHII(29), υCHIII(17)

3064

0.23

3.06

3037

1.17

12.44

-

-

-

υCHII(56), υCHI(37)

3059

17.08 61.55

3029

14.99 105.83

-

-

-

υCHIII(81)

3033

0.24

19.52

3010

0.21

3012

3012

3011

υCHIII(98)

3009

62.39 11.19

2969

43.05 8.52

2972

2971

2972

υasCH2(96)

3007

68.27 129.92

2957

10.17 89.60

-

-

-

υasCH2(97)

2991

19.48 218.26

2954

31.87 119.26

-

2953

-

υasCH2Pz(85)

2990

30.66 41.86

2950

54.52 88.29

2947

-

-

υasCH2(89)

2986

55.77 31.55

2946

3.18

47.55

2947

-

-

υasCH2Pz(89)

2984

68.41 76.71

2944

8.13

59.21

-

-

-

υasCH2Pz(31),

26.69

υasCH2(60) 2980

7.60

40.76

2941

46.06 45.61

-

-

-

υasCH2(85)

2978

13.53 13.69

2939

15.42 60.73

-

2935

2929

υasCH2Pz(66), υasCH2(24)

2976

20.81 38.80

2918

23.12 143.73

2922

2915

2911

υasCH2Pz(80)

2937

39.25 135.44

2902

40.69 65.10

-

-

-

υsCH2(96)

2905

78.72 111.76

2867

87.90 205.75

2870

-

2883

υsCH2(92)

2868

65.00 63.14

2842

61.48 94.83

2842

2844

2848

υsCH2(95)

1

2856

199.59 131.47

2806

223.63 21.91

2810

-

2815

υsCH2Pz(46), υsCH2(47)

2846

31.89 16.59

2795

81.37 207.78

-

-

-

υsCH2Pz(45), υsCH2(47)

-

2792

-

υsCH2Pz(93)

28.23 37.32

-

-

-

υsCH2Pz(83)

2780

47.37 38.27

-

-

-

υsCH2Pz(95)

40.62 26.18

2774

45.63 36.31

-

-

-

υsCH2(88)

2.01

1611

2.56

1620

1622

1628

υC=C(57),

2844

23.99 18.40

2793

6.23

2821

109.20 195.00

2784

2808

54.03 21.67

2797 1626

675.40

198.41

535.57

υRingII(15) 1579

11.17 38.67

1569

11.71 47.10

1569

1571

1592

υRingII(46), δCHII(11), υRingI(12)

1577

13.61 158.89

1567

12.32 154.43

1569

-

1570

υRingI(54), υRingII(20)

1558

8.87

233/65

1542

6.38

180.60

-

-

-

υRingI(24), υRingII(52)

1550

6.00

11.94

1542

5.36

94.18

-

-

-

υRingII(19), υRingI(58)

1482

1.41

12.18

1467

15.68 40.13

1470

1465

1475

δCH2(79), υRingI(11)

1477

5.51

8.84

1474

21.70 29.59

1458

7.10

11.31

1458

-

-

δCH2(92)

1454

2.91

9.57

-

-

-

υRingII(54), δCH2(11), δCHI(14)

1472

10.76 2.43

1453

11.52 5.69

-

-

-

δCH2(73), δCH2Pz(14)

1463

12.20 8.24

1446

61.68 9.36

-

1445

-

δCH2(13), δCH2Pz(66)

1458

90.38 15.13

1446

7.81

7.35

-

1445

-

υRingI(62), δCH2Pz(12)

1457

5.17

10.93

1438

12.80 4.37

-

-

-

δCH2(53), δCH2Pz(22)

1456

27.12 6.46

1434

2.71

7.17

1435 2

-

-

δCH2(26),

δCH2Pz(59) 1452

2.50

10.26

1431

47.14 5.95

-

-

-

δCH2(21), δCH2Pz(71)

1446

1.99

3.43

1430

3.87

10.04

-

-

-

δCH2(20), δCH2Pz(69)

1444

5.49

2.46

1426

2.44

4.66

-

-

-

δCH2(54), δCH2Pz(33)

1437

43.92 7.16

1419

5.26

1.67

-

1418

-

υRingII(48), δCHIII(30)

1408

14.28 0.81

1409

12.13 1.56

1406

-

1407

δCHII(20), υRingI(52)

1393

5.79

12.69

1393

0.97

6.01

-

-

-

δCHIII(40), δCHI(14), δCH2(18)

1389

0.42

6.58

1383

1.03

15.98

1385

-

-

δCH2(38), δCH2Pz(33)

1379

6.43

8.66

1379

0.38

3.62

-

-

-

δCH2(22), δCH2Pz(49)

1371

40.93 4.79

1370

2.39

2.61

-

-

-

δCH2(44), δOH(39)

1368

4.00

0.79

1360

4.09

1.34

1360

-

-

δCH2(43), δCH2Pz(23)

1360

3.51

3.80

1352

0.90

2.40

-

-

-

δCH2(29), δCH2Pz(29)

1354

24.41 3.09

1346

54.48 1.52

1342

-

-

δCH2(65), δCH2Pz(34)

1338

5.89

1.92

1331

9.41

1.63

-

-

-

δCH2(21), δCH2Pz(51)

1329

7.27

3.39

1317

13.43 0.80

1318

-

-

δCH2(23), δCH2Pz(50)

1329

1.11

147.45

1306

12.76 5.35

-

1310

1309

υRingII(41), υRingI(27)

1319

6.72

7.08

1293

23.40 31.34

-

-

-

δCH2(40), υRingII(11),

3

υRingI(13) 1314

4.46

13.79

1290

4.95

87.26

-

-

-

υRingII(29), υRingI(48)

1301

39.56 14.58

1287

50.25 21.75

-

-

-

δCH2(40), υCO(44)

1296

22.28 14.74

1284

19.95 17.34

-

1284

-

δCH2(40), δCH2Pz(17), υCO(12)

1292

14.94 29.83

1278

3.88

4.47

-

-

δCH2Pz(57),

-

δCH2(13), υCN(10) 1284

31.49 13.14

1268

28.68 18.38

1270

1267

-

δCH2(45), υCN(11), δCHI(15)

1278

2.30

1.06

1261

1.23

1.49

-

-

-

δCHI(42), υRingI(10), δCHIII(14)

1272

32.75 1.74

1254

7.74

2.90

-

-

-

δCH2Pz(44), δCH2(18), υRingI(14)

1263

2.45

9.43

1252

6.20

3.31

1248

1248

-

δCH2(46), δCH2Pz(24)

1251

9.48

9.22

1243

14.49 5.73

-

-

-

δCH2(25), δCHII(18), δCH2Pz(23)

1239

13.29 12.32

1226

6.55

8.14

1230

1228

1224

δCHII(51), δCHIII(12), δCH2Pz(14)

1231

4.02

54.18

1218

20.92 12.14

-

-

-

δCHIII(47), δCHI(11), υRingI(10)

1219

36.96 16.40

1208

33.15 14.50

-

1206

-

υCN(32), δCH2(13), υRingI(18)

4

1207

55.09 27.27

1201

22.12 62.84

-

-

-

υCN(42), δCH2(12), υCC(20)

1198

4.07

12.99

1196

42.72 33.22

1198

-

-

δCH2(21), υCC(12), δCH2Pz(18), δCHI(15)

1192

19.35 15.01

1180

0.39

5.37

-

-

-

υCC(21), δCH2Pz(23), δCHI(25)

1185

8.43

6.66

1179

9.13

15.32

-

-

-

δCH2Pz(66), δCHI(16)

1173

49.69 1.42

1172

38.94 4.51

-

1165

1165

δCH2(27), δOH(18), δCH2Pz(19)

1161

0.33

7.45

1148

43.01 2.29

1149

-

-

δCHII(37), υPz(36)

1160

0.16

2.71

1139

2.52

8.17

1139

-

-

δCHII(16), δCHI(67)

1145

1.58

17.82

1138

0.94

1.92

-

-

-

υCC(46), δCHII(15), δCHI(13)

1143

2.19

4.27

1137

0.47

13.00

-

-

-

υPz(12), δCH2(38), δCHII(13), δCHI(11)

1131

71.84 6.19

1125

61.44 3.02

1122

1124

1127

υPz(41), υCN(38)

1122

53.01 4.40

1116

27.35 12.52

-

1112

-

υPz(49), υCN(15), δCH2Pz(13)

1118

13.49 7.55

1107

33.31 6.85

-

-

-

υPz(15), υCN(17), δCH2Pz(41)

5

1111

8.66

30.74

1101

3.54

36.73

1103

-

-

υCN(43), δCHI(41)

1102

16.33 5.30

1095

7.70

3.57

-

-

-

υCN(40), δCHII(39)

1096

29.31 15.43

1088

30.85 5.64

1082

1080

-

υCN(10), δCH2(11), υCC(18), υPz(20)

1076

20.42 2.71

1065

11.26 2.73

1062

-

-

δCH2(37), υCC(38), υPz(12)

1073

5.53

2.91

1052

6.68

3.42

-

-

-

υPz(36), δCH2Pz(35), υCC(10)

1057

0.73

2.76

1041

17.22 3.05

1041

1044

1041

δCH2(13), υCC(33), υPz(15)

1043

3.76

0.35

1039

42.22 1.88

1041

-

-

δCH2(11), δCH2Pz(66)

1042

7.94

1.29

1032

1.12

1.86

-

-

-

υCN(15), δCHI(60)

1030

13.22 10.75

1028

10.29 9.60

-

-

-

υRingII(38), δCHII(42)

1024

6.86

84.12

1024

2.41

97.78

-

-

-

υRingII(32), υRingI(45)

1023

24.51 11.84

1018

1.74

1.35

-

1015

-

υPz(11), υCC(45)

1003

19.82 4.87

1001

6.65

7.03

1005

-

-

υPz(39), υCN(17), υCC(14)

990

2.06

7.91

989

40.65 3.64

-

991

-

γCHIII(54), γCHII(21)

989

78.49 1.02

961

1.11

7.19

965

-

965

δPz(12), δCH2Pz(41),

6

υCN(12) 985

0.18

0.44

949

0.68

4.87

-

-

-

γCHII(50), γCHI(30)

981

0.75

2.91

947

0.58

3.82

-

-

-

γCHI(41), γCHII(14), γCHIII(25)

955

7.53

11.99

946

0.91

3.95

940

942

-

υCC(31), γCHI(14), γCHII(15), δCH2(11)

948

2.02

1.73

920

2.22

0.90

-

-

-

γCHII(73), υPz(15)

944

2.29

3.47

918

2.01

0.63

-

-

-

γCHI(73), υPz(20)

914

2.97

0.73

915

39.30 1.41

912

-

-

υPz(37), γCHII(14), γCHI(15)

904

7.60

5.47

906

10.20 3.04

-

909

-

υCC(19), υPz(15), δCH2(40)

896

13.50 8.83

889

9.25

5.03

889

889

-

δRingI(28), δRingII(19), δRingIII(12)

874

4.56

6.69

858

9.37

2.03

861

862

-

γCHII(63), γCHI(12)

870

4.15

3.26

850

5.05

5.97

-

-

-

γCHI(51), γCHII(15)

859

7.79

4.76

849

17.29 1.68

-

848

-

γCHI(23), υCC(16), δCH2(25)

849

12.88 8.08

842

1.39

1.98

-

-

-

γCHI(21), υCC(16), δCH2(28)

836

1.52

2.62

831

2.31

1.00

834 7

-

-

δCH2Pz(64),

δCH2(12) 824

4.15

1.99

822

5.36

1.94

-

818

-

δRingIII(30), δCH2(18), γCHIII(21)

803

61.59 18.14

795

4.51

2.35

-

794

797

γCHIII(42), δRingIII(12), δCH2(14)

791

25.98 8.99

787

26.31 4.85

788

-

-

γCHIII(13), δCH2(40), δRingIII(16)

772

7.48

3.48

774

19.74 20.72

772

770

-

δRingI(25), υPz(12), δRingII(21)

763

56.90 1.21

756

16.51 4.68

757

-

-

γCHI(57), γCHII(12)

760

29.43 2.67

750

13.42 11.24

-

-

-

γCHI(30), γCHII(48)

747

57.81 3.50

740

23.65 3.65

-

744

-

υPz(11), δCH2(36), γCHI(10), γCHII(18)

735

0.49

6.63

739

56.13 6.21

-

-

-

τRingI(25), τRingII(26), τRingIII(15)

725

5.60

22.11

726

4.09

0.65

720

724

715

υPz(35), υCN(13), γCHI(11), γCHII(13)

711

1.65

22.59

698

6.21

26.80

-

700

-

τRingI(23), τRingII(23), γCHIII(24)

697

0.91

2.56

691

0.22

1.23

-

688

685

δRingIII(36), δRingI(16), δRingII(14)

8

667

3.49

16.46

671

2.94

15.20

660

-

-

δRingI(27), δRingII(36)

617

1.26

2.67

618

0.81

0.92

616

-

-

δRingI(32), δRingII(17), δPz(15)

599

3.43

1.24

600

0.76

0.95

597

598

-

δPz(25), δRingI(20), δRingII(16)

580

0.44

8.80

572

1.03

5.33

-

-

-

τRingI(20), τRingII(16), τRingIII(33)

572

5.29

10.05

568

4.05

8.16

-

-

-

τRingI(15), τRingII(24), τRingIII(17)

561

109.90 1.15

556

5.24

2.79

-

-

-

τOH(88)

557

3.51

528

1.71

0.91

532

520

-

δRingI(12),

2.53

δRingII(16), δRingIII(31) 540

1.18

2.97

503

6.47

1.76

508

499

495

δCH2(31), τRingI(10), δCH2(15)

526

2.72

0.85

488

2.47

1.09

490

-

-

τRingI(20), δCH2(25), τRingII(10), τRingIII(11)

492

4.69

0.92

486

2.28

1.76

-

-

-

τRingII(25), τRingIII(20), δPz(12)

479

12.97 1.79

479

29.56 2.43

476

474

475

δPz(33), δRingI(11), δRingII(14)

477

10.37 2.02

461

4.42

0.54

459

456

-

δPz(46), δRingI(15), δRingII(10)

9

463

1.76

1.66

439

10.10 5.25

-

-

435

δRingI(18), δRingII(21), τRingII(15)

440

8.29

5.96

422

0.84

1.11

424

-

-

τRingI(24), τRingII(22), τRingIII(20)

413

2.95

1.04

415

0.26

7.26

412

-

416

γCN(34), τPz(19)

403

0.33

1.99

380

0.73

2.42

-

385

387

τPz(39), γCN(32), δPz(10)

383

0.01

8.22

378

0.18

3.63

-

-

-

τRingI(28), τRingII(28), τRingIII(15)

375

3.27

0.97

368

1.56

1.19

-

-

-

δCN(23), δPz(12), δCH2(15)

357

1.58

2.69

353

5.76

0.24

-

356

-

δPz(33), δCH2(17), τCN(11)

343

10.30 1.22

334

0.07

2.01

-

331

330

τCN(10), δCN(10), τPz(29)

331

5.22

2.32

320

1.15

5.01

-

-

-

δRingIII(26), γCN(17), τRingI(10)

319

2.80

7.64

311

1.53

2.32

-

307

-

δRingIII(33), τRingII(19), τRingI(17)

312

2.96

1.39

288

0.53

1.17

-

280

-

δRingIII(24), γCN(16), δPz(15)

289

0.36

1.71

255

0.24

0.53

-

-

-

δPz(31), δCN(19),

10

τRingIII(12) 254

2.85

1.71

252

3.40

1.56

-

-

-

τRingIII(36), δCN(15), τPz(13)

242

0.99

0.49

247

1.65

2.75

-

240

240

τPz(65)

238

0.79

1.10

227

5.94

0.74

-

224

-

τPz(22), γCN(32), τCH2(10)

221

0.15

0.14

214

1.81

1.42

-

-

-

τCH2(19), δCN(15), τRingIII(19)

212

1.38

1.36

191

108.43 1.87

-

-

-

δCH2(30), τRingIII(16), τRingI(12)

169

0.91

1.66

165

7.84

1.07

-

162

160

δCH2(22), τRingIII(26), τRingI(10)

157

2.35

3.06

152

0.94

2.30

-

-

-

τRingIII(36), τRingI(14), γCN(10)

142

3.10

0.32

141

8.96

1.09

-

135

134

τCO(35), τPz(16), τOH(11)

116

1.54

9.31

117

2.12

8.77

-

115

-

τRingIII(28), τCO(14), τRingI(12), τRingII(14)

89

1.75

1.08

93

0.51

0.55

-

93

-

τRingIII(26), τCO(15), τOH(12)

87

0.36

1.99

87

1.58

1.98

-

-

-

τRingIII(27), τCO(14), τOH(13)

74

1.51

1.99

77

2.13

1.61

11

-

-

τCO(24),

τOH(17), τCH2(10) 55

1.57

11.75

55

0.38

12.55

-

-

-

τCH2(12), τRingIII(42)

48

1.64

2.88

43

1.16

0.73

-

-

-

τCH2(28), τOH(20), τRingIII(11), γCN(11)

38

1.80

1.32

39

0.91

1.46

-

-

-

τPz(16), τCN(15), τCH2(13), γCN(17)

22

0.06

3.52

23

0.04

2.32

-

-

-

τCH2(22), γCN(56)

16

0.52

4.96

18

0.23

3.57

-

-

-

τCN(17), τCH2(50)

10

0.67

2.03

10

0.46

1.64

-

-

-

τCH2(50), γCN(34)

-----------------------------------------------------------------------------------------------------------------------a

υ-stretching; δ-in-plane deformation; γ-out-of-plane deformation; τ-torsion; The C5-C6-C8-C10-C12-C14,

C20-C22-C24-C26-C28-C19, N2-C5-C14-C15-C17-C19-C28 and piperazine rings are designated as RingI, RingII, RingIII and Pz; as-asymmetric; s-symmetric; potential energy distribution is given in brackets in the assignment column; IRI-IR intensity in KM/Mole ; RA-Raman activity in Å4/a.m.u.

12

Table 3. PASS prediction of activity spectrum, Pa is the probability of the molecule to be active and Pi is the probability to be inactive. Pa

Pi

Activity

0.910

0.004

Anaphylatoxin receptor antagonist

0.900

0.003

Glycosylphosphatidylinositol phospholipase D inhibitor

0.891

0.005

Aldehyde oxidase inhibitor

0.821

0.026

Phobic disorders treatment

0.806

0.016

Antineurotic

0.792

0.004

CYP2E1 inhibitor

0.788

0.014

Beta-adrenergic receptor kinase inhibitor

0.788

0.014

G-protein-coupled receptor kinase inhibitor

0.790

0.021

Antieczematic

0.776

0.009

Carboxypeptidase Taq inhibitor

0.769

0.003

CYP2C18 substrate

0.763

0.004

X-methyl-His dipeptidase inhibitor

0.736

0.004

Skeletal muscle relaxant

0.726

0.007

CYP2D substrate

0.719

0.007

CYP2D6 substrate

0.705

0.005

Choline-phosphate cytidylyltransferase inhibitor

0.701

0.003

Antidepressant, Imipramin-like

0.704

0.062

Aspulvinone dimethylallyltransferase inbibitor

Highlights *

IR, Raman and SERS spectra and NBO analysis were reported.

*

The wavenumbers are calculated theoretically using Gaussian09 software.

*

The wavenumbers are assigned using PED analysis.

*

Molecular docking is reported.

Graphical abstract