Synthesis, spectroscopic characterization, anti-urease activities of a novel bisphosphoramidate, a combined experimental and computational study
Accepted Manuscript Synthesis, spectroscopic characterization, anti-urease activities of a novel bisphosphoramidate, a combined experimental and compu...
Accepted Manuscript Synthesis, spectroscopic characterization, anti-urease activities of a novel bisphosphoramidate, a combined experimental and computational study Nasrin Fallah, Khodayar Gholivand, Mohammad Yousefi, Parviz Aberoomand Azar PII:
S0022-2860(18)30807-X
DOI:
10.1016/j.molstruc.2018.06.106
Reference:
MOLSTR 25393
To appear in:
Journal of Molecular Structure
Received Date: 11 March 2018 Revised Date:
27 May 2018
Accepted Date: 27 June 2018
Please cite this article as: N. Fallah, K. Gholivand, M. Yousefi, P. Aberoomand Azar, Synthesis, spectroscopic characterization, anti-urease activities of a novel bisphosphoramidate, a combined experimental and computational study, Journal of Molecular Structure (2018), doi: 10.1016/ j.molstruc.2018.06.106. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Synthesis, spectroscopic characterization, anti- urease activities of a novel bisphosphoramidate, a combined experimental and computational study
Nasrin Fallaha, Khodayar Gholivandb, *, Mohammad Yousefia,
a
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Parviz Aberoomand azara,
Department of Chemistry, Science and Research Branch, Islamic Azad University, Tehran, Iran.
b
Department of Chemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran
Methylenebis(N,N'-dibenzylphosphoramidate) (MBDPA) is synthesized by 1:8 mole ratio reaction of Methylenebis(phosphonic dichloride) and benzylamine. MBDPA characterized by FT-IR, 1H NMR, 13C NMR, 31
PNMR spectroscopy. Also, The IC50 value for MBDPA is 5.23 nM that show very strong urease inhibitory
activity. The geometry optimization is performed using the B3LYP and PBE1PBE density functional methods
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with two basis sets; 6-311 G (d, p) and 6-311+G(d,p). 1H, 13C and 31PNMR chemical shifts have been calculated by using the gauge independent atomic orbital (GIAO) method. The infrared spectra of MBDPA are calculated and compared with the experimentally observed ones. The assignments are determined on the basis of the
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potential energy distribution of the all vibrational modes. Results of this study showed that there is a good correlation between the experimental data and computational results. The electronic structure is studied by
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analyzing the frontier molecular orbitals and molecular electrostatic potential for the prediction of stability and activity of the MBDPA.
Keywords: bisphosphoramidate; urease inhibitor; B3LYP; PBE1PBE; chemical shift; electronic structure.
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ACCEPTED MANUSCRIPT 1. Introduction Phosphorus is a very important element in organic, bioorganic and inorganic chemistry and phosphoramidates are the compounds contain P=O and P-N functional groups. Chemistry of mono and bisphosphoramidates and their derivatives have been a subject of
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growing interest for the last two decades [1]. Thus, in this work, we are synthesis Methylenebis(N,N'-dibenzylphosphoramidate) (MBDPA) and characterized by FT-IR, 1H NMR, 13C NMR, 31P NMR spectroscopy. Also, we calculated IR and NMR spectra and NBO
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analysis with density functional theory (DFT). Ft-IR and NMR spectra could be fairly good reproduced by theoretical method applied here. Additionally, charge distributions and NBO
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analysis of MBDPA predicts that the oxygen atoms of the title compound are the most negative site to an electrophilic attacked and a good candida for reacting with urease enzyme, because urease (jack bean urease) is a nickel-containing metalloenzyme which two nickel ions within its active site that catalyzes the hydrolysis of urea to form ammonia and
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carbamate [2,3]. Therefore, the measurement of urease inhibitory activity was carried out and the title compound shows strong urease inhibitory activity.
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2. Experimental details
2.1. General considerations
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Infrared spectra were recorded in KBr discs in the region 4000–400 cm−1 on a
Thermo Nicolet NEXUS 870 FT-IR spectrometer.1H,
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recorded on a Bruker Avance DRX 500 spectrometer. 1H and determined relative to internal TMS, and
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31
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P NMR spectra were
C chemical shifts were
P chemical shifts relative to 85% H3PO4 as an
external standard. Melting points were obtained with an electrothermal instrument. 2.2. Synthesis of [(PhCH2NH)2P(O)CH2P(O)(PhCH2NH)2]
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ACCEPTED MANUSCRIPT Methylenebis(phosphonic dichloride) was very moisture sensitive, therefore reaction was performed in the absence of water, under argon gas and dry solvent. All reagents were purchased from Sigma-Aldrich and Merck companies were used as received.
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A solution of benzylamine (0.8572 g, 8 mmol) in CH2Cl2 was added at 273 °K to a solution of Methylenebis(phosphonic dichloride) (0.250g, 1 mmol) in CH2Cl2. After 4 h stirring the solvent was removed in vacuum and the resulting white powder was washed with
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distilled water. The synthesis pathway of the compound is represented below.
2.3. Urease inhibitory activity Urease (urea amidohydrolase; E.C.3.5.1.5) is a metalloenzyme containing two nickel atoms which catalyze the hydrolysis of urea so as to form ammonia and carbamate. The carbamate produced, automatically decomposes to produce a second molecule of ammonia and carbon dioxide. High concentrations of ammonia which follow from these reactions as well as the accompanying pH elevation, have important negative implications for both human and animal health, and for agriculture [4]. The use of inhibitors for controlling the activity of 3
ACCEPTED MANUSCRIPT urease could counteract these negative effects. In the past few years, a few phosphinicamide were studied for their urease inhibitory activity, and have recently had a breakthrough [5]. We previously reported the urease inhibitory activities of some phosphinicamide and Phosphorhydrazide [3]. For urease inhibitory activity, all chemicals used were of analytical
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grade from Merck Co. Sodium nitroprusside and urease (EC 3.5.1.5) from Jack beans were purchased from Sigma-Aldrich Co. Ultra-pure water was used throughout the experiments.
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Potassium phosphate buffer (100 mM), pH=7.4, was prepared in distilled water.
3. Computational details
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In the present work, quantum chemical calculations were carried out at DFT levels using the Gaussian 09 suites of programs [6]. The structure of MBDPA was optimized using the B3LYP and PBE1PBE density functional employing, 6-311G(d,p) and 6-311+G(d,p) basis sets in the gas phase. All calculations were performed assuming C1 point group
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symmetry for title compound. All computed structures were confirmed as energy by calculating the vibrational frequencies using second derivative analytic methods and confirming the absence of imaginary frequencies. The isotropic shielding (σiso) values were
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[7].
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calculated based on the gauge-independent atomic orbital (GIAO) technique in the gas phase
Calculated shielding values for each atom in molecule were shifted relatively from a
frequency of standard compounds: tetramethylsilane (TMS) for hydrogen and carbon atoms and orthophosphoric acid (H3PO4) for phosphorus atom. In order to compare theoretical values with experimental results, we also computed the absolute shielding constants for TMS and H3PO4 using the same set of quantum chemical calculations. The 1H, and 13C chemical shifts (δ) were then calculated by subtracting the calculated isotopic value of the shielding tensor σiso,cal from that of TMS: δ= σiso, TMS - σiso,cal. and
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ACCEPTED MANUSCRIPT chemical shifts (δ) were calculated by: δ= σiso, H3PO4 - σiso,cal. Also, 1H, 13C and 31P chemical shifts of structure were simulated using the Gauss View software [8]. The NBO [9] analyses have been performed to compare the electronic features of the gas-phase structures of the compound at the B3LYP/6-311+G(d,p)and PBE1PBE/6-
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311+G(d,p) level. All band assignments were performed by the potential energy distribution calculated by PBE1PBE and B3LYP using the VEDA 4 program[10]. The statistical analysis
Microsoft Excel version 2013.
4. Results and Discussion 4.1. Structural study
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was carried out using MATLAB version R2010a.[11] and all charts were plotted using the
The optimized structure and atom numbering of MBDPA are shown in Figure 1. The
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optimized structure was compared with the previously published similar molecules. The selected most relevant calculated bond lengths, bond angles and dihedral angles at different levels of theory are listed in Tables 1-3. These data reveal that the estimated bond lengths at
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different DFT levels are almost independent of the method and basis set used. The following
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results are referred to the B3LYP/6-311+G(d,p) and PBE1PBE/6-311+G(d,p) levels of theory.
The computed bond length of phosphoryl group are 1.4947– 1.4996 Å at the B3LYP/6-311+G(d,p)level and 1.4915–1.4933 Å at the PBE1PBE/6-311+G(d,p) level. The experimental P=O bond lengths in mono and bisphosphoramidate are reported 1.476– 1.469 Å in the published literature [12,13]. The computed bond length of P-N are 1.6748, 1.6812,
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ACCEPTED MANUSCRIPT 1.6708, 1.6879 Å at the B3LYP/6-311+G(d,p)level and 1.6653, 1.6735, 1.6646, 1.6782 Å at the PBE1PBE/6-311+G(d,p) level and the experimental value reported in the published literature 1.6571, 1.6184, 1.6158, 1.676(2) [10-13]. The B3LYP/6-311+G(d,p) calculations predict the P-C bond lengths are 1.8476-1.8387 Å, and 1.8306- 1.8309 Å at the PBE1PBE/6-
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311+G(d,p) and the experimental value reported in the published 1.7990(14)- 1.8048(15) Å, [14] respectively. Figure 2 shows the correlations between the experimental and calculated bond lengths. As is evident, a good linear correlation (correlation coefficient= 0.9878) was
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observed between the B3LYP/6-311+G(d,p) results and experimental data.
The computed bond angles for O2-P1-N51 (113.2072°), O2-P1-N53 (118.7465°), O3-
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P4-N49 (121.0186°), O3-P4-N55 (110.5819°), at the B3LYP level, which are confirmed by the experimental data of 114.88 (9)°, 113.17 (8)°, [13]. (Table 2). The calculated dihedral angles at B3LYP level for O2-P1-N51-C66 (28.5837°), O2-P1-N53-C63 (-50.4682°), O3-P4-
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N49-C60 (36.3634°), O3-P4-N55-C57 (-28.8961°), respectively, which are in experimental
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values of -47.97(19) –178.63(12) [13]. It is evident from Tables 1-3, the optimized bond lengths and bond angles were
slightly dissimilar than the literature values because the molecular states were different during the experimental and theoretical process. The isolated molecule is considered as in gas phase during theoretical calculation, while many packing molecules were treated as in the condensed phase during the experimental measurements. 4.2. Vibrational assignments
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ACCEPTED MANUSCRIPT The experimental and theoretical simulated FT-IR spectra of MBDPA are presented in Figure S1 and S2 of Supporting Information. The FT-IR vibrational pattern includes many stretching, bending and torsional vibrations that creates many signals. In this research, The
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computational methods were used for analysis of the interpretation of vibrational spectra.
The theoretical calculations shows MBDPA has the C1 point group symmetry. This
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compound consists of 71 atoms. The number of vibration normal modes of MBDPA are 207, 70 modes of vibrations are stretching, bending 69, torsion 63 and 5 vibrational modes are out-
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of-plane. The theoretical frequencies and infrared intensities of title compound calculated at the B3LYP/6-311+G(d,p) and PBE1PBE/6-311+G(d,p) levels are presented in Tables 4 and 5.
4.2.1. N–H vibrations
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In phosphoramidates, N–H stretching vibration appears at 3200–3500 cm-1 [16]. The
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FT-IR bands for the N–H stretching vibrations are observed at 3301 cm-1. The theoretical
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calculated N–H stretching vibration appears at 3556 cm-1 at the B3LYP/6-311+G(d,p) level which is in good agreement with the experimental data. The calculated N–H stretching vibrations appear at 3604 cm-1 by the PBE1PBE/6-311+ G (d, p). As evidenced by the Table 4, these modes number are 204-207. 4.2.1.C–H vibrations The C–H stretching vibration in alkanes and aromatic structures are in the regions of 2850–3000 cm-1 and 3000–3100 cm-1, respectively [17]. The title compound was contained 8 aliphatic and 20 aromatic C–H. bonds. There are 30 vibrational modes for C–H stretching
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ACCEPTED MANUSCRIPT (Table 4). The FT-IR bands for the C–H stretching vibrations are observed at 3190 cm-1 and 3036 cm-1. The calculated C–H stretching vibration appears at 3185 and 3066 cm-1 at the B3LYP/6-311+G(d,p). The calculated C–H stretching vibrations appear at 3213 and 3083 cm1
by the PBE1PBE/6-311+G(d,p).
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As seen from Tables 4, 5 there are many vibrational modes for H–C–C and H–C–H bending vibration. The calculated H–C–C bending vibration appears at 1647 to 1050 cm-1 and 1676 to 1061 cm-1 at the B3LYP/6-311+G(d,p) and the PBE1PBE/6-311+G(d,p) level,
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respectively. The calculated H–C–H bending vibration appears at 1531 to 1429 cm-1 and 1539 to 1430 cm-1 at the B3LYP/6-311+G(d,p) and the PBE1PBE/6-311+G(d,p),
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respectively. Bending vibrations of H–C–P have occurred in 109, 110, 112 and 115 mode numbers (Table 5). 4.2.3. P=O vibrations
The absorption at 1100-1300 cm-1 is assigned to the P=O stretching absorption bands
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[18]. In this structure, the P=O stretching vibration is seen at 1167 cm-1. But the calculated P=O stretching vibration appears at 122, 123, 128 and 130 mode numbers that in 122, 123 mode numbers infrared intensities higher than 128 and 130 mode numbers at the B3LYP/6-
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311+G(d,p) and the PBE1PBE/6-311+G(d,p), respectively which are in good agreement with
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the experimental data.(Tables 4, 5) 4.2.4.P–N vibrations
The P–N stretching absorption of phosphoramidates occurs in the region of 730-
930cm-1[19]. According to the Table 5 the calculated P–N stretching vibration appears at 744 to 897cm-1 using the B3LYP/6-311+G(d,p) and at the PBE1PBE/6-311+G(d,p) level appears at 752 to 911 cm-1 that the most of them overlap with C–C stretching vibrations and C–C–C bending vibrations. Nevertheless, these bands are observed weak in the FT-IR. 4.2.5. Other vibrations
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ACCEPTED MANUSCRIPT In the low wavenumber range (below 600 cm-1) the bending and torsion vibrations are expected to occur. As seen in Table 5 the maximum number of vibrational modes refer to C– C–C bending vibration and C–C–C–C torsion vibration, because the number of carbon atoms is higher than other atoms and any vibrational motion in the molecule results in the transfer of
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vibration to these bonds. According to the Table 5 there are many vibrational modes for C– C–C, P–C–N, O–P–N, C–N–P, and N–P–N bending vibration that often overlap with torsion vibration. However, in the FT-IR spectra, weak bands are observed. Although most of the
4.3. Chemical shift analysis The experimental 1H,
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shown a good way to analyze the structure of molecules.
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FT-IR spectroscopic devices cannot scan a range of fewer than 400 cm-1, But calculation was
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P NMR spectra of MBDPA are presented in Figure
S3-S7.of Supporting Information. The experimental and theoretical chemical shifts of 1H nuclei in MBDPA are listed in Table 6. It is obtained that the 1H chemical shifts (with respect
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to TMS) occur at 2.2767–9.7759 and 0.9032–9.1079 ppm at the B3LYP/6-311+G(d,p) and PBE1PBE/6-311+G(d,p) levels, respectively, whereas the experimental shifts are in the range of 1.22–8.128 ppm. The N–H protons (H50, H52, H54, H56) experimentally resonate at
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1.22–1.27 ppm as a doublet which these chemical shifts are theoretically predicted in the range of 1.8956–2.3284 and 1.2178–2.3352 ppm at the B3LYP/6-311+G(d,p) and
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PBE1PBE/6-311+G(d,p), respectively. The multiplet signal in the range of 2.107–2.203 ppm is assigned to methylene protons between two phosphoryl groups (H70, H71). This chemical shift theoretically occurs at 2.4418–2.6844 and 0.9932–2.2013 ppm at the B3LYP/6311+G(d,p) and PBE1PBE/6-311+G(d,p) levels of theory, respectively. The multiplet signal in the range of 3.928–4.035 ppm is assigned to methylene protons for the benzyl groups (H58, H59, H61, H62, H64, H65, H67, H68). This chemical shift occurs theoretically at 4.2051–5.8304 and 3.305–4.7273 ppm at the B3LYP/6311+G(d,p) and PBE1PBE/6-311+G(d,p) levels of theory, respectively. Twenty protons have 9
ACCEPTED MANUSCRIPT remained, which are related to the aromatic protons. The multiplet at 6.828–8.128 ppm corresponds to the phenyl ring protons that are calculated at 7.8256−9.2759 and 7.082−9.1079 ppm by B3LYP/6-311+G(d,p) and PBE1PBE/6-311+G(d,p), respectively. In the last two rows of Table 6, the two parameters R2 and RMSE are presented. The
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value of the squared correlation coefficients (R2) and root-mean-square error (RMSE) were calculated in MATLAB, version R2010a [9]. The squared correlation coefficients values obtained at the B3LYP/6-311+G(d,p) (0.9707) level are slightly larger than those at the other
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levels. This indicates that for the system under study, the B3LYP/6-311+G(d,p) provides more reliable 1H chemical shifts than the B3LYP/6-311G(d,p) and the two PBE1PBE levels.
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On the other hand, the root-mean-square error (RMSE) values obtained at the PBE1PBE/6-311G(d,p) (0.5010) level are slightly smaller than those at the other levels. The RMSE of the calculated 1H NMR chemical shifts is calculated to be 1.0267, 1.0020 and 0.5581 at the B3LYP/6-311G(d,p), B3LYP/6-311+G(d,p) and PBE1PBE/6-311+G(d,p)
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levels, respectively. However, in the statistical data analyses, the most robust technique among the others that which has the highest values of R2 and the lowest RMSE values.
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In summary, there are three classes of carbons in the MBDPA molecule, including:
methylene carbon between two phosphoryl groups, methylene carbons for the benzyl groups, and phenyl ring carbons. The
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C chemical shifts (with respect to TMS) are assigned in the
range of 42.83−153.19 and 39.08−148.51 ppm by B3LYP/6-311+G(d,p) and PBE1PBE/6311+G(d,p), respectively, while the corresponding experimental data are observed in the range of 88.524−141.09 ppm (Figure 4 and Table 7). The largest deviation between the calculated and experimental
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CNMR chemical shifts (δexp-δcal) are seen for C69, C57, C60,
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311+G(d,p) (0.9767) level are to a small degree larger than those at the other levels. This indicates that for the system under study, the B3LYP/6-311+G(d,p) provides more reliable 13
C chemical shifts than the B3LYP/6-311G(d,p) and PBE1PBE levels. Moreover, the root-
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mean-square error (RMSE) values acquired at the B3LYP/6-311G(d,p) (18.2112) level are slightly smaller than those at the other levels.
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The RMSE of the calculated 1H NMR chemical shifts is calculated to be 0.5010– 1.0267 at the B3LYP, and PBE1PBE levels. Besides, the average absolute standard deviation of
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C chemical shifts is 18.2112–19.5461 at the B3LYP and PBE1PBE levels. This clearly
indicates the larger deviation of
C chemical shifts from the experimental data than that of
H chemical shifts, because in the title compound, the 1H chemical shifts are assigned in the
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range of 1.22–8.128 ppm and the
C chemical shifts are assigned in the range of
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88.524−141.09 ppm.
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P NMR is a predominant method for studying phosphorus-containing compounds.
Chemical shifts in
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PNMR normally depend on the concentration of the taster, the solvent
used, and the presence of other chemical compounds. The reason is that the external standard does not take into account the bulk properties of the sample. It is possible to evaluate and correlate the magnitude and orientation of the chemical shielding anisotropy tensor with differences in: (I) the bond order of the adjacent bonds, (II)
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ACCEPTED MANUSCRIPT the charge on the atom x, (III) the a-b-x bond angles, dihedral angles and (IV) the a-x distances (that "a" is a target nucleus and "x" another atom that influence on "a"). There are two phosphors in MBDPA that appear to have the same chemical environments. Obviously, the 31P NMR spectrum of MBDPA shows only one signal at 22.8407 ppm that is multiplet,
slight difference in the 31P chemical shifts.( Table 8)
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P chemical shifts (with respect to H3PO4) are assigned in the range of
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The
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which was affected by the factors mentioned above. The result of the calculations shows a
10.5737−11.1336 and 7.8582−7.9337 ppm by B3LYP/6-311+G(d,p) and PBE1PBE/6-
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311+G(d,p), respectively, while the corresponding experimental data are observed in 22.8407 ppm (Figure 5 and Table 8) The minor deviation between the calculated and experimental 31P NMR chemical shifts (δexp-δcal) are observed for B3LYP/6-311+G(d,p) calculation method and the major deviation for PBE1PBE/6-311+G(d,p).
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The relationship between the experimental and computed chemical shifts of 1H and 13C NMR
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is shown in Figure 6. As is evident, the correlation between the experimental and calculated chemical shifts is better for
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C atoms than for 1H atoms. This discrepancy is reasonable as
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the 1H chemical shifts more quickly responded to solvent effects [20, 21] As seen in Figure 6, the squared correlation coefficients (R2) values obtained at the B3LYP level are slightly larger than those at the PBE1PBE level. This indicates that for the system under study, the B3LYP provides more reliable
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C chemical shifts than the PBE1PBE. This may be due to
the proper description of the structure of this molecule by the former density functional.
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ACCEPTED MANUSCRIPT 4.4. Frontier molecular orbital analysis The molecular orbital energy diagram for the HOMOs, the LUMOs and bond gap for MBDPA are presented in Figure 7. It is well-known that chemical stability of a molecule is principally affected by the frontier orbitals [22]. The highest occupied molecular orbital
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(HOMO) characterizes electron-donating ability, while the lowest unoccupied molecular orbital (LUMO) represents electron accepting ability. As a result, it is expected that the energy difference between the HOMO and LUMO (HOMO-LUMO energy gap) displays the
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chemical activity of the molecule. The calculated energy gap of the title compound is -5.943 and -6.033 eV at the B3LYP and PBE1PBE levels, respectively. Therefore, a relatively
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smaller amount of kinetic stability is predicted for this molecule at the B3LYP level. Note that the relatively higher energy gap between the HOMO and LUMO of MBDPA at the PBE1PBE level, compared to that at the B3LYP, can be associated with the large destabilization of the LUMO (Figure7).
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Selected atomic charges of MBDPA calculated by natural population analysis (NPA)
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are presented in Figure 8 and Table 9.
All atoms: O2, O3, N49, N51, N53, N55, C57, C60, C63, C66, and C69 are
negatively charged. P1 and P4 and carbon's other atoms are positively charged. The largest value of negative charge is located on the O2 and O3 atoms, ca. -1.096 and -1.101 (-1.089 and-1.106) at the B3LYP/6-311+G(d,p) (PBE1PBE/6-311+G(d,p)) level. On the other hand, the P1 and P4 atoms have the most positive charge 2.204 and 2.198 (2.203 and 2.199) at the B3LYP/6-311+G(d,p) (PBE1PBE/6-311+G(d,p)) level.
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According to Table 9, the calculated dipole moment value of the title compound at the B3LYP is less than PBE1PBE levels. For a given density functional, the addition of diffuse
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functions to the 6-311G(d,p) basis set has a significant influence on the calculated dipole moment value. The interaction between two molecules is expressed via the interaction between atoms. It expresses the interaction energy as the sum of three terms: the columbic or
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electrostatic interaction, the attractive charge-transfer term (due to the mixing of filled orbitals on one molecule with empty orbitals on the others) and the closed-shell repulsion
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[22]. Therefore, in the nucleophilic/electrophilic reactions, dipole moment value ‘charge controlled’ and bond gap ‘orbital controlled’ play an important role in the occurrence of a reaction. 4.5. MEP analysis
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In a molecule, it is very important to identify nucleophilic and electrophilic sites so as to predict its reactivity. Molecular electrostatic potential (MEP) is an advanced method to
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identify the sites for the electrophilic and nucleophilic attack in a molecule [23]. The positive area of the MEP is indicative of a nucleophilic site, and the negative area is associated with
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an electrophilic site. The values of the electrostatic potential at the surface are indicated by various colors: red represents areas of most negative electrostatic potential; green corresponds to areas of approximately zero potential; and blue corresponds to an area of most positive electrostatic potential. The incremental arrangement of potential is red < orange
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ACCEPTED MANUSCRIPT The electrophilic, nucleophilic color range in MBDPA is – 6.333 to 6.333 e-2. One can see that the most negative region(red color) on the MEP map of MBDPA is associated with the lone-pairs of the oxygen atom. This indicates that the oxygen atom of this molecule is the most reactive site to interact favorably with an acidic reagent in the protonation reaction or an
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appropriate ligand for the complexation reaction. On the other hand, the MEP of MBDPA shows the presence of negative regions (yellow color area) around the carbon atoms of phenyl groups, which clearly indicates the propensity of these sites for the formation of
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intermolecular interactions with potential electron-rich sites (e.g. π-π interactions). The corresponding contour map is graphically represented in Figure 9.
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4.6. Anti urease activity
The concentration that induces an inhibition halfway between the minimum and maximum response of compound (relative IC50) was specified to monitor the inhibition effect of various concentrations of MBDPA in the assay. The IC50 value for the title compound is
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5.23nM (0.00523 µM). It is significant that the urease inhibitory activity of the title compound is very superior to that of the acetohydroxamic acid co-assayed as a positive
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5. Conclusion
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reference with the IC50 value of 41.27 ± 0.17 µM. [24]
The main conclusions obtained in this study can be summarized as follows: 1.
The title compound; MBDPA, was synthesized under argon gas and dry solvent via reaction of methylenebis(phosphonic dichloride) and benzylamine. The structure of MBDPA was characterized by FT-IR, 1H, 13C NMR and 31P NMR.
2.
Full geometry optimization of MBDPA, was performed by the B3LYP and PBE1PBE methods with 6-311G(d,p) and 6-311+G(d,p) basis sets. The comparison between the calculated and experimental values indicated that the 15
ACCEPTED MANUSCRIPT B3LYP/6-311+G(d,p) can predict the bond lengths, bond angles and dihedral angles of MBDPA better than the PBE1PBE/6-311+G(d,p) method. This can be expected to be of particular use in inferring complete information which cannot be inferred crystallographically. In addition, it is noted that the calculated frequencies by B3LYP method are all in
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3.
good agreement with the experimental vibrational frequencies. 4.
The result of the comparison between the experimental and computed chemical
calculated chemical shifts is better for
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shifts of 1H and 13C NMR is shown the correlation between the experimental and C atoms than for 1H atoms and the
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squared correlation coefficients (R2) values obtained at the B3LYP level are slightly larger than those at the PBE1PBE level. The results of the study show that for the system under study, the B3LYP provides more reliable 13C chemical shifts than the PBE1PBE.
The molecular electrostatic potential map of MBDPA indicates that the oxygen
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5.
atoms are the most reactive sites to electrophilic attack. 6.
The measurement of urease inhibitory activity was carried out. It is remarkable
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that the urease inhibitory activity of the title compound is very superior to that of
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the acetohydroxamic acid co-assayed as a positive reference.
Acknowledgement
The financial supports of this work was provided by Tarbiat Modares University, Tehran, Iran and by the Research Office of Science and Research Branch, Islamic Azad University, Tehran, Iran
References
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ACCEPTED MANUSCRIPT [23] A. Rajavel, A. Aditya Prasad, T. Jeyakumar, Polymorphs of 4-isopropylbenzylidine thiophene-2carbohydrazide: Crystal growth and density functional theory computations, Journal of Molecular Structure, 1130(2) (2017) 138-149. [24] W.N. Fishbein, P.P. Carbone,Urease Catalysis: II. INHIBITION OF THE ENZYME BY HYDROXYUREA HYDROXYLAMINE, AND ACETOHYDROXAMIC ACID, J Biol Chem. 240(6)
O +
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Cl
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Cl Cl
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N H
CH2Cl2, 273 K
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(1965) 2407-2414.
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Scheme 1. The synthetic pathway for the title compound.
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Fig. 1. The optimized structure and atom numbering of methylenebis(N,N'-dibenzylphosphoramidate)
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Fig. 2. Linear correlations between the experimental and calculated bond lengths of MBDPA
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Fig. 3. The deviation of calculated 1H chemical shifts from the corresponding experimental values
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Fig. 4. The deviation of calculated 13C chemical shifts from the corresponding experimental values
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Fig. 5. The deviation of calculated 31P chemical shifts from the corresponding experimental values
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Fig. 6. Correlation between the experimental and calculated 1H and 13C chemical shifts of MBDPA
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Fig. 7. Molecular orbitals energy(in eV) diagrams of the HOMO and LUMO'S for MBDPA
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Fig. 8. The atomic charges of MBDPA calculated by natural population analysis at the B3LYP/6-311+G(d,p)level. (Hydrogen atoms were omitted for clarity, atomic charge negative, zero and positive are presented with, red < black < green color, respectively )
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Fig. 9. (a,b) Molecular electrostatic potential maps, solid and transparent form. (c) Contour maps of ESP
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Table 1. The selected calculated bond lengths (Å) of title compound at different levels of theory B3LYP/ 6-311+G(d,p) 1.4947 1.6748 1.6812 1.8476 1.4996 1.6708 1.6879 1.8387 1.3967 1.4013 1.5183 1.3957 1.3919 1.392 1.3962 1.4687 1.4912 1.4726 1.4773
3455 w 3424 m 3401 m 3301 s 3262 m 3239 s 3208 s 3193 s 3185 s 3178 s 3170 s 3162 s 3154 s 3147 s 3139 s 3131 s 3124 m 3116 m 3108 m 3100 m 3093 m 3085 m 3077 s 3070 s 3062 s 3054 s 3046 s 3039 s 3023 s 3000 m 2985 m 2969 s 2954 s 2915 s 1658 w 1635 w 1627 w 1619 w 1612 w 1604 w 1596 w 1589 w 1581 w 1573 w 1565 w 1542 w 1511 w 1496 w 1488 w 1481 w 1473 w
1465 s 1457 s 1450 s 1442 s 1434 s 1427 m 1419 m 1411 w 1403 w 1396 w 1388 w 1380 w 1373 w 1365 w 1249 w 1241 w 1234 w 1226 w 1211 m 1203 m 1195 m 1187 s 1180 s 1172 s 1164 s 1157 s 1133 m 1126 m 1118 s 1110 s 1103 s 1095 m 1087 m 1079 m 1072 m 1064 m 1056 1049 w 1033 w
Abbreviations: m, medium; s, strong; w, weak; υ, stretching; δ, bending; τ, torsion; γ, out-of-plane torsion. † Band assignments calculated by PBE1PBE and B3LYP using the VEDA 4 program.
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Table 5. Experimental infrared of MBDPA and the theoretical harmonic frequencies (υ, cm-1), infrared intensities (A), calculated for MBDPA by the B3LYP and PBE1PBE methods with the 6/311+G(d,p) basis set. Exp. IR 1025 w
979 w 971 w 964 w 956 w 941 w 933 w 925 w 918 w 910 w 902 w 837 w 825 w 817 w 809 w 802 w 763 w 756 w 748 w 740 w 734 w 732.w 725 w 717 w 702 w 694 w 686 w 678 w 609 w 601 w 594 w 586 w 578 w 516 w 509 w
Abbreviations: m, medium; s, strong; w, weak; υ, stretching; δ, bending; τ, torsion; γ, out-of-plane torsion. † Band assignments calculated by PBE1PBE and B3LYP using the VEDA 4 program.
H50 H52 H54 H 56 H 70 H 71 H 58 H 59 H 61 H 62 H 64 H 65 H 67 H 68 H 44 H 11 H 33 H 20 H 47 H 36 H 14 H 24 H 48 H 42 H 46 H 37 H 15 H 26 H 13 H 25 H 35 H9 H 22 H 31 R2 RMSE
A novel bisphosphoramidate was synthesized and characterized. Computational studies via B3LYP and PBE1PBE method were used with two different basis sets.
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For biological evaluation, Electrophilic and nucleophilic sites are identified.
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The IC50 value is 5.23nM that was shown strong urease inhibitory activity.
