Synthesis, density functional theory (DFT) studies and urease inhibition activity of chiral benzimidazoles

Abstract

A variety of benzimidazole by the heterocyclization of orthophenylenediamine were synthesized in 69–86% yields. The synthesized compounds 3a-f and 6a-f were characterized and further investigated as jack bean urease inhibitors. Density functional theory (DFT) studies were performed utilizing the basis set B3LYP/6-31G (d, p) to acquire perception into their structural properties. Frontier molecular orbital (FMO) analysis of all compounds 3a–f and 6a-f was computed at the same level of theory to get a notion about their chemical reactivity and stability. The mapping of the molecular electrostatic potential (MEP) over the entire stabilized molecular geometry indicated the reactive centers. They exhibited urease inhibition activity with IC50 between 22 and 99 μM. Compounds containing withdrawing groups on the benzene ring (3d, 6d) were not showing significant urease inhibition. The value obtained for 3a, 3b, 3f had shown their significant urease inhibition for both theoretical and experimental. Notably, the compound having S-configuration (3a) (22.26 ± 6.2 μM) was good as compared to its R enantiomer 3f (31.42 ± 23.3 μM). Despite this, we elaborated the computational studies of the corresponding compounds, to highlight electronic effect which include HOMO, LUMO, Molecular electrostatic potential (MEP) and molecular docking.

Organic chemistry; Pharmaceutical chemistry; Theoretical chemistry, Benzimidazole; Urease inhibition; Density functional theory; HOMO; LUMO; Molecular docking

1. Introduction

Benzimidazoles are well known nitrogen-containing bioactive compounds. They showed a variety of activities including anti-diabetic [1], antimicrobial [2], antifungal [3], antiviral [4], anti-cancer activity [5a,b] and anthelminthic [6]. Recent studies in medicinal chemistry have demonstrated them as potential drug motifs for the pharmaceutical industry [7]. They have also well-known applications in chemo-sensing [8], fluorescence [9], crystal engineering [10], corrosion science [11a,b] and asymmetric catalysis [12]. The bioactivity of drug molecule depends predominantly on their interaction with drug targets, such as proteins, enzymes, nucleic acids and the biological membranes [13a,b]. The efficacy and of the imidazole can be impute to its H-bond donor and acceptor ability as well as its binding affinity with metals [14] (see Figure 1).

Figure 1.

Biologically active drug molecules containing benzimidazoles like omeprazole (used for gastroesophageal diseases), pimobendan (vasodilator), albendazole (parasitic worm infestations), tibendazole, (antifungal agent), lansoprazole (Anti acid) and pantoprazole (proton pump inhibitor) [1, 2, 3, 4, 5, 6, 7].

Urease enzymes belongs to a class of hetero-polymeric enzymes. It is found in excess quantities in beans like soya beans, jack beans, and some other plant seeds [15]. The active site of urease comprises of two nickel(II) atoms (Ni⁺²) [16]. Urease converts urea into NH₃ and carbon dioxide by hydrolysis [17a,b]. It has been reported that ureases are responsible for pathogenicity such as stomach cancer, peptic ulceration, hepatic coma and urinary stones [18]. Besides in agriculture, high urease activity results in serious environmental and commercial losses by releasing abnormally enormous amounts of NH₃ in atmosphere [19a,b]. The detailed mechanism of urease suggested that the nickel atoms are playing key role in their activity [20].

Different compounds have been studied for their urease inhibition response [21]. However, benzimidazoles as jackbean urease inhibitors and their DFT studies have rarely ever found in the literature. In this context, some chiral derivatives of benzimidazoles of benzimidazoles were synthesized, several quantum chemical descriptors in order to interpret various molecular properties such as electronic structure, stability, reactivity, in the interest of determining how they could have an impact on our understanding of the experimental observations and describing various aspects of chemical binding. Finally, they have been tested for their Jack Bean Urease inhibition activity. The comparative inhibitory activity of the most potent enantiomer was also calculated.

2. Experimental

2.1. General

All chemicals for the synthesis of chiral benzimidazole derivatives (3a-f and 6a-f) and Jack Bean Urease were purchased from Sigma Co. Büchi melting point B-540 was used for measurement of melting points while Nicolet iS10 spectrophotometer was used for IR spectra. The NMRs of all compounds were run on an Advance 400 MHz NMR spectrometer in DMSO and CDCl₃ solutions. EIMS were recorded on JEOL MSR mass spectrometer.

2.2. Synthesis

2.2.1. General procedure for synthesis of 3a~f

Equimolar amount (0.0073mol) of o-phenylenediamine (different derivatives) and L-lactic acid were taken in 4N HCl (30.0 ml) and refluxed for 6 h. Whereas, reaction was monitored by thin layer chromatography. Reaction mixture was filtered and neutralized by liquid ammonia (NH₄OH) to get solid product, which is then purified by flash column using 8:2 of eluents n-hexane and ethyl acetate.

2.2.2. General procedure for synthesis of 6a~f

Naproxenoyl chloride (4a-f) was prepared according to given procedure [22]: 5.0 ml of POCl₃ was added to a solution of 0.01 mol naproxen in 30 ml of m-xylene. The reaction mixture was refluxed for 3 h, the settled down and cooled precipitates were filtered off, washed by ether, and on drying gave a colorless crystal (mp 81 °C). Obtained Yield 73 %; ¹H NMR: δ ppm = 1.62 (d, 3H, J = 6.6 Hz, CH₃), 3.84 (q, 1H, J = 6.5, CH), 3.93 (s, 3H, OCH₃), 7.12–7.74 (m, 6H, Ar). ¹³CNMR; 17.53 (CH₃), 46.31(CH), 54.45 (OCH₃), 106.30, 119.00, 126.13, 126.30, 127.11, 129.01, 129.61, 134.04, 135.20, 157.70, 179.31.

Equimolar quantity of naproxenoyl chloride (4a-f) (0.0073 mol) and orthophenylenediame (different derivatives) were taken in 4N HCl (60 ml) were heated underneath reflux for 6 h. Reaction mixture was filtered off and neutralized with NH₄OH to induce the solid compound, which is then purified by silica gel column.

2.2.3. General procedure for separation of enantiomers 3a, 3f and 6a, 6f

Desired chiral benzimidazole was dissolved (10 mg/L) in the solvent system of the mobile phase (n-hexane: iso-propanol, 95/5). Each sample (20 μl) was then injected into HPLC with the mobile phase including the reverse phase (CH₃CN/H₂O) and normal phase (n-hexane/isopropanol). HPLC system comprised of two pumps (an intelligent pump L-200, L-6000 pump), UV-Vis detector L-4200 and an integration system. The chiral stationary phase was LiChroCART 250-4 (S,S)-Whelk-O1 (5 mm) and LiChroCART 250-4 (R,R)-Whelk-O1 (5mm) with (3R,4R)-and (3S,4S)-4-(3,5-dinitrobenzamido)-1,2,3, 4-tetrahydro-phenanthrene as packing materials. The wavelength used for UV-Vis was set at 210 nm while the chromatography was done at room temperature. To separate the racemate, the samples were injected hundreds of times under a given condition. The pure enantiomer was collected, concentrated under a vacuum evaporator to dry and stored [23].

2.2.4. General procedure for urease inhibition studies [24].

The urease inhibitory activity of synthesized chiral benzimidazoles 3a-f and 4a-b was determined by measuring the amount of ammonia produced by the indophenols method described by Weather burn. The reaction mixtures, comprising 20 μL of enzyme (Jack bean urease, 5 U/mL) and 20 μLof test compounds in 50 lL buffer (100 micro molar urea, 0.01 MK₂HPO₄, 1 micro molar EDTA and 0.01 M Lithium dichloride, pH 8.15), were incubated for 10 min, at 37 °C in 96-well plate. Briefly, 40 μL each of phenol reagents (1%, w/v phenol and 0.005%, w/v sodium nitroprusside) and 70 μL of alkali reagent (0.5%, weight by volume NaOH and 0.1% active chloride, sodium hypochloride) were added to each well. The absorbance at 625.0 nm was measured after 30.0 min, using a microplate reader (OPTI Max, Tunable). All these reactions were performed in atriplicate. The urease inhibition activities were calculated according to the following formula:

Urease inhibition activity (%) = (OD_control – OD_sample×100)/OD_control

Where OD_control and OD_sample represents the optical densities in the absence and presence of sample respectively. Thiourea was used as the standard inhibitor for urease inhibition.

2.3. Characterization

2.3.1. (R or S)-1-(1H-benzo[d] imidazol-2-yl)ethanol (3a and 3f)

(85%), m.p 182 °C, Rf: 0.4 (n hexane: ethyl acetate) 3:1; IR: ⱱ: 2942 (=C–H), 2843 (-CH₃), 1615 (C=N), 3500 (O–H), 3305(N–H). ¹HNMR (400 MHz, CDCl₃) δ ppm: 1.52 (s, 3H, CH₃, J = 6.8 Hz), 4.96 (q, 1H, J = 6.8), 7.3 (m, 4H, Ar–H), 12.2 (s, 1H, NH); ¹³CNMR, (75 MHz) δ ppm: 70 (chiral carbon),141.5 (C=N), 138.9, 115. 123 (Ar); HRMS (ESI): m/z calculated for C₉H₉N₂O [M⁺]; 163.0871, found 163.0874 (M⁺ + H).

2.3.2. (±)-1-(6-chloro-1H-benzo[d] imidazol-2-yl) ethanol (3b)

(80%), m.p 233 °C; IR: ⱱ: 3500 (O–H), 1615 (C=N), 3300 5 (NH), 2952 (CH), 2982 (C-H_Ar), 700 (C–Cl). ¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.49 (d, 3H, CH₃), 1.48–1.50 (d, J = 6.8 Hz), 4.92 (q, 1H, CH) 4.91–4.93(J = 6.8), 7.75 (d, 1H, Ar), range (d, J = 8 Hz), 7.61 (s, 1H, Ar), 7.14 (d, 1H, Ar), 7.13–7.45 (d, J = 8 Hz), 12.2 (s, 1H, NH); ¹³CNMR, (75 MHz) δ ppm: 70.4 (chiral carbon),143.9 (C=N imidazole), 134.8-112.5 (Ar); HRMS (ESI): m/z calculated for C₉H₉ClN₂O [M⁺ + H⁺], 197.0482 found 197.0484.

2.3.3. (±)-1-(6-bromo-1H-benzo[d]imidazol-2-yl)ethanol (3c)

(82%), m.p 193 °C; IR: ⱱ: 1615(C=N), 3500 (O–H), 3320(N–H), 1550 (C–Br), 1335 (C–N).¹HNMR (400 MHz, CDCl₃) δ ppm: 1.52 (d, 3H, CH₃), 1.46–1.63 (d, J = 6.8 Hz), 4.99 (q, 1H, CH) 4.95–5.01 (J = 6.8), 7.66 (d, 2H, Ar), range (s, J = 3.2 Hz),8.08 (d, 2H, Ar), range (d, J = 6.0 Hz), 8.38 (s, 1H, Ar), 12.20 (s, 1H, NH), 3.33 (s, 1H, OH); ¹³CNMR, (75 MHz) δ ppm: 23.3(CH₃) 69.8 (chiral carbon),143.5 (C=N imidazole), 134.8-112.5 (Ar); HRMS (ESI): m/z calculated for C₉H₉BrN₂O [M⁺ + Na⁺], 262.9796; found, 262.9799.

2.3.4. (±)-1-(6-nitro-1H-benzo[d] imidazol-2-yl)ethanol (3d)

(78%), m.p 186 °C: IR: ⱱ: 1615 (C=N), 3500 (O–H), 3320(N–H), 1550 (N–O), 1335 (C–N). ¹HNMR (400 MHz, CDCl₃) δ ppm: 1.52 (d, 3H, CH₃), 1.51–1.52 (d, J = 6.8 Hz), 4.99 (q, 1H, CH) 4.97–5.01 (J = 6.8), 7.66 (d, 2H, Ar), range (d, J = 3.2 Hz), 8.08 (d, 2H, Ar–H), range (d, J = 6.0 Hz), 8.38 (s, 1H, Ar–H), 12.20 (s, 1H, NH), 3.33 (s, 1H, OH); ¹³CNMR, (75 MHz) δ ppm: 69.7 (chiral carbon),142.21 (C=N imidazole), 145.0-110.5 (Ar–H); HRMS (ESI): m/z calculated for C₉H₉N₃O3 [M⁺ + NH₄⁺], 225.0988 found 225.0986.

2.3.5. (±)-1-(6-methyl-1H-benzo[d]imidazol-2-yl)ethanol (3e)

(86%), m.p 195 °C; IR: ⱱ: 1615 (C=N), 3500 (O–H), 3320 (N–H), 1335 (C–N). ¹HNMR (400 MHz, DMSO) δ ppm: 1.51 (d, 3H, CH₃), 1.47–1.49 (d, J = 6.8 Hz), 2.5 (s, 3H, CH₃) 4.99 (q, 1H, CH) 4.85–4.93 (J = 6.8), 7.66 (d, 1H, Ar–H), 8.08 (d, 1H, Ar), 8.38 (s, 1H, Ar–H), 12.20 (s, 1H, NH), 3.33 (s, 1H, O–H); ¹³CNMR, (75 MHz) δ ppm: 70 (chiral carbon), 141 (C=N imidazole), 138.8, 115.2 (Ar–H), 23.2 MHz (CH₃), 25 (CH₃); HRMS (ESI): m/z calculated for C₁₀H₁₂N₂O [M⁺ + Na⁺], 199.0847; found, 199.0847 (M⁺ + Na⁺).

2.3.6. (S/R)-1-(2-methoxynaphthalen-6-yl)ethyl)-1-H-benzo[d]imidazole (6a and 6f)

(73%), m.p 212 °C; IR: ⱱ: 2940 (C=C–H), 1615 (C=N), 3320 (N–H), 1325 (C–O), 2980 (C-H_Ar). ¹HNMR (400 MHz, CDCl₃) δ ppm: 1.76 (d, 3H, CH₃), 1.75–1.78 (d, J = 6.8 Hz), 3.87 (s, 3H, OCH₃), 4.48 (q, 1H, CH) 4.95-4.52 (J = 6.8), 7.70 (d, 2H, Ar), range (d, J = 6.2 Hz), 7.26 (d, 2H, Ar), range (d. J = 6.0 Hz), 7.47 (s, 1H, Ar), 7.57 (d, 1H, Ar), range (d, J = 3.2 Hz), 7.04 (d, 1H, Ar), range (d, J = 3.2 Hz), 7.07 (s, 1H, Ar), 7.38 (d, 1H, Ar); ¹³CNMR, (75 MHz) δ ppm: 70 (chiral carbon),141.5 (C=N imidazole), 152.2-115.3 (Ar); HRMS (ESI): m/z calculated for C₂₀H₁₈N₂O [M⁺ + H⁺], 303.1497; found, 303.1499.

2.3.7. (±)-6-chloro-2-((R)-1-(2-methoxynaphthalen-6-yl)ethyl)-1H-benzo[d]imidazol (6b)

(70%), m.p 224 °C; IR: ⱱ: 2980 (C=C–H), 1615 (C=N), 3500 (O–H), 3320(N–H). ¹HNMR (400 MHz, CDCl₃) δ ppm: 3.91 (s, 3H, OCH₃), 1.76 (d, 3H, CH₃), 1.56–1.57(d, J = 6.8 Hz), 4.48 (q, 1H, CH) 1.76–1.79(J = 6.8), 7.70 (d, 1H, Ar), range (d, J = 6.2 Hz), 7.26 (d, 1H, Ar–H), range (d, J = 6.0 Hz), 7.47 (s, 1H, Ar–H), 7.57 (d, 1H, Ar–H), range (d, J = 3.2), 7.04 (d, 1H, Ar–H), range (d, J = 3.2 Hz), 7.07 (s, 1H, Ar–H), 7.38 (d, 1H, Ar–H), range (d, J = 2.0 Hz), 7.35 (d, 1H, Ar–H), range (d, J = 2.0 Hz), 12.3 (s, 1H, NH), 7.67 (s, 1H, Ar–H), 3.7 (s, 3H, OCH₃); ¹³CNMR, (75 MHz) δ ppm: 70MHz (chiral carbon),141.5 (C=N imidazole), 156.2, 105.4 (Ar–H), 130.3 (C–Cl). HRMS (ESI): m/z calculated for C₂₀H₁₇ClN₂O [M⁺ + H⁺], 337.1108; found, 337.1109.

2.3.8. (±)-6-bromo-2-((R)-1-(2-methoxynaphthalen-6-yl)ethyl)-1H-benzo[d]imidazol (6c)

(69%), m.p 202 °C; IR: ⱱ: 2976 (C=C–H), 1614 (C=N), 3500 (O–H), 3323(N–H). ¹HNMR (400 MHz, DMSO) δ ppm: 1.71 (d, 3H, CH₃), 1.55–1.56(d, J = 6.7 Hz), 3.90 (s, 3H, OCH₃), 4.48 (q, 1H, CH) 1.75–1.78(J = 6.7), 7.70 (d, 1H, Ar–H), range (d, J = 6.2 Hz), 7.16 (d, 1H, Ar), range (d, J = 6.0 Hz), 7.32 (s, 1H, Ar), 7.35 (d, 1H, Ar), range (d, J = 3.2), 7.01 (d, 1H, Ar), range (d, J = 3.2 Hz), 7.01 (s, 1H, Ar), 7.29 (d, 1H, Ar), range (d, J = 2.0 Hz), 7.26 (d, 1H, Ar), range (d, J = 2.0 Hz), 12.3 (s, 1H, NH), 7.67 (s, 1H, Ar), 3.3 (s, 3H, OCH₃); ¹³CNMR, (75 MHz) δ ppm: 70MHz (chiral carbon),140.1 (C=N imidazole), 153.2, 105.4 (Ar), 117.5 (C–Br). HRMS (ESI): m/z calculated for C₂₀H₁₇BrN₂O [M⁺ + H⁺], 381.0603; found, 381.0605.

2.3.9. (±)-6-Nitro-2-((R)-1-(2-methoxynaphthalen-6-yl)ethyl)-1H-benzo[d]imidazol (6d)

(69%), m.p 210 °C; IR: ⱱ: 2988 (C=C–H), 1612 (C=N), 3500 (O–H), 3331(N–H). ¹HNMR (400 MHz, CDCl₃) δ ppm: 3.91 (s, 3H, OCH₃), 1.69 (d, 3H, CH₃), 1.55–1.56(d, J = 6.7 Hz), 4.38 (q, 1H, CH) 1.75–1.78(J = 6.7), 7.70 (d, 1H, Ar), range (d, J = 6.2 Hz), 7.16 (d, 1H, Ar), range (d, J = 6.0 Hz), 7.32 (s, 1H, Ar), 7.35 (d, 1H, Ar), range (d, J = 3.2), 7.01 (d, 1H, Ar), range (d, J = 3.2 Hz), 7.01 (s, 1H, Ar), 7.29 (d, 1H, Ar), range (d, J = 2.0 Hz), 7.26 (d, 1H, Ar), range (d, J = 2.0 Hz), 12.3 (s, 1H, NH), 7.67 (s, 1H, Ar), 3.31 (s, 3H, OCH₃); ¹³CNMR, (75 MHz) δ ppm: 70 (chiral carbon),141.1 MHz (C=N imidazole), 154.6, 105.4 (Ar), 144.5 (C–NO₂). HRMS (ESI): m/z calculated for C₂₀H₁₇N₃O₃ [M⁺ + Na⁺], 370.1168; found, 370.1165.

2.3.10. (±)-6-methyl-2-((R)-1-(2-methoxynaphthalen-6-yl)ethyl)-1H-benzo[d] imidazol (6e)

(69%), m.p 212 °C; IR: ⱱ: 2988 (C=C–H), 1612 (C=N), 3500 (O–H), 3331(N–H). ¹HNMR (400 MHz, CDCl₃) δ ppm: 1.69 (d, 3H, CH₃), 1.55–1.56(d, J = 6.5 Hz), 3.91 (s, 3H, OCH₃), 4.38 (q, 1H, CH) 1.75–1.78(J = 6.6), 7.71 (d, 1H, Ar), range (d, J = 6.2 Hz), 7.16 (d, 1H, Ar), range (d, J = 6.1 Hz), 7.32 (s, 1H, Ar), 7.35 (d, 1H, Ar), range (d, J = 3.2), 7.01 (d, 1H, Ar), range (d, J = 3.2 Hz), 7.01 (s, 1H, Ar), 7.29 (d, 1H, Ar), range (d, J = 2.0 Hz), 7.26 (d, 1H, Ar), range (d, J = 2.0 Hz), 12.3 (s, 1H, NH), 7.67 (s, 1H, Ar), 3.31 (s, 3H, OCH₃); ¹³CNMR, (75 MHz) δ ppm: 70 (chiral carbon),141.6 (C=N imidazole), 152.6, 105.4 (Ar), 22.6 (C–CH₃). HRMS (ESI): m/z calculated for C₂₁H₂₀N₂O [M⁺ + H⁺], 317.1654; found, 317.1650.

3. Results and discussions

Numerous synthetic routes have been used for the preparation of benzimidazoles however our recent investigations involving hetrocyclization of orthophenylenediammine are very encouraging [25a,b,c]. Thus in our initial attempt we have synthesized the chiral derivatives of benzimidazole by the hetrocyclization of orthophenylenediammine with lactic acid and secondly, evaluate their urease inhibition studies (see Tables 1 and 2).

Table 1.

Synthesis and urease inhibition studies of benzimidazoles derivatives 3a-3f.

compound	R	% yield	IC50 ± SEM (μM)
3a	H (R config.)	85	22 ± 6.2
3b	Cl	80	38 ± 18.2
3c	Br	82	46 ± 8.3
3d	NO2	78	66 ± 27.4
3e	CH3	86	36 ± 9.2
3f	H (S-config.)	85	31 ± 23.3

Table 2.

Synthesis and urease inhibition studies of benzimidazoles derivatives 6a-6f.

compound	R	% yield	IC50 ± SEM (μM)
6a	H (R config)	73	38 ± 1.3
6b	Cl	70	36 ± 1.7
6c	Br	69	51 ± 9.3
6d	NO2	69	99 ± 9.4
6e	CH3	69	44 ± 2.4
6f	H (S-config.)	73	39 ± 2.4

Chiral benzimidazoles were obtained in a yield range 70–86%. The FT-IR spectrum displayed several characteristic bands round about 3305.08, 3152.47 2982.12, 2952.46, 1605, and 1145 cm⁻¹. More importantly the presence of characteristic peak C=N at 1605-1620 cm⁻¹ has provided a strong evidence of formation imidazole ring of benzimidazole [26a,b,c]. While the remaining observed vibration bands were assigned to (NH) stretching (3305.08), (Ar–H) stretching vibrations (2982.12), (CH) stretching vibrations (2952.46), (C=N) stretching vibration (1605) and (OH) stretching vibration (3400cm⁻¹) [27a,b,c].

In the ¹H NMR and ¹³CNMR we have found the characteristic peaks of benzimidazoles (Figure S4 to S7 supporting information). The most downfield signal around 12.2 ppm for NH, methyl group at 1.51 ppm as a doublet due to ‘AB’ spin system coupling with one neighboring proton, the peaks of aromatic ring ranging from 7.10-7.42 ppm. The molecular masses of the synthesized compounds were determined by their EIMS.

3.1. DFT studies

3.1.1. Geometry optimization

Currently, computational methods attract a lot of attention to the study of the SAR of compounds, and DFT study is one of the most prominent calculation approach due to its accuracy and less time duration. The energy and geometries of 3a-f (Figure 2) and 6a-f (Fig. S1) were optimized at the B3LYP/6-31G (d, p) level of DFT by Gaussian 09 program [28a,b,c]. In addition, investigation like MEP and FMOs carried out on optimized geometries [29].

Figure 2.

Optimized structures of compounds 3a-f at B3LYP/6-31G (d, p); Atom color code: green, chlorine; red, oxygen; gray, carbon; brown, bromine blue, nitrogen; white, hydrogen.

3.1.2. Frontier molecular orbital (FMO) analysis

This method is widely employed to explain the electronic as well as the optical properties of compounds. The main participants during molecular interactions, are the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). These values used to determine the kinetic stability and chemical reactivity of molecules. The energy of LUMO energy level represents electron-accepting abilities while HOMO shows electron donating ability.

We depict the HOMO-LUMO energy diagram (Fig. S2 and Fig. S3) show that electron rich region (red), electron scale region (green). The populations analysis revealed that iso-densities are mainly concentrated on imidazole and aromatic moieties. The HOMO-LUMO energy gaps are summarized in Table 3. This energy gap for 3a, 3f is found to be the maximum and equal to 5.61 eV, while the least band gap (4.48 eV and 3.71 eV,) was determined for 3d, 6d. The iso-density in HOMO of 3a, 3f is mainly spread only to the rings, which reflects less conjugation. This observation indicates that the HOMO-LUMO gap in 3a, 3f should display the highest energy among all products studied, and indeed this was the case.

Table 3.

HOMO and LUMO energies along with HOMO-LUMO energy gap of products 3a-f and 6a-f.

Entry	HOMO (a.u)	LUMO (a.u)	HOMO/LUMO (ΔE/Ev)
3a	-0.21439	-0.00815	5.61
3b	-0.21869	-0.02077	5.38
3c	-0.21706	-0.02119	5.33
3d	-0.24394	-0.07919	4.48
3e	-0.20726	-0.00459	5.51
3f	-0.21438	-0.00814	5.61
6a	-0.20685	-0.04243	4.47
6b	-0.20979	-0.04580	4.55
6c	-0.20984	-0.04580	4.46
6d	-0.21473	-0.07855	3.71
6e	-0.20505	-0.04174	4.44
6f	-0.20663	-0.04241	4.47

In 3e, the density on the HOMO spread over the entire scaffold (including the CH₃ substituent) in the same region as compared to 3a, 3f; therefore, its energy gap is comparable to that of 3a, 3f (i.e., of 5.51 eV). This slightly smaller energy gap in 3e is expected due to the presence of planarity and the electron donating-effect of the methyl group (see Figure 3).

Figure 3.

Frontier molecular orbitals (HOMO-LUMO) of compounds 3a-f at B3LYP/6-31G (d, p) base set level. (FMO for 6a-f are included in figure S2).

3.1.3. Molecular electrostatic potential (MEP)

$$V\left(r\right)=\sum \frac{Z_A}{\left|R_A-r\right|}-\int \frac{\rho \left(r^{\prime }\right)}{\left|r^{\prime }-r\right|}d{r}^{\prime }$$ (1)

Z_A is the nuclear charge present at a distance R_A and ρ(r') representing electron density. The molecular electrostatic potential (MEP) mapping utilize DFT techniques can be beneficial in biochemistry to decide drug-receptor interactions. The MEP is associated chemically active sites of a molecule and additionally improves the perception of molecular reactivity, electrophilic reactions, substituent effects, inter and intra-molecular relations. The increasing electrostatic potential in the following order: red, yellow, blue and green. According to color scale, electron rich (red), slightly electron rich (yellow) which is due to the lone pair of oxygen atom, electron deficient (blue) region of the molecule was surrounded over the hydrogen atoms. Which is due to the electronegative nitrogen atom being attached with the hydrogen atoms. The neutral electrostatic potential (green) envelopes over the π-system of the ring. The electrophilic region of the molecule was predicted around the hydrogen atoms of benzene and imidazole ring. The reactive sites of electrophilic and nucleophilic attacks are investigated for all products 3a~f, 6a~f was computed by MEP the use of B3LYP/6-31G (d, p) at 0.02 a.u is values for the Map surface are proven in Figure 4 and Fig S3. MEP analysis revealed that the negative potential is concentrated on the N atoms on the imidazole rings (3a~f, 6a~f) and oxygen atoms of the NO₂ (3d, 6d) moiety (attached to the benzene ring) and as a result this is the preferred site for electrophilic attack, In addition, the positive charge regions are located on the H atoms on the imidazole (NH group) ring. The negative (-ve) and positive (+ve) potential values of each compound are listed in Table 4.

Figure 4.

Calculated MEP maps of compounds 3a-f at B3LYP/6-31G (d,p). (MEP maps of compounds 6a-f is in figure S3).

Table 4.

Values of -ve and +ve potential of products 3a–f, 6a-f computed at the DFT/B3LYP/6-31G (d,p) level.

Entry	Compound	Negative potential (a.u)	Positive potential (a.u)
1	3a	-0.06386	0.06386
2	3b	-0.06747	0.06747
3	3c	-0.06808	0.06808
4	3d	-0.07353	0.07353
5	3e	-0.06284	0.06284
6	3f	-0.06346	0.06346
7	6a	-0.05524	0.05524
8	6b	-0.05396	0.05396
9	6c	-0.05360	0.05360
10	6d	-0.05997	0.05997
11	6e	-0.05647	0.05647
12	6f	-0.05765	0.05765

From the results in Table 4, it is clear that both potential of all testing compounds are seen as about in a similar range and no noteworthy distinction was seen. The minimal value was observed for 6c (-0.05360 a.u. to 0.05360 a.u), whereas the highest value was observed for 3d (-0.07353 a.u. to 0.07353 a.u).

3.2. Jack bean urease inhibition study

The current work accounts the study of several chiral benzimidazole derivatives (3a~f and 6a~f) as inhibitors of jack bean ureases. All of the benzimidazoles showed variety of urease inhibitory orders.

As the above experimental values of compounds 3a, 3b and 3f revealed that they exhibited good enzyme inhibitory activity with IC₅₀ 0.064 and 0.058l M respectively, while benzimidazoles containing no withdrawing groups on benzene ring are quite effective. The compound 3a (22.26 ± 6.2) acted as good urease inhibitor while the experimental values of 3d (99 ± 19.4) indicates that the withdrawing moiety (NO₂) on benzimidazole decreases its urease inhibition activity while IC₅₀ of reference thiourea is 20.92 M. Another key thing to remember was the structure of acid used in Scheme 1. The smaller acid moiety (Lactic acid) containing benzimidazoles (3a~f) were more effective than the one having bulky acid naproxen (6a~f).

Scheme 1.

General scheme for the synthesis of chiral derivatives of benzimidazoles.

Imidazole ring has central importance in urease inhibition thus any substitution on side phenyl ring of imidazole can affect the urease inhibition activity of benzimidazole. Based on our research we propose that the chiral benzimidazole 3a, 3b and 3f can serve as good competitive inhibitors.

3.3. Molecular docking studies of chiral benzimidazole with urease enzyme (3LA4)

The compounds 3a and 6d along with reference compound thiourea were studied by using docking AutoDock Tools v1.5.6 for protein-ligand docking the virtual screening tool via VINA module. Results are obtained in the form of binding affinity (kcal/mol) which shows the strength of binding interactions between ligand and receptor (Table 5).

Table 5.

Binding affinity of ligands with receptor urease enzyme.

Sr.No.	Ligands	Binding Affinity Kcal/mol
1	3a +3LA4	-4.8
2	6d +3LA4	-8.6
3	Tiourea+3LA4 (standard)	-3.3

As we observed that the order of in-silico and in-vitro results comply with each other. The compound 3a which is the most potent among our synthesized compounds has shown better binding energy (-4.8 Kcal/mol) as compared to least potent compound 6d (-8.6 Kcal/mol) (Figure S8). Docking studies revealed that, Hydrogen bond formations were considered as most important for perfect firm fitting of ligand within the active site of enzyme [30]. In the binding model of the most potent compound 3a, three hydrogen bonds were noticed between residues GLU742, LEU13 and PRO74 with hydroxyl and N–H groups of ligands (Figure 5).

Figure 5.

Protein-ligand Interaction between chiral benzimdazole 3a and active residue of enzyme (3LA4) where Vander Waal forces, hydrogen bondingunfavorable doner-doner and pi alkyl. (Docking for reference thiourea is attached in Figure S9).

4. Conclusion

Chiral derivatives of benzimidazoles have been synthesized to validate their role in jack bean urease inhibition. All of the above synthesized compounds showed significant inhibition. The compound 3a, 3b and 3f were found to be most potent among all above synthesized compounds. Computational studies also enabled us to understand the electronic effects on different substituted chiral benzimidazole. The most responsive sites for nucleophilic and electrophilic attack were predicted by the MEP analysis. On the other hand, FMOs analysis shows that the molecule energy gap for 3a, 3f is found to be the maximum and equal to 5.61 eV, which influences the biological activity of the compound. While the least band gap (4.48 eV and 3.71 eV,) was determined for 3d, 6d were not showing significant urease inhibition, because of its low kinetic stability and high chemical reactivity. In addition, docking studies were performed to elaborate the interaction of ligands with residues of active enzyme which was consistent with experimental results. During this study we have found the effect of chirality on urease inhibition whereas the detailed impact of chirality and synthesis of molecules after docking studies is a project underway.

Declarations

Author contribution statement

Naghmana Rashid: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.

Hasil Aman: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Mohammad Zaman Ashraf: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Nadaraj Sathishkumar: Analyzed and interpreted the data.

Hsin-Tsung Chen: Analyzed and interpreted the data; Wrote the paper.

Aamna Bibi: Contributed reagents, materials, analysis tools or data.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing interest statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Appendix A. Supplementary data

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