Synthesis and exploration of QSAR model of 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one as potential antibacterial agents

Abstract

Present communication deals with the synthesis of novel 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one derivatives under phase transfer catalysis (PTC) conditions using benzyl triethyl ammonium chloride (BTEAC) as PTC. It also elicits the studies on in vitro antimicrobial evaluation of synthesized compounds against a representative genera of gram-negative and gram-positive bacteria i.e., Bacillus subtilis, Staphylococcus aureus, Pseudomonas diminuta and Escherichia coli. All the compounds have been found to manifest profound antimicrobial activity. Moreover, extensive quantitative structure-activity relationship (QSAR) studies have been performed to deduce a correlation between molecular descriptors under consideration and the elicited biological activity. A tri-parametric QSAR model has been generated upon rigorous statistical treatment.

Keywords::

• Phase transfer catalysis
• benzimidazole
• quantitative structure-activity relationship (QSAR)
• antibacterial activity

Introduction

The fight against bacterial infection is one of the great success challenges of medicinal chemistry. Multiple drug-resistant micro-organisms^1,2 are becoming common causes of infections in the acute and long-term care. The emergence of these resistant microorganisms has created a major concern and there exists an urgent need of antibacterial agents in structural classes distinct from known antibacterial agents. A systematic perusal of literature reveals that benzimidazole skeleton has gained a reputation as a drug of privileged sub-structure owing to its extensive use in medicinal chemistry^3–6. Interest in benzimidazole containing structures stems from their widespread occurrence in molecules that display a plethora of useful biological properties. They are characterized by a broad spectrum of activity against roundworms (nematodes), an ovicidal effect, and a wide safety margin. Current clinical example includes astemizole, esomeprazole, albendazole, mebendazole, fenbendazole, oxfendazole, oxibendazole, albendazolesulfonamide, thiabendazole, thiophanate, febantel, nelotrimin, and triclabendazole, carbendazim, fuberidazole^7–12. Nowadays, in view of the affinity displayed by them towards a variety of enzymes and protein receptors, compounds containing benzimidazole sub-structure are being recognized as a drug of choice in the current drug design scenario.

The advent of high throughput screening technologies has impacted significantly on the methodologies that are used for the synthesis of a number of medicinal compounds. The implementation in the laboratory of these synthetic technologies to increase the number of molecules generated by chemists is now a pre-requisite to competitive advantage in the field. Several publications describing synthesis of benzimidazole in solution phase and on solid supports have appeared in the literature^13–17. Thus, keeping in view the ever-increasing demand of new antibiotic agents, it was thought of our interest to design the synthesis of a novel series of benzimidazoles i.e., 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (Figure 1) under phase transfer catalysis (PTC) conditions.

Figure 1.  Structure of synthesized 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r).

Moreover, in order to explore the medicinal potential of these synthesized compounds, comprehensive QSAR studies have been performed to establish a correlation between the various physicochemical parameters and the elicited antibacterial behaviour.

Systematic perusal of literature reveals that a number of methods have been reported for the synthesis of different benzimidazole derivatives under variable experimental conditions^18–22. However, most of the existing methods to design benzimidazole skeleton requires the insertion of a carbon into a precursor with ortho heteroatoms on a benzene ring. Moreover, most of the methods have not been found to be quite accessible from the viewpoints of both yield and economics of the reaction. Thus, in order to cater the needs associated with synthetic aspects, herein, we would like to present unique approach to synthesize benzimidazole derivatives under PTC conditions considering insertion-cyclisation sequence onto azo functionality of the precursor.

Thus an important preparative methodology for the synthesis of 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r) has been delineated incorporating substituted aniline derivatives, urea and ethyl 3-oxobutanoate as the substrates. In this strategy, 1H-benzimidazol-2-amine (1), obtained by the reaction of 1,2-phenylenediamine dihydrochloride with cyanogen bromide, was treated with substituted azo-coupled products i.e., ethyl 3-oxo-2-(phenyldiazenyl)butanoate (2a–r). Furthermore, the obtained precursor compound 2-methyl-3-(phenyldiazenyl) pyrimido[1,2-a] benzimidazol-4(3H)-one (3a–r) was then treated with 3-methylbut-1,2-dien-1-ylidene, generated in situ as per Scheme 1, under PTC conditions to obtain 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazole-4(3H)-one (4a–r) (Figure 1) as the final product (Scheme 2).

Scheme 1.  Generation of 3-methylbuta-1,2-dien-1-ylidene (dimethylvinylidene carbene) intermediate.

Scheme 2.  An overview of synthesis of 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r).

From the viewpoint of mechanistic considerations, the formation of 4a–r could be rationalized by an initial trap of dimethylvinylidene carbene by 2-methyl-3-(phenyldiazenyl)pyrimido[1,2-a] benzimidazol-4(3H)-one at−N=N- to form three membered ring, which is thermally labile compared with the carbocyclic analogue because of much smaller bond dissociation energy²³ of N–N bond than C–C bond. Hence, it is rearranged to 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazole-4(3H)-one (4a–r).

Materials and methods

All the chemicals and other reagents used were of AR grade purity. All the synthesized compounds were characterized by IR, ¹H-NMR, ¹³C NMR, FAB-Mass spectroscopy and elemental analyses. Melting points were determined on an electro-thermal apparatus by open capillary method and are uncorrected. The NMR spectra were recorded on Bruker DRX300 instrument (300 MHz) in CDCl₃ using TMS as an internal standard. Chemical shifts are reported in δ (ppm) values. All IR spectra were run on Shimadzu 460 spectrophotometer in KBr Discs; frequencies are reported in cm⁻¹. FAB mass spectra (FAB-MS) were recorded on a JEOL SX 102 Mass Spectrometer. To ascertain the purity of all the synthesized compounds, analytical thin layer chromatography was performed on (E Merck silica gel G0.50 mm plate no. 5700) using acetonitrile, methanol, and water (50:30:20, v/v) as eluting system. Elemental analyses were performed on Elementar Vario EL-III Carlo-Erba-1108 equipment.

Synthesis of 1H-benzimidazol-2-amine (1²⁴)

A solution of 1,2-phenylenediamine dihydrochloride (0.45 g, 2.5 mmol) in 5 mL of water was cooled to 0°C and treated with a solution of cyanogen bromide (0.60 mL, 5 M in acetonitrile, 3.0 mmol) and solid NaHCO₃ (0.41 mg, 4.9 mmol). The solution was stirred at ambient temperature for 40–45 h. The mixture was made basic with 1 M aqueous Na₂CO₃ and the solution was concentrated under reduced pressure. The residue was triturated with hot ethanol, and the ethanolic solution was filtered and concentrated under reduced pressure to obtain the compound 1 in appreciable yield.

1H-benzimidazol-2-amine (1): Yield 85%; mp 135–136°C; Anal. Calcd. for C₇H₇N₃ (R=H): C, 63.14; H, 5.30; N, 31.56%; Found: C, 63.10; H, 5.28; N, 31.53%; IR (υ cm⁻¹): 3045 (C-H, sp²), 3210 (NH, bonded), 3175 (NH, free), 1654 (C=N), 1626, 1586, 1444 (C…C, ring str) 958, 859, 742 (sub. phenyl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 4.0 (s, 2H, NH₂), 5.0 (s, NH), 7.6–7.9 (m, 4H, Ar-H); ¹³C NMR (CDCl₃) δ (ppm): 117.41, 124.34, 136.66, 158.62; FAB-MS: 134 (M+H)⁺.

General procedure for synthesis of ethyl 3-oxo-2-[(E)-phenyldiazenyl]butanoate (2a-r²⁵)

Different substituted coupled products were synthesized as the precursors taking ethyl 3-oxobutanoate as the active methylene compound. Pertinent aniline derivatives (0.01 M) were dissolved in 4 mL HCl and 4 mL distilled water and kept at freezing temperature. To this, an aqueous solution of sodium nitrite (0.69 g, 0.01 M) in 5 mL of distilled water was added dropwise with continuous stirring, keeping the temperature in the vicinity of 0–5°C. Meanwhile, in another beaker ethyl 3-oxobutanoate (1 mL), sodium acetate (7.0 g) and ethyl alcohol (25 mL) were taken and cooled in an ice bath. Now the diazotized solution (Scheme 2) was added to this solution dropwise with thorough stirring under temperature-controlled conditions. The reaction mixture was kept for overnight period, filtered through suction, washed well with water and dried in vacuum. Fine yellow crystals of ethyl 3-oxo-2-[(E)-phenyldiazenyl]butanoate (2a–r) were obtained.

Ethyl 3-oxo-2-[(E)-phenyldiazenyl]butanoate (2a): Yield 82%; mp 145–146°C; Anal. Calcd. for C₁₂H₁₄N₂O₃: C, 62.11; H, 5.76; N, 19.06%; Found: C, 62.08; H, 5.71; N, 19.01%; IR (υ cm⁻¹): 3050 (C-H, sp²), 2945 (C-H, sp³), 2010 (N=N), 1750 (C=O, ester), 1618, 1580, 1449 (C…C, ring str), 1118 (C-O), 958, 859, 742 (sub. phenyl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.1 (t, 3H, CH₂CH₃, J = 6.5 Hz), 2.0 (s, 3H, CH₃), 5.4 (q, 2H, CH₂CH₃, J = 6.5 Hz), 6.1 (s, CH), 7.2-7.6 (m, 5H, Ar-H); ¹³C NMR (CDCl₃) δ (ppm): 21.24, 27.42, 86.14, 122.62, 125.10, 129.25, 150.20, 172.02; FAB-MS: 235 (M+H)⁺.

General procedure for synthesis of 2-methyl-3-(phenyldiazenyl)pyrimido[1,2-a]benzimidazol-4(3H)-one (3a–r)

An equimolar (0.01 M) quantity of 1H-benzimidazol-2-amine (1) and ethyl 3-oxo-2-[(E)-phenyldiazenyl] butanoate (2a–r) was taken in a 250 cc RB flask and 10 mL freshly prepared sodium ethoxide was added to this. The mixture was heated under reflux condition for 30 min. The solid product thus formed was cooled, dehydrated by anhydrous sodium sulphate, filtered and recrystallized from ethanol. The obtained yield was 70–86%.

2-methyl-3-(phenyldiazenyl) pyrimido[1,2-a] benzimidazol-4(3H)-one (3a): Yield 78%; mp 163–164°C; Anal. Calcd. for C₁₇H₁₃N₅O: C, 67.32; H, 4.32; N, 32.09%; Found: C, 67.26; H, 4.28; N, 23.05%; IR (υ cm⁻¹): 3055 (C-H, sp²), 2940 (C-H, sp³), 2051 (N=N), 1621 (C=C/C=N), 1620, 1532, 1445 (C…C, ring str), 955, 850, 745 (sub. phenyl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 2.4 (s, 3H, CH₃), 5.6 (s, CH), 7.30 -7.61 (m, 9H, Ar-H); ¹³C NMR (CDCl₃) δ (ppm): 20.20, 70.62, 114.20, 115.24, 122.64, 123.25, 125.20, 130.18, 137.02, 139.56, 141.54, 159.22, 165.50; FAB-MS: 304 (M+H)⁺.

General procedure for synthesis of 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r²⁶)

In a 100 mL three-necked bolt-head flask, fitted with a dropping funnel and a mechanical stirrer, a mixture of 50% of aqueous potassium hydroxide (15 mL), benzyltriethylammonium chloride (BTEAC) (0.57 g, 2.5 mmol) and benzene (5 mL) was taken and stirred thoroughly for 30 min. To this, a pertinent 2-methyl-3-(phenyldiazenyl)pyrimido[1,2-a] benzimidazol-4(3H)-one (3a–r) (2.5 mmol) was added slowly and stirred for further 5–7 h under nitrogen atmosphere. While stirring was going on, 3-chloro-3-methyl-1-butyne (25 mmol) (Scheme 1) in benzene (5 mL) was added slowly to the mixture. The contents were diluted with water (120 mL), followed by extraction with ether (120 mL) to afford crude product. It was purified on an alumina column (benzene as eluent) so as to finally obtain 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r). Their purity was further ascertained upon performing TLC resolution on E Merck silica gel-G plates using acetonitrile, methanol, and water (50:30:20, v/v) as eluting system. An overview of all the synthetic steps is depicted in Scheme 2.

2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a): Yield 76%; mp 122–123°C; Anal. Calcd. for C₂₂H₁₉N₅O: C, 71.53; H, 5.18; N, 18.96%; Found: C, 71.50; H, 5.15; N, 18.96%; IR (υ cm⁻¹): 3085 (C-H, sp²), 2952 (C-H, sp³), 1720 (C=O), 1631 (C=C/C=N), 1620, 1522, 1440 (C…C, ring str), 952, 853, 741 (sub. phenyl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.85 (s, 6H, 2 × CH₃, isopropenyl), 2.02 (s, 3H, CH₃), 4.54 (s, CH, pyrimidine), 5.13 (s, CH, methine), 7.27 (m, 4H, H11, H12, H21, H22), 7.66 (m, 4H, H10, H13, H20, H23); ¹³C NMR (CDCl₃) δ (ppm): 17.44 (C24), 20.25 (C28), 24.27 (C27), 65.82 (C3), 110.02, 114.12, 115.28, 123.02, 123.26, 130.74, 134.28, 138.91, 139.24 (aromatic ring), 141.64 (C6, C16), 144.66 (C26), 165.16 (C4), 169.57 (C2); FAB-MS: 370 (M+H)⁺.

2-methyl-3-[4-Chloro-2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4f): Yield 79%; mp 126–127°C; Anal. Calcd. for C₂₂H₁₈N₅ ClO: C, 65.43; H, 4.49; N, 17.34%; Found: C, 65.40; H, 4.46; N, 17.31%; IR (υ cm⁻¹): 3052 (C-H, sp²), 2950 (C-H, sp³), 1725 (C=O), 1621 (C=C/C=N), 1605, 1550, 1440 (C—C, ring str), 952, 840, 755 (sub.phenyl), 6.3 (C-Cl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.82 (s, 6H, 2 × CH₃, isopropenyl), 1.99 (s, 3H, CH₃), 4.62 (s, CH, pyrimidine), 5.82 (s, CH, methine), 7.18 (d, 1H, H21), 7.24 (t, 1H, H22), 7.24 (d, 2H, H11, H12), 7.62 (d, 2H, H10, H13), 7.48 (d, 1H, H23); ¹³C NMR (CDCl₃) δ (ppm): 17.46 (C24), 19.22 (C28), 25.29 (C27), 65.34 (C3), 113.35, 114.13, 115.28, 120.74, 123.07, 124.12, 124.49, 130.75, 138.32, 138.90, 140.37 (aromatic ring), 141.50 (C6, C16), 143.52 (C26), 164.74 (C4), 167.88 (C2); FAB-MS: 405 (M+H)⁺.

2-methyl-3-[4-methyl-2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4i): Yield 84%; mp 122–123°C; Anal. Calcd. for C₂₃H₂₁N₅O: C, 72.04; H, 5.52; N, 18.26%; Found: C, 72.00; H, 5.49; N, 18.22%; IR (υ cm⁻¹): 3062 (C-H, sp²), 2971 (C-H, sp³), 1726 (C=O), 1629 (C=C/C=N), 1612, 1525, 1437 (C…C, ring str), 954, 849, 743 (sub. phenyl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.80 (s, 6H, 2 × CH₃, isopropenyl), 1.95 (s, 3H, CH₃), 2.12 (s, 3H, ArCH₃), 4.52 (s, CH, pyrimidine), 5.75 (s, CH, methine), 7.17 (t, 1H, H22), 7.44 (d, 1H, H23), 7.56 (d, 1H, H21), 7.24 (d, 2H, H11, H12), 7.68 (d, 2H, H10, H13); ¹³C NMR (CDCl₃) δ (ppm): 17.02 (Ar-CH₃), 18.06 (C24), 20.12 (C28), 24.89 (C27), 66.14 (C3), 112.25, 114.13, 115.22, 122.95, 123.04, 124.18, 126.24, 130.77, 138.82, 139.08, 140.37 (aromatic ring), 142.33 (C6, C16), 144.11 (C26), 165.64 (C4), 168.28(C2); FAB-MS, 384 (M+H)⁺.

2-methyl-3-[4-hydroxy-2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4j): Yield 83%; mp 120–121°C; Anal. Calcd. for C₂₂H₁₉N₅O₂: C, 68.56; H, 4.97; N, 18.17%; Found: C, 68.53; H, 4.95; N, 18.15%; IR (υ cm⁻¹): 3345 (O-H), 3056 (C-H, sp²), 2925 (C-H, sp³), 1718 (C=O), 1620 (C=C/C=N), 1595, 1521, 1450 (C…C, ring str), 950, 852, 744 (sub. phenyl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.85 (s, 6H, 2 × CH₃, isopropenyl), 1.97 (s, 3H, CH₃), 4.67 (s, CH, pyrimidine), 5.78 (s, CH, methine), 6.25 (s, 1H, OH), 6.95(d,1H, H21), 7.08 (t, 1H, H22), 7.15 (d, 1H, H23), 7.29 (d, 2H, H11, H12), 7.68 (d, 2H, H10, H13). ¹³C NMR (CDCl₃) δ (ppm): 17.48 (C24), 19.22 (C28), 25.25 (C27), 66.18 (C3), 105.62, 107.84, 121.82, 123.05, 123.18, 114.15, 115.28, 130.74, 138.91, 140.39, 145.08 (aromatic ring), 141.58 (C6, C16), 143.53 (C26), 164.70 (C4), 168.22 (C2); FAB-MS: 386 (M+H)⁺.

2-methyl-3-[6-methyl-2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4k): Yield 76%; mp 121–22°C; Anal. Calcd. for C₂₃H₂₁N₅O: C, 72.04; H, 5.52; N, 18.26%; Found: C, 72.02; H, 5.49; N, 18.25%; IR (υ cm⁻¹): 3068 (C-H, sp²), 2979 (C-H, sp³), 1721 (C=O), 1625 (C=C/C=N), 1615, 1528, 1441 (C…C, ring str), 952, 847, 746 (sub. phenyl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.85 (s, 6H, 2 × CH₃, isopropenyl), 1.98 (s, 3H, CH₃), 2.32 (s, 3H, ArCH₃), 4.64 (s, CH, pyrimidine), 5.83 (s, CH, methine), 7.24 (d, 2H, H11, H12), 7.43 (s, 1H, H23), 7.49 (d, 1H, H21), 7.58 (d, 1H, H21), 7.68 (d, 2H, H10, H13); ¹³C NMR (CDCl₃) δ (ppm): 18.06 (C24), 21.42 (Ar-CH₃), 20.24 (C28), 24.96 (C27), 66.92 (C3), 114.13, 115.12, 115.34, 123.04, 126.04, 130.74, 131.22, 132.84, 138.82, 138.88, 140.37 (aromatic ring), 141.84 (C6, C16), 143.78 (C26), 165.25 (C4), 169.28(C2); FAB-MS: 384 (M+H)⁺.

2-methyl-3-[4-nitro-2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4m): Yield 73%; mp 124–25°C; Anal. Calcd. for C₂₂H₁₉N₆O₃: C, 63.76; H, 4.38; N, 20.28%; Found: C, 63.73; H, 4.35; N, 20.25 %; IR (υ cm⁻¹): 3080 (C-H, sp²), 2959 (C-H, sp³), 1725 (C=O), 1625 (C=C/C=N), 1612, 1520, 1440 (C…C, ring str), 1331 (NO₂) 952, 855, 745 (sub. phenyl); ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.80(s, 6H, 2 × CH₃, isopropenyl), 1.94 (s, 3H, CH₃), 4.64 (s, CH, pyrimidine), 5.23 (s, CH, methine), 7.23 (d, 2H, H11, H12), 7.45 (t, 1H, H22), 7.62 (d, 2H, H10,H13), 7.95 (d, 1H, H23), 8.18(d,1H, H21); ¹³C NMR (CDCl₃) δ (ppm): 17.43 (C24), 19.28 (C28), 25.27 (C27), 64.88 (C3), 114.23, 115.26, 118.65, 121.32, 123.02, 123.95, 130.75, 133.08, 137.06, 138.90, 139.87 (aromatic ring), 141.53 (C6, C16), 143.53 (C26), 164.66 (C4), 166.28 (C2); FAB-MS: 415 (M+H)⁺.

In vitro antibacterial assay

Broth micro-dilution assay

The newly synthesized compounds were screened for their antibacterial activity against Pseudomonas diminuta, Escherichia coli, Bacillus subtilis and Staphylococcus aureus using broth micro-dilution method. Approximately 10⁹ cells of respective bacterial cells were inoculated in 10 mL Mueller-Hinton broth. Each synthesized compound (200 µg) was added and mixed well by shaking at 200 rpm and the plates are incubated at 37°C for 48–72 h. After every 6-h interval, 1 mL of cell culture were withdrawn and subjected to serial dilution (10⁻¹–10⁻⁶) and spread on nutrient agar plates and the plates were incubated at 37^°C for 24 h. The bacterial colonies were counted by colony form unit (CFU) in each dilution to find out the antibacterial activity of the tested compounds.

The MIC (µg/mL) of a compound was determined according to the lowest concentration that inhibited visible growth of bacteria after incubation at 37°C for 24 h. IC₅₀ (µg/mL) represents that inhibitory concentration of the compound, at which the 50% inhibition of the growth of bacteria occurred in vitro. IC₅₀ is also converted to the pIC₅₀ scale (-log IC₅₀), in which higher values indicate exponentially greater potency. Mueller-Hinton broth was used as the test medium. Each experiment in the antibacterial assays was replicated twice in order to define the MIC and IC₅₀ values. Ampicillin was used as standard antibacterial agent.

Results and discussion

In order to screen these synthesized benzimidazoles as more potent lead compounds, QSAR study was performed on substituted 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r) derivatives. The structure of more active member of the series is shown in Figure 2. Fifty percent inhibitory concentration [IC₅₀] of the substituted 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r) was calculated and converted to the negative logarithmic scale (pIC₅₀) (Table 1). A number of physicochemical parameters viz., Connolly accessible area (CAA), Connolly molecular area (CMA), molar refractivity (MR), dipole-dipole energy (DDENE), non-1,4 VDW energy (NVDW), VDW 1,4 energy (VDW) were calculated by the Chem office 6.0 using Chem. 3D Pro package²⁷. Geometry optimization was done by PM3 method incorporated in MOPAC²⁸ server of the ChemOffice 6.0 program. Single point energies calculation for the lead compounds was done by RHF (Restricted Hartree-Fock: closed shell) wave function. HyperChem. Release 8.0.3 Molecular Modeling System²⁹ was used for the calculation of van der Waal surface volume (WVOL), van der Waal surface area (WSA), surface area grid (SAG), hydrophobicity (logP), molecular refractivity (REF), molecular polarizability (P), and molecular orbital energies (HOMO and LUMO). Initial energy minimizations were done by employing molecular mechanics methods. These initially optimized geometries were optimized by PM3 method employing Fletcher-Reeves (conjugate gradient) algorithm with a termination RMS gradient of 0.0001 kcal/(Å mol).

Table 1.  The in vitro antimicrobial inhibition activities of 2-methyl-3-[2-(2-methylprop-1-en-1-yl)-1H-benzimidazol-1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one (4a–r).

S. No.	Entry	R	pIC50
			P. diminuta	B. subtilis	E. Coli	S. aureus
1	4a	-H	−3.0934	−3.2574	−3.1746	−3.3222
2	4b	4-OC2H5	−1.1761	−1.3617	−1.2553	−1.4472
3	4c	6-C2H5	−1.5563	−1.7243	−1.6532	−1.7634
4	4d	6-NO2	−2.1303	−2.2900	−2.2041	−2.3617
5	4e	4-OCH3	−2.2304	−2.3802	−2.3222	−2.4698
6	4f	4-Cl	−2.4232	−2.5843	−2.5119	−2.6532
7	4g	6-OH	−3.1761	−3.3426	−3.2565	−3.4108
8	4h	4-COOH	−2.4624	−2.6021	−2.5441	−2.6902
9	4i	4-CH3	−2.5315	−2.6928	−2.6021	−2.7559
10	4j	4-OH	−3.1303	−3.3118	−3.2041	−3.3598
11	4k	6-CH3	−2.5740	−2.7380	−2.6628	−2.8162
12	4l	6-OCH3	−2.3324	−2.4843	−2.4393	−2.5563
13	4m	4-NO2	−1.9542	−2.1139	−2.0607	−2.1903
14	4n	4-C2H5	−1.3424	−1.5441	−1.4472	−1.6232
15	4o	4-Br	−1.7634	−1.9294	−1.8751	−2.0253
16	4p	5-CH3	−2.6021	−2.7076	−2.6284	−2.8062
17	4q	5-Cl	−2.5575	−2.6857	−2.5911	−2.7324
18	4r	5-OCH3	−2.3617	−2.4698	−2.3802	−2.5441
19	A	--	−3.2175	−3.3802	−3.2742	−3.4150

A, Ampicilline.

Figure 2.  PM3 optimized geometry of more active 2-methyl-3-[2-(2-methylprop-1-en-1-yl)- 1H-benzimidazol -1-yl]pyrimido[1,2-a]benzimidazol-4(3H)-one.

All the statistical analyses were carried out by the computer program Valstat³⁰. Correlation analysis between molecular descriptors (calculated by ChemOffice and HyperChem packages), and biological activity was performed. The dataset were divided into test set and training set containing three and fifteen compounds, respectively. All possible combinations of parameters were considered for the QSAR study. Multiple regression analysis was carried out using in vitro antimicrobial inhibition activities as the dependent variable and all molecular descriptors as independent variables in all possible combinations. The statistical quality of the regression equations was justified on the basis of significant statistical parameters viz., percentage of explained variance (% EV), variance ratio (F), and standard error of estimate (SEE). All the final equations have regression coefficients, intercepts and variance ratio (F) significant to more than 95% confidence level. Use of more than one variable in the multivariate equation was justified by an autocorrelation study. The predictive powers of the equations were validated by the leave one out (LOO) cross validation method. Predicted residual sum of square (PRESS), total sum of squares (SSY) and cross-validated r² (r²_CV) were considered for the validation of the models.

Tri-parametric QSAR model for activity against P. diminuta

1

n = 18, F = 273.089, PRESS = 0.311, SSY = 5.406, PRESS/SSY = 0.058, r²_CV = 0.942, % EV = 93.805, SEE = 0.144, r²_bsp = 0.989, Bootstrapping std = 0.006, Chance < 0.001, Q² = 0.967, S_PRESS = 0.121, SDEP = 0.104

Contribution of parameters to model is MR: CMA: DDENE:: 85.306: 12.958: 1.000. In the above equation % EV, F, SEE, PRESS, SSY, r²_CV, r²_bsp, S_PRESS are percentage of explained variance, ratio between the variances of observed and calculated activities, standard error of estimate, predicted residual sum of squares, total sum of squares, cross validated r², Bootstrapping r² and standard deviation error of prediction, respectively.

Equation (1) explains up to 93.805% of the variation of the activities. However, MR is the key factor to control biological activity, which can be attributed to steric interaction occurring in polar species. It is related to molar volume (V) and the refractivity index (η) according to the relation:

which reveals that MR is a measure of bulk due to its relation with molar volume. However, it also contains a polarizability component η. Hence, the positive coefficient of MR corresponds to higher activity of these compounds. On the other hand, negative coefficient of the CMA signifies the importance of Connolly molecular area in the activity. Lower value of this index corresponds to higher activity of these compounds.

Further, PRESS is a cross-validation parameter whose value less than SSY (sum of the squares of response value) points out that the model predicts better than Chance and can be considered statistically important. To be a reasonable QSAR model, PRESS/SSY ratio should be smaller than 0.4 and its value smaller than 0.11 indicates an excellent model. The data indicate that for all the proposed models, this ratio is < 0.1 suggesting all of them to be excellent models. Furthermore, the value of cross-validated correlation coefficient (r²_CV) and Bootstrapping r² are further supporting the predictive power of these explaining models.

Moreover, the compounds have also been screened against E. coli, B. subtilis and S. aureus bacteria and the tri-parametric QSAR models are presented in the equation 2, 3, and 4, respectively.

2

n = 18, F = 295.238, PRESS = 0.057, SSY = 4.670, PRESS/SSY = 0.012, r²_CV = 0.988, % EV = 98.679, SEE = 0.062, r²_bsp = 0.989, Bootstrapping std = 0.007, Chance < 0.001, Q² = 0.971, S_PRESS = 0.111, SDEP = 0.095

Contribution of parameters to model is MR: CMA: DDENE:: 83.414: 11.981: 1.000.

3

n = 18, F = 327.158, PRESS = 0.052, SSY = 4.677, PRESS/SSY = 0.011, r²_CV = 0.989, % EV = 98.801, SEE = 0.059, r²_bsp = 0.990, Bootstrapping std = 0.006, Chance < 0.001, Q² = 0.969, S_PRESS = 0.115, SDEP = 0.099

Contribution of parameters to model is MR: CMA: DDENE:: 85.306: 12.958: 1.000.

4

n = 18, F = 282.885, PRESS = 0.054, SSY = 4.630, PRESS/SSY = 0.012, r²_CV = 0.988, % EV = 98.736, SEE = 0.060, r²_bsp = 0.990, Bootstrapping std = 0.006, Chance < 0.001, Q² = 0.964, S_PRESS = 0.123, SDEP = 0.106

Contribution of parameters to model is MR: CMA: DDENE:: 89.055: 12.034: 1.000.

In all the above models, MR term has significant contribution as supported by statistics of the analysis. Thus, it is revealed from all the proposed models deduced through equations 1-4 that the MR is the main governing factor. Moreover, increase in the percentage contribution of CMA also signifies the importance of Connolly molecular area (CMA).

Conclusion

A variety of benzimidazole derivatives have been successfully synthesized in appreciable yields and screened in vitro for their antimicrobial activities against both strains of gram-positive and gram-negative bacteria. Moreover, quantitative structure-activity relationship studies have been performed to explore their inherent antimicrobial potential. All the synthesized compounds have shown excellent antimicrobial activities. It is evident from the developed QSAR model that MR, CMA, and DDENE parameters were the main governing physicochemical factors for the displayed antimicrobial activities. Such a QSAR evaluation would open up future perspectives to use these compounds as new lead compounds in drug discovery processes.

Declaration of interest

We are thankful to the Defence Research Development Organization (DRDO), New Delhi (India), and Central Drugs Research Institute (CDRI), Lucknow (India), for providing financial assistance and spectro-analytical facilities, respectively.