Design, Synthesis and Microbiological Evaluation of Novel Compounds as Potential Staphylococcus aureus Phenylalanine tRNA Synthetase Inhibitors

Abstract

AS THE RESISTANCE of Staphylococcus aureus to antibiotics represents a major threat to global health, anti-infectives with novel mechanisms must be developed. Novel compounds were generated as potential phenylalanine tRNA synthetase (PheRS) inhibitors based on the published homology model of S. aureus PheRS to aid the design process using Molecular Operating Environment (MOE) software. PheRS was selected as it is structurally unique enzyme among the aminoacyl-tRNA synthetases (aaRS), it is considerably different from human cytosolic and human mitochondrial aaRS and it is essential and conserved across bacterial species. The designed compounds were synthesized according to different clear schemes. The compounds were confirmed by ¹H NMR, ¹³C NMR, HRMS and/or microanalysis, and they were microbiologically evaluated.

Introduction

Staphylococcus aureus (S. aureus) commonly colonizes human skin and mucosa without causing any infections. However, if there is an opportunity for the bacteria to enter the body, through broken skin or a medical procedure, they can cause illnesses which range from mild to life-threatening infections. As they include skin and wound infections, infected eczema, abscesses or joint infections, infections of the heart valves (endocarditis), pneumonia and bacteraemia (blood stream infection). These severe infections acquired either in health-care facilities or in the community [1]. Certain strains of S. aureus developed resistance known as methicillin resistant Staphylococcus aureus (MRSA). At present, less than 90% of S. aureus strains are resistant to most penicillin derivatives [2] and ordinary antimicrobial agents like drugs from the family of aminoglycosides, macrolides, chloramphenicols, tetracyclines and fluoroquinolones so known as multidrug resistant Staphylococcus aureus [3].

Increased resistance of MRSA to anti-infective drugs is a threat to global health; so anti-infectives with novel mechanisms must be developed. Our potential target in the drug development for the treatment of MRSA infections is phenylalanine tRNA synthetase which is considered as the most complex and large enzyme of aminoacyl-tRNA synthetases (aaRSs).

Aminoacyl-tRNA synthetases (aaRSs) (also known as aminoacyl-tRNA ligases) are essential enzymes for protein biosynthesis, playing a crucial role in the genetic code translation [4,5]. AaRSs catalyze the attachment of an amino acid to its cognate tRNA molecule in a two-step reaction. Firstly, cognate amino acids react with ATP forming aminoacyl-adenylate, through a covalent linkage between the 5′-phosphate group of ATP and the carboxyl end of the amino acid. Secondly, the activated forms of the amino acids are subsequently attached to 2′-OH or 3′-OH of the evolutionarily invariant 3′-adenosine terminus of the cognate tRNA molecule by esterification. The resulting aminoacyl-tRNA acts as a substrate for protein biosynthesis which occurs on ribosomes [4]. The aaRSs are categorized into two classes according to the structural features of the enzymes. Class I enzymes contain a Rossman fold in the catalytic core and two conserved motifs, called HIGH and KMSKS. Class II enzymes have an antiparallel β-sheet with three conserved motifs in the catalytic centre. To date only one drug, mupirocin, which inhibits a specific type of aaRS (IleRS), has been licensed as a topical antibiotic for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) [6].

Phenylalanine tRNA synthetase (PheRS) is a unique enzyme of the aaRS family, as it is an (αβ)₂ heterotetrameric enzyme composed of two small alpha subunits and two larger beta subunits. According to the structure, PheRS is classified as a class II aaRS as its catalytic domain is built around antiparallel β-sheet but functionally it resembles class I because it aminoacylates the 2′ OH of the terminal ribose of tRNA where class II aminoacylate the 3′ OH [7,8]. The natural substrate (phenylalanyl-adenylate) was considered as a template for the design of novel potential compounds against S. aureus PheRS (Fig. 1).

Fig. 1.

Structures of the natural substrate phenylalanyl-adenylate.

Experimental

Chemistry

All employed reagents and solvents were of general purpose or analytical grade and purchased from Fluka, Acros, Alfa-Aeser chemicals and Sigma-Aldrich Chemical Company. Solvents were dried over molecular sieves (4 Å). Flash column chromatography was performed with silica gel 60 (Merck 40–60 nm, 230–400 mesh) and Thin layer chromatography (TLC) was performed on precoated silica gel plates (Merck Kiesegel 60 F₂₅₄) with visualization by UV light (254 nm) and/or vanillin stains. Melting points were determined using Gallenkamp as an electrothermal instrument and they are uncorrected. ¹H and ¹³C-NMR spectra were recorded on a Bruker Advance DPX500 spectrometer operating at 500 MHz and 125 MHz, respectively. Accurate mass spectroscopic analysis was performed at the EPSRC National Mass Spectrometry Centre (Swansea, UK) and at Medac Ltd., Chobham Business Centre, Surrey, UK. Elemental analysis was performed at Medac Ltd., Chobham Business Centre, Chertsey Road, Surrey, UK.

General method for the synthesis of methyl 3-(1H-benzimidazol-1-yl)propanoate (4a) and methyl 3-(1H-indol-1-yl)propanoate (4b) [9]

To a stirred solution of methylacrylate (3) (3 eq.) and DBU (1 eq.) in acetonitrile (1.2 mL/mmol) benzimidazole (1) or indole (2) (2 eq.) was added. The reaction mixture stirred at room temperature for 6 h in case of benzimidazole and heated at 50 °C for 6 h in case of indole. The solvent was evaporated under reduced pressure.

Methyl 3-(1H-benzimidazol-1-yl)propanoate (4a) (C₁₁H₁₂N₂O₂ M.wt 204.23)

The product was purified by flash column chromatography using dichloromethane: methanol, the product was collected at 98: 2 % v/v. Yield: 8.56 g (99 %) as a yellow oil.

TLC

10 % methanol in dichloromethane, R_f = 0.61

¹H NMR (CDCl₃) δ

8.03 (s, 1H, CH-imidazole), 7.83 (t, J = 7.4 Hz, 1H, Ar), 7.42 (t, J = 7.6 Hz, 1H, Ar), 7.32 (m, 2H, CH-Ar), 4.52 (t, J = 6.5 Hz, 2H, CH₂), 3.67 (s, 3H, CH₃), 2.89 (t, J = 6.3 Hz, 2H, CH₂).

¹³C NMR (CDCl₃) δ

171.04 (C=O), 143.51 (C), 143.27 (CH-imidazole), 133.30 (C), 123.27 (CH-Ar), 122.41 (CH-Ar), 120.44 (CH-Ar), 109.34 (CH-Ar), 52.13 (CH₃), 40.38 (CH₂), 34.17 (CH₂).

Methyl 3-(1H-indol-1-yl)propanoate (4b) (C₁₂H₁₃NO, M.wt 203.24)

The product was purified by flash column chromatography using n-hexane: ethylacetate, the product was collected at 90 : 10 % v/v. Yield: 6.94 g (99.9 %) as a yellow oil.

TLC

hexane: ethyl acetate, 6: 1, v/v, R_f = 0.44

¹H NMR (CDCl₃) δ

7.85 (d, J = 7.9 Hz, 1H, Ar), 7.50 (d, J = 8.2 Hz, 1H, Ar), 7.44 (t, J = 7.1 Hz, 1H, Ar), 7.37 (t, J = 7.5 Hz, 1H, Ar), 7.27 (d, J = 3.2 Hz, 1H, Ar), 6.70 (d, J = 3.1 Hz, 1H, Ar), 4.52 (t, J = 6.9 Hz, 2H, CH₂), 3.79 (s, 3H, CH₃), 2.90 (t, J = 6.9 Hz, 2H, CH₂).

¹³C NMR (CDCl₃) δ

171.78 (C=O), 135.87 (C), 128.95 (C), 128.13 (CH-Ar), 121.83 (CH-Ar), 121.25 (CH-Ar), 119.72 (CH-Ar), 109.34 (CH-Ar), 101.78 (CH-Ar), 51.96 (CH₃), 41.88 (CH₂), 34.83 (CH₂).

General method for the synthesis of 3-(1H-Benzimidazol-1-yl)propane hydrazide [10,11] (5a) and 3-(1H-Indol-1-yl)propane hydrazide [12] (5b)

To a stirred solution of methyl 3-(1H-benzimidazol-1-yl)propanoate (4a) or methyl 3-(1H-indol-1-yl)propanoate (4b) (1 eq.) in methanol (1 mL/mmol), hydrazine monohydrate (5 eq.) was added. The reaction mixture was stirred for 3h at room temperature, then evaporation of the solvent under vacuum and co-evaporation with diethyl ether to afford solid product. The product was purified by recrystallization form aqueous ethanol.

3-(1H-Benzimidazol-1-yl)propane hydrazide [10,11] (5a) (C₁₀H₁₂N₄O, M.wt 204.23)

Yield

4.2 g (82 %) as yellowish crystals. [Lit. ¹⁰ 66 %, Lit. ¹¹ 91 % as a white solid].

Melting Point (°C)

116–118 [Lit. ¹⁰ 264–266 °C],

TLC

10 % methanol in dichloromethane, R_f = 0.57

¹H NMR (DMSO-d₆) δ

9.08 (br s, 1H, NH, D₂O-exchangeable), 8.13 (s, 1H, CH-imidazole), 7.65 (d, J = 8.0 Hz, 1H, Ar), 7.60 (d, J = 8.0 Hz, 1H, Ar), 7.26 (t, J = 7.2 Hz, 1H, Ar), 7.20 (t, J = 7.2 Hz, 1H, Ar), 4.48 (t, J = 6.6 Hz, 2H, CH₂), 3.93 (br s, 2H, NH₂, D₂O-exchangeable), 2.61 (t, J = 6.7 Hz, 2H, CH₂).

¹³C NMR (DMSO-d₆) δ

169.33 (C=O), 144.50 (CH-imidazole), 143.83 (C), 134.08 (C), 122.73 (CH-Ar), 121.92 (CH-Ar), 119.85 (CH-Ar), 110.87 (CH-Ar), 44.49 (CH₂), 32.12 (CH₂).

3-(1H-Indol-1-yl)propane hydrazide [12] (5b) (C₁₁H₁₃N₃O, M.wt 203.25)

Yield

3.5 g (85 %) as pale-yellow crystals.

Melting Point (°C)

94–96

TLC

10 % methanol in dichloromethane, R_f = 0.65

¹H NMR (DMSO-d₆) δ

9.04 (s, 1H, NH, D₂O-exchangeable), 7.53 (d, J = 7.9 Hz, 1H, Ar), 7.47 (d, J = 8.3 Hz, 1H, Ar), 7.29 (t, J = 2.6 Hz, 1H, Ar), 7.14 (t, J = 7.4 Hz, 1H, Ar), 7.02 (t, J = 7.4 Hz, 1H, Ar), 6.41 (d, J = 2.3 Hz, 1H, Ar), 4.41 (t, J = 6.8 Hz, 2H, CH₂), 4.15 (s, 2H, NH₂, D₂O-exchangeable), 2.54 (t, J = 6.8 Hz, 2H, CH₂).

¹³C NMR (DMSO-d₆) δ

169.63 (C=O), 135.95 (C), 129.19 (CH-Ar), 128.59 (C), 121.48 (CH-Ar), 121.40 (CH-Ar), 119.42 (CH-Ar), 110.20 (CH-Ar), 101.78 (CH-Ar), 41.84 (CH₂), 34.78 (CH₂).

Synthesis of 2-(3-(1H-benzimidazol-1-yl) propanoyl) hydrazine-1-carbothioamide (6a) and 2-(3-(1H-indol-1-yl)propanoyl)hydrazine-1 - carbothioamide (6b)

A solution of potassium thiocyanate (15.13 mmol) in the least amount of distilled water (2 mL), HCl (1.5 mL) was added dropwise, followed by slow addition of a methanolic solution of 3-(substituted)propane hydrazide (5a, b) (10.05 mmol). The reaction mixture was stirred at room temperature overnight. The resulting yellow solid was collected by filtration and washed several times with water. The product was used in the next step without further identification or purification.

Synthesis of 3-(Substituted)-N′-(4-(4-substituted phenyl)thiazol-2-yl)propane hydrazide (8a-c)

Equimolarsolutions of2-(3-(1H-benzimidazol-1-yl)propanoyl)hydrazine-1-carbothioamide (6a) or 2-(3-(1H-indol-1-yl)propanoyl)hydrazine-1-carbothioamide (6b) and appropriate 2-bromo-4′-substituted acetophenone (7a) or (7b) in absolute ethanol (20 mL/mmol) was heated under reflux overnight. The solvent was evaporated under vacuum. The product was purified by flash column chromatography, followed by preparative TLC for final purification.

3-(1H-benzimidazol-1-yl)-N’-(4-(4-chlorophenyl)thiazol-2-yl) propane hydrazide (8a) (C₁₉fl₁₆ClN₅OS, M.wt 397.88)

Synthesized using 2-bromo-4′-chloroacetophenone (7a) (0.195 g, 0.835 mmol). The product was purified by flash column chromatography using dichloromethane: methanol, the product was collected at 93: 7 % v/v, followed by re-crystallization from ethanol then preparative TLC for final purification using 90 % dichloromethane: 10 % methanol. Yield: 125 mg (38 %) as a brown solid.

TLC

10 % methanol in dichloromethane, R_f = 0.75

¹H NMR (DMSO-d₆) δ

10.28 (s, 1H, NH, D₂O-exchangeable), 9.50 (s, 1H, NH, D₂O-exchangeable), 8.15 (s, 1H, CH-imidazole), 7.82 (d, J = 8.2 Hz, 2H, Ar), 7.66 (t, J = 9.0 Hz, 2H, Ar), 7.44 (d, J = 8.2 Hz, 2H, Ar), 7.24 (m, 3H, 2 × Ar and CH-thiazole), 4.56 (t, J = 6.5 Hz, 2H, CH₂), 2.80 (t, J = 6.5 Hz, 2H, CH₂).

¹³C NMR (DMSO-d₆) δ

172.66 (C=O), 170.26 (C), 149.76 (C), 144.57 (CH-imidazole), 143.93 (C), 134.07 (C), 133.94 (C), 133.41 (C), 132.33 (2 × CH-Ar), 129.08 (2 × CH-Ar), 121.97 (CH-Ar), 121.87 (CH-Ar), 119.90 (CH-Ar), 110.92 (CH-Ar), 104.35 (CH-thiazole), 40.59 (CH₂), 33.93 (CH₂).

3-(1H-Benzimidazol-1-yl)-N’-(4-(4-cyanophenyl)thiazol-2-yl)propane hydrazide (8b) (C₂₀H₁₆N₆OS, M.wt 388.45)

Synthesized using 2-bromo-4′-cyanoacetophenone (7b) (0.43 g, 1.89 mmol). The product was purified by flash column chromatography using dichloromethane: methanol, the product was collected at 95 : 5 % v/v, followed by preparative TLC for final purification using 90 % dichloromethane : 10 % methanol. Yield: 153 mg (21 %) as a yellow solid.

TLC

10 % methanol in dichloromethane, R_f = 0.55

¹H NMR (DMSO-d₆) δ

10.32 (s, 1H, NH, D₂O-exchangeable), 9.60 (s, 1H, NH, D₂O-exchangeable), 8.15 (s, 1H, CH-imidazole), 7.97 (d, J = 8.4 Hz, 2H, Ar), 7.83 (d, J = 8.4 Hz, 2H, Ar), 7.66 (dd, J = 8.0, 13.3 Hz, 2H, Ar), 7.51 (s, 1H, CH-thiazole), 7.25 (m, 2H, Ar), 4.55 (t, J = 6.3 Hz, 2H, CH₂), 2.81 (t, J = 6.2 Hz, 2H, CH₂).

¹³C NMR (DMSO-d₆) δ

172.83 (C=O), 170.29 (C), 149.27 (C), 144.57 (CH-imidazole), 143.94 (C), 139.15 (C), 134.08 (C), 133.10 (2 × CH-Ar), 126.64 (2 × CH-Ar), 121.98 (CH-Ar), 121.87 (CH-Ar), 119.91 (CH-Ar), 119.46 (CN), 110.92 (CH-Ar), 110.02 (C), 107.40 (CH-thiazole), 40.59 (CH₂), 33.92 (CH₂).

N’-(4-(4-Cyanophenyl)thiazol-2-yl)-3-(1H-indol-1-yl)propanehydarzide (8c) (C₂₁H₁₇N₅OS, M.wt 387.46)

Synthesized using 2-bromo-4′-cyanoacetophenone (7b) (0.26 g, 1.14 mmol). The product was purified by flash column chromatography using petroleum ether: ethylacetate, the product was collected at 40: 60 % v/v. Yield: 172 mg (39 %) as a yellow solid.

TLC

petroleum ether : ethyl acetate 1: 4, v/v, R_f = 0.66

¹H NMR (DMSO-d₆) δ

10.29 (s, 1H, NH, D₂O-exchangeable), 9.59 (s, 1H, NH, D₂O- exchangeable), 8.00 (d, J = 8.2 Hz, 2H, Ar), 7.85 (d, J = 8.2 Hz, 2H, Ar), 7.57 (d, J = 7.5 Hz, 2H, Ar), 7.52 (s, 1H, CH-thiazole), 7.50 (d, J = 7.5 Hz, 2H, Ar), 7.33 (s, 1H, Ar), 7.15 (t, J = 7.2 Hz, 1H, Ar), 7.03 (t, J = 7.2 Hz, 1H, Ar), 6.43 (s, 1H, Ar), 4.47 (t, J = 6.6 Hz, 2H, CH₂), 2.72 (t, J = 6.6 Hz, 2H, CH₂).

¹³C NMR (DMSO-d₆) δ

172.89 (C=O), 170.57 (C), 149.25 (C), 139.15 (C), 135.95 (C), 133.11 (2 × CH-Ar), 129.04 (CH-Ar), 128.67 (C), 126.63 (2 × CH-Ar), 121.42 (CH-Ar), 120.90 (CH-Ar), 119.51 (CH-Ar), 119.46 (CN), 110.22 (CH-Ar), 110.06 (C), 107.41 (CH-thiazole), 101.25 (CH-Ar), 41.31 (CH₂), 34.53 (CH₂).

Synthesis of 5-(2-(1H-benzimidazol-1-yl)ethyl)-1,3,4-thiadiazol-2-amine (9) (C₁₁H₁₁N₅S, M.wt 245.07)

Potassium thiocyanate (1.07 g, 11.02 mmol) was dissolved in the least amount of water (2 mL) then hydrochloric acid (1 mL) was added dropwise. The aforementioned mixture was added to a methanolic solution of 3-(1H-benzimidazol-1-yl)propane hydrazide (5a) (1.5 g, 7.34 mmol). The reaction mixture was stirred overnight at room temperature, followed by solvent evaporation under vacuum. The resulting solid was added portionwise to H₂SO₄ (5 mL) with continuous stirring. The reaction mixture was stirred for 2 h, then slowly poured into crushed ice with stirring and neutralized with ammonia solution. The resulting pale brown solid was collected by filtration under vacuum. The product was pure enough to proceed to further reaction. Yield: 1.5 g (88 %) as a pale-brown solid.

Melting Point (°C)

182–184

TLC

10 % methanol in dichloromethane, R_f = 0.37

¹H NMR (DMSO-d₆) δ

8.16 (s, 1H, CH- imidazole), 7.63 (t, J = 7.5 Hz, 2H, Ar), 7.26 (t, J = 7.3 Hz, 1H, Ar), 7.21 (t, J = 7.5 Hz, 1H, Ar), 7.3 (s, 2H, NH₂, D₂O-exchangeable), 4.60 (t, J = 6.7 Hz, 2H, CH₂), 3.40 (t, J = 6.7 Hz, 2H, CH₂).

¹³C NMR (DMSO-d₆) δ

169.15 (C), 154.69 (C), 144.61 (CH-imidazole), 143.89 (C), 134.12 (C), 122.81 (CH-Ar), 121.99 (CH-Ar), 119.92 (CH-Ar), 110.90 (CH-Ar), 43.68 (CH₂), 30.32 (CH₂)

[ESI-HRMS]

Calculated mass: 246.0726 [M+H]⁺, Measured mass: 246.0720 [M+H]⁺.

General method for the synthesis of N-(5-(2-(1H-benzimidazol-1-yl)ethyl)-1,3,4-thiadiazol-2-yl)-substituted benzamide (11a, b)

To a solution of 5-(2-(1H-benzimidazol-1-yl)ethyl)-1,3,4-thiadiazol-2-amine (9) (1 eq.) in dry dichloromethane (10 mL/mmol), triethylamine (10 eq.) was added. The reaction mixture was cooled to 0 °C, followed by addition of 3,5-dimethoxybenzoyl chloride (10a) or 4-fluorobenzoyl chloride (10b) (1.1 eq.) in dry dichloromethane (10 mL) dropwise over 30 min. Then, the reaction mixture was stirred at room temperature overnight. Solvent was evaporated under pressure and the resulting solid was extracted with dichloromethane (50 mL/mmol) and saturated aqueous sodium bicarbonate (3 × 25 mL/mmol). The organic layer was dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure. The product was purified by flash column chromatography using gradient elution of dichloromethane: methanol, the product was collected at 96: 4 % v/v.

N-(5-(2-(1H-benzimidazol-1-yl)ethyl)-1,3,4-thiadiazol-2-yl)-3,5-dimethoxybenzamide (11a) (C₂₀H₁₉N₅O₃S, M.wt 409.46)

Yield

0.15 g (56 %) as a white solid.

Melting Point (°C)

200–202

TLC

10 % methanol in dichloromethane, R_f = 0.6

¹H NMR (DMSO-d₆) δ

12.95 (s, 1H, NH, D₂O-exchangeable), 8.18 (s, 1H, CH-imidazole), 7.66 (t, J = 8.3 Hz, 2H, Ar), 7.25 (m, 4H, Ar), 6.76 (s, 1H, Ar), 4.72 (t, J = 6.8 Hz, 2H, CH₂), 3.82 (s, 6H, 2 × CH₃), 3.62 (t, J = 6.8 Hz, 2H, CH₂).

13C NMR (DMSO-d6) δ

165.10 (C=O), 161.11 (C), 160.94 (2 × C), 160.17 (C), 144.57 (C), 143.90 (CH-imidazole), 134.33 (C), 133.83 (C), 129.11 (CH-Ar), 122.84 (CH-Ar), 122.04 (CH-Ar), 119.95 (CH-Ar), 110.91 (CH-Ar), 106.47 (CH-Ar), 105.79 (CH-Ar), 55.06 (CH₃), 43.76 (CH₂), 29.94 (CH₂).

[ESI-HRMS]

Calculated mass: 410.1281 [M+H]⁺, Measured mass: 410.1278 [M+H]⁺.

N-(5-(2-(1H-benzimidazol-1-yl)ethyl)-1.3.4-thiadiazol-2-yl)-4fluorobenzamide (11b) (C₁₈H₁₄FN₅OS, M.wt 367.40)

Yield

0.4 g (45 %) as a yellow solid.

Melting Point (°C)

210–212

TLC

10 % methanol in dichloromethane, R_f = 0.8

¹H NMR (DMSO-d₆) δ

13.03 (s, 1H, NH, D₂O-exchangeable), 8.17 (t, J = 7.5 Hz, 3H, 2 Ar and CH-imidazole), 8.01 (d, J = 8.8 Hz, 1H, Ar), 7.66 (d, J = 8.0 Hz, 1H, Ar), 7.27 (m, 4H, Ar), 4.72 (t, J = 6.8 Hz, 2H, CH2), 3.62 (t, J = 6.8 Hz, 2H, CH₂).

¹³C NMR (DMSO-d₆) δ

166.84 (C=O), 166.38 (C), 164.39 (C), 161.07 (C), 143.90 (CH- imidazole), 143.88 (C), 134.07 (C), 132.61 (CHAr), 132.54 (CH-Ar), 128.67 (C), 122.84 (CH-Ar), 122.04 (CH-Ar), 119.94 (CH-Ar), 116.18 (CH-Ar), 116.00 (CH-Ar), 110.91 (CH-Ar), 43.73 (CH₂), 29.96 (CH₂).

[ESI-HRMS]

Calculated mass: 368.0976 [M+H]⁺, Measured mass: 368.0977 [M+H]⁺.

General procedures for the synthesis of 5-(3-(1H-Benzimidazol-1-yl)propyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (14) and 5-(3-(6-Amino-9H-purin-9-yl)propyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (15)

To a suspension of 2-(4-(1H-benzimidazol-1-yl)butanoyl)-N-phenylhydrazine-1-carbothioamide (12) or 2-(4-(6-amino-9H-purin-9-yl)butanoyl)-N-phenylhydrazine-1-carbothioamide (13) (1 eq.) in ethanol (15 mL/mmol) was added 2N aqueous NaOH (5 mL/mmol) dropwise with continuous stirring. The reaction mixture was stirred at room temperature for 5 h. Evaporation of ethanol under reduced pressure. The solution was neutralized by the addition of HCl dropwise until the formation of a white precipitate. The precipitate was collected by filtration under vacuum.

5-(3-(1H-Benzimidazol-1-yl)propyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (14) (C₁₈H₁₇N₅S, M.wt 335.43)

The product was purified by flash column chromatography using dichloromethane: methanol, the product was collected at 96: 4 % v/v. Yield: 0.204 g (72 %) as a white solid.

Melting Point (°C)

98–100

TLC

10 % methanol in dichloromethane, R_f = 0.52

¹H NMR (DMSO-d₆) δ

13.61 (br s, 1H, NH, D₂O-exchangeable), 8.04 (s, 1H, CH-imidazole), 7.92 (d, J = 6.9 Hz, 1H, Ar), 7.48 (d, J = 6.5 Hz, 3H, Ar), 7.31 (3H, J = 6.7 Hz, Ar), 7.20 (t, J = 3.0 Hz, Ar), 4.30 (t, J = 6.7 Hz, 2H, CH₂), 2.48 (t, J = 6.9 Hz, 2H, CH₂), 2.21 (quin., J = 6.9 Hz, 2H, CH2).

¹³C NMR (DMSO-d₆) δ

168.90 (C), 151.07 (C), 142.98 (CH-imidazole), 144.00 (C), 133.47 (C), 133.06 (C), 130.22 (2 × CH-Ar), 130.01 (2 × CH-Ar), 127.65 (CH-Ar), 123.26 (CH-Ar), 122.50 (CH-Ar), 120.59 (CH-Ar), 109.44 (CH-Ar), 43.43 (CH₂), 25.58 (CH₂), 22.79 (CH₂).

[ESI-HRMS]

Calculated mass: 336.1277 [M+H]⁺, Measured mass: 336.1284 [M+H]⁺.

5-(3-(6-Amino-9H-purin-9-yl)propyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (15) (C₁₆H₁₆N₈S, M.wt 352.42)

The product was purified by re-crystallization from aqueous ethanol. Yield: 2.3 g (97 %) as white crystals.

Melting Point (°C)

280–282

TLC

10 % methanol in dichloromethane, R_f = 0.4

¹H NMR (DMSO-d₆)δ

13.73 (s, 1H, NH, D₂O-exchangeable), 8.09 (s, 1H, CH-imidazole), 8.03 (s, 1H, CH-pyrimidine), 7.49 (d, J = 6.5 Hz, 3H, Ar), 7.35 (d, J = 7.6 Hz, 2H, Ar), 7.17 (s, 2H, NH₂, D₂O-exchangeable), 4.15 (t, J = 6.6 Hz, 2H, CH₂), 2.41 (t, J = 7.3 Hz, 2H, CH₂), 2.06 (quin., J = 6.8 Hz, 2H, CH₂).

¹³C NMR (DMSO-d₆) δ

172.91 (C), 161.15 (C), 157.56 (CH-pyrimidine), 156.48 (C), 154.68 (C), 141.33 (CH-imidazole), 138.76 (C), 134.60 (2 × CH-Ar), 134.51 (2 × CH-Ar), 133.35 (CH-Ar), 123.94 (C), 47.13 (CH₂), 30.68 (CH₂), 27.77 (CH₂).

[ESI-HRMS]

Calculated mass: 353.1291 [M+H]⁺, Measured mass: 353.1291 [M+H]⁺.

General procedure for the synthesis of 1-(3-(4-phenyl-5-(substituted thio)-4H-1,2,4-triazol-3-yl)propyl)1H-benzimidazole (16a,b).

To a mixture of 5-(3-(1H-benzimidazol-1-yl) propyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (14) (1 eq.) and anhydrous potassium carbonate (1.5 eq.) in dry DMF (10 mL/0.5 mmol) iodoethane or 1-iodopropane (1 eq.) in dry DMF (5 mL/0.5 mmol) was added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum and the residue was dissolved in ethyl acetate (100 mL/0.5 mmol) and washed with water (3 × 50 mL/0.5 mmol). The organic layer was dried over anhydrous MgSO₄ and concentrated under vacuum.

1-(3-(5-(Ethylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)propyl)-1H-benzimidazole (16a) (C₂₀H₂₁N₅S, M.wt 363.48)

The product was purified by flash column chromatography using gradient elution of dichloromethane: methanol, the product was collected at 94: 6 % v/v. Yield: 0.2 g (84 %) as a yellowish solid.

Melting Point (°C)

118–120

TLC

10 % methanol in dichloromethane, R_f = 0.6

¹H NMR (CDCl₃) δ

7.85 (s, 1H, CH-imidazole), 7.78 (d, J = 6.9 Hz, 1H, Ar), 7.48: 7.42 (m, 3H, Ar), 7.29 (m, 3H, Ar), 7.04 (d, J = 7.1 Hz, 1H, Ar), 4.41 (t, J = 6.7 Hz, 2H, CH₂), 3.23 (q, J = 7.3 Hz, 2H, CH₂), 2.48 (t, J = 6.9 Hz, 2H, CH₂), 2.29 (quin., J = 6.6 Hz, 2H, CH₂), 1.41 (t, J = 7.3 Hz, 3H, CH₃).

¹³C NMR (CDCl₃) δ

154.13 (C), 151.90 (C), 143.86 (C), 143.04 (CH-imidazole), 133.73 (C), 132.90 (C), 130.09 (2 × CH-Ar), 130.02 (2 × CH-Ar), 126.82 (CH-Ar), 122.93 (CH-Ar), 122.10 (CH-Ar), 120.39 (CH-Ar), 109.68 (CH-Ar), 43.40 (CH₂), 26.88 (CH₂), 21.79 (CH₂), 14.77 (CH₃).

Microanalysis

Theoretical: %C: 66.09, %H: 5.82, %N: 19.26, Found: %C: 65.64, %H: 5.74, %N: 19.09.

1-(3-(4-Phenyl-5-(propylthio)-4H-1,2,4-triazol-3-yl)propyl)1H-benzimidazole (16b) (C₂₁H₂₃N₅S, M.wt: 377.51)

The product was purified by flash column chromatography using gradient elution of dichloromethane: methanol, the product was collected at 97: 3 % v/v. Yield: 0.27 g (80 %) as a yellow oil.

TLC

10 % methanol in dichloromethane, R_f = 0.6

¹H NMR (CDCl₃) δ

7.86 (s, 1H, CH-imidazole), 7.76 (d, 1H, J = 6.7, Ar), 7.41: 7.47 (m, 3H, Ar), 7.29 (d, J = 7.6, 1H, Ar), 7.22: 7.26 (m, 3H, Ar), 7.02 (d, J = 7.0, 2H, Ar), 4.38 (t, J = 6.7, 3H, CH₂), 3.17 (t, J= 7.3, 3H, CH₂), 2.47 (t, J = 6.7, 3H, CH₂), 2.27 (quin., J = 6.6 Hz, 2H, CH₂), 1.75 (m, 2H, CH₂), 0.98 (t, J = 7.2, 3H, CH₃)

¹³C NMR (CDCl₃) δ

154.12 (C), 152.12 (C), 143.69 (C), 143.02 (CH-imidazole), 133.69 (C), 132.88 (C), 130.08 (2 × CH-Ar), 130.01 (2 × CH-Ar), 126.82 (CH-Ar), 122.96 (CH-Ar), 122.14 (CH-Ar), 120.29 (CH-Ar), 109.72 (CH-Ar), 43.43 (-CH₂), 34.42 (CH₂), 26.30 (CH₂), 22.75 (CH₂), 21.79 (CH₂), 13.24 (CH3).

[ESI-HRMS]

Calculated mass: 378.1747 [M+H]⁺, Measured mass: 378.1749 [M+H]⁺.

General procedure for the synthesis of 9-(3-(4-phenyl-5-(substituted thio)-4H-1,2,4-triazol-3-yl)propyl)-9H-purin-6-amine (17a,b)

To a mixture of 5-(3-(6-amino-9H-purin-9-yl) propyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole- 3-thione (15) (1 eq.) and anhydrous potassium carbonate (1.5 eq.) in dry DMF (10 mL/0.5 mmol) iodoethane or 1-iodopropane (1 eq.) in dry DMF (5 mL/0.5 mmol) was added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum and the residue was dissolved in ethyl acetate (100 mL/0.5 mmol) and washed with water (3 × 50 mL/0.5 mmol). The organic layer was dried over anhydrous MgSO₄ and concentrated under vacuum.

9-(3-(5-(Ethylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)propyl)-9H-purin-6-amine (17a) (C₁₈H₂₀N₈S, MW 380.47)

The product was purified by flash column chromatography using gradient elution of dichloromethane: methanol, the product was collected at 92: 8 % v/v. Yield: 0.6 g (86 %) as yellow crystals.

Melting Point (°C)

180–182°C

TLC

10% methanol in dichloromethane, R_f = 0.3

¹H NMR (DMSO-d₆) δ

8.09 (s, 1H, CH-pyrimidine), 8.04 (s, 1H, CH-imidazole), 7.51 (m, 3H, Ar), 7.35 (m, 2H, Ar), 7.18 (s, 2H, NH₂), 4.17 (t, J = 6.8, 2H, CH₂), 3.04 (q, J = 7.4 Hz, 2H, CH₂), 2.52 (masked by DMSO peak, CH₂), 2.12 (quin., J = 6.7 Hz, 2H, CH₂), 1.26 (t, J = 7.4, 3H, CH₃).

¹³C NMR (DMSO-d₆) δ

156.40 (C), 154.97 (C), 152.78 (CH-Ar), 150.25 (C), 149.93 (C), 141.20 (CH-Ar), 133.46 (C), 130.28 (2 × CH-Ar), 130.22 (2 × CH-Ar),127.65 (CH-Ar), 119.23 (C), 42.62 (CH₂), 27.03 (CH₂), 26.85 (CH₂), 22.36 (CH₂), 15.228 (CH₃).

Microanalysis

Theoretical: %C: 56.82, %H: 5.30, %N: 29.44, Found: %C: 56.33, %H: 5.22, %N: 28.99.

9-(3-(4-Phenyl-5-(propylthio)-4H-1,2,4-triazol-3-yl)propyl)-9H-purin-6-amine (17b) (C₁₉H₂₂N₈S M.wt 394.50)

The product was purified by flash column chromatography using gradient elution of dichloromethane: methanol, the product was collected at 94: 6 % v/v. Yield: 0.64 g (88 %) as white crystals.

Melting Point (°C)

174–178

TLC

10 % methanol in dichloromethane, R_f = 0.58

¹H NMR (DMSO-d₆) δ

8.10 (s, 1H, CH-pyrimidine), 8.05 (s, 1H, CH-imidazole), 7.51 (m, 3H, Ar), 7.35 (m, 2H, Ar), 7.20 (s, 2H, NH₂), 4.17 (t, J = 6.7, 2H, CH₂), 3.02 (t, J = 6.9, 2H,² CH₂), 2.52 (t, J = 7.6, 2H, CH₂), 2.12 (quin., J = 6.6 Hz, 2H, CH₂), 1.62 (m, 2H, CH₂), 0.90 (t, J = 7.3, 3H, CH₃).

¹³C NMR (DMSO-d₆) δ

156.38 (C), 154.96 (C), 152.76 (CH-Ar), 150.38 (C), 149.92 (C), 141.21 (CH-Ar), 133.47 (C), 130.28 (2 × CH-Ar), 130.22 (2 × CH-Ar), 127.65 (CH-Ar), 119.22 (C), 42.62 (CH₂), 34.54 (CH₂), 26.85 (CH₂), 22.79 (CH₂), 22.36 (CH₂), 13.35 (CH₃).

Microanalysis

Theoretical: %C: 57.85, %H: 5.62, %N: 28.40, Found: %C: 57.46, %H: 5.58, %N: 28.29.

Molecular modeling

Docking studies were performed using MOE software [13] utilizing the homology model for S. aureus phenylalanine tRNA synthetase enzyme having phenylalanyl adenylate as a ligand [14]. Ligands were built using MOE and then the energy was minimized for each ligand, creating a ligand database. All minimizations were performed with MOE to a RMSD gradient of 0.01 Kcal/mol/Å with MMFF94 forcefield, and partial charges were automatically calculated. The ligands were docked using the MOE default setting: Placement: Triangular Matcher, Rescoring 1: London ΔG, 30 poses were constructed for each compound and the best scoring model-ligand complexes were selected. The consequent ligand interactions within the constructed model were visualized using the MOE ligand interaction simulation.

Microbiological evaluation

The synthesized compounds were evaluated for MIC against S. aureus SH1000 sensitive strain according to CSLI guidelines 2012 [15].

Moreover, compounds 11b, 16a, 17a and 17b were evaluated for their antimicrobial activity alongside comparator agent ampicillin (Amp) against a variety of clinically important pathogens. Isolates tested included clinical and NCTC/ATCC control organisms; S. aureus (including methicillin, tetracycline, erythromycin/clindamycin and vancomycin resistance), Klebsiella pneumoniae (including 3^rd generation cephalosporin and carbapenem resistance), Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis, Acinetobacter baumannii, Enterococcus faecalis and faecium (including vancomycin resistance) and E. coli. Minimum Inhibitory Concentrations (MICs) were determined using microbroth dilution, the “gold standard”, international standard ISO 10776–1[16].

P aeruginosa PheRS and S. pneumonia PheRS enzymes assay

The inhibitory activity (IC₅₀) of the final compounds was determined using the tRNA aminoacylation assay adapted to a scintillation proximity assay (SPA) [17]. Test compounds were dissolved in 100% DMSO to a concentration of 3.3 mM. To determine IC₅₀ values the test compounds (2 μl) were serially diluted across 10 wells on the assay plates resulting in final assay concentrations ranging from 200 μM to 0.4 μM. Briefly, the compounds were equilibrated by the addition of 33 μL of the protein/substrate mix: 50 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 1.25 mM ATP, 1 mM spermine, 1 mM DTT, 100 μM [³H]Phe (75 cpm/pmol), and 0.08 μM P aeruginosa PheRS or 0.2 μM S. pneumonia PheRS. Control reactions contained only DMSO with no compound. This mixture was incubated at ambient temperature for 15 min and then reactions were started by the addition of 15 μl E. coli tRNA (80 μM total tRNA or 2 μM tRNA^Phe), followed by incubation for 1 h at 37 °C. Reactions were stopped by the addition of 5 μl of 0.5 M EDTA. 400 μg of yttrium silicate (Ysi) poly-L-ly sine coated SPA beads (Perkin-Elmer) in 150 μl of 300 mM citric acid were added and allowed to incubate at room temperature for 1 h. The plates were analyzed using a 1450 Microbeta (Jet) liquid scintillation/luminescent counter (Wallac). The curve fits and IC₅₀ values were determined using the Sigmoidal Dose-Response Model in XLfit 5.3 (IDBS).

Results and Discussion

Chemistry

The sequence of the reactions followed in the preparation of the designed compounds is summarized in Schemes 1–3.

Scheme 1.

Reagents and conditions: (i) DBU, CH₃CN, rt or 50 °C, 6h (ii) hydrazine monohydrate, MeOH, rt, 3 h (iii) KSCN, Conc. HCl, MeOH, rt, overnight (iv) EtOH, reflux, overnight.

Scheme 3.

Reagents and conditions: (i) (a) 2N NaOH, 5 h, rt, (b) Conc. HCl (ii) Alkyl halide, anhyd. K2CO3, DMF, rt, overnight.

The synthesis of methyl esters (4a) and (4b) were carried out via an aza-Michael addition reaction. Hydrazinolysis of the methyl ester compounds (4a,b) produced hydrazides [18] (5a,b) which were confirmed by the disappearance of the CH₃ peak from both ¹H NMR and ¹³C NMR spectra. ¹H NMR spectrum showed two singlet signals at ~ 9.0 ppm and ~ 4.0 ppm for the protons of NH and NH₂ groups, respectively. Several attempts were investigated to convert (4a, b) directly to (6a, b) without the hydrazinolysis step according to literature [19,20]. These trials involved refluxing the ester compounds 4a, b with thiosemicarbazide in the presence of either acetone, EtOH with few drops of AcOH or EtOH with a few drops of dimethylsulfoxide (DMSO). However, all attempts were unsuccessful, based on ¹H NMR analysis. Therefore, an alternative pathway was taken, based on the reaction of the hydrazides (5a, b) with KSCN through nucleophilic addition reaction [21]. Compounds 8a-c having 1,3-thiazole ring were achieved by the reaction of 6a or 6b and appropriate 2-bromo-4′-substituted acetophenone (7a) or (7b) in EtOH under reflux overnight [22,23]. After column chromatography purification, it was found that ¹H NMR showed a few impurities. So, compound 8a was further purified by re-crystallization. However, the compound changed color during the heating of the re-crystallization process, and TLC showed several spots, indicating that the compound is heat sensitive. Preparative TLC was utilized for final purification of compound 8a. For compounds 8b and 8c, fast column chromatography was done to avoid any decomposition in the desired compounds, then purified with preparative TLC. ¹H and ¹³C NMR confirmed the structures with the singlet CH-thiazole peak at approximately 7.5 ppm in the ¹H NMR. The yields of this reaction were very low. Mass spectroscopy or microanalysis was not conducted for the structures of the prepared compounds owing to the instability (Scheme 1).

5-(2-(1H-benzimidazol-1-yl)ethyl)-1,3,4-thiadiazol-2-amine (9) was achieved by the reaction of a methanolic solution of 3-(1H-benzimidazol-1-yl)propane hydrazide (5a) with KSCN in acidic solution through a nucleophilic addition reaction [24,25]. The acidic condition afforded the cyclization of the 1,3,4-thiadiazole ring. ¹H NMR spectrum showed downfield singlet signal at 7.03 ppm corresponding to the two protons of the primary amino group and more carbon atom at 154.69 ppm appeared in ¹³C NMR spectrum. The synthesis of 11a, b was achieved through the nucleophilic substitution reaction of 9 with benzoyl chloride derivatives (10a) or (10b) in CH₂Cl₂/Et₃N. The low product yield was presumably owing to the reduced nucleophilicity of the primary amino group (Scheme 2).

Scheme 2.

Reagents and conditions: (i) KSCN, HCl, MeOH, rt, overnight (ii) Conc. H₂SO₄, 2h, aqueous NH (iii) CH₂C_l2, Et₃N, 0 °C - rt, overnight.

Thiosemicarbazide (12, 13) cyclization in alkaline medium resulted in the formation of 1,2,4-triazoles (14, 15). This is because N-4, in alkaline medium, is more nucleophilic than the sulfur of the thiocarbonyl group and oxygen of carbonyl group producing 1,2,4-trizoles [26–28]. Treatment of 14 or 15 with potassium carbonate as a base and an appropriate alkyl halide gave the desired thioethers (16 and 17) in very good yields (Scheme 3).

Molecular modeling evaluation

These series were developed to include either adenine or a biarylmimic (benzimidazole or indole) to represent the adenyl moiety of phenylalanyl adenylate (Table 1). The ‘adenyl’ portion was linked, through a 3–5 atom linker that spans the hydrophobic channel, to a heterocyclic 5-membered ring having either thiol or nitrogen or both to make H-bonds with the key binding amino acid residues (His172, Ser174, Gln214 and/or Glu216), and finally the remainder of the compound, which may be aliphatic or aromatic, to fill the large hydrophobic pocket and may contributed with H-bond interactions.

TABLE 1.

3D and 2D models of binding interactions of phenylalanyl-adenylate in S. aureus PheRS activE SITE ^[14]

The compounds were investigated through docking studies. The thiol group, amino group and/or carbonyl group for compounds 8a - c showed interactions with the following binding residues in the active site: Ser174, Gln214 and Gly288. The amide group in compounds 11a and 11b interacted with Ser174 and Ala311 and the thiol group of 1,3,4-thiadiazolyl moiety interacted with His172. As observed for compounds 16a, 17a and 17b, the thiol group formed a H-bond with acidic Glu216 and for compound 16b, Gln214 formed a H-bond with the 1,2,4-triazole moiety (Table 2).

TABLE 2.

2D models of binding interactions of compounds in S. aureus PheRS active site using MOE

Microbiological evaluation

Compounds 8a-c were not subjected to microbiological evaluation because of their instability. Nevertheless, none of the tested compounds showed inhibitory activity (MIC ≥ 128 μg/mL) against S. aureus SH1000 sensitive strains. The synthesized compounds were microbiologically tested for P aeruginosa PheRS and S. pneumonia PheRS inhibition through aminoacylation assay due to the availability of these enzymes and their high degree of similarity with S. aureus PheRS. Analysis of the synthesized series resulted in only compound 34a (IC₅₀ 199 μM) exhibiting moderate inhibitory activity (Table 3).

TABLE 3.

MIC (μg/mL) values against S. aureus sensitive SH1000 strain and IC₅₀ data against S. pneumonia PheRS and P. aeruginosa PheRS (μM).

	MIC (μg/mL) S. aureus Sensitive SH1000	S. pneumonia PheRS IC50(μM)	P. aeruginosa PheRS IC50(μM)
11a	>128	345	742
11b	>128	685	907
16a	>128	561	343
16b	>128	390	375
17a	>128	619	199
17b	>128	686	294

Moreover, compounds (11b, 16a, 17a and 17b) showed no inhibitory activity (MIC ≥ 128 μg/mL) against the tested bacteria. However, moderate inhibitory activity (32–64 μg/mL) was observed with 11b and 17a against E. faecalis sensitive and vanA and vanB resistant strains (Table 4).

TABLE 4.

Broad microbiological evaluation of 11b, 16a, 17a and 17b (MIC (μg/mL) determination).

Organism	Amp	lib	16a	17a	l7b
S. aureus ATCC 29213 sensitive	4	>128	>128	>128	>128
S. aureus NCTC 12493 mecA resistant	>128	128	>128	128	>128
K. pneumoniae 21856 sensitive	>128	>128	>128	>128	>128
P. mirabilis NCTC 10975 sensitive	>128	>128	>128	>128	>128
P. aeruginosa ATCC 27853 sensitive	>128	>128	>128	>128	>128
S.enteritidis8204 sensitive	8	>128	>128	>128	>128
A.baumannii572 sensitive	>128	>128	>128	>128	>128
B.cepacia NCTC 10661 sensitive	>128	>128	>128	>128	>128
E. faecalisATCC 29212 sensitive	2	64	>128	>128	>128
E. faecalis NCTC 12201 vanA resistant	16	32	>128	64	>128
E. faecalis ATCC 51299 vanB resistant	8	64	>128	64	>128
E. faecium 16568 sensitive	4	-	>128	-	>128
E. coli ATCC 25922 sensitive	8	>128	>128	>128	>128

Conclusion

In summary, three novel series were designed and synthesized depending on the natural substrate, phenylalanyl-adenylate. All designed compounds make H-bonds with the key amino acid residues allowing the orientation of the compounds in the adenylate and amino acid binding sites. As the compounds in thiazole series are unstable, the future work should be optimized for the structure for further investigation of compound stability and yield. Analysis of the thiadiazole and triazole series resulted in only compound 17a (IC₅₀ 199 μM) exhibiting moderate inhibitory activity against P. aeruginosa PheRS. Compounds 11b and 17a showed moderate inhibitory activity (32–64 μg/mL) against E. faecalis sensitive and vanA and vanB resistant strains.