# Evaluation of NovelN-(Dibenzylcarbamothioyl)benzamide Derivatives as Antibacterial Agents by Using DFT and Drug-Likeness Assessment

Authors: Huda Misral; Suhaila Sapari; Tajudin Rahman; Nazlina Ibrahim; Bohari M. Yamin; Siti Aishah Hasbullah
Journal: Journal of Chemistry
 (2018)

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## Abstract

Isomers of monothioureas,2a–2d, derived from the reaction of disubstituted benzoyl isothiocyanate and dibenzylamine were synthesised and characterised by using elementary analysis CHNS and IR, 1H NMR, and 13C NMR spectroscopies. The compounds were screened for their in vitro antibacterial activity by using selected Gram-positive bacteria, and moderate inhibition activity was displayed for compound 2b with the value of inhibition zone 11 ± 0.8 mm at a concentration of 50 mg/ml. The outcomes of Lipinski’s rule of five assessment appeared to be in agreement with all compounds as they adhered to most of the rules, in which they can be preliminarily classified as active drug-like. The frontier molecular orbitals (HOMO and LUMO) for halogen-substituted 3,4-dichloro (2a) and 3,4-difluoro (2b) were also determined by applying the computational method of density functional theory (DFT) to determine their relationship as a molecular descriptor in antibacterial activities. The value of LUMO energy for compound 2b (1.8229 eV) is lower than that of compound 2a (1.8492 eV) which indicates higher antibacterial activities.


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## Body

## 1. Introduction

In recent times, the occurrence of microbial infections has tremendously raised in many countries around the world due to antimicrobial resistance [1]. This phenomenon has led to the design of novel antimicrobial as well as antibiotic divergent from the current classes of compounds [2]. Specifically, the growth of the new classes of antibacterial agents and modification to the known drugs must be conducted in such a way that would induce them to preserve their physiological action, but a reduction in their resistance towards the agents [3]. Thiourea is known as a versatile compound that has been intensely synthesised due to its ability to undergo structural modification [4]. Having two units of reactive primary amine group has led thiourea to be a suitable precursor for the synthesis of many derivatives in new compounds [5]. Oxygen, nitrogen, and sulphur atoms of thiourea derivatives provide a multitude of bonding possibilities that may contribute to a broad spectrum of thiourea applications in the pharmaceutical field, such as antiparasitic, anticancer, antioxidant, antibacterial, antifungal, antimalarial, and anti-HIV [6–8]. Furthermore, Mandava et al. explained in his research that the amide and indole groups at the moiety of thiourea showed considerable interaction with active site amino acid of ribosyltranferase [9]. In the literature survey and to the best of our knowledge, the disubstituted carbonyl thiourea derived from the secondary amines is still in scarcity [10].In this study, the synthesis and characterisation of novel disubstituted carbonyl monothioureas (Figure1) are reported after conducting related antibacterial screening. The frontier molecular orbitals (HOMO and LUMO) were also determined via the computational method of DFT to determine their relationship as a molecular descriptor in antibacterial activities.Figure 1
Antibacterial activity of compound2b towards bacterium Bacillus subtilis.


## 2. Experimental

### 2.1. Physical Measurement

All synthesis processes were carried out by using the conventional method of reflux, and no preventative measure was taken to exclude air or moisture. Chemical and solvents were purchased from Sigma-Aldrich or Merck and directly used without further purification. Infrared spectra were recorded by using FTIR Perkin Elmer Model Spectrum GX in the range of 400–4000 cm−1. The 1H NMR and 13C NMR spectra of the samples were verified by using a spectrometer NMR model Joel EX 400 MHz in d6-DMSO4.


### 2.2. General Synthesis of Thiourea Derivatives

A mixture of disubstituted 3,4-difluorobenzoyl chloride (1a) (0.317 g, 0.0018 mol) and NH4SCN (0.1370 g, 0.0018 mol) in 10 ml acetone was stirred at room temperature for 15 minutes. The mixture was filtered and directly uses “in situ” into a round bottom flask containing dibenzylamine (0.355 g, 0.0018 mol). The solution was refluxed for 3 hours [11]. The solution was filtered and poured into a beaker containing ice cubes to form a precipitate, which was then filtered, and the precipitate was proceeded for characterisation. The procedure was repeated for the synthesis of 2b–2d using 3,4-dichlorobenzoyl chloride (1b) (0.377 g, 0.0018 mol), 2-chloro-4-fluorobenzoyl chloride (1c) (0.0.612 g, 0.0018 mol), and 2-chloro-5-fluorobenzoyl chloride (1d) (0.3430 g, 0.0018 mol).


#### 2.2.1. 3,4-Difluoro-N-(dibenzylcarbamothioyl)benzamide (2a)

Percentage yield, 74%; Mp: 365.7–366.3°C, (found: C, 63.08; H, 4.42; N, 6.54%; C22H18F2N2OS requires C, 66.65; H, 4.58; N, 6.54%); IR (KBr pellets) ν (cm−1): 3170, 1690, 1186. 1H NMR (DMSO-d6, 600 MHz) δ (ppm): 4.68, 5.24 (s, 4H, 2 × C-H2); 7.19–7.90 (m, 8H, Ar-H); 11.08 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz) δ (ppm): 56.2, 55.2 (2 × CH2), 118.2–153.5 (Ar-C), 162.8 (C=O), 183.4 (C=S).


#### 2.2.2. 3,4-Dichloro-N-(dibenzylcarbamothioyl)benzamide (2b)

Percentage of yield, 65%; Mp: 365.4–366.5°C, (found: C, 62.80; H, 4.77; N, 7.91%; C22H18Cl2N2OS requires C, 61.54; H, 4.22; N, 6.53%); IR (KBr pellets) ν (cm−1): 3171, 1697, 1185. 1H NMR (DMSO-d6, 600 MHz): δ 4.71, 5.24 (2 × s, 4H, CH2); δ 7.05–7.87 (8 × m, 8H, Ar); δ 8.54 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 56.9, 55.9 (2 × CH2), δ 126.9–135.6 (Ar), δ 162.4 (C=O), δ 180.6 (C=S) [12].


#### 2.2.3. 2-Chloro-4-fluoro-N-(dibenzylcarbamothioyl)benzamide (2c)

Percentage of yield, 70%; Mp: 366.4–367.3°C, (found: C, 64.03; H, 3.71; N, 7.94%; C22H18Cl2N2OS requires C, 64.00; H, 4.40; N, 6.79%); IR (KBr pellets) ν (cm−1): 3271, 1702, 1189. 1H NMR (DMSO-d6, 600 MHz): δ 4.83, 5.20 (2 × s, 4H, CH2); δ 7.22–7.94 (8 × m, 8H, Ar); δ 11.29 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 55.9, 55.2 (2 × CH2), δ 114.9–162.2 (Ar), δ 163.8 (C=O), δ 182.4 (C=S).


#### 2.2.4. 2-Chloro-5-fluoro-N-(dibenzylcarbamothioyl)benzamide (2d)

Percentage of yield, 63%; Mp: 366.3–367.5°C, (found: C, 64.75; H, 4.84; N, 6.91%; C22H18Cl2N2OS requires C, 64.00; H, 4.40; N, 6.79%); IR (KBr pellets) ν (cm−1): 3179, 1708, 1190. 1H NMR (DMSO-d6, 600 MHz): δ 4.81, 5.20 (2 × s, 4H, CH2); δ 7.14–7.42 (8 × m, 8H, Ar); δ 8.70 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 56.3, 55.9 (2 × CH2), δ 117.7–134.6 (Ar), δ 161.9 (C=O), δ 180.4 (C=S) [13].


### 2.3. Computational Method

Computational-assisted geometry optimisation, conformational analysis, and total energy calculation were carried out in the gas phase with Gaussian 09 suite of program using Beck’s three-parameter hybrid method, B3LYP, with 6-31G (d, p) basis set to gain better comprehension regarding the molecular structure of the thiourea derivatives [14, 15].


### 2.4. Antibacterial Screening

Antibacterial screening was carried out using the disc diffusion technique with strains of bacteria from Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis). The control positive was chloramphenicol (50 mg/ml), while DMSO (100% of concentration) was the control negative. The bacteria were cultured in nutrient broth and left for 18–24 hours to grow. The nutrient agar and nutrient broth were in aseptic condition to prevent the bacteria from air, which may affect the results. The thioureas were dissolved in DMSO with 5 different concentrations: 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, and 3.125 mg/ml. Then, the single colony of bacteria was picked from the nutrient agar and placed into the nutrient broth. By using a micropipette, 5 μl of each compound with different concentrations was dropped onto the filter paper (6 mm diameter). The inhibition result of the bacteria was noted after 18–24 hours of incubation at 37°C–48°C. The bacteria tests were performed in triplicate, and the results are shown in Table 1 [16].Table 1
Antibacterial screening of compounds2a, 2b, 2c, and 2d.
Compound
Gram-positive bacteria (mm)/concentration of sample (mg/mL)
Staphylococcus aureus
Enterococcus faecalis
Bacillus subtilis
2a
—
—
—
2b
11 ± 0.8 (50 mg/ml)
—
8 ± 0.4 (50 mg/ml)
2c
—
—
—
2d
—
—
—
Positive control (chloramphenicol)
30.1 ± 0.0 (50 mg/ml)
20.0 ± 0.0 (50 mg/ml)
32.8 ± 0.0 (50 mg/ml)
“—” denotes no activity.


## 2.1. Physical Measurement

All synthesis processes were carried out by using the conventional method of reflux, and no preventative measure was taken to exclude air or moisture. Chemical and solvents were purchased from Sigma-Aldrich or Merck and directly used without further purification. Infrared spectra were recorded by using FTIR Perkin Elmer Model Spectrum GX in the range of 400–4000 cm−1. The 1H NMR and 13C NMR spectra of the samples were verified by using a spectrometer NMR model Joel EX 400 MHz in d6-DMSO4.


## 2.2. General Synthesis of Thiourea Derivatives

A mixture of disubstituted 3,4-difluorobenzoyl chloride (1a) (0.317 g, 0.0018 mol) and NH4SCN (0.1370 g, 0.0018 mol) in 10 ml acetone was stirred at room temperature for 15 minutes. The mixture was filtered and directly uses “in situ” into a round bottom flask containing dibenzylamine (0.355 g, 0.0018 mol). The solution was refluxed for 3 hours [11]. The solution was filtered and poured into a beaker containing ice cubes to form a precipitate, which was then filtered, and the precipitate was proceeded for characterisation. The procedure was repeated for the synthesis of 2b–2d using 3,4-dichlorobenzoyl chloride (1b) (0.377 g, 0.0018 mol), 2-chloro-4-fluorobenzoyl chloride (1c) (0.0.612 g, 0.0018 mol), and 2-chloro-5-fluorobenzoyl chloride (1d) (0.3430 g, 0.0018 mol).


### 2.2.1. 3,4-Difluoro-N-(dibenzylcarbamothioyl)benzamide (2a)

Percentage yield, 74%; Mp: 365.7–366.3°C, (found: C, 63.08; H, 4.42; N, 6.54%; C22H18F2N2OS requires C, 66.65; H, 4.58; N, 6.54%); IR (KBr pellets) ν (cm−1): 3170, 1690, 1186. 1H NMR (DMSO-d6, 600 MHz) δ (ppm): 4.68, 5.24 (s, 4H, 2 × C-H2); 7.19–7.90 (m, 8H, Ar-H); 11.08 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz) δ (ppm): 56.2, 55.2 (2 × CH2), 118.2–153.5 (Ar-C), 162.8 (C=O), 183.4 (C=S).


### 2.2.2. 3,4-Dichloro-N-(dibenzylcarbamothioyl)benzamide (2b)

Percentage of yield, 65%; Mp: 365.4–366.5°C, (found: C, 62.80; H, 4.77; N, 7.91%; C22H18Cl2N2OS requires C, 61.54; H, 4.22; N, 6.53%); IR (KBr pellets) ν (cm−1): 3171, 1697, 1185. 1H NMR (DMSO-d6, 600 MHz): δ 4.71, 5.24 (2 × s, 4H, CH2); δ 7.05–7.87 (8 × m, 8H, Ar); δ 8.54 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 56.9, 55.9 (2 × CH2), δ 126.9–135.6 (Ar), δ 162.4 (C=O), δ 180.6 (C=S) [12].


### 2.2.3. 2-Chloro-4-fluoro-N-(dibenzylcarbamothioyl)benzamide (2c)

Percentage of yield, 70%; Mp: 366.4–367.3°C, (found: C, 64.03; H, 3.71; N, 7.94%; C22H18Cl2N2OS requires C, 64.00; H, 4.40; N, 6.79%); IR (KBr pellets) ν (cm−1): 3271, 1702, 1189. 1H NMR (DMSO-d6, 600 MHz): δ 4.83, 5.20 (2 × s, 4H, CH2); δ 7.22–7.94 (8 × m, 8H, Ar); δ 11.29 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 55.9, 55.2 (2 × CH2), δ 114.9–162.2 (Ar), δ 163.8 (C=O), δ 182.4 (C=S).


### 2.2.4. 2-Chloro-5-fluoro-N-(dibenzylcarbamothioyl)benzamide (2d)

Percentage of yield, 63%; Mp: 366.3–367.5°C, (found: C, 64.75; H, 4.84; N, 6.91%; C22H18Cl2N2OS requires C, 64.00; H, 4.40; N, 6.79%); IR (KBr pellets) ν (cm−1): 3179, 1708, 1190. 1H NMR (DMSO-d6, 600 MHz): δ 4.81, 5.20 (2 × s, 4H, CH2); δ 7.14–7.42 (8 × m, 8H, Ar); δ 8.70 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 56.3, 55.9 (2 × CH2), δ 117.7–134.6 (Ar), δ 161.9 (C=O), δ 180.4 (C=S) [13].


## 2.2.1. 3,4-Difluoro-N-(dibenzylcarbamothioyl)benzamide (2a)

Percentage yield, 74%; Mp: 365.7–366.3°C, (found: C, 63.08; H, 4.42; N, 6.54%; C22H18F2N2OS requires C, 66.65; H, 4.58; N, 6.54%); IR (KBr pellets) ν (cm−1): 3170, 1690, 1186. 1H NMR (DMSO-d6, 600 MHz) δ (ppm): 4.68, 5.24 (s, 4H, 2 × C-H2); 7.19–7.90 (m, 8H, Ar-H); 11.08 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz) δ (ppm): 56.2, 55.2 (2 × CH2), 118.2–153.5 (Ar-C), 162.8 (C=O), 183.4 (C=S).


## 2.2.2. 3,4-Dichloro-N-(dibenzylcarbamothioyl)benzamide (2b)

Percentage of yield, 65%; Mp: 365.4–366.5°C, (found: C, 62.80; H, 4.77; N, 7.91%; C22H18Cl2N2OS requires C, 61.54; H, 4.22; N, 6.53%); IR (KBr pellets) ν (cm−1): 3171, 1697, 1185. 1H NMR (DMSO-d6, 600 MHz): δ 4.71, 5.24 (2 × s, 4H, CH2); δ 7.05–7.87 (8 × m, 8H, Ar); δ 8.54 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 56.9, 55.9 (2 × CH2), δ 126.9–135.6 (Ar), δ 162.4 (C=O), δ 180.6 (C=S) [12].


## 2.2.3. 2-Chloro-4-fluoro-N-(dibenzylcarbamothioyl)benzamide (2c)

Percentage of yield, 70%; Mp: 366.4–367.3°C, (found: C, 64.03; H, 3.71; N, 7.94%; C22H18Cl2N2OS requires C, 64.00; H, 4.40; N, 6.79%); IR (KBr pellets) ν (cm−1): 3271, 1702, 1189. 1H NMR (DMSO-d6, 600 MHz): δ 4.83, 5.20 (2 × s, 4H, CH2); δ 7.22–7.94 (8 × m, 8H, Ar); δ 11.29 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 55.9, 55.2 (2 × CH2), δ 114.9–162.2 (Ar), δ 163.8 (C=O), δ 182.4 (C=S).


## 2.2.4. 2-Chloro-5-fluoro-N-(dibenzylcarbamothioyl)benzamide (2d)

Percentage of yield, 63%; Mp: 366.3–367.5°C, (found: C, 64.75; H, 4.84; N, 6.91%; C22H18Cl2N2OS requires C, 64.00; H, 4.40; N, 6.79%); IR (KBr pellets) ν (cm−1): 3179, 1708, 1190. 1H NMR (DMSO-d6, 600 MHz): δ 4.81, 5.20 (2 × s, 4H, CH2); δ 7.14–7.42 (8 × m, 8H, Ar); δ 8.70 (s, 1H, NH). 13C NMR (DMSO-d6, 150 MHz): δ 56.3, 55.9 (2 × CH2), δ 117.7–134.6 (Ar), δ 161.9 (C=O), δ 180.4 (C=S) [13].


## 2.3. Computational Method

Computational-assisted geometry optimisation, conformational analysis, and total energy calculation were carried out in the gas phase with Gaussian 09 suite of program using Beck’s three-parameter hybrid method, B3LYP, with 6-31G (d, p) basis set to gain better comprehension regarding the molecular structure of the thiourea derivatives [14, 15].


## 2.4. Antibacterial Screening

Antibacterial screening was carried out using the disc diffusion technique with strains of bacteria from Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis). The control positive was chloramphenicol (50 mg/ml), while DMSO (100% of concentration) was the control negative. The bacteria were cultured in nutrient broth and left for 18–24 hours to grow. The nutrient agar and nutrient broth were in aseptic condition to prevent the bacteria from air, which may affect the results. The thioureas were dissolved in DMSO with 5 different concentrations: 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, and 3.125 mg/ml. Then, the single colony of bacteria was picked from the nutrient agar and placed into the nutrient broth. By using a micropipette, 5 μl of each compound with different concentrations was dropped onto the filter paper (6 mm diameter). The inhibition result of the bacteria was noted after 18–24 hours of incubation at 37°C–48°C. The bacteria tests were performed in triplicate, and the results are shown in Table 1 [16].Table 1
Antibacterial screening of compounds2a, 2b, 2c, and 2d.
Compound
Gram-positive bacteria (mm)/concentration of sample (mg/mL)
Staphylococcus aureus
Enterococcus faecalis
Bacillus subtilis
2a
—
—
—
2b
11 ± 0.8 (50 mg/ml)
—
8 ± 0.4 (50 mg/ml)
2c
—
—
—
2d
—
—
—
Positive control (chloramphenicol)
30.1 ± 0.0 (50 mg/ml)
20.0 ± 0.0 (50 mg/ml)
32.8 ± 0.0 (50 mg/ml)
“—” denotes no activity.


## 3. Results and Discussion

The compounds2a–2d were synthesised in good yield (63–74%) by the reaction between disubstituted benzoyl chlorides (1a–1d) and ammonium thiocyanate to give the disubstituted benzoyl isothiocyanate as the intermediate which is later to be reacted with dibenzylamine (Scheme 1). The formations of products were confirmed by NMR, IR, and elemental analysis.Scheme 1
Reaction scheme to synthesis thiourea derivatives2a, 2b, 2c, and 2d.


### 3.1. Antibacterial Analysis

The activity of compounds2a–2d against bacteria was observed after 24 hours of inhibition at 37°C–42°C. At concentration of 25.0, 12.5, 6.25, and 3.13 mg/ml, the compound showed no inhibition activity except for concentration of 50 mg/ml. Gram-positive bacteria of Staphylococcus aureus and Bacillus subtilis except Enterococcus faecalis exhibited positive results towards compound 2b, which contradicted to compounds 2a, 2c, and 2d, as no inhibition was recorded, as shown in Table 1. Figure 1 illustrates the antibacterial activity of compound 2b towards bacteria Bacillus subtilis. 100% concentration of DMSO was used as a negative control and shows no clear expansion which indicates no inhibition of bacterial activity.Overall, the synthesised compound2b displayed a significant inhibition activity compared to compounds 2a, 2c, and 2d. It was clear from the study that the disubstituted halogen compound (3,4-chloro, 2b) on the phenyl group showed comparatively potent activity than the 3,4-fluoro (2a), 2-chloro-4-fluoro (2c), and 2-chloro-5-fluoro (2d) substituted. This is due to the size of chlorine atom that is bigger than that of fluorine, thus exerting a significant impact on the London dispersion force and also the lipophilicity [17, 18]. In general, the addition of halogen substituents (in particular, Cl, Br, and I) increases the lipophilicity of a molecule. Larger and heavier atoms or molecules demonstrate stronger dispersion forces than smaller and lighter ones. As the London dispersion force increases, the lipophilicity interaction increases and as a result, penetration of the compounds into the bacterial membrane is eased [19]. Furthermore, the position of the chlorine atom at the meta-para (2b) position generally correlates well but not for ortho-substituted ones (2c and 2d) due to the ortho steric effect and ortho polar effects which resulted to no antibacterial activity for compounds 2c and 2d that in contrast to 2b [20].Besides, in reference to Lipinski’s rule of five regarding drug-likeness, all of the compounds appeared to adhere to most of the rules with only one violation, where lipophilicity (log P) value exceeded 5 (Table2). This, however, concludes that compounds 2a–2d can be classified as drug-like [21]. The evaluation of Lipinski’s rule on the designated compound is crucial as the preliminary study on the ability of the compound to act as a drug, which then can be further carried out and tested in various related analyses. In detail, thiourea 2b displayed the highest lipophilicity value (6.6), when compared to 2a, 2c, and 2d which is 5.8 that corresponded to the result of antibacterial analysis. The higher the lipophilicity, the higher is the inhibition of the compound towards the bacteria. Further structural modification, including changing of substituents group, might be necessary for the other compounds 2a, 2c, and 2d to refine their ability as antibacterial agents for them to display exceptional criteria as a drug from Lipinski’s rule evaluation.Table 2
Evaluation of Lipinski’s rule of five for compounds2a, 2b, 2c, and 2d.
Lipinski rule/compounds
2a
2b
2c
2d
1. Log P (lipophilicity) < 5
5.8
6.6
5.8
5.8
2. Hydrogen bond donor < 5
1
1
1
1
3. Hydrogen bond acceptor < 10
2
2
2
2
4. Molecular weight < 500 g/mol
396.46
429.36
412.91
412.91In addition, this antibacterial study is also supported by the computational calculation of density functional theory (DFT) at level theory of hybrid functional B3LYP/6-31G (d, p) basis set method to determine the band gap energy of HOMO-LUMO and the value of LUMO energy. The comparative study was conducted between (3,4-dichloro)2a and (3,4-di-fluoro) 2b which have similar position for halogen substituted. The results reported in Table 3 show that the value of the LUMO energy for compound 2b (1.8229 eV) is lower than that of 2a (1.8492 eV), which is in agreement to the antibacterial result. Compound 2b exhibited higher antibacterial activity, when compared to compound 2a, as tabulated in Table 1. This is related to the lowest energy value of LUMO for compound 2b, which contributed to its higher antibacterial activity as well as the size of the halogen (Cl) substituent [17, 22]. The collected data from the frontier molecular orbitals (HOMO-LUMO) analysis of thioureas 2a and 2b are recorded in Table 3 and illustrated in Figure 2.Table 3
List of HOMO-LUMO energy of compounds2a and 2b.
Compounds
HOMO (eV)
LUMO (eV)
HOMO-LUMO (eV)
2a
5.8295
1.8492
3.980
2b
5.9541
1.8229
4.131Figure 2
Frontier molecular orbitals (HOMO and LUMO) of thioureas2a and 2b.
(a)
(b)


## 3.1. Antibacterial Analysis

The activity of compounds2a–2d against bacteria was observed after 24 hours of inhibition at 37°C–42°C. At concentration of 25.0, 12.5, 6.25, and 3.13 mg/ml, the compound showed no inhibition activity except for concentration of 50 mg/ml. Gram-positive bacteria of Staphylococcus aureus and Bacillus subtilis except Enterococcus faecalis exhibited positive results towards compound 2b, which contradicted to compounds 2a, 2c, and 2d, as no inhibition was recorded, as shown in Table 1. Figure 1 illustrates the antibacterial activity of compound 2b towards bacteria Bacillus subtilis. 100% concentration of DMSO was used as a negative control and shows no clear expansion which indicates no inhibition of bacterial activity.Overall, the synthesised compound2b displayed a significant inhibition activity compared to compounds 2a, 2c, and 2d. It was clear from the study that the disubstituted halogen compound (3,4-chloro, 2b) on the phenyl group showed comparatively potent activity than the 3,4-fluoro (2a), 2-chloro-4-fluoro (2c), and 2-chloro-5-fluoro (2d) substituted. This is due to the size of chlorine atom that is bigger than that of fluorine, thus exerting a significant impact on the London dispersion force and also the lipophilicity [17, 18]. In general, the addition of halogen substituents (in particular, Cl, Br, and I) increases the lipophilicity of a molecule. Larger and heavier atoms or molecules demonstrate stronger dispersion forces than smaller and lighter ones. As the London dispersion force increases, the lipophilicity interaction increases and as a result, penetration of the compounds into the bacterial membrane is eased [19]. Furthermore, the position of the chlorine atom at the meta-para (2b) position generally correlates well but not for ortho-substituted ones (2c and 2d) due to the ortho steric effect and ortho polar effects which resulted to no antibacterial activity for compounds 2c and 2d that in contrast to 2b [20].Besides, in reference to Lipinski’s rule of five regarding drug-likeness, all of the compounds appeared to adhere to most of the rules with only one violation, where lipophilicity (log P) value exceeded 5 (Table2). This, however, concludes that compounds 2a–2d can be classified as drug-like [21]. The evaluation of Lipinski’s rule on the designated compound is crucial as the preliminary study on the ability of the compound to act as a drug, which then can be further carried out and tested in various related analyses. In detail, thiourea 2b displayed the highest lipophilicity value (6.6), when compared to 2a, 2c, and 2d which is 5.8 that corresponded to the result of antibacterial analysis. The higher the lipophilicity, the higher is the inhibition of the compound towards the bacteria. Further structural modification, including changing of substituents group, might be necessary for the other compounds 2a, 2c, and 2d to refine their ability as antibacterial agents for them to display exceptional criteria as a drug from Lipinski’s rule evaluation.Table 2
Evaluation of Lipinski’s rule of five for compounds2a, 2b, 2c, and 2d.
Lipinski rule/compounds
2a
2b
2c
2d
1. Log P (lipophilicity) < 5
5.8
6.6
5.8
5.8
2. Hydrogen bond donor < 5
1
1
1
1
3. Hydrogen bond acceptor < 10
2
2
2
2
4. Molecular weight < 500 g/mol
396.46
429.36
412.91
412.91In addition, this antibacterial study is also supported by the computational calculation of density functional theory (DFT) at level theory of hybrid functional B3LYP/6-31G (d, p) basis set method to determine the band gap energy of HOMO-LUMO and the value of LUMO energy. The comparative study was conducted between (3,4-dichloro)2a and (3,4-di-fluoro) 2b which have similar position for halogen substituted. The results reported in Table 3 show that the value of the LUMO energy for compound 2b (1.8229 eV) is lower than that of 2a (1.8492 eV), which is in agreement to the antibacterial result. Compound 2b exhibited higher antibacterial activity, when compared to compound 2a, as tabulated in Table 1. This is related to the lowest energy value of LUMO for compound 2b, which contributed to its higher antibacterial activity as well as the size of the halogen (Cl) substituent [17, 22]. The collected data from the frontier molecular orbitals (HOMO-LUMO) analysis of thioureas 2a and 2b are recorded in Table 3 and illustrated in Figure 2.Table 3
List of HOMO-LUMO energy of compounds2a and 2b.
Compounds
HOMO (eV)
LUMO (eV)
HOMO-LUMO (eV)
2a
5.8295
1.8492
3.980
2b
5.9541
1.8229
4.131Figure 2
Frontier molecular orbitals (HOMO and LUMO) of thioureas2a and 2b.
(a)
(b)


## 4. Conclusion

The compounds2a–2d were successfully synthesised and fully characterised via spectroscopic analyses. From the antibacterial study, only thiourea 2b exhibited moderate inhibition activity against selected Gram-positive bacteria, which are 11 ± 0.8 mm (50 mg/ml) towards bacteria Staphylococcus aureus and 8 ± 0.4 mm (50 mg/ml) towards bacteria Bacillus subtilis, in comparison to chloramphenicol as the control positive. Theoretical calculation unveiled that the antibacterial activity has some relationship with the LUMO energy value. The lower the LUMO value, the higher the antibacterial activity. Assessment from Lipinski’s rule of five depicted that all of the valuable compounds can be classified as drug-like, thus suggesting an exceptional starting point for the compounds to be explored in future development. This study offers good insight towards selective applications in the antibacterial field as it highlights the remarkable characteristics of a good antibacterial agent. Therefore, further detailed study, including modification of the substituents and the fragments of the compounds as antibacterial agents, is deemed necessary.



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*Source: 9176280-2018-11-22.xml*