Molecular Design, Synthesis, and Biological Evaluation of 2-Hydroxy-3-Chrysino Dithiocarbamate Derivatives

Abstract

Antimicrobial Evaluation In order to find the potent antimicrobial agent among the synthesized compounds (3a–k), they were assessed for their in vitro antibacterial activity against Staphylococcus epidermis (MTCC 96) and Bacillus subtilis (MTCC 441) as Gram-positive, and Escherichia coli (MTCC 443) and Pseudomonas aeruginosa (MTCC 741) as Gram-negative bacteria. First target is β-Ketoacyl-acyl-carrier protein (ACP) synthase III, also known as FabH or KAS III (pdb id: 1HNJ), due to its important and regulatory role in bacterial fatty acid biosynthesis (FAB) [59,60], and found in both Gram-positive and Gram-negative bacteria. The published crystal structure of ITZ bound within the active site cavity of CYP51 (PDB ID:5EQB) served as a useful template for generating proposed binding modes with respect to antifungal activity. The docking results showed that the synthetic analogues (3a–k) were bound in the active sites of the enzymes by forming combination of hydrogen bonds, hydrophobic, and van der Waals interactions.

1. Introduction

In the recent past, many pharmaceutical products have been designed and developed using plant based lead compounds like polyphenols [1]. Among them, flavonoids are the extensively studied biologically active compounds possessing strong antioxidant property and having potential health benefits in the prevention of cardiovascular disorders, and considered safe with a low toxicity [2,3,4,5,6]. Flavonoids also exhibit a wide variety of biological activities like antibacterial, anti-inflammatory, anti-diabetic, anti-allergic, antiviral, vasodilatory, and anticancer activities [7,8,9,10,11,12,13,14,15,16,17] and have potential to be developed or modified as effective drug candidates. Heterocyclic moieties linked to chromone system have enormous applications in pharmacological fields like antimicrobial [8,9,10,11,12,13,14,15,16,17,18,19,20,21], anti-inflammatory [22,23], anti-cancer [24,25,26,27], and anti-oxidant [28] activities.

Among the flavones class, chrysin (5,7-dihydroxy flavone) is an important biologically active compound. It is found in many medicinal plants, honey, propolis, mushrooms, and mainly isolated from an Indian medicinal plant ‘oroxylumindicum’ [29,30]. Chrysin is reported to exhibit various biological activities, which includes antibacterial [31], anti-inflammatory [32] anti-allergic [33], antioxidant [34], and anticancer [35] activities. Several attempts have been made to synthesize the structural derivatives of chrysin and to study their biological activities [36,37,38]. These studies indicated that synthetic analogues of chrysin are found to have more potent biological activities than standard drugs.

Epoxides are most useful intermediates for variety of synthetic reactions in organic synthesis due to ring strain. They can undergo regioselective ring opening from the less hindered terminal carbon side of the epoxide ring with a wide variety of nucleophiles by SN² reaction. The dithiocarbamate nucleophiles generated in situ from CS₂ and amines open the epoxide ring from the terminal carbon side [39,40,41] to afford 2-hydroxy dithiocarbamates, which are found to have a wide variety of applications in organic synthesis [42,43,44,45], pharmaceuticals [46,47,48,49], and agriculture [50,51,52].

In continuation to our studies of the synthetic modifications of chrysin and their biological screening [53,54], we herein report the synthesis of new 2-hydroxy-3-chrysino dithiocarbamate derivatives (3a–k). Antimicrobial studies were carried out to find the best drug candidate among the synthesized compounds (3a–k). Molecular modeling studies were also performed on these analogues (3a–k) to find the binding interaction to support the antibacterial activities. As per our knowledge, this is the first report of synthesis and antimicrobial activity studies of 2-hydroxy-3-chrysino dithiocarbamate derivatives.

2. Results 2.1. Chemistry

In our present study, our aim is to enhance the biological activity of chrysin by linking it to 2-hydroxy dithiocarbamates at C (7) position. This was achieved in two steps. Epoxy-methyl group was linked to chrysin at its C (7) position by reacting with epichlorohydrin in presence of K₂CO₃ in DMF solvent at 50 °C which gave the epoxide (2). This epoxide derivative (2) was made to react with a mixture of CS₂ and secondary amine in acetonitrile at 60 °C to give the designed 2-hydroxy-3-chrysino dithiocarbamate derivatives (3a–k) (Scheme 1) in moderate to good yields (Table 1). The synthetic analogues (3a–k) were well characterized by ¹H NMR, ¹³C NMR, and HRMS-ESI spectral analysis and the spectra of (3a–k) can be found in the supplementary materials. Formation of the designed compounds was identified by ESI-HRMS spectra with their [M + H] m/z values. The characteristic peak of the methine proton of all the synthesized compounds with chemical shift at δ = 4.15–4.16ppm (CHOH, ddd, J = 15.3, 9.5, 5.3 Hz, 1H) in ¹H NMR spectra indicated the formation of the designed 2-hydroxy dithiocarbamate derivatives (3a–k).

2.2. Pharmacology

2.2.1. Antimicrobial Evaluation

In order to find the potent antimicrobial agent among the synthesized compounds (3a–k), they were assessed for their in vitro antibacterial activity against Staphylococcus epidermis (MTCC 96) and Bacillus subtilis (MTCC 441) as Gram-positive, and Escherichia coli (MTCC 443) and Pseudomonas aeruginosa (MTCC 741) as Gram-negative bacteria. In vitro antifungal activities were also evaluated against Saccharomyces cerevisiae (MTCC 170) and Candida albicance (MTCC 3017) for the synthesized compounds (3a–k). To determine these preliminary antimicrobial activities, agar diffusion method [55,56] was used with penicillin and itrazole as the reference drugs to compare antibacterial and antifungal activities, respectively.

For all the synthesized compounds, the average diameter zone of inhibition round the disk in mm was recorded against the selected bacterial and fungal strains. For the selected compounds, which were showing remarkable growth in inhibition zones, minimum inhibitory concentration (MIC) in μg/mL was also measured using the two fold serial dilution method [57,58]. Most of the synthesized compounds exhibited considerable activity against the selected microorganisms and the findings of these antimicrobial studies are presented Table 2.

The compounds 3d, 3f, and 3j manifested excellent antibacterial activity with zone of inhibition of >20 mm. The compounds 3e, 3g, and 3h exhibited significant activity. The compounds 3a, 3b, 3c, 3i, and 3k showed least activity with respect to the reference drug penicillin. The MIC values of 3d, 3f, 3g, and 3j also reinforce the inhibitory activity. The compounds 3d, 3f, and 3h showed remarkable antifungal activity with inhibitory zone > 20 mm. The compounds 3j and 3k also exhibited moderate activity, which is also supported by MIC values.

2.2.2. Molecular Docking Studies

In addition to the synthesis and antimicrobial screening of 2-hydroxy-3-chrysino dithiocarbamate derivatives (3a–k), molecular docking studies were also performed to elucidate the observed antimicrobial results using Molegro virtual docker (MVD-2013 (6.0)) software. These docking studies predict the drug likeness of ligands, which will give the substitutional and configurational necessities for optimum receptor pit which are essential to have best pharmacophore activity. From PDB Bank RSCB, 3D-structures of selected proteins of E. coli FabH (pdb id: 1HNJ) and S. cerevisiae (pdb id: 5EQB) were taken with an X-ray resolution range of 1.46 Å and 2.59 Å, respectively. First target is β-Ketoacyl-acyl-carrier protein (ACP) synthase III, also known as FabH or KAS III (pdb id: 1HNJ), due to its important and regulatory role in bacterial fatty acid biosynthesis (FAB) [59,60], and found in both Gram-positive and Gram-negative bacteria. The enzyme FabH is found to initiate the elongation cycle of fatty acid [61,62]. It was also observed that via product inhibition, FabH is involved in the regulation of the biosynthetic pathway [63]. Some of the earlier synthesized C (7) modified chrysin derivatives were also found to inhibit FabH as antibiotics [38]. The second target is Lanosterol 14-alpha demethylase with intact transmembrane domain bound to itraconazole (S. cerevisiae) for showing antifungal activity. It acts as oxidoreductase inhibitor. Its organism is S. cerevisiae (MTCC 170). The published crystal structure of ITZ bound within the active site cavity of CYP51 (PDB ID:5EQB) served as a useful template for generating proposed binding modes with respect to antifungal activity.

Investigations were carried out to evaluate the interaction between the ligands and the receptor, their fitness function ability of transferase, oxido-reductase proteins with different inhibitors. The active site pocket of these proteins consist of Arg36, Trp32, Thr28, Arg151, Ser29, Asp27, Ile55, Gly152, Gly209, Asn210, Lys214, Phe213, Ala208, Met207, Arg249, Thr37, Arg56, and Ile156 amino acid residues. In the active site regions of 1HNJ protein, Thr81, Gly306, Phe304, Gly305, Asn210, Arg249, Ala246, Leu142, Cys112, Val212, Met207, Lys214, Gly306, and Leu189 amino acid residues can play important roles. In 5EQB protein, Met25, Ser61, Ile62, Pro63, Leu69, and Lys24 amino acid residues can play important roles.

Three-dimensional conformations of the synthesized 2-hydroxy-3-chrysino dithiocarbamate derivatives (3a–k) were generated. These structures were then docked into the active site of protein structures of E. coli FabH (1HNJ) and S. cerevisiae (5EQB) using the Molegro virtual docking software package. This will give the binding interaction of the ligand with the proteins and understanding of the possible mechanism of action. By knowing the putative binding site and docked poses with Cys–His–Asn residues of E. coli FabH (1HNJ) and S. cerevisiae (5EQB), enzymes were generated based on their binding energy with manual inspection. The synthesized analogues (3a–k) with the binding site interactions of the synthesized analogues (3a–k) with E. coli FabH (1HNJ) and S. cerevisiae (5EQB) enzymes are shown in Figure 1.

The molecules having optimum lipophilicity, maximum H-bonding ability with minimum clashes are needed to dock for good fit in the active site region of the target receptor. For the docked compounds, binding affinity values were found to be in terms of negative binding energy kcal mol⁻¹. The ligands with relatively more negative binding energy will be more potent in binding with the protein. Docking data for all the synthesized compounds (3a–k) were generated and presented in Table 3 and Table 4. The docking results showed that the synthetic analogues (3a–k) were bound in the active sites of the enzymes by forming combination of hydrogen bonds, hydrophobic, and van der Waals interactions. The molecular docking results showed good binding score values for all the synthesized molecules compared to standard drugs.

Based on the Moldockscore [Grid] (kcal/mol), it is clear that from the synthesized 2-hydroxy-3-chrysino dithiocarbamate derivatives (3a–k), the compounds 3d–h and 3j showed greater binding affinity with the protein of E. coli of FabH (1HNJ) compared to standard drug penicillin. All the synthesized compounds (3a–k) showed lower binding affinity with the protein S. cerevisiae (5EQB) compared to standard drug itrazole.

2.2.3. Adsorption, Distribution, Metabolism, and Excretion (ADME)-Profile

The properties like adsorption, distribution, metabolism, and excretion (ADME) are important for any compound to be developed as a successful drug. In our present study, the Molinspiration online property calculation toolkit [64] was used to determine the properties like molecular volume (MV), molecular weight (MW), logarithm of partition coefficient (m_iLogP), number of hydrogen bond acceptors (n-ON), number of rotatable bonds (n-ROTB), and Lipinski’s rule of five [65]. Absorption (% ABS) was calculated by: %ABS = 109 − [0.345 × Total Polar Surface Area (TPSA)] [66]. Drug-likeness model score is computed by using the Molsoft software [67], which is a collective property of physico-chemical properties, pharmacokinetics and pharmacodynamics. The pharmacokinetic parameters were calculated for the known inhibitors, synthesized analogues (3a–k), and the standard drugs, shown in Table 5.

For any molecule likely to be developed as an orally active drug candidate, it should not exhibit more than one violation from the following four criteria: m_iLogP (octanol-water partition coefficient) ≤ 5, molecular weight ≤ 500, number of hydrogen bond acceptors ≤ 10, and number of hydrogen bonds ≤ 5. It is observed that the synthesized 2-hydroxy dithiocarbamates linked chrysin derivates (3a–k) exhibited good % absorption (% ABS) ranging from 70.12% to 80.31%. All the synthesized compounds, except 3d, 3f, and 3h of 3a–k obeyed Lipinski’s rule of five (number of hydrogen bond acceptors (n-ON) ≤ 10) and obeyed the requirement to be an orally active drug candidate. Hence, the synthesized derivatives (3a–k) have good potential for subsequent development in drug discovery.

3. Materials and Methods 3.1. Chemistry

3.1.1. General

Melting points of the synthesized compounds were determined in open capillaries and are uncorrected. ¹H NMR spectra were recorded on Bruker-500 (500 MHz) spectrometer (Bruker, Fallanden, Switzerland), using deutero-chloroform (CDCl₃) as solvent and tetramethylsilane (TMS) as an internal standard. ¹³C NMR spectra were obtained with Bruker-500 (125 MHz) spectrometer by using CDCl₃ + DMSO-d6 as solvent. Chemical shifts are given in parts per million (δ) and coupling constants (J) in Hz. Mass spectra were recorded on LC-QTOF MS mass spectrometer and given in mass units (m/z). Thin layer chromatography (TLC) was performed on Merck silica gel 60 F₂₅₄ precoated aluminum sheets.

3.1.2. Synthesis of 5-hydroxy-7-(oxiran-2-ylmethoxy)-2-phenyl-4H-chromen-4-one (2):

To a solution of chrysin 1 (4 mmol) in DMF (12 mL), was added K₂CO₃ (20 mmol) and the mixture was stirred for 20 min at room temperature. Epichlorohydrin (20 mmol) was then added drop wise to above mixture. The reaction mixture was heated at 50 °C for 6 h. After completion of reaction, the mixture was poured into the ice water. The precipitate and the extractions were combined and subjected to column chromatography (silica gel; eluent: PE:EA = 10:1) to afford 2 as pale yellow solid (780 mg, 64%).

m.p: 165–167 °C; ¹H NMR (500MHz, CDCl₃) δ 12.73 (s, 1H), 7.89–7.86 (m, 2H), 7.53 (dd, J = 17.2, 6.6 Hz, 3H), 6.67 (s, 1H), 6.53 (d, J = 2.2 Hz, 1H), 6.38 (d, J = 2.2 Hz, 1H0, 4.34 (dd, J = 11.0, 2.9 Hz, 1H), 4.01 (dd, J = 11.0, 5.9 Hz, 1H), 3.39 (dt, J = 6.8, 2.8 Hz, 1H), 2.98–2.93 (m, 1H), 2.79 (dd, J = 4.8, 2.6 Hz, 1H0; ¹³C NMR (125 MHz, CDCl₃) δ 182.4, 164.3, 164.07, 162.2, 157.72, 131.8, 131.2, 129.8, 126.3, 106.0, 105.9, 98.6, 93.3, 69.2, 49.2, 44.5; HRMS (ESI): m/z calcd. for C₁₈H₁₄O₅ [M + H]⁺ 311.0919, found 311.0920.

3.1.3. Typical Procedure for the Synthesis of 3a–k

To a solution of secondary amine (1.5 mmol) in ACN (10 mL), CS₂ (3 mmol) was added drop wise. The reactions mixture was then stirred at room temperature for 30 min. To this reaction mixture, 2 (1 mmol) and LiBr (0.04 mmol) were added and then stirred at 60 °C for an appropriate time as monitored by TLC. After completion, the reaction mixture was diluted with ice cold water and extracted with EtOAc. Then, evaporation of EtOAc gave a crude residue which was further purified by column chromatography (silica gel, ethyl acetate/hexane as eluent) to afford the designed products (3a–k).

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy) propyl piperidine-1-carbodithioate (3a): m.p: 134–136 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.71 (s, 1H), 7.89 (dd, J = 8.1, 1.5 Hz, 2H), 7.59–7.49 (m, 3H), 6.67 (s, 1H), 6.56 (d, J = 2.2 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 4.40–4.35 (m, 1H), 4.31 (s, 2H), 4.15 (ddd, J = 15.3, 9.5, 5.4 Hz, 1H), 3.95 (s, 2H), 3.85 (dd, J = 14.6, 4.3 Hz, 2H), 3.68 (dd, J = 14.6, 6.8 Hz, 2H), 3.28 (d, J = 4.4 Hz, 1H), 1.73 (br.s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 195.5, 182.4, 164.4, 164.1, 162.2, 157.7, 131.8, 131.3, 129.1, 126.3, 106, 105.9, 98.8, 93.2, 71, 69.5, 39.5, 24.2; HRMS (ESI): m/z calcd. for C₂₄H₂₅NO₅S₂ [M + H]⁺ 472.1252, found 472.1258.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl pyrrolidine-1-carbodithioate (3b): m.p: 144–146 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.71 (s,1H), 7.89 (dd, J = 8.1, 1.5 Hz, 2H), 7.59–7.49 (m, 3H), 6.67 (s, 1H), 6.56 (d, J = 2.2 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 4.40–4.35 (m, 1H), 4.31 (s, 2H), 4.15 (ddd, J = 15.3, 9.5, 5.4 Hz, 1H), 3.95 (s, 2H), 3.85 (dd, J = 14.6, 4.3 Hz, 2H), 3.68 (dd, J = 14.6, 6.8 Hz, 2H), 3.28 (d, J = 4.4 Hz, 1H), 1.73 (br.s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 195.5, 182.4, 164.4, 164.1, 162.2, 157.7, 131.8, 131.3, 129.1, 126.3, 106, 105.9, 98.8, 93.2, 71, 69.5, 39.5, 24.2; HRMS (ESI): m/z calcd. for C₂₃H₂₃NO₅S₂ [M + H]⁺ 458.1096, found 458.1106.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl morpholine-4-carbodithioate (3c): m.p: 146–150 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.71 (s, 1H), 7.89 (dd, J = 8.1, 1.5 Hz, 2H), 7.57–7.50 (m, 3H), 6.68 (s, 1H), 6.55 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.42–4.35 (m, 2H), 4.15 (ddd, J = 15.4, 9.6, 5.3 Hz, 1H), 4.02 (br.s., 2H), 3.85 (dd, J = 14.5, 4.3 Hz, 1H), 3.79 (s, 4H), 3.69 (dd, J = 14.5, 7.0 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 197.5, 182.5, 164.3, 164.1, 162.2, 157.7, 131.9, 131.3, 129.1, 126.3, 106.0, 105.9, 98.7, 93.2, 71.0, 69.3, 39.4. HRMS (ESI): m/z calcd. for C₂₃H₂₃NO₆S₂ [M + H]⁺ 474.1045, found 474.1043.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl 4-benzylpiperazine-1-carbodithiote (3d): m.p: 154–156 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.71 (s, 1H), 7.89 (dd, J = 8.1, 1.5 Hz, 2H), 7.59–7.49 (m, 3H), 7.37–7.30 (m, 5H), 6.68 (s, 1H), 6.55 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.37 (s, 3H), 4.15 (ddd, J = 15.4, 9.5, 5.3 Hz, 1H), 3.99 (s, 2H), 3.84 (dd, J = 14.6, 4.3 Hz, 1H), 3.67 (dd, J = 14.6, 6.9 Hz, 1H), 3.55 (s, 2H), 3.18 (s, 1H), 2.56 (br.s., 4H); ¹³C NMR (125 MHz, CDCl₃) δ 196.6, 182.4, 164.4, 164.1, 162.2, 157.7, 137.2, 131.8, 131.3, 129.1, 128.4, 127.4, 126.3, 106, 105.9, 98.8, 93.2, 71, 69.4, 62.4, 52.3, 39.5; HRMS (ESI): m/z calcd. for C₃₀H₃₀N₂O₅S₂ [M + H]⁺ 563.1674, found 563.1686.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl thiomorpholine-4-carbodithioate (3e): m.p: 168–170 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.72 (s, 1H), 7.89 (dd, J = 8.1, 1.5 Hz, 2H), 7.58–7.50 (m, 3H), 6.68 (s, 1H), 6.56 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.64 (br.s, 2H), 4.41–4.34 (m, 2H), 4.32 (br.s, 1H), 4.15 (ddd, J = 15.4, 9.5, 5.3 Hz, 1H), 3.85 (dd, J = 14.5, 4.3 Hz, 1H), 3.68 (dd, J = 14.5, 6.9 Hz, 1H), 3.07 (d, J = 4.5 Hz, 1H), 2.78 (s, 4H); ¹³C NMR (125 MHz, CDCl₃+DMSO-d₆) δ 196.7, 182.4, 164.5, 164.1, 162.1, 157.7, 131.8, 131.2, 129.1, 126.3, 105.9, 105.8, 98.8, 93.2, 71.2, 68.9, 29.6, 27.2; HRMS (ESI): m/z calcd. for C₂₃H₂₃NO₅S₃ [M + H]⁺ 490.0817, found 490.0822.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl 4-(4-fluorophenyl) piperazine-1-carbodithiate (3f): m.p: 146–148 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.72 (s, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.54 (m, 3H), 7.03–6.95 (m, 2H), 6.88 (dd, J = 9.1, 4.5 Hz, 2H), 6.68 (s, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 4.52 (br.s, 2H), 4.45–4.36 (m, 1H), 4.16 (ddd, J = 15.3, 9.5, 5.3 Hz, 1H), 3.86 (dd, J = 14.6, 4.3 Hz, 1H), 3.70 (dd, J = 14.6, 4.3 Hz, 1H), 3.26–3.18 (m, 4H), 3.13 (d, J = 4.5 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 197.2, 182.4, 164.3, 164.1, 162.2, 158.7, 157.7, 156.8, 146.88 (d, J = 2.4 Hz), 131.8, 131.3, 129.1, 126.3, 118.46 (d, J = 7.8 Hz), 115.9, 115.7, 106, 105.9, 98.7, 93.2, 71, 69.3, 49.9, 39.3; HRMS (ESI): m/z calcd. for C₂₉H₂₇FN₂O₅S₂ [M + H]⁺ 567.1424, found 567.1439.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl 4-(pyridine-2-yl) piperazine-1-carbodithioate (3g): m.p: 152–154 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.71 (s, 1H), 8.20 (dd, J = 4.9, 1.2 Hz, 1H), 7.89 (dd, J = 8.0, 1.5 Hz, 2H), 7.59–7.48 (m, 4H), 6.71–6.69 (m, 1H), 6.68 (d, J = 2.5 Hz, 1H), 6.63 (d, J = 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 4.49 (s, 1H), 4.40 (s, 1H), 4.18 (td, J = 11.9, 6.0 Hz, 2H), 4.15 (ddd, J = 15.3, 9.5, 5.4 Hz, 1H), 3.86 (dt, J = 18.6, 9.3 Hz, 1H), 3.70 (m, 6H), 3.19 (s, 1H); ¹³C NMR (125 MHz, CDCl₃ + DMSO-d₆) δ 196.6, 182.3, 164.9, 164, 161.9, 158.4, 157.7, 147.8, 137.7, 131.9, 131.1, 129.1, 126.3, 113.8, 107, 105.7, 105.6, 98.9, 93.3, 71.8, 68.1, 44.2; HRMS (ESI): m/z calcd. for C₂₈H₂₇N₃O₅S₂ [M + H]⁺ 550.1470, found 550.1492.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl 4-(4-methoxyphenyl) piperazine-1-carbodithioate (3h): m.p: 151–153 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.71 (s, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.59–7.47 (m, 3H), 6.90 (d, J = 9.1 Hz, 2H), 6.85 (d, J = 9.1 Hz, 2H), 6.67 (s, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 4.51 (br. s, 2H), 4.39 (dd, J = 10.2, 4.6 Hz, 1H), 4.16 (ddd, J = 15.3, 9.5, 5.3 Hz, 1H), 3.86 (dd, J = 14.6, 4.3 Hz, 1H), 3.77 (s, 3H), 3.70 (dd, J = 14.6, 6.9 Hz, 1H), 3.21–3.15 (m, 5H); ¹³C NMR (125 MHz, CDCl₃) δ 197, 182.4, 164.4, 164.1, 162.2, 157.7, 154.6, 144.5, 131.8, 131.3, 129.1, 126.3, 118.9, 114.6, 106, 105.9, 98.8, 93.2, 71, 69.4, 55.6, 50.5, 39.6; HRMS (ESI): m/z calcd. for C₃₀H₃₀N₂O₆S₂ [M + H]⁺ 579.1624, found 579.1627.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7yl)oxy)propyl cis-3,5-dimethylmorpholine-4-carbodithioate (3i): m.p: 181–183 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.72 (s, 1H), 7.89 (dd, J = 8.1, 1.5 Hz, 2H), 7.63–7.43 (m, 3H), 6.68 (s, 1H), 6.55 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 5.45 (s, 1H), 4.51 (s, 1H), 4.44–4.33 (m, 1H), 4.15 (ddd, J = 15.4, 9.5, 5.3 Hz, 1H), 3.85 (dd, J = 14.5, 4.3 Hz, 1H), 3.68 (dd, J = 14.5, 6.9 Hz, 3H), 3.13 (d, J = 4.4 Hz, 1H), 2.95 (s, 1H), 2.79 (s, 1H), 1.26 (s, 3H), 1.25 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 196.9, 182.4, 164.3, 164.1, 162.3, 157.7, 131.8, 131.3, 129.1, 126.3, 106, 105.9, 98.7, 93.2, 71, 69.3, 39.3, 18.5; HRMS (ESI): m/z calcd. for C₂₅H₂₇NO₆S₂ [M + H]⁺ 502.1358, found 502.1366.

tert-Butyl 4-(((2-hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl)thio) carbonothioyl)piperazine-1-carboxylate (3j): m.p: 140–142 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.72 (s, 1H), 7.89 (d, J = 6.6 Hz, 2H), 7.61–7.48 (m, 3H), 6.68 (s, 1H), 6.56 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.38 (dd, J = 10.5, 5.7 Hz, 2H), 4.15 (ddd, J = 15.4, 9.5, 5.3 Hz, 1H), 4.00 (br.s, 2H), 3.85 (dd, J = 14.6, 4.3 Hz, 1H), 3.68 (dd, J = 14.6, 7.0 Hz, 1H), 3.60–3.54 (m, 4H), 3.11 (d, J = 4.4 Hz, 1H), 1.48 (s, 9H); ¹³C NMR (125 MHz, CDCl₃) δ 197.5, 182.4, 164.3, 164.1, 162.2, 157.7, 154.4, 131.6, 131.3, 129.1, 126.3, 106, 105.9, 98.7, 93.2, 80.7, 71, 69.3, 39.6, 28.3; HRMS (ESI): m/z calcd. for C₂₈H₃₂N₂O₇S₂ [M + H]⁺ 573.1729, found 573.1731.

2-Hydroxy-3-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)propyl diethyl carbamodithioate (3k): m.p: 158–160 °C; ¹H NMR (500 MHz, CDCl₃) δ 12.71 (s, 1H), 7.89 (d, J = 6.7 Hz, 2H), 7.60–7.47 (m, 3H), 6.68 (s, 1H), 6.56 (d, J = 2.2 Hz, 1H), 6.40 (d, J = 2.2 Hz, 1H), 4.38 (dd, J = 11.2, 5.2 Hz, 1H), 4.15 (ddd, J = 15.3, 9.5, 5.4 Hz, 1H), 4.05 (dt, J = 10.8, 6.7 Hz, 2H), 3.82 (m, 3H), 3.67 (dd, J = 14.7, 6.8 Hz, 1H), 3.35 (d, J = 4.4 Hz, 1H), 1.33 (t, J = 7.1 Hz, 3H), 1.29 (t, J = 7.1 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 195.7, 182.4, 164.4, 164, 162.2, 157.7, 131.8, 131.3, 129, 126.3, 106, 105.9, 98.8, 93.2, 71, 69.5, 50.2, 47.1, 39.5, 12.5, 11.5; HRMS (ESI): m/z calcd. for C₂₃H₂₅NO₅S₂ [M + H]⁺ 460.1252, found 460.1256.

3.2. Preparation of the Protein and the Ligand for Docking Simulations

Chemdrawsuite [68] was used to generate the optimized structures of the synthesized analogues (3a–k) and energy was minimized using OPLS 2005 force field [69] through Ligprep module of Schrodinger Suite 2013 [70]. Docking simulations were carried on the optimized structures of the synthesized compounds using .sdf format.

The crystal structure of E. coli FabH with accession number (1HNJ) was retrieved from the Protein Data Bank (PDB) for docking simulations. Molegro Virtual Docker, version 5.5, was used to perform the docking. Protein structure for the docking procedure was prepared by removing the solvent molecules and structural parameters of ligands like hybridization, bond order, precise hydrogen atoms were assigned using Molegro Virtual Docker software. Based on the requirement, charges were assigned. Detect cavities option was used to obtain possible binding sites in preparation tools and five cavities were obtained. The cavity around the anion binding site (volume of 177 ų) was used for docking calculations and further modified using side chain minimization. Grid-based Mol-Dock score (GRID) function was used to carry docking calculations with a grid resolution of 0.20 Å. Based on the Mol Dock score and Rerank score, the best ligand poses were chosen. The docking calculations were performed with a dual processor, Windows 7 based computer with 4 GB RAM and each docking process took 10–15 min. COOT graphical program [71] was used to perform molecular alignment with ALIGN program. Protein-ligand interaction studies were performed using Accelrys Discovery Studio v3.5 [72].

3.3. Docking Simulation of the Synthesized Compounds

Molgro Virtual Docker 2010.4.0 molecular docking program predicts the interaction of the molecules with a protein receptor. The structure based virtual screening of the compounds was carried based on Mol Dock scoring function (MolDock Score) derived from the Piecewise Linear Potential (PLP) scoring functions [73]. Further, the total energy was minimized using Melder Mead Simplex Minimization using non-grid force field and H-bond directionality [74]. Protein–receptor interactions with the compound were evaluated based on internal electrostatic, hydrogen bond interactions and sp²–sp² torsions and binding affinity. The synthesized compounds with highest binding affinity against E. coli FabH protein (1HNJ), S. cerevisiae (5EQB) were selected as a function of Moldock score.

3.4. Biological Activity and ADME Properties of Compounds

For all the synthesized compounds, drug-likeness was evaluated using Lipinski filters and biological activity was predicted using Molinspiration webserver (©Molinspiration Cheminformatics 2018).

3.5. Softwares, Suites, and Webservers

MarvinSketch 5.6.0.2 (1998–2011, Copyright © Chem Axon Ltd.) was used to design the compounds and they were docked using Molegro Virtual Docker 2010.4.0.0. For the molecular visualizations, Accelrys Discovery Studio^® Visualiser 3.5.0.12158 (Copyright © 2005-12, Accelrys Software Inc.) was used and various solubility parameters were calculated by applying QikProp module of Schrodinger suite 2013.

Computer-aided drug design was used for developing potential E. coli FabH protein (1HNJ), S. cerevisiae (5EQB) organism inhibitors, which enable the prediction of the ligand-binding site and to suggest possible interactions with the ligands. Molecular docking simulations were performed based on the binding model for the synthesized analogues (3a–k) with the proteins of E. coli FabH and S. cerevisiae. Putative interactions proposed by the best docked position were used as a template to evaluate the drug candidates. The active site of E. coli FabH generally contains Cys–His–Asn catalytic triad tunnel which is sustained in various bacteria and is important in the regulation of chain elongation and substrate binding. The interaction between Cys and substrate plays a key role in substrate binding, since the alkyl chain of CoA is broken by Cys of the catalytic triad of E. coli FabH.

4. Conclusions

In conclusion, a series of novel 2-hydroxy-3-chrysino dithiocarbamate analogues (3a–k) were synthesized in moderate to good yields and assessed for their in vitro antimicrobial activities. These antimicrobial studies indicated that most of the derivatives manifested moderate to good biological activities compared to the standard drugs penicillin and itrazole. Among the synthesized analogues, 3d, 3f, and 3j showed remarkable antimicrobial activities. In addition to the antimicrobial screening, molecular modeling studies were also performed to support these biological activities, providing further insight into the interactions of the synthesized ligands with the protein of E. coli FabH and S. cerevisiae. These docking scores are in good correlation with the experimental antimicrobial results. We hope these studies will be useful in developing the new drug entities as potential chemotherapeutic agents in controlling the microbial epidemics.

Supplementary Materials

Supporting information available online, includes 1H NMR, 13C NMR, and HRMS-ESI spectra of the synthesized compounds.

Author Contributions

P.R. performed the experiments and analyzed the data, V.S.R. executed molecular docking studies, and Y.-A.H. performed the biological activity experiments. P.M.R. and A.H. wrote the paper. All authors contributed and accepted the final version of the manuscript.

Funding

This research was funded by Buddhist Tzu-Chi General Hospital, Tzu-Chi University, grant number "TCMRC-P-107012" and the Ministry of Science and Technology (MOST 108-2113-M-320-001), Taiwan.

Acknowledgments

The authors thank IICT scientists Dr. K. Suresh Babu and Dr. M. Srinivasa Rao for their assistance in chemical characterization and anti-microbial screening.

Conflicts of Interest

The authors declare no conflict of interest.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

References

1. Newman, D.J.; Cragg, G.M.; Snader, K.M. Natural products as sources of new drugs over the period 1981-2002. J. Nat. Prod. 2003, 66, 1022-1037.

2. Liu, R.H. Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Amer. J. Clin. Nutr. 2003, 78, 517-520.

3. Surh, Y.H. Cancer chemoprevention with dietary phytochemicals. Nat. Rev. Cancer 2003, 3, 768-780.

4. De Kok, T.M.; van Breda, S.G.; Manson, M.M. Mechanisms of combined action of different chemo preventive dietary compounds: A review. Eur. J. Nutr. 2008, 47, 51-59.

5. Park, S. Cyclic glucans enhance solubility of bioavailable flavonoids. Molecules 2016, 21, 1556.

6. Sobeh, M.; Petruk, G.; Osman, S.; El Raey, M.A.; Imbimbo, P.; Monti, D.M.; Wink, M. Isolation of myricitrin and 3,5-di-O-mehtyl gossypetin from syzygiumsamarangense and evaluation of their involvement in protecting keratinocytes against oxidative stress via activation of the Nrf-2 pathway. Molecules 2019, 24, 1839.

7. Yang, Y.; Zhang, T. Antimicrobial activities of tea polyphenol on phytopathogens: A review. Molecules 2019, 24, 816.

8. Lin, J.K.; Tsai, S.H.; Lin, S.Y. Anti-inflammatory and antitumor effects of flavonoids and flavanoids. Drugs Future 2001, 26, 145-152.

9. Mierziak, J.; Kostyn, K.; Kulma, A. Flavonoids as important molecules of plant interactions with the environment. Molecules 2014, 19, 16240-16265.

10. Mahomoodally, M.F.; Fakim, A.G.; Subratty, A.H. Antimicrobial activities and phytochemical profiles of endemic medicinal plants of Mauritius. Pharm. Biol. 2005, 43, 237-242.

11. Pandey, A.K. Anti-staphylococcal activity of a pan-tropical aggressive and obnoxious weed parthenium histerophorus: An in vitro study. Natl. Acad. Sci. Lett. 2007, 30, 383-386.

12. Bohm, B.A. Introduction to Flavonoids; Gordon & Breach: Amsterdam, The Netherlands, 1998.

13. Harborne, J.B.; Baxter, H. (Eds.) The Handbook of Natural Flavonoids; Wiley: Chichester, UK, 1999; Volume 1&2.

14. Havsteen, B.H. The biochemistry and medical significance of the flavonoids. Pharm. Ther. 2002, 96, 67-202.

15. Skibola, C.F.; Smith, M.T. Potential health impacts of excessive flavonoid intake. Free Rad. Biol. Med. 2000, 29, 375-383.

16. Cazalolli, L.H.; Zanatta, L.; Alberton, E.H.; Figueiredo, M.S.R.B.; Folador, P.; Damazio, D.G.; Pizzolatti, F.; Silva, F.R.M.B. Flavonoids: Prospective drug candidates. Mini. Rev. Med. Chem. 2008, 8, 1429-1440.

17. Lalou, C.; Basak, P.; Mohantra, B.C.; Banik, R.; Dinda, B.; Khatib, A.M. Inhibition of tumor cells by the flavonoid furin inhibitor isolated from oroxylumindicum. Curr. Med. Chem. 2013, 20, 583-591.

18. Suresh Babu, K.; HariBabu, T.; Srinivas, P.V.; Sastry, B.S.; Hara Kishore, K.; Murthy, U.S.N.; Rao, J.M. Synthesis and in vitro study of novel 7-O-acyl derivatives of Oroxylin A as antibacterial agents. Bioorg. Med. Chem. Lett. 2005, 15, 3953-3956.

19. Prakash, O.; Kumar, R.; Prakash, V. Synthesis and antifungal activity of some new 3-hydroxy-2-(1-phenyl-3-aryl-4-pyarazolyl) chromones. Eur. J. Med. Chem. 2008, 43, 435-440.

20. Losgen, S.; Magull, J.; Schulz, B.; Draeger, S.; Zeeck, A. Isofusidienols: Novel chromone-3-oxepines produced by the endophytic fungus Chalara sp. Eur. J. Org. Chem. 2008, 4, 698-703.

21. Ali, E.S.T.; Abdel-Aziz, S.A.G.; El-Shaaer, H.M.; Hanafy, F.I.; El-Fauomy, A.Z. Synthesis of some new 4-oxo-4H-chromene derivatives bearing nitrogen heterocyclic systems as antifungal agents. Turk. J. Chem. 2008, 32, 365-374.

22. Hutter, J.A.; Salman, M.; Stavinoha, W.B.; Satangi, N.; Williams, R.F.; Streeper, R.T.; Weintraub, S.T. Anti-inflammatory C-glucosyl chromone from aloe barbadensis. J. Nat. Prod. 1996, 59, 541-543.

23. Mathias, L.; Silva, D.; Bernadete, P.; Mors, B.W.; Parente, J.P. Isolation and structural elucidation of a novel rotenoid from the seeds of clitoriafairchildiana. Nat. Prod. Res. 2005, 19, 325-329.

24. Birt, D.F.; Hendrich, S.; Wang, W. Dietary agents in cancer prevention: Flavonoids and isoflavonoids. Pharmacol. Ther. 2001, 90, 157-177.

25. Huang, W.; Ding, Y.; Miao, Y.; Liu, M.Z.; Li, Y.; Yang, G.F. Synthesis and antitumor activity of novel dithiocarbamate substituted chromones. Eur. J. Med. Chem. 2009, 44, 3687-3696.

26. Shaw, A.Y.; Chang, C.Y.; Liau, H.H.; Lu, P.J.; Chen, H.L.; Yang, C.N.; Li, H.Y. Synthesis of 2-styrylchromones as a novel class of antiproliferative agents targeting carcinoma cells. Eur. J. Med. Chem. 2009, 44, 2552-2562.

27. Pisco, L.; Kordian, M.; Peseke, K.; Feist, H.; Michalik, D.; Estrada, E.; Carvalho, J.; Hamilton, G.; Rando, D.; Quincoces, J. Synthesis of compounds with antiproliferative activity as analogues of prenylated natural products existing in Brazilian propolis. Eur. J. Med. Chem. 2006, 41, 401-407.

28. Kuroda, M.; Uchida, S.; Watanabe, K.; Mimaki, Y. Chromones form the tubers of eranthiscilicica and their antioxidant activity. Phytochemistry 2009, 70, 288-293.

29. Ly, C.; Yockell, L.J.; Ferraro, Z.M.; Arnason, J.T.; Ferrier, J.; Gruslin, A. The effects of dietary polyphenols on reproductive health and early development. Hum. Reprod. Update 2015, 21, 228-248.

30. Guo, K.; Liang, Z.; Liu, L.; Li, F.; Wang, H. Flavonoids intake and risk of prostate cancer: A meta-analysis of observational studies. Andrologia 2016, 48, 1175-1182.

31. Qais, N.; Rahman, M.M.; Rashid, M.A.; Koshino, H.; Nagasawa, K.; Nakata, T. A new C-benzylated chalcone from Desmos chinensis. Fitoterpia 1996, 67, 511-514.

32. Fishkin, R.J.; Winslow, J.T. Endotoxin-induced reduction of social investigation by mice: Interaction with amphetamine and anti-inflammatory drugs. Psychopharmacology (Berl.) 1997, 132, 335-341.

33. Pearce, F.L.; Befus, A.D.; Bienenstock, J. Mucosal mast cells: III. Effect of quercetin and other flavonoids on antigen-induced histamine secretion from rat intestinal mast cells. J. Allergy. Clin. Immunol. 1984, 73, 819-823.

34. Hecker, M.; Preiss, C.; Klemm, P.; Busse, R. Inhibition by antioxidants of nitric oxide synthase expression in murine macrophages: Role of nuclear factor kB and interferon regulatory factor 1". Br. J. Pharmcol. 1996, 118, 2178-2184.

35. Cardenas, M.; Marder, M.; Blank, V.C.; Roguin, L.P. Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines. Bioorg. Med. Chem. 2006, 14, 2966-2971.

36. Wei, F.; Jubo, W.; Liqin, Y.; Li, Z.; Na, L.; Qidong, Y.; Qinglong, G.; Zhiyu, L. Synthesis and biological evaluation of 7-O-modified oroxylin A derivatives. Bioorg. Med. Chem. Lett. 2012, 22, 1118-1121.

37. Kun, H.; Wei, W.; Hong, C.; Sha, S.P.; Jie, R. Synthesis and cytotoxicity of novel chrysin derivatives. Med. Chem. Res. 2011, 20, 838-846.

38. Li, H.Q.; Shi, L.; Li, Q.S.; Liu, P.G.; Luo, Y.; Zhao, J.; Zhu, H.L. Synthesis of C(7) modified chrysin derivatives designing to inhibit β-ketoacyl carrier protein synthase III (FabH) as antibiotics. Bioorg. Med. Chem. 2009, 17, 6264-6269.

39. Bonini, C.; Righi, G. Regio- and chemoselective synthesis of halohydrins by cleavage of oxiranes with metal hydrides. Synthesis 1994, 3, 225-238.

40. Azizi, N.; Pourhasan, B.; Aryanasab, F.; Saidi, M.R. A simple and novel eco-friendly process for the one-pot synthesis of dithiocarbamates from amines, carbon disulphide and epoxides. Synlett 2007, 8, 1239-1242.

41. Azizi, N.; Gholibeglo, E.; Maryami, M.; Nayeri, S.D.; Bolourtchian, S.M. Ultrasound mediated efficient ring opening of epoxides by in situ generated dithiocarbamates in green reaction media. C. R. Chim. 2013, 16, 412-418.

42. Morf, P.; Raimondi, F.; Nothofer, H.G.; Schnyder, B.; Yasuda, A.; Wessels, J.M.; Jung, T.A. Dithiocarbamates: Functional and versatile linkers for the formation of self-assembled monolayers. Langmuir 2006, 22, 658-663.

43. McClain, A.; Hsieh, Y.L. Synthesis of polystyrene-supported dithiocarbamates and their complexation with metal ions. J. Appl. Polym. Sci. 2004, 92, 218-225.

44. Dunn, A.D.; Rudorf, W.D. Carbon Disulphide in Organic Chemistry; Ellis Hordwood: Chichester, UK, 1989; pp. 226-367.

45. Griffin, T.S.; Woods, T.S.; Klayman, D.L. Thioureas in the synthesis of Heterocycles. In Advances in Heterocyclic Chemistry; Academic Press Inc., Elsevier: Amsterdam, The Netherlands, 1975; Volume 18, pp. 99-158.

46. Liao, S.; Raung, S.; Chen, C. Japanese encephalitis virus stimulates superoxide dismutase activity in rat glial cultures. Neurosci. Lett. 2002, 324, 133-136.

47. Gursoy, A.; Ates, O.; Karali, N.; Cesur, N.; Kiraz, M. Synthesis and antifungal activity of new carbamodithioc acid esters derived from 3-acetylcoumarin. Eur. J. Med. Chem. 1996, 31, 643-646.

48. Ronconi, L.; Marzano, C.; Zanello, P.; Corsini, M.; Miolo, G.; Macca, C.; Trevisan, A.; Fregona, D. Gold (III) dithiocarbamate derivatives for the treatment of cancer: Solution chemistry, DNA binding and hemolytic properties. J. Med. Chem. 2006, 49, 1648-1657.

49. Bandari, S.K.; Kammari, B.R.; Madda, J.; Kommu, N.; Lakkadi, A.; Vuppala, S.; Tigulla, P. Synthesis of new chromeno-carbamodithioate derivatives and preliminary evaluation of their antioxidant activity and molecular docking studies. Bioorg. Med. Chem. Lett. 2017, 27, 1256-1260.

50. Thorn, G.D.; Ludwig, R.A. The Dithiocarbamates and Related Compounds; Elsvier: Amsterdam, The Netherlands, 1962.

51. Nace, H.R. The preparation of olefins by the pyrolysis of xanthates: The chugaev reaction. Org. React. 1962, 12, 57.

52. Rafin, C.; Veignie, E.; Sanchole, M.; Postel, D.; Len, C.; Villa, P.; Ronco, G. Synthesis and antifungal activity of novel bisdithiocarbamate derivatives of carbohydrates against fusarium oxysporum f. sp. lini. J Agric. Food Chem. 2000, 48, 5283-5287.

53. Ramesh, P.; Sanjeeva Reddy, C.; Suresh Babu, K.; Muralidhar Reddy, P.; SrinivasaRao, V.; Parthasarathy, T. Synthesis, characterization and molecular docking studies of novel 2-amino 3-cyano pyrano [2,3H]chrysin derivatives as potential antimicrobial agents. Med. Chem. Res. 2015, 24, 3696-3709.

54. Suresh Babu, K.; HariBabu, T.; Srinivas, P.V.; Hara Kishore, K.; Rao, J.M. Synthesis and biological evaluation of novel C (7) modified chrysin analogues as antibacterial agents. Bioorg. Med. Chem. Lett. 2006, 16, 221-224.

55. Patel, M.A.; Bhila, V.G.; Patel, N.H.; Patel, A.K.; Brahmbhatt, D.I. Synthesis, characterization and biological evaluation of some pyridine and quinoline fused chromenone derivatives. Med. Chem. Res. 2012, 21, 4381-4388.

56. Shridhar, R.; Perumal, P.T.; Etti, S.; Shanmugam, G.; Ponnuswamy, M.N.; Prabavathy, V.R.; Mathivanan, N. Design, synthesis and anti-microbial activity of 1H-pyrazole carboxylates. Bioorg. Med. Chem. Lett. 2004, 14, 6035-6040.

57. Keche, A.P.; Hatnapure, G.D.; Tale, R.T.; Rodge, A.H.; Birajdar, S.S.; Kamble, V.M. Synthesis, anti-inflammatory and antimicrobial evaluation of novel N1-(quinolin-4yl)ethane-1,2-diamine phenyl urea derivatives. Med. Chem. Res. 2013, 22, 1480-1487.

58. Shamroukh, A.H.; Zaki, M.E.A.; Morsy, E.M.H.; Abdel Motti, F.M.; Abdel Megeid, F.M.E. Synthesis, isomerization and antimicrobial evaluation of some pyrazolopyranotriazolopyrimindine derivatives. Arch. Pharm. 2007, 340, 345-351.

59. Khandekar, S.S.; Daines, R.A.; Lonsdale, J.T. Bacterial β-Ketoacyl-acyl carrier protein synthases as targets for antibacterial agents. Curr. Protein Pept. Sci. 2003, 4, 21-29.

60. Heath, R.J.; Rock, C.O. The claisen condensation in biology. Nat. Prod. Rep. 2002, 19, 581-596.

61. Tsay, J.T.; Oh, W.; Larson, T.J.; Jakowski, S.; Rock, C.O. Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) form Escherichia coli K-12. J. Biol. Chem. 1992, 267, 6807-6814.

62. Clough, R.C.; Matthis, A.L.; Barnum, S.R.; Jaworski, J.G. Purification and characterization of 3-ketoacyl-acyl carrier protein synthase III from spinach. A condensing enzyme utilizing acetyl-coenzyme A to initiate fatty acid synthesis. J. Biol. Chem. 1992, 267, 20992-20998.

63. Heath, R.J.; Rock, C.O. Regulation of fatty acid elongation and initiation by acyl-acyl carrier protein in Escherichia coli. J. Biol. Chem. 1996, 271, 1833-1836.

64. Molinspiration Cheminformatics, Bratislava, Slovak Republic. Available online: http://www.molinspiration.com/services/properties.html (accessed on 22 April 2010).

65. Lipinski, C.A.; Lombardo, L.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimated solubility and permeability in drug discovery and development settings. Adv. Drug Deliv.Rev. 2001, 46, 3-26.

66. Zhao, Y.H.; Abraham, M.H.; Le, J. Rate-limited steps of human oral absorption and QSAR studies. Pharm. Res. 2002, 19, 1446-1457.

67. Drug-Likeness and Molecular Property Prediction. Available online: http://www.molsoft.com/mptop (accessed on 11 April 2019).

68. Devi, A.M.; Gopinath, G.; Srinivas, B.; Venu, S.; Madhavi, M.; Sudha, N.S.; Sudhakar, K.; Rani, A.R.; Rao, S.S. Virtual screening of RAGE inhibitors using molecular docking. Bioinformation 2016, 12, 124-130.

69. Jorgensen, W.L.; Tirado-Rives, J. Potential energy functions for atomic level simulations of water and organic and bimolecular systems. Proc. Nat. Acad. Sci. USA 2005, 102, 6665-6670.

70. LigPrep; Version 2.3; Schrodinger, L.L.C.: New York, NY, USA, 2009.

71. Brown, A.; Long, F.; Nicholis, R.A.; Toots, J.; Emsley, P.; Murshudov, G. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D Biol. Crystallogr. 2015, 1, 136-153.

72. Brooks, R.R.; Brooks, C.L.; Mackerell, A.D., Jr.; Nilsson, L.; Petrella, R.J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S.; et al. CHARMM: The biomolecular simulation program. J. Comp. Chem. 2009, 30, 1545-1614.

73. Thomsen, R.; Christensen, M.H. MolDock: A new technique for high-accuracy molecular docking. J. Med. Chem. 2006, 49, 3315-3321.

74. Nelder, J.A.; Mead, R. A simplex method for function minimization. Comput. J. 1965, 7, 308-313.