Antimicrobial Activity of Some Novel Armed Thiophene Derivatives and Petra/Osiris/Molinspiration (POM) Analyses

Tetrasubstituted 2-acetylthiophene derivative 5 was synthesized and then condensed with various nitrogen nucleophiles such as 5-amino-1,2,4-triazole, 2-aminobenzimidazole, aniline or p-chloroaniline to afford the corresponding iminothiophene derivatives 6–8a,b. Condensation of thiophene 5 with malononitrile as carbon nucleophile afforded compound 9, which underwent nucleophilic addition with DMF-DMA to afford compound 10. The newly synthesized products were characterized by elemental analysis, IR, MS, 1H-13C-NMR and CHN analysis and then evaluated for their antimicrobial activity. Results of the in vitro antibacterial activity showed that thiophene derivative 7 was found to be more potent than the standard drug gentamicin against Pseudomonas aeruginosa. Some of these compounds showed potential antimicrobial activities. Molecular docking and Osiris/Molinspiration analyses show the crucial role and impact of substituents on bioactivity and indicate the unfavorable structural parameters in actual drug design: more substitution with electronic donor group doesn’t guarantee more effective bioactivity. This study should greatly help in an intelligent and a controlled pharmacomodulation of antibiotics.

Prompted by these findings and as a continuation of our research program on the chemistry of thiophene derivatives [25][26][27][28], we report herein the synthesis using simple experimental procedures of some novel thiophene derivatives obtained using the readily accessible 2-acetylthiophene derivative 5 and incorporating triazole, benzimidazole, and substituted aniline moieties. The antimicrobial activities of the synthesized compound were also investigated. A comparison between experiment and theoretical predictions of the antibacterial activity has enabled us to identify alternative combined pharmacophore sites structures. The nature of pharmacophore site assignment of the thiophene compounds was based on their docking and Petra/Osiris/Molinspiration (POM) analyses.
Prompted by these findings and as a continuation of our research program on the chemistry of thiophene derivatives [25][26][27][28], we report herein the synthesis using simple experimental procedures of some novel thiophene derivatives obtained using the readily accessible 2-acetylthiophene derivative 5 and incorporating triazole, benzimidazole, and substituted aniline moieties. The antimicrobial activities of the synthesized compound were also investigated. A comparison between experiment and theoretical predictions of the antibacterial activity has enabled us to identify alternative combined pharmacophore sites structures. The nature of pharmacophore site assignment of the thiophene compounds was based on their docking and Petra/Osiris/Molinspiration (POM) analyses.

Synthesis
Preparation of the target compound 5 started with the reaction of ethyl benzoylacetate (1) with phenyl isothiocyanate in the presence of K 2 CO 3 and DMF as a solvent to afford the intermediate ketene N,S-acetal salt 2. Treatment of the potassium salt 2 with chloroacetone gave the desired ethyl 5-acetyl-4-phenyl-2-(phenylamino)thiophene-3-carboxylate (5) in good yield (Scheme 1).
Prompted by these findings and as a continuation of our research program on the chemistry of thiophene derivatives [25][26][27][28], we report herein the synthesis using simple experimental procedures of some novel thiophene derivatives obtained using the readily accessible 2-acetylthiophene derivative 5 and incorporating triazole, benzimidazole, and substituted aniline moieties. The antimicrobial activities of the synthesized compound were also investigated. A comparison between experiment and theoretical predictions of the antibacterial activity has enabled us to identify alternative combined pharmacophore sites structures. The nature of pharmacophore site assignment of the thiophene compounds was based on their docking and Petra/Osiris/Molinspiration (POM) analyses.

Synthesis
Preparation of the target compound 5 started with the reaction of ethyl benzoylacetate (1) with phenyl isothiocyanate in the presence of K2CO3 and DMF as a solvent to afford the intermediate ketene N,S-acetal salt 2. Treatment of the potassium salt 2 with chloroacetone gave the desired ethyl 5-acetyl-4-phenyl-2-(phenylamino)thiophene-3-carboxylate (5) in good yield (Scheme 1). Data are expressed as mean ± SD. Table 2. Antifungal activity of the synthesized thiophene derivatives 5, 7, 8a-b, 9 and 10.
Results of the in vitro antibacterial activity revealed that compounds 7, 8a and 8b had the best antibacterial activity against all tested bacteria (Table 1, Figures 2 and 3). It is widely known that Gram-negative bacteria are more resistant to antimicrobial agents than are Gram-positive ones [29]. This intrinsic resistance is attributed to the lipopolysaccharide outer membrane which acts as an efficient permeability barrier [30,31]. This, on the other hands, makes the results of the synthesized thiophene derivatives against Pseudomonas aeruginosa very promising. Also, the results in Table 1 revealed that thiophene derivative 7 was found to be more potent than the standard drug gentamicin against Pseudomonas aeruginosa. Data from the antifungal evaluation have shown that compounds 7, Scheme 3. Synthesis of thiophene derivatives 9 and 10.

Antimicrobial Evaluation
We investigated in vitro antibacterial activity of the newly synthesized compounds against two Gram-positive and two Gram-negative bacteria known to cause infections in humans. The antifungal activity of these compounds was also evaluated against four fungal species. Diameter of the inhibition zone was used as a criterion for the antimicrobial activity using a well diffusion agar method. We also included standard antimicrobial agents in the same assay to compare the potency of the tested compounds. The results are depicted in Tables 1 and 2.  Results of the in vitro antibacterial activity revealed that compounds 7, 8a and 8b had the best antibacterial activity against all tested bacteria (Table 1, Figures 2 and 3). It is widely known that Gram-negative bacteria are more resistant to antimicrobial agents than are Gram-positive ones [29]. This intrinsic resistance is attributed to the lipopolysaccharide outer membrane which acts as an efficient permeability barrier [30,31]. This, on the other hands, makes the results of the synthesized thiophene derivatives against Pseudomonas aeruginosa very promising. Also, the results in Table 1 revealed that thiophene derivative 7 was found to be more potent than the standard drug gentamicin against Pseudomonas aeruginosa. Data from the antifungal evaluation have shown that compounds 7, 8a, 8b and 10 were also active against the four tested fungal species (Figure 4, Table 2). The results suggest that the new skeletons possessing benzimidazole and thiophene moieties may provide valuable leads for the synthesis and development of novel antimicrobial agents.

Molecular Docking
Molecular docking is used to predict the binding mode of ligands within the binding site of target proteins [32]. To validate and specify the target protein for the antifungal and antibacterial activity of the newly synthesized tetrasubstituted thiophene derivatives, like in a previous study, nine different target proteins i.e., dihydrofolate reductase (DHFR) (PDB ID 4HOF), secreted aspartic protease (PDB ID 3Q70) and N-myristoyl transferase (PDB ID 1IYL) from Candida albicans were

Molecular Docking
Molecular docking is used to predict the binding mode of ligands within the binding site of target proteins [32]. To validate and specify the target protein for the antifungal and antibacterial activity of the newly synthesized tetrasubstituted thiophene derivatives, like in a previous study, nine different target proteins i.e., dihydrofolate reductase (DHFR) (PDB ID 4HOF), secreted aspartic protease (PDB ID 3Q70) and N-myristoyl transferase (PDB ID 1IYL) from Candida albicans were

Molecular Docking
Molecular docking is used to predict the binding mode of ligands within the binding site of target proteins [32]. To validate and specify the target protein for the antifungal and antibacterial activity of the newly synthesized tetrasubstituted thiophene derivatives, like in a previous study, nine different target proteins i.e., dihydrofolate reductase (DHFR) (PDB ID 4HOF), secreted aspartic protease (PDB ID 3Q70) and N-myristoyl transferase (PDB ID 1IYL) from Candida albicans were selected as antifungal targets, whereas for anti-bacterial targets dihydrofolate reductase (PDB ID 3FYV), gyrase B (PDB ID 4URM) and sortase A (PDB ID 2MLM) from Staphyllococcus aureus and rhomboid protease (PDB ID 3ZMI) from Escherichia coli were downloaded from the Protein Data Bank. All the tetrasubstituted thiophene derivatives were docked into the active site of these nine selected target proteins. After analysis of the docking results it was found that these compounds showed good interactions with two target proteins, i.e., dihydrofolate reductase from C. albicans and rhomboid protease from E. coli, while showing very poor interaction or no interactions with the rest of the target proteins. Therefore, the binding mode of these compounds within the active sites of dihyrofolate reductase and rhomboid protease were studied in more detail.
The docking results showed that all the compounds fit well in the binding pockets of dihydrofolate reductase and rhomboid protease target proteins. In the case of compound 5, good interactions with the dihydrofolate reductase and rhomboid protease were observed. As shown in Figure 5, compound 5 established two hydrogen bonds with important active site residues (Gly20 and Ile112) and a number of hydrophobic and van der Waals (VDW) interactions with active site residues (Ala11, Met25 and Phe36 etc.) of dihydrofolate reducatase. Bank. All the tetrasubstituted thiophene derivatives were docked into the active site of these nine selected target proteins. After analysis of the docking results it was found that these compounds showed good interactions with two target proteins, i.e., dihydrofolate reductase from C. albicans and rhomboid protease from E. coli, while showing very poor interaction or no interactions with the rest of the target proteins. Therefore, the binding mode of these compounds within the active sites of dihyrofolate reductase and rhomboid protease were studied in more detail.
The docking results showed that all the compounds fit well in the binding pockets of dihydrofolate reductase and rhomboid protease target proteins. In the case of compound 5, good interactions with the dihydrofolate reductase and rhomboid protease were observed. As shown in Figure 5, compound 5 established two hydrogen bonds with important active site residues (Gly20 and Ile112) and a number of hydrophobic and van der Waals (VDW) interactions with active site residues (Ala11, Met25 and Phe36 etc.) of dihydrofolate reducatase. Furthermore, the phenyl ring of the compound formed arene-cation interactions with the active site residue Thr58. Similarly, the docking conformation of compound 5 in the active site of rhomboid protease showed good interactions with the active site residues of this protein. Compound 5 formed three hydrogen bonds with active site residues (Trp157, Met249 and His254) and hydrophobic and VDW interactions with the active site residues (Phe153, Ala239 and Phe245 etc.) of rhomboid protease ( Figure 6). Compound 5 also interacted with active site residue Tyr 205 via arene-arene interactions. This strong hydrogen bonding and hydrophobic interactions with the active site residues of dihydrofolate reductase and rhomboid protease might be one of the reasons for good antifungal and antibacterial activities shown by this compound in the series. Like compound 5, good interactions for compounds 8a, 8b and 9 were observed with the active site residues of dihydrofolate reductase. With compounds 7 and 10 poor interactions were observed with the active site residues of dihydrofolate reductase. For example, as shown in Figure 7 only one hydrogen bond was established between and antibacterial activities shown by this compound in the series. Like compound 5, good interactions for compounds 8a, 8b and 9 were observed with the active site residues of dihydrofolate reductase. With compounds 7 and 10 poor interactions were observed with the active site residues of dihydrofolate reductase. For example, as shown in Figure 7 only one hydrogen bond was established between compound 7 and the active site residues of the target protein. The poor interactions and comparatively low antifungal activities of compounds 7 and 10 might be due to the presence of a more bulky moiety attached to the thiophene ring as compare to compound 5. Similarly, poor interactions of compounds 7, 8a, and 10 with the active site residues of rhomboid protease were observed. As shown in Figure 8, compound 7 which showed less anti-bacterial activity in the series established two hydrogen bonds and few hydrophobic interactions as compared to compound 5 ( Figure 6). Overall the docking results showed that the bulky moieties attached to the thiophene ring of compounds 7, 8a and 10 might be one of the reasons for their poor interactions and low antifungal and antibacterial activities as compared to compound 5.  The docking results are unable to explain the good antifungal activity of compound 8b with a similar bulky group attached to the thiophene ring of the compound as 8a. On the other hand, we have noted the crucial role and the impact of the electronic effect of chlorine atom of the 4-Cl-phenyl group of 8b, displaying an electro-donor mesomeric effect (+M), on the antifungal activity (10.9 < % inhibition < 21.5) and (12.3 < % inhibition < 22.8 ) for compounds 8b and 8a respectively ( Table 2).  The docking results are unable to explain the good antifungal activity of compound 8b with a similar bulky group attached to the thiophene ring of the compound as 8a. On the other hand, we have noted the crucial role and the impact of the electronic effect of chlorine atom of the 4-Cl-phenyl group of 8b, displaying an electro-donor mesomeric effect (+M), on the antifungal activity (10.9 < % inhibition < 21.5) and (12.3 < % inhibition < 22.8 ) for compounds 8b and 8a respectively ( Table 2).  The docking results are unable to explain the good antifungal activity of compound 8b with a similar bulky group attached to the thiophene ring of the compound as 8a. On the other hand, we have noted the crucial role and the impact of the electronic effect of chlorine atom of the 4-Cl-phenyl group of 8b, displaying an electro-donor mesomeric effect (+M), on the antifungal activity (10.9 < % inhibition < 21.5) and (12.3 < % inhibition < 22.8 ) for compounds 8b and 8a respectively ( Table 2). The docking results are unable to explain the good antifungal activity of compound 8b with a similar bulky group attached to the thiophene ring of the compound as 8a. On the other hand, we have noted the crucial role and the impact of the electronic effect of chlorine atom of the 4-Cl-phenyl group of 8b, displaying an electro-donor mesomeric effect (+M), on the antifungal activity (10.9 < % inhibition < 21.5) and (12.3 < % inhibition < 22.8 ) for compounds 8b and 8a respectively ( Table 2).  This means that any supplementary negative surcharge on the nitrogen atom will not beneficial for a perfect cis geometric structure of the antifungal pharmacophore site. This is the most notable result of this work, see Figure 9. This means that any supplementary negative surcharge on the nitrogen atom will not beneficial for a perfect cis geometric structure of the antifungal pharmacophore site. This is the most notable result of this work, see Figure 9.

POM Analyses
One of the practical problems associated with clinical drugs is the existence of various side effects. For a molecule to be potential drug, besides having a good biological activity, it must have good pharmacokinetic properties in human biological systems. To access the pharmacokinetic profile of the tested compounds, we employed well established in silico tools such as Osiris, Petra, and Molinspiration, validated with about 7000 drug molecules available in databases [33].

Osiris Calculations
Structure-based design is now a fairly routine procedure and many potential drugs do not qualify for clinic trials because of ADME-Tox liabilities. One very important class of enzymes, responsible for many ADMET problems, are the P450 cytochromes. Inhibition of these or production of unwanted metabolites can result in many adverse drug reactions. Of the most important program, Osiris is already available online [33] and very useful for its design/prediction of various activities. In our recent publications [34][35][36][37][38][39][40][41][42][43] on the drug design of various pharmacophore sites by using spiro-heterocyclic structures, we have predicted activity and/or inhibition with increasing success in

POM Analyses
One of the practical problems associated with clinical drugs is the existence of various side effects. For a molecule to be potential drug, besides having a good biological activity, it must have good pharmacokinetic properties in human biological systems. To access the pharmacokinetic profile of the tested compounds, we employed well established in silico tools such as Osiris, Petra, and Molinspiration, validated with about 7000 drug molecules available in databases [33].

Osiris Calculations
Structure-based design is now a fairly routine procedure and many potential drugs do not qualify for clinic trials because of ADME-Tox liabilities. One very important class of enzymes, responsible for many ADMET problems, are the P450 cytochromes. Inhibition of these or production of unwanted metabolites can result in many adverse drug reactions. Of the most important program, Osiris is already available online [33] and very useful for its design/prediction of various activities. In our recent publications [34][35][36][37][38][39][40][41][42][43] on the drug design of various pharmacophore sites by using spiro-heterocyclic structures, we have predicted activity and/or inhibition with increasing success in two targets, Mycobacterium tuberculosis and HIV. This is done by using a combined electronic/structure docking procedure. The remarkable mutagenicity of divers synthetic molecules classified in the CELERON (Allschwil, Switzerland) database [33], can be used to quantify the role of various organic groups in promoting or interfering the way a drug can associate with DNA [33].
The OSIRIS Property Explorer data shown on this page is an integral part of Actelion's in-house substance registration system. It allows drawing chemical structures and also calculates various drug-relevant properties whenever a structure is valid. Prediction results are color coded in which the red color shows high risks with undesired effects like mutagenicity or a poor intestinal absorption and a green color indicates drug-conforming behaviour ( Table 3).
Although two combined antibacterial and antifungal pharmacophore sites coexist in these molecules ( Figure 10), the selected compounds of series 5-10 showed low to moderate drug scores (DS < 0.50) as compared with the standard drugs used, confirmed by Osiris (Table 3) and Molinspiration (Table 4) calculations. This low bioactivity is due to low hydrosolubility which has a direct impact on bioavailability. In fact most of tested compounds suffer of high cLogP (cLogP must <5) as it was described by the Lipinski "rule of five").

Molinspiration Calculations
cLogP (octanol/water partition coefficient) is calculated by the methodology developed by Molinspiration and Osiris as a sum of fragment-based contributions and correction factors [44]. The method is very robust and is able to process practically all organic-and organometallic-based molecules. Molecular Polar Surface Area Total Polar Surface Area (TPSA) is calculated by the methodology published by Ertl et al. [44] as a sum of fragment contributions. Oand N-centered polar fragments are considered. PSA has been shown to be a very good descriptor characterizing drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability and blood-brain barrier penetration. Prediction results of the tested compounds of series 5-10 with molecular properties (TPSA, GPCR ligand and Ion Channel Modulation (ICM)) are recorded in Table 4. Most of tested compounds have one NH-O or N-HO interactions, too low in comparison with number of NH/O of standard drugs (SD1 and SD2). This explains why most of compounds represent low bioavailability. Drug likeness of series 5-10 seems correct and in same range of standard drugs (negative values).
As a result of this information, some prospective leads are under construction in our laboratories ( Figure 11). We must avoid the problem of solubility by incorporating more hydroxyl groups on various aryl and heterocyclic groups.  [44]. The method is very robust and is able to process practically all organic-and organometallic-based molecules. Molecular Polar Surface Area Total Polar Surface Area (TPSA) is calculated by the methodology published by Ertl et al. [44] as a sum of fragment contributions. O-and N-centered polar fragments are considered. PSA has been shown to be a very good descriptor characterizing drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability and blood-brain barrier penetration. Prediction results of the tested compounds of series 5-10 with molecular properties (TPSA, GPCR ligand and Ion Channel Modulation (ICM)) are recorded in Table 4. Most of tested compounds have one NH---O or N---HO interactions, too low in comparison with number of NH/O of standard drugs (SD1 and SD2). This explains why most of compounds represent low bioavailability. Drug likeness of series 5-10 seems correct and in same range of standard drugs (negative values).
As a result of this information, some prospective leads are under construction in our laboratories ( Figure 11). We must avoid the problem of solubility by incorporating more hydroxyl groups on various aryl and heterocyclic groups.

Antibacterial Activity
In vitro antibacterial screening tests of the synthesized compound was carried out against four bacterial strains: two Gram-positive (Streptococcus pneumonia and Bacillis subtilis) and two Gram-negative (Pseudomonas aeruginosa and Escherichia coli). The disc diffusion method [45,46] was used in this assay and each experiment was performed in triplicate. Readings of the zone of inhibition, which are shown in Table 1, represent the mean value of three readings. Ampicillin and gentamicin were used as standard drugs in this assay. The data obtained show that the compounds have good antibacterial activity. The antibacterial activity and inhibition zone around the tested compound can be caused by their bactericide effects (killing the bacteria) or by their bacteriostatic effects (inhibiting multiplication of bacteria by blocking their active sites on surface or inside bacterial cell).

Antifungal Activity
The synthesized thiophene derivatives 5, 7, 8a-b, 9 and 10, were subjected to in vitro antifungal activity against four fungal strains (Aspergillus fumigates, Syncephalastrum racemosum, Geotricum candidum and Candida albicans) using the disk dilution method [47]. The results are shown in Table 2. Amphotericin B was used as a standard drug. Percentage inhibition value of biological active samples was evaluated by following formula: PIFG " rGDTC{GDCsˆ100 PIFG: Percent inhibition of fungal growth. GDTC: Growth diameter in test compound (mm). GDC: Growt diameter in control (mm).
It is concluded from the antibacterial and antifungal activities that the compounds show good antibacterial and antifungal activities so they may be used as potential antimicrobial drug leads.

Conclusions
Armed derivatives of thiophene were synthesized in moderate to excellent yield of 6-7 is not "excellent" using ethyl 5-acetyl-4-phenyl-2-(phenyl-amino)thiophene-3-carboxylate (5) as principal precursor. The tested compounds, thiophene analogs armed at different positions namely at C3, C5 and substituted at C2 and C3 in the thiophene subunit were evaluated for their in vitro antimicrobial activity against selected pathogenic bacteria and fungus. The preliminary structure-activity relationship (SAR) analysis suggested that the introduction of appropriate substituent groups at position 5 of the thiophene ring enhanced the antibacterial activities of these compounds. Most of the newly synthesized products have promising antimicrobial activity, comparable to the first line standard drugs. Although we did our best to build the most efficient bioactive leads, extensive synthesis in the analogue series of compounds 5-10 is still needed in order to revise some of the structural shortcomings of the pharmacophoric sites and to produce more efficient drugs on the basis of a POM-guided better understanding of the relationship between the physicochemical properties and biological activity observed for these compounds.