Synthesis and Biological Evaluation of New Pyridothienopyrimidine Derivatives as Antibacterial Agents and Escherichia coli Topoisomerase II Inhibitors

The growing resistance of bacteria to many antibiotics that have been in use for several decades has generated the need to discover new antibacterial agents with structural features qualifying them to overcome the resistance mechanisms. Thus, novel pyridothienopyrimidine derivatives (2a,b–a,b) were synthesized by a series of various reactions, starting with 3-aminothieno[2,3-b]pyridine-2-carboxamides (1a,b). Condensation of compounds 1a,b with cyclohexanone gave 1’H-spiro[cyclohexane-1,2’-pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidin]-4’(3’H)-ones (2a,b), which in turn were utilized to afford the target 4-substituted derivatives (3a,b–8a,b). In vitro antibacterial activity evaluations of all the new compounds (2a,b–8a,b) were performed against six strains of Gram-negative and Gram-positive bacteria. The target compounds showed significant antibacterial activity, especially against Gram-negative strains. Moreover, the compounds (2a,b; 3a,b; 4a,b; and 5a,b) that exhibited potent activity against Escherichia coli were selected to screen their inhibitory activity against Escherichia coli topoisomerase II (DNA gyrase and topoisomerase IV) enzymes. Compounds 4a and 4b showed potent dual inhibition of the two enzymes with IC50 values of 3.44 µΜ and 5.77 µΜ against DNA gyrase and 14.46 µΜ and 14.89 µΜ against topoisomerase IV, respectively. In addition, docking studies were carried out to give insight into the binding mode of the tested compounds within the E. coli DNA gyrase B active site compared with novobiocin.


Introduction
Nowadays, the danger of infectious diseases is again on the rise because of the persistent evolution of antibiotic resistance, which will, over time, be a significant threat to health worldwide [1,2].
Antimicrobial-resistant infections are predicted to cause millions of deaths in the coming decades, unless suitable actions are taken to overcome this risk [3]. Thus, there is an urgent need to discover

Chemistry
The synthesis of the target pyridothienopyrimidines (2a,b-8a,b) was achieved via various reactions, as depicted in Scheme 1; Scheme 2. The starting 3-amino-6-phenylthieno [2,3-b]pyridine-2-carboxamides 1a,b were prepared [44,45] and underwent a cyclocondensation reaction with cyclohexanone in refluxing N,N-dimethyformamide containing anhydrous zinc chloride to produce 1'H-spiro[cyclohexane-1,2'-pyrido [3',2':4,5]thieno [3,2-d]pyrimidin]-4'(3'H)-ones 2a,b. The chemical structures of 2a,b were confirmed by their 1 H NMR spectra, which revealed signals at 1.21-2.02 ppm, corresponding to the 5CH 2 protons of the spiro cyclohexane ring and two signals corresponding to the 2NH groups of the pyrimidinone ring at 4.53 and 7.90 ppm and 4.90 and 7.88 ppm for 2a and 2b, respectively. Furthermore, the 13 C NMR spectra of 2a,b displayed three signals at 21.6-36.2 ppm of the 5CH 2 carbons and a signal corresponding to the spiro carbon of 2a at 69.9 ppm and a signal at 69.8 ppm for that of 2b. Subsequently, treatment of 2a,b with a refluxing POCl 3 /PCl 5 mixture gave 4'-chloro derivatives 3a,b. Then the target 1'H-spiro[cyclohexane-1,2'-pyrido [3',2':4,5]thieno [3,2-d]pyrimidin]-4'-amines 4a-e were obtained by carrying out a nucleophilic substitution reaction between 4'-chloro derivatives 3a,b and different amines (furan-2-amine, 1-methylpiperazine, morpholine, 4-acetylaniline and 4-fluoroaniline) in boiling DMF (Scheme 1). In the 1 H NMR spectrum of 4b, the protons of the N-methylpiperazinyl moiety were verified by three signals at 2.21, 2.59 and 3.75 ppm. The 13 C NMR spectrum of 4c also showed the carbons of a morpholine ring as two signals at 45.5 and 66.4 ppm, corresponding to the 2CH 2 N and 2CH 2 O moieties, respectively. Upon treatment of 2a,b with phosphorus pentasulfide in refluxing pyridine, the pyrido [3',2':4,5]thieno [3,2-d]pyrimidine]-4'(3'H)-thiones 5a,b were formed. IR spectra of 5a,b displayed new bands at 1246.2 cm −1 and 1243.2 cm −1 , corresponding to the C=S groups of 5a and 5b, besides the disappearance of the C=O bands of 2a,b at 1658.2 and 1659.4 cm −1 , respectively. The signal at 181.3 ppm in the 13 C NMR spectrum of 5a and at 182.1 ppm in the 13 C NMR spectrum of 5b also assisted the presence of the C=S carbon. Subsequent reaction of 4'(3'H)-thiones 5a,b with ethyl 2chloroacete in DMF containing a catalytic amount of anhydrous sodium carbonate afforded the formation of ethyl 2-(pyrido [3',2':4,5]thieno [3,2-d]pyrimidin-4'-yl)thio)acetates 6a,b via S-alkylation at position 4. The steric hindrance of the bulky spiro cyclohexane moiety was a possible explanation for the forbidding of N-alkylation at position 1. 1 H NMR spectra of 6a showed a singlet signal at 3.89 ppm of SCH2 protons, while the CH3 and CH2O protons of the ester group were represented by the triplet signal at 1.07 ppm and quartet signal at 4.08 ppm, respectively. The 13 C NMR spectrum of 6b also confirmed the occurrence of S-alkylation through the disappearance of the C=S signal at 182.1 ppm, adding to the presence of four new signals at 32.8, 168.7, 61.5 and 14.1 ppm, corresponding to the carbon of SCH2 and the three carbons (C=O, OCH2 and CH3) of the ester group, respectively. Then, the condensation reaction of the esters 6a,b with hydrazine hydrate in refluxing ethanol gave the acetohydrazide derivatives 7a,b. Furthermore, N'-(arylidene)-acetohydrazide derivatives 8a,b were obtained by carrying out a nucleophilic addition-elimination reaction between 7a and an aromatic aldehyde (4-N,N-dimethylaminobenzaldehyde and/or thiophene-2-carbaldehyde) in refluxing glacial acetic acid (Scheme 2). Upon treatment of 2a,b with phosphorus pentasulfide in refluxing pyridine, the pyrido [3',2': 4,5]thieno [3,2-d]pyrimidine]-4'(3'H)-thiones 5a,b were formed. IR spectra of 5a,b displayed new bands at 1246.2 cm −1 and 1243.2 cm −1 , corresponding to the C=S groups of 5a and 5b, besides the disappearance of the C=O bands of 2a,b at 1658.2 and 1659.4 cm −1 , respectively. The signal at 181.3 ppm in the 13 C NMR spectrum of 5a and at 182.1 ppm in the 13 C NMR spectrum of 5b also assisted the presence of the C=S carbon. Subsequent reaction of 4'(3'H)-thiones 5a,b with ethyl 2-chloroacete in DMF containing a catalytic amount of anhydrous sodium carbonate afforded the formation of ethyl 2-(pyrido [3',2':4,5]thieno [3,2-d]pyrimidin-4'-yl)thio)acetates 6a,b via S-alkylation at position 4. The steric hindrance of the bulky spiro cyclohexane moiety was a possible explanation for the forbidding of N-alkylation at position 1. 1 H NMR spectra of 6a showed a singlet signal at 3.89 ppm of SCH 2 protons, while the CH 3 and CH 2 O protons of the ester group were represented by the triplet signal at 1.07 ppm and quartet signal at 4.08 ppm, respectively. The 13 C NMR spectrum of 6b also confirmed the occurrence of S-alkylation through the disappearance of the C=S signal at 182.1 ppm, adding to the presence of four new signals at 32.8, 168.7, 61.5 and 14.1 ppm, corresponding to the carbon of SCH 2 and the three carbons (C=O, OCH 2 and CH 3 ) of the ester group, respectively. Then, the condensation reaction of the esters 6a,b with hydrazine hydrate in refluxing ethanol gave the acetohydrazide derivatives 7a,b. Furthermore, N'-(arylidene)-acetohydrazide derivatives 8a,b were obtained by carrying out a nucleophilic addition-elimination reaction between 7a and an aromatic In the 1 H NMR spectrum of 7a, beside the absence of ester signals, the hydrazide moiety was represented by two signals at 4.62 ppm and 9.18 ppm, corresponding to the NH2 and NH groups, respectively. The 1 H NMR spectrum of N'- (4-(dimethylamino)benzylidene derivative 8a also supported the presence of the arylidine moiety by a signal at 3.18 ppm for the six protons dimethylamino group (N(CH3)2) and a signal at 8.43 ppm for the proton of the azomethine group (CH=N), as well the signals corresponding to new aromatic protons. Moreover, the 13 C NMR spectrum of 8a showed a signal at 44.7 ppm, corresponding to the two carbons of the N(CH3)2 group. The chemical structures of the new pyridothienopyrimidine derivatives (2a,b-8a,b) were confirmed by IR, 1 H NMR, 13 C NMR and mass spectra, in addition to the correct results of their elemental microanalyses (Supplementary Materials: NMR spectra of compounds 2a,b-8a,b; Figures S1-S28).

Antibacterial Activity
The results of the in vitro antibacterial activity evaluation (MIC values in μg/mL) of the target pyridothienopyrimidine compounds (2a,b-8a,b), listed in Table 1 and Figure 2, revealed the significant activity of the majority of these compounds against the tested Gram-positive bacteria (Staphylococcus aureus 25923, Bacillus cereus 33018 and Bacillus subtilis 6633) and Gram-negative bacteria (Escherichia coli 8739, Salmonella typhimurium 14028 and Pseudomonas aeruginosa 27853) compared with amoxicillin trihydrate as a reference drug. In particular, the activity of the target compounds against the Gram-negative strains were more potent than their activity against the Grampositive strains. In turn, pyrido[3',2':4,5]thieno[3,2-d]pyrimidin]-4'(3'H)-ones 2a,b showed potent activity against all tested Gram-negative strains (MIC = 15.63 μg/mL), equal to that of the reference drug, but they had no activity against all tested Gram-positive strains at the highest used concentration (125 μg/mL). The 4'-chloro derivatives 3a,b also revealed inhibition activity against Gram-negative strains, the same as amoxicillin, with enhancement in the activity against the Gram-positive strains, Scheme 2. Synthesis of the pyridothienopyrimidine derivatives 5a,b-8a,b.
In the 1 H NMR spectrum of 7a, beside the absence of ester signals, the hydrazide moiety was represented by two signals at 4.62 ppm and 9.18 ppm, corresponding to the NH 2 and NH groups, respectively. The 1 H NMR spectrum of N'-(4-(dimethylamino)benzylidene derivative 8a also supported the presence of the arylidine moiety by a signal at 3.18 ppm for the six protons dimethylamino group (N(CH 3 ) 2 ) and a signal at 8.43 ppm for the proton of the azomethine group (CH=N), as well the signals corresponding to new aromatic protons. Moreover, the 13 C NMR spectrum of 8a showed a signal at 44.7 ppm, corresponding to the two carbons of the N(CH 3 ) 2 group. The chemical structures of the new pyridothienopyrimidine derivatives (2a,b-8a,b) were confirmed by IR, 1 H NMR, 13 C NMR and mass spectra, in addition to the correct results of their elemental microanalyses (Supplementary Materials: NMR spectra of compounds 2a,b-8a,b; Figures S1-S28).

Antibacterial Activity
The results of the in vitro antibacterial activity evaluation (MIC values in µg/mL) of the target pyridothienopyrimidine compounds (2a,b-8a,b), listed in Table 1 and Figure 2, revealed the significant activity of the majority of these compounds against the tested Gram-positive bacteria (Staphylococcus aureus 25923, Bacillus cereus 33018 and Bacillus subtilis 6633) and Gram-negative bacteria (Escherichia coli 8739, Salmonella typhimurium 14028 and Pseudomonas aeruginosa 27853) compared with amoxicillin trihydrate as a reference drug. In particular, the activity of the target compounds against the Gram-negative strains were more potent than their activity against the Gram-positive strains. In turn, pyrido [3',2':4,5]

thieno[3,2-d]pyrimidin]-4'(3'H)-ones 2a,b
showed potent activity against all tested Gram-negative strains (MIC = 15.63 µg/mL), equal to that of the reference drug, but they had no activity against all tested Gram-positive strains at the highest used concentration (125 µg/mL). The 4'-chloro derivatives 3a,b also revealed inhibition activity against Gram-negative strains, the same as amoxicillin, with enhancement in the activity against the Gram-positive strains, especially against S. aureus (MIC = 15.63 µg/mL). Further increases in the potency of antibacterial activity against Gram-positive strains were observed in the 4'-amine derivatives 4a-e, especially against B. subtilis (MIC = 15.63 µg/mL). The antibacterial activity of 4a-e against the Gram-negative strains varied from potent to moderate, with MIC values ranging from 7.81 to 31.25 µg/mL. Moreover, 4'-(4-methylpiperazin-1-yl) derivative 4b showed potent activity against all the tested bacterial strains, with MIC values ranging from 7.81 to 15.63 µg/mL, which was equal in potency or more potent than that of amoxicillin. The conversion of 4'(3'H)-ones 2a,b to 4'(3'H)-thiones 5a,b also enhanced the antimicrobial activity against Gram-positive bacteria and showed inhibition activity ranging from potent to moderate against the three tested microorganisms, with MIC values ranging from 15.63 to 31.25 µg/mL. On the reverse, the esters 6a,b showed dramatic lowering in their activity; they were inactive against the Gram-positive strains and gave moderate or weak activity against the Gram-negative strains (MIC = 31.25 µg/mL and 62.5 µg/mL). However, the transformation of the esters 6a,b to acetohydrazide derivatives 7a,b and then to their arylidene derivatives 8a,b revealed an improvement in the antibacterial activity against Gram-positive strains, with MIC values ranging from 31.25 to 62.5 µg/mL. Moreover, 7a,b and 8a,b showed moderate activity against all tested Gram-negative strains (MIC = 31.25 µg/mL).

DNA Gyrase and Topoisomerase IV Inhibitory Activity
The target compounds (2a,b; 3a,b; 4a,b; and 5a,b) that showed the most potent activity against the tested Gram-negative bacteria, especially against E. coli, were chosen to evaluate their in vitro inhibitory activity of E. coli DNA gyrase and topoisomerase IV. The results of the DNA gyrase supercoiling and topoisomerase IV decatenation assays (IC 50 values in µM) of these compounds are in Table 2, showing the potent dual inhibition of the 4'-amine derivatives 4a;b against both enzymes compared with the two reference inhibitors (ciprofloxacin and novobiocin).

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH 2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47].

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47].

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47].

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47].

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47]

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47].

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47]

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47]. 12

Molecular Docking Studies
To explore the binding modes of the newly synthesized pyridothienopyrimidines (2a,b; 3a,b; 4a,b; and 5a,b) with the active site of E. coli DNA gyrase B, a molecular docking simulation was accomplished using MOE. Firstly, novobiocin (the original co-crystallized ligand) was re-docked in the active site of E. coli DNA gyrase B kinase ((PDB code: 1AJ6) [46,47] (Figure 3) and revealed a score energy of −80 kcal/mol at a root mean square deviation (RMDS) value equal to 0.81 Å. As reported in docking of novobiocin, having a coumarin core linked to oxan-4-yl moiety, the protons of the hydroxyl group of oxan-4-yl and NH2 of the carbamate group formed hydrogen bonds within the active site of DNA gyrase B kinase via the backbone of Asp46 and the side chain of Asp73. Furthermore, the coumarin scaffold shared fixation through an arene-cation interaction with the essential amino acid Arg76 [37,47].
14. 23 17.24 Then, the target compounds (2a,b; 3a,b; 4a,b; 5a,b) were docked into the ATP-active sites of E. Then, the target compounds (2a,b; 3a,b; 4a,b; 5a,b) were docked into the ATP-active sites of E. coli DNA gyrase B and the docking results are listed in Table 3. By comparing the energy scores and the binding orientations of the target compounds with that of the original ligand novobiocin, it can be seen that all derivatives displayed promising energy scores ranging, from −5.25 to −6.99 kcal/mol for E. coli DNA gyrase B, as well as a noticeable binding affinity between the pyridine scaffold and the essential amino acid Arg76 via an arene-cation interaction. The 4'-(4-methyl-piperazin-1-yl)-derivative 4b, which displayed the most potent inhibitory activity, gave the highest binding affinity to DNA gyrase B, with an energy score of −6.99 kcal/mol. Moreover, the N-(furan-2-yl)-4'-amine derivative 4a, which came after 4b in inhibition potency, displayed the second-best docking score, with a binding energy of −6.64 kcal/mol. Thus, compounds 4a and 4b were selected as the ligand examples against the structure of E. coli DNA gyrase B (PDB ID code: 1AJ6); the obtained docking models are illustrated in Figure 4; Figure 5. Table 3. Docking results of compounds 2a,b; 3a,b; 4a,b; and 5a,

General Consideration
Melting points were determined by open glass capillary tubes using an Electro thermal IA9100 digital melting point apparatus and were uncorrected. Elemental microanalyses were carried out at the Micro Analytical Unit at Cairo University and were found within ± 0.5%. 1 H NMR and 13 C NMR spectra were recorded on a Bruker High Performance Digital FT-NMR Spectrometer Advance III (400/100 MHz) in the presence of TMS as the internal standard. Infrared spectra were recorded by using the KBr disc technique on a Jasco FT/IR-6100, Fourier transform, Infrared spectrometer (Japan) at the cm⁻ 1 scale. The ESI-mass spectra were measured using an Advion Compact Mass Spectrometer (CMS) NY, USA. Follow-up of the reactions and checking the purity of the compounds were made by TLC on silica gel aluminum sheets (Type 60, F 254, Merck, Darmstadt, Germany) and the spots were illustrated by exposure to UV analysis lamp at λ 254/366 nm or by iodine vapor. The nomenclature of the new synthesized compounds is according to the IUPAC system. The starting 3amino-6-phenylthieno[2,3-b]pyridine-2-carboxamides 1a,b were prepared as per reported methods [44,45]  Regarding to the binding of 4a with E. coli DNA gyrase B, there was an H-bond donor between the NH proton of amino-2-furan scaffold with the side chain of Thr165 (distance: 2.15 Å), and it exhibited one arene-cation interaction with Arg76 ( Figure 4). However, the pyridine and phenyl moieties of 4b demonstrated two arene-cation interactions with the key amino acid Arg76. Furthermore, the nitrogen of piperazine moiety of 4b formed with the side chain of Asn46 a favorable hydrogen bonding (distance: 2.99 Å) ( Figure 5).

General Consideration
Melting points were determined by open glass capillary tubes using an Electro thermal IA9100 digital melting point apparatus and were uncorrected. Elemental microanalyses were carried out at the Micro Analytical Unit at Cairo University and were found within ± 0.5%. 1 H NMR and 13 C NMR spectra were recorded on a Bruker High Performance Digital FT-NMR Spectrometer Advance III (400/100 MHz) in the presence of TMS as the internal standard. Infrared spectra were recorded by using the KBr disc technique on a Jasco FT/IR-6100, Fourier transform, Infrared spectrometer (Japan) at the cm −1 scale. The ESI-mass spectra were measured using an Advion Compact Mass Spectrometer (CMS) NY, USA. Follow-up of the reactions and checking the purity of the compounds were made by TLC on silica gel aluminum sheets (Type 60, F 254, Merck, Darmstadt, Germany) and the spots were illustrated by exposure to UV analysis lamp at λ 254/366 nm or by iodine vapor. The nomenclature of the new synthesized compounds is according to the IUPAC system. The starting 3-amino-6-phenylthieno[2,3-b]pyridine-2-carboxamides 1a,b were prepared as per reported methods [44,45]  dilution was performed to obtain the required concentrations (125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.97, µg/mL) for the target compounds and the reference drug. The minimum concentration of the sample that showed no growth of the tested microorganism (MIC) were specified; then the MIC values for the compounds and standard drug were listed (see Table 1).

DNA Gyrase Supercoiling and Topoisomerase IV Decatenation Inhibition Assays
Eight of the target compounds (2a, 2b, 3a, 3b, 4a, 4b, 5a, and 5b) were chosen to evaluate their inhibitory activity against bacterial type II topoisomerase enzymes. The assay kits of E. coli DNA gyrase supercoiling and E. coli topoisomerase IV decatenation were provided by TopoGEN, Inc. (Port Orange, FL) and the assays were performed according to established protocols obtained from the supplier [49]. The new compounds and two standard inhibitors (ciprofloxacin and novobiocin) were dissolved in DMSO and serially diluted at concentrations of 100, 10, 1 and 0.1 µM, and then assayed in reaction mixtures in three different replicate runs. For DNA gyrase supercoiling: the final reaction volume was 20 µL, which included 35 mM Tris pH 7, 2 mM DTT, 24 mM KCl, 4 mM MgCl 2 , 1.8 mM spermidine, 0.1 mg/mL acetylated BSA, 6.5% (w/v) glycerol, 1 mM ATP, 0.1 mg/mL album and 0.2 mg pBR322 substrate. While, for topoisomerase IV decatenation: the final reaction volume was 20 µL, containing 40 mM Tris pH 7.5, 10 mM DTT, 6 mM MgCl 2 , 100 mM potassium glutamate, 1 mM ATP, 50 mg/mL acetylated BSA and 0.2 mg kDNA substrate. The reactions were initiated by addition of 2 U of E.coli DNA gyrase or E.coli topoisomerase IV (TopoGen), and 3 µL of inhibitor solution in 10% DMSO, and then were incubated with shaking for 30 min at 37 • C. All of the reactions were terminated by the addition of 10 mL of a 3X gel-loading buffer (final concentration: 6 mM EDTA, 1.2% SDS, 0.02% bromophenol blue, and 10% glycerol blue), after which 20 mL of this was loaded on a 1% agarose, TAE (0.01 M EDTA pH 8.3, 40 mM Tris-acetate) gel that was then run at 60 V for 3 h. The gel was stained by (0.5 mg/L) ethidium bromide in TAE for 30 min and then de-stained in water for 20 min. Fluorescent images were taken at a wavelength of 300 nm on a UV transilluminator imaging system. The fluorescence intensity of the supercoiled plasmid reaction product and the decatenation product, in the case of gyrase and topo IV, were quantitated using ImagQuant software (Molecular Dynamics, Sunnyvale, CA, USA). The results as IC50 values (concentration of the tested compound that leads to 50% inhibition of enzyme activity) for all samples were determined by nonlinear regression analysis in GraphPad Prism [49,50]. The average IC 50 values (µM) of the triplicate experiments were calculated for the target compounds and the two reference antibiotics and then listed in Table 2.

Molecular Docking Study
The molecular docking simulation study was done using Molecular Operating Environment (MOE ® ) 2008.10 software [51]. The crystal structures of E. coli DNA gyrase B complexed with their ligand novobiocin (PDB codes: 1AJ6 and 1S14) [46,47] were retrieved from the Protein Data Bank. At the beginning, the co-crystallized ligand was re-docked into the assigned active E. coli DNA gyrase B enzyme to evaluate the root mean square deviation value. Then, the molecular docking procedure was performed for the newly synthesized compounds (2a,b; 3a,b; 4a,b; 5a,b) into the ATP-binding site of E. coli DNA gyrase B (PDB code: 1AJ6 and 1S14), following the reported method [37].

Conclusions
This work included synthesis of novel 4-substituted-1'H-spiro[cyclohexane-1,2'-pyrido [3',2':4,5] thieno [3,2-d]pyrimidin] compounds (2a,b-8a,b) and the in vitro evaluation of these compounds against six strains of both Gram-positive and Gram-negative bacteria compared with amoxicillin trihydrate as a reference drug. The tested pyridothienopyrimidine compounds (2a,b-8a,b) showed significant antibacterial activity, especially against Gram-negative strains, with MIC values ranging from 7.81 to 62.5 µg/mL . Compounds 2a,b; 3a,b; and 5a,b gave potent activity against all tested Gram-negative bacteria, equal to that of the reference drug, with an MIC value of 15.63 µg/mL. The N-(furan-2-yl)-4'-amine derivative 4a also displayed potent activity against all Gram-negative strains, besides its potent to moderate activity against Gram-positive strains. Moreover, 4'-(4-methyl-piperazin-1-yl)-derivative 4b was the most active compound compared with the reference drug; it gave potent activity against all the tested bacterial strains, with MIC values ranging from 7.81 to 15.63 µg/mL. In turn, the antibacterial activity of compounds 6a,b; 7a,b and 8a,b varied from weak, with an MIC value of 62.5 µg/mL, to moderate, with an MIC value of 31.25µg/mL, against the tested microorganisms. Thus, compounds 2a,b; 3a,b; 4a,b and 5a,b, because they were the most potent compounds against E. coli, were selected to evaluate their in vitro inhibitory activity of E. coli topoisomerase II enzymes (DNA gyrase and topoisomerase IV). The tested compounds showed dual inhibition of the two enzymes and their inhibitory activity varied from potent to moderate compared with the two reference antibiotics (ciprofloxacin and novobiocin). Furthermore, compound 4b displayed dual inhibition, and was more potent than the two references, with IC 50 values of 3.44 µM and 14.46 µM against DNA gyrase and topoisomerase IV, respectively. Furthermore, 4a came after 4b in inhibition potency with IC 50 values of 5.77 µM for DNA gyrase and 14.89 µM for topoisomerase IV. In addition, docking studies were performed with compounds 2a,b; 3a,b; 4a,b and 5a,b, to illustrate their binding mode in the active site of DNA gyrase B compared with that of novobiocin. The docking results of the tested compounds were compatible with their inhibitory potency and gave binding scores ranging from −5.25 to −6.99 kcal/mol.
The results of this study pointed to the importance of pyridothienopyrimidine compounds as a promising heterocyclic sector that, with further study and development, can provide new antimicrobial agents competent of facing the increasing antimicrobial resistance.