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Article

Synthesis, Docking Studies, and In Vitro Evaluation of Some Novel Thienopyridines and Fused Thienopyridine–Quinolines as Antibacterial Agents and DNA Gyrase Inhibitors

by
Eman M. Mohi El-Deen
1,*,
Eman A. Abd El-Meguid
2,
Sherifa Hasabelnaby
3,
Eman A. Karam
4 and
Eman S. Nossier
5
1
Department of Therapeutic Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt
2
Department of Chemistry of Natural and Microbial Products, National Research Centre, Dokki, Cairo 12622, Egypt
3
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan, Cairo 11795, Egypt
4
Microbiology Chemistry Department, National Research Centre, Dokki, Cairo 12622, Egypt
5
Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11754, Egypt
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(20), 3650; https://doi.org/10.3390/molecules24203650
Submission received: 11 September 2019 / Revised: 3 October 2019 / Accepted: 6 October 2019 / Published: 10 October 2019
(This article belongs to the Special Issue Chemical Biology of Antimicrobial Resistance)
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Abstract

:
A series of novel thienopyridines and pyridothienoquinolines (3a,b–14) was synthesized, starting with 2-thioxo-1,2-dihydropyridine-3-carbonitriles 1a and 1b. All compounds were evaluated for their in vitro antimicrobial activity against six bacterial strains. Compounds 3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14 showed significant growth inhibition activity against both Gram-positive and Gram-negative bacteria compared with the reference drug. The most active compounds (4a, 7a, 9b, and 12b) against Staphylococcus aureus were also tested for their in vitro inhibitory action on methicillin-resistant Staphylococcus aureus (MRSA). The tested compounds showed promising inhibition activity, with the performance of 12b being equal to gentamicin and that of 7a exceeding it. Moreover, the most promising compounds were also screened for their Escherichia coli DNA gyrase inhibitory activity, compared with novobiocin as a reference DNA gyrase inhibitor. The results revealed that compounds (3a, 3b, 4a, 9b, and 12b) had the highest inhibitory capacity, with IC50 values of 2.26–5.87 µM (that of novobiocin is equal to 4.17 µM). Docking studies were performed to identify the mode of binding of the tested compounds to the active site of E. coli DNA gyrase B.

Graphical Abstract

1. Introduction

Recently, infections of resistant bacteria have become very common [1,2] and many pathogens, mainly multi-drug resistant organisms (MDRO), have become resistant to different classes of antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA), which causes serious infections in hospitals [3,4]. This growing risk of antimicrobial resistance has led to the insistent need to discover new antimicrobial agents with structural features different from those of existing antibiotics [5].
The DNA gyrase enzyme has a crucial role in bacterial cell viability through its initiation of DNA replication and introduction of negative supercoils into DNA during replication [6,7]. The principal family of prokaryotic DNA-gyrase-targeted drugs is represented by fluoroquinolones, which result in the accumulation of double-strand DNA breaks, leading to bacterial cell death [8,9]. Although resistance to fluoroquinolones has appeared in the last few years, DNA gyrase remains an attractive target due to its presence across all microbes [10,11,12]. Targeting DNA gyrase with an inhibitor is a new strategy for dealing with antimicrobial resistance by disrupting DNA synthesis, leading to cell death and reducing the development of resistance [13,14].
In addition, thieno[2,3-b]pyridine derivatives have attracted great interest due to their various pharmacological activities, including anti-inflammatory [15], kinase inhibitory [16,17], anticancer [18,19,20], and antimicrobial activities [21,22,23,24,25]. Quinoline derivatives also possess important biological activities, such as anti-HIV [26,27], antimalarial [28], antihypertensive [29], and antimicrobial activities [30,31]. Furthermore, a quinolone moiety is one of the key building elements for many naturally occurring bioactive compounds [32]. Strongly effective quinolone antibiotics such as ciprofloxacin and ofloxacin selectively inhibit bacterial DNA gyrase [33], but these antibiotics are now being subjected to increasing incidences of microbial resistance, and need continuous development [34].
Therefore, as part of our research into the development of novel potential antimicrobial compounds, a series of thieno[2,3-b]pyridine derivatives was synthesized and cyclized to afford tetracyclic pyridothienoquinolines, which have both thieno[2,3-b]pyridine and quinoline bicyclic systems in the same framework, to provide effective antibacterial agents with novel structural features able to overcome bacterial resistance. The antimicrobial activity of these new compounds was evaluated by in vitro screening of their antibacterial and DNA gyrase inhibition activity. Docking studies of the most active compounds then gave insight to their binding styles at the active sites of DNA gyrase.

2. Results and Discussion

2.1. Chemistry

The synthesis of new thienopyridines and fused thienopyridine–quinolines (3a,b–14) is outlined in Scheme 1, Scheme 2 and Scheme 3. The reaction of the starting compounds 2-thioxo-1,2-dihydropyridine-3-carbonitriles 1a,b [35] with 2-chloroacetamide in N,N-dimethylformamide containing anhydrous sodium carbonate provided 2-((3-cyanopyridin-2-yl)thio)acetamides 2a,b, which underwent a base-catalyzed intramolecular cyclization by refluxing with sodium methoxide in methanol to give 3-aminothieno[2,3-b]pyridine -2-carboxamides 3a,b. IR and 1H NMR spectra of 3a,b confirmed their structures by the disappearance of C≡N bands and SCH2 signals, which appeared in the IR and 1H NMR spectra of 2a,b at 2210 and 2211 cm−1 and at 4.00 and 4.01 ppm, respectively. Moreover, the 1H NMR spectra of 3a and 3b showed additional signals at 5.97 and 6.20 ppm, respectively, corresponding to NH2 groups. Further condensation of 3a,b with cyclohexanone in glacial acetic acid afforded 3-(cyclohexylideneamino)-thieno[2,3-b]pyridine-2-carboxamides 4a,b. Upon refluxing of 4a,b with phosphorous oxychloride, a cyclocondensation reaction was achieved to give 6,7,8,9- tetrahydropyrido [3′,2′:4,5]thieno[3,2-b]quinolin-10-amines 5a,b (Scheme 1). The 1H NMR spectra of 4a,b revealed three signals at 0.86–1.98 ppm, corresponding to the 5CH2 protons of cyclohexylidene moiety alongside the basic signals of amide–NH2 and aromatic H. The 1H NMR spectra of 5a,b showed three signals at 1.82–2.76 ppm corresponding to the 4CH2 protons and NH2 signal at 5.53 and 6.21 ppm, respectively. In the 13C NMR spectra of 5a,b, the 4CH2 carbons were verified by four signals at 22.75–33.20 ppm.
The tetracyclic amines 5a,b acted as key intermediates for synthesis of a series of fused thienopyridine derivatives (6a,b–14). Treatment of the amines 5a,b with chloroacetyl chloride in 1,4-dioxane containing drops of triethylamine gave chloroacetamide derivatives 6a,b, which were reacted with hydrazine hydrate in boiling ethanol to afford hydrazinylacetamide derivatives 7a,b The bands of acetamide C=O of 6a,b and 7a,b were revealed in their IR spectra in the 1659–1677 cm−1and 1654–1656 cm−1 regions, respectively. The signal of the CH2 protons of the chloroacetamide moiety appeared at 4.45 and 4.46 ppm in the 1H NMR spectra of 6a,b and shifted upfield to 3.67 and 3.84 ppm in the 1H NMR spectra of 7a,b, confirming the presence of CH2N from the acetohydrazinyl moiety. In addition, the 13C NMR spectrum of 6b showed two signals at 43.13 ppm and 167.21 ppm, attributed to the two carbons of CH2Cl and C=O, respectively. Next, condensation reaction of 7a with aromatic aldehydes, such as 3,4-dimethoxybenzaldehyde and 4-methylbenzaldehyde, in refluxing glacial acetic acid produced 3,4-dimethoxybenzylidene derivative 8a and 4-methylbenzylidene derivative 8b, respectively. The 1H NMR spectrum of 8a confirmed arylidine formation, with signals at 3.85 ppm and 8.18 ppm corresponding to the protons of 3OCH3 and CH=N, respectively. Chloroacetamide derivative 6a was reacted with morpholine and/or 1-methylpiperazine in N,N-dimethylformamide under reflux to give 2-morpholinoacetamide derivative 9a and 2-(4-methylpiperazin-1-yl) acetamide derivative 9b (Scheme 2). The presence of a morpholine moiety in the structure of 9a was supported by its 1H NMR spectrum, which revealed two signals at 2.49–2.69 ppm and 3.64 ppm, corresponding to the protons of 2CH2N and 2CH2O, respectively. The 13 C NMR spectrum of 9a also showed two signals at 62.12 ppm for the 2CH2N carbons, and at 67.11 ppm for 2CH2O carbons.
In addition, reaction of 5a with benzenesulfonyl chloride in pyridine at reflux temperature gave benzenesulfonamide derivative 10. The IR spectrum of 10 showed a band corresponding to sulfonamide NH at 3424 cm−1, and two bands at 1335 cm−1 and 1170 cm−1 due to sulfonamide SO2. Upon refluxing of amine 5a with phenyl isocyanate in absolute ethanol, phenylurea derivative 11 was formed by nucleophilic addition reaction; the 1H NMR spectrum of 11 showed two signals at 9.08 ppm and 10.15 for the 2NH of the phenylurea moiety. Amine 5a was also condensed with 4-(dimethylamino)benzaldehyde and/or thiophene-2-carboxaldehyde in boiling glacial acetic acid to produce the Schiff bases 12a and 12b, respectively. The structures of 12a,b were supported by IR spectra which revealed the absence of the NH2 bands, which belong to the amino group of 5a. Furthermore, the 1H NMR spectra of 12a showed two signals at 2.93 ppm and 8.30 ppm besides the signals of the expected aromatic protons, attributed to N(CH3)2 and CH=N protons, respectively. Reaction of 5b with ethyl acetoacetate in dimethylsulfoxide in the presence of anhydrous sodium carbonate gave oxobutanamide derivative 13, which in turn was treated with hydrazine hydrate in boiling N,N-dimethyformamide to afford N-(5-methyl-4H-pyrazol-3-yl)-10-amine derivative 14 (Scheme 3). The chemical structures of the newly synthesized compounds (2a,b–14) were confirmed by 1H NMR, 13C NMR, and mass spectra (Supplementary Materials, Figures S1–S53), in addition to the IR spectra and correct elemental microanalyses.

2.2. Antimicrobial Activity Evaluation

2.2.1. Antibacterial Activity

The results of the in vitro antibacterial screening (inhibition zone in mm, MIC in µg/mL) of the novel thienopyridines (3a,b, and 4a,b) and tetracyclic pyridothienoquinolines (5a,b–14) against Gram-negative bacteria (Escherichia coli 8739, Salmonella typhimurium 14028, Pseudomonas aeruginosa 27853) and Gram-positive bacteria (Bacillus subtilis 6633, Bacillus cereus 33018, Staphylococcus aureus 25923), represented in Table 1 and Figure 1, revealed that compounds 3a,b, 4a, 5b, 6b, 7a, 9b, 12b, and 14 had significant activity compared with amoxicillin trihydrate. The most active of these compounds were the tetracyclic 2-(4-methylpiperazin-1-yl)acetamide derivative 9b and 1-(thiophen-2-yl)methanimine derivative 12b. They had an MIC = 15.63 µg/mL against the six bacterial strains, which equalled the effect of amoxicillin trihydrate. Moreover, 2-hydrazinylacetamide derivative 7a revealed higher inhibition activity against Gram-positive strains, especially against S. aureus, with an inhibition zone of 19 mm and an MIC value of 15.63 µg/mL, the same as the reference drug. The 4-(4-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide derivative 3a and tetracyclic amine 5b were mostly active against Gram-negative bacteria. In addition, amides 3b and 4a, chloroacetamide derivative 6b, and pyrazolylamine derivative 14 showed variable activity against the different bacterial strains, ranging from potent to moderate. For example, 4a, which had potent activity against E. coli and S. aureus, had only moderate activity against P. aeruginosa. Compounds 4b, 5a, 7a, 6a, 9a, 12a, and 13 possessed moderate inhibitory activity against some of the tested organisms. The remaining compounds (8a,b, 10, and 11) were mostly inactive.

2.2.2. Antimicrobial Resistance Activity against MRSA

The novel compounds which showed highest inhibitory activity against Staphylococcus aureus (4a, 7a, 9b, and 12b) were selected to be screened for their inhibitory activity against methicillin-resistant Staphylococcus aureus, compared with gentamicin as a reference antibiotic. The results of inhibition zones in mm are presented in Table 2, revealed that Schiff base 12b and hydrazide derivative 7a were the most potent compounds against MRSA; their inhibition zones were 15 mm and 18 mm, respectively. The inhibition zone of gentamicin was 15 mm, so 12b and 7a had inhibition activity equipotent to gentamicin or exceeded it, respectively. Additionally, compounds 4a and 9b showed good activity against MRSA, with inhibition zones near to that of the reference.

2.2.3. Escherichia coli DNA gyrase Inhibition Activity

Targeting of DNA gyrase with an inhibitor has been considered as new strategy for developing antimicrobial agents that can deal with antimicrobial resistance. Thus, the most active antibacterial compounds (3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14) were selected to evaluate their in vitro inhibition activity against DNA gyrase from Escherichia coli, compared with novobiocin as a reference inhibitor. The evaluation results, presented in Table 3, showed that thienopyridine carboxamides 3a and 4a were the most active among the tested compounds with IC50 = 2.26 and 3.69 µM, and were also more potent than novobiocin (IC50 = 4.17 µM). Moreover, carboxamide derivative 3b and tetracyclic methanimine derivative 12b showed promising inhibition activity (IC50 = 4.50 and 4.60 µM) near that of Novobiocin. Methylpiperazinyl derivative 9b and chloroacetamide derivative 6b showed good inhibition with IC50 = 5.78 and 5.95 µM, respectively. From the compounds’ IC50 values, it was clear that the presence of carboxamide, methanimine, methylpiperazinyl, and chloroacetamide moieties in their structure supported inhibition activity against DNA gyrase.

2.2.4. Molecular Docking Studies

One of the most important strategies in structure-based drug design, is molecular docking, as it can give an idea of the binding modes of the novel molecules in the binding site of the suitable target, which is a key step in drug design [36,37]. In this research work, docking studies were performed to give insight into the mode of binding between the enzyme active binding site and the novel bioactive compound, in addition to possible interactions and the docking score.
To correlate the observed potencies and structure activity relationship (SAR) of our newly synthesized derivatives, which had thieno[2,3-b]pyridine or the fused tetrahydroquinoline system giving their possible binding modes and interactions within the active site of E. coli DNA gyrase B, docking simulation was carried out. The co-crystallized ligand novobiocin was re-docked in the active site of E. coli DNA gyrase B (PDB code: 1AJ6) [38,39] and exhibited and energy score of −6.30 kcal/mol, with a root-mean-square deviation (RMDS) value equal to 9.2. The most active 10 compounds were docked into the ATP-active site of E. coli DNA gyrase B using the 3D protein structure (PDB ID: 1AJ6). The docking results obtained for the investigated derivatives are recorded in Table 4.
Novobiocin consists of a coumarin core linked to an oxan-4-yl moiety. This ligand binds to E. coli DNA gyrase B kinase via formation of two hydrogen bonds, one between the hydroxyl proton of oxan-4yl moiety and the backbone of Asp46, and the other between the sidechain of Asp73 and the protons of NH2 of the carbamate group in novobiocin. Moreover, the coumarin scaffold establishes an arene–cation interaction with Arg76 in the ligand (Figure 2).
It was clear from the docking data that compounds 3a, 3b, 4a, 9b, and 12b, with the highest E. coli DNA gyrase B inhibitory activities, showed the best binding style. These thieno[2,3-b]pyridine derivatives exhibited hydrogen bonds between the nitrogen of the pyridine and the side chain of Thr165. Another hydrogen bond appeared between the oxygen of the carboxamide group and Arg136 (Figure 3, Figure 4, Figure 5 and Figure 6, respectively).
Fusion of the thieno[2,3-b]pyridine scaffold with a tetrahydroquinoline moiety maintained the improved potency in 9b and 12b, which could be attributed to the presence of the important Asn46 residue via two hydrogen bonds with the nitrogen of pyridine and oxygen of acetamide in 9b (distance: 2.79 and 2.46 Å) and one hydrogen bond with nitrogen of methanimine group in 12b (distance: 2.98 Å). Additionally, the oxygen of the methoxy group in 12b formed a favorable hydrogen bond with the side chain of Arg76 (distance: 2.80 Å). As show in Figure 5a,b and Figure 6a,b.
Finally, the docking results in Table 4, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6 of the screened compounds (3a, 3b, 4a, 5b, 6a, 6b, 7a, 9b, 12b, and 14), and their E. coli DNA gyrase B inhibitory activities confirmed that the presence of carboxamide, acetamide, and methanimine groups led to enhancement of the inhibitory activities of these thieno[2,3-b]pyridine-based and tetrahydroquinoline-based compounds by forming significant H-bonds and other interactions inside the ATP-binding cavity.

3. Experimental

3.1. Chemistry

3.1.1. General Information

Melting points were recorded in open glass capillary tubes using an Electro thermal IA9100 digital melting point apparatus and were uncorrected. Elemental microanalyses were carried out at the Microanalytical Unit in Cairo University, and were found to within ±0.5%. Infrared spectra were recorded on a Jasco FT/IR-6100, Fourier transform infrared spectrometer (Tokyo, Japan) at cm−1 scale using the KBr disc technique. 1H NMR and 13C NMR spectra were recorded on a Bruker High-Performance Digital FT-NMR Spectrometer Advance III (400/100 MHz, Billerica, MA, USA) in the presence of TMS as internal standard. The mass spectra were measured using a Model (ISQ LT) mass spectrometer using Thermo X-CALIBUR SOFTWARE. Follow ups of the reactions and checks of 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 detected by exposure to a UV analysis lamp at λ 254/366 nm for a few seconds and by iodine vapor. The chemical names given for the prepared compounds were according to the IUPAC system. The starting 2-thioxo-1,2-dihydropyridine-3-carbonitriles 1a,b were prepared by following the literature method [35].

3.1.2. Synthesis of 2-((3-Cyanopyridin-2-yl)thio)acetamides 2a,b

A mixture of compounds 1a,b (0.1 mol) and 2-chloroacetamide (9.35, 0.1 mol) in N,N-dimethylformamide (100 mL) containing anhydrous sodium carbonate (15 g) was heated at 80 °C with stirring for 3 h. The reaction mixture was poured into an ice–water mixture. The formed precipitate was collected by filtration, washed with water, dried, and recrystallized from DMF/H2O to produce compounds 2a,b.
2-((3-Cyano-6-(furan-2-yl)-4-(4-methoxyphenyl)pyridin-2-yl)thio)acetamide (2a), pale yellow solid, (82% yield), m.p. 246 °C. IR (KBr, ν max cm−1): 3407, 3195 (NH2), 2924, 2850 (CH), 2210 (CN), 1670 (C=O), 1601 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 3.85 (s, 3H,OCH3), 4.01 (s, 2H, SCH2), 6.78 (m, 1H, Ar-H), 7.14 (d, 2H, J = 8.7 Hz, Ar-H), 7.26 (s, 1H, Ar-H), 7.51 (s, 2H, NH2, D2O exchangeable), 7.69 (d, 2H, J = 8.7 Hz, Ar-H), 7.72 (d, 1H, J = 7.0 Hz, Ar-H), 7.99 (d, 1H, J = 7.0 Hz, Ar-H); MS m/z (%) 365 (M+, 39), 349 (9), 321 (100), 307 (11), 292 (10), 278(15), 100 (10). Anal. Calcd. For C19H15N3O3S (365.41): C, 62.45; H, 4.14; N, 11.50%. Found: C, 62.11; H, 4.51; N, 11.88%.
2-((3-Cyano-6-(furan-2-yl)-4-(thiophen-2-yl)pyridin-2-yl)thio)acetamide (2b), brown solid, (81% yield), m.p. 242 °C; IR (KBr, ν max cm−1): 3378, 3196 (NH2), 2925 (CH), 2211 (CN), 1661 (C=O), 1601 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 4.00 (s, 2H, SCH2), 6.69-7.60 (m, 5H, Ar-H), 7.71 (s, 2H, NH2, D2O exchangeable), 7.93–8.03 (m, 2H, Ar-H); MS m/z (%) 341 (M+, 35), 325 (5), 299 (13), 297 (100), 269 (14), 251 (6), 108 (5), 58 (9), 44(64). Anal. Calcd. For C16H11N3O2S2 (341.40): C, 56.29; H, 3.25; N, 12.31%. Found: C, 56.58; H, 3.49; N, 12.52%.

3.1.3. Synthesis of 3-Amino-thieno[2,3-b]pyridine-2-carboxamides 3a,b

Compounds 2a,b (20 mmol) were added to a solution of 0.2 M MeONa in MeOH (40 mL), and the reaction mixture was refluxed for 4 h. The precipitate formed after cooling was isolated by filtration, washed with ethanol, dried, and recrystallized from ethanol to give products 3a,b.
3-Amino-6-(furan-2-yl)-4-(4-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (3a), yellow solid, (74% yield), m.p. 220 °C; IR (KBr, ν max cm−1): 3462, 3310, 3256 (NH), 2940 (CH), 1656 (C=O), 1592 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 3.86 (s, 3H, OCH3), 5.97 (s, 2H, NH2, D2O exchangeable), 6.71 (s, 1H, Ar-H), 7.14 (d, 2H, J = 7.6 Hz, Ar-H), 7.23 (m, 1H, Ar-H), 7.34 (d, 1H, J = 5.2 Hz, Ar-H), 7.50 (d, 2H, J = 8.4 Hz, Ar-H), 7.51 (s, 2H, NH2, D2O exchangeable), 7.91 (s, 1H, Ar-H); 13C-NMR (DMSO-d6, 100 MHz): δ = 55.80 (OCH3), 106.19, 111.35, 113.18, 114.71, 116.99, 129.65, 130.48, 131.35, 145.61, 146.20, 152.65, 154.18, 161.19, 162.20 (Ar–C), 167.30 (C=O); MS m/z (%) 365 (M+, 20), 321 (9), 323 (100), 296 (16), 266 (29), 238 (19), 191 (15), 44 (14). Anal. Calcd. For C19H15N3O3S (365.41): C, 62.45; H, 4.14; N, 11.50%. Found: C, 62.09; H, 4.47; N, 11.24%.
3-Amino-6-(furan-2-yl)-4-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxamide (3b), yellow solid, (71% yield), m.p. 253°C; IR (KBr, ν max cm−1): 3462, 3311, 3257 (NH), 2927 (CH), 1654 (C=O), 1594 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 6.20 (s, 2H, NH2, D2O exchangeable), 6.72 (m, 1H, Ar-H), 7.28-7.44 (m, 4H, Ar-H), 7.62 (s, 2H, NH2, D2O exchangeable), 7.89–7.93 (m, 2H, Ar-H); 13C-NMR (DMSO-d6, 100 MHz): δ = 107.04, 111.70, 113.27, 117.72, 121.22, 128.54, 129.63, 130.01, 136.68, 140.34, 145.84,146.02, 147.82, 152.30, 160.37 (Ar–C), 167.21 (C=O); MS m/z (%) 341 (M+, 91), 325 (14), 323 (100), 281 (20), 83 (4), 44 (12). Anal. Calcd. For C16H11N3O2S2 (341.40): C, 56.29; H, 3.25; N, 12.31%. Found: C, 55.96; H, 3.49; N, 12.66%.

3.1.4. Synthesis of 3-(Cyclohexylideneamino)-thieno[2,3-b]pyridine-2-carboxamides 4a,b

A mixture of 3-amino-thieno[2,3-b]pyridine-2-carboxamides 3a,b (10 mmol) and cyclohexanone (1.47 g, 15 mmol) in glacial acetic acid (20 mL) was refluxed for 2 h. The reaction mixture was concentrated, poured onto cold water, and the obtained solid was collected by filtration and recrystallized from acetone to give compounds 4a,b.
3-(Cyclohexylideneamino)-6-(furan-2-yl)-4-(4-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (4a), yellow-green solid, (77% yield), m.p. 283–285 °C. IR (KBr, ν max cm−1): 3418, 3264 (NH), 3045, 2928, 2856 (CH), 1645 (C=O), 1605 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 0.86–1.03 (m, 2H, CH2), 1.10–1.24 (m, 2H, CH2), 1.36–1.48 (m, 4H, 2CH2), 1.91–1.96 (m, 2H, CH2), 3.85 (s, 3H, OCH3), 6.73 (s, 1H, Ar-H), 7.16 (d, 2H, J = 10.4 Hz, Ar-H), 7.38 (d, 1H, J = 5.2 Hz, Ar-H),), 7.56 (d, 2H, J = 8.0 Hz, Ar-H), 7.62 (s, 2H, NH2, D2O exchangeable), 7.89–7.93 (m, 2H, Ar-H); 13C-NMR (DMSO-d6, 100 MHz): δ = 21.66 (CH2), 24.55 (2CH2), 36.15 (2CH2), 55.99 (OCH3), 105.42, 111.62, 113.24, 114.61, 116.40, 121.20, 128.73, 130.54, 142.76, 145.73, 147.62, 147.92, 152.64, 160.56, 161.33 (Ar–C, C=N), 162.65 (C=O); MS m/z (%) 445 (M+, 26), 416 (8), 403 (29), 402 (100), 389 (8), 349 (12), 319 (10), 277 (9), 54(7). Anal. Calcd. For C25H23N3O3S (445.54): C, 67.40; H, 5.20; N, 9.43%. Found: C, 67.72; H, 5.54; N, 9.77%.
3-(Cyclohexylideneamino)-6-(furan-2-yl)-4-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carboxamide (4b), yellow-green solid, (73% yield), m.p. 320-322 °C. IR (KBr, ν max cm−1): 3462, 3311 (NH), 2927, 2856 (CH), 1654 (C=O), 1594 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 0.96–1.11 (m, 4H, 2CH2), 1.42–1.53 (m, 4H, 2CH2), 1.98 (m, 2H, CH2), 6.72 (m, 1H, Ar-H), 7.32 -7.55 (m, 3H, Ar-H), 7.71 (s, 2H, NH2, D2O exchangeable), 7.92–7.97 (m, 3H, Ar-H);); MS m/z (%) 421 (M+, 100), 392 (5), 378 (43), 366 (4), 325 (30), 268 (11), 159 (13). Anal. Calcd. For C22H19N3O2S2 (421.53): C, 62.69; H, 4.54; N, 9.97%. Found: C, 62.36; H, 4.25; N, 10.25%.

3.1.5. Synthesis of 6,7,8,9-Tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-amines 5a,b

A solution of compounds 4a,b (10 mmol) in phosphorous oxychloride (25 mL) was refluxed for 3 h. After cooling, the reaction mixture was poured into an ice–water mixture and aqueous 10% NaOH solution was added to pH 7. The precipitate formed was isolated by filtration, washed with water, and recrystallized from ethanol to give free amines 5a,b.
2-(Furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-amine (5a), beige solid, (68% yield), m.p. 231 °C. IR (KBr, ν max cm−1): 3338, 3216 (NH), 2927(CH), 1601 (C=N); 1H-NMR (CDCl3, 400 MHz): δ = 1.83-1.89 (m, 4H, 2CH2), 2.60 (t, 2H, CH2), 2.74 (t, 2H, CH2), 3.93 (s, 3H, OCH3), 5.53 (br.s, 2H, NH2, D2O exchangeable), 6.59-7.73 (m, 8H, Ar-H); 13C-NMR (DMSO-d6, 100 MHz): δ = 22.78 (CH2), 22.98 (CH2), 23.86 (CH2), 33.20 (CH2), 55.67 (OCH3), 110.64, 112.97, 113.09, 117.21, 126.55, 129.54, 132.13, 137.70, 145.24, 147.40, 149.18, 152.70, 157.13, 160.15 (Ar–C); MS m/z (%) 427 (M+, 93), 426 (100), 412 (9), 498 (13), 484 (8), 482(18), 368 (4), 355 (5). Anal. Calcd. For C25H21N3O2S (427.52): C, 70.24; H, 4.95; N, 9.83%. Found: C, 70.56; H, 5.22; N, 9.56%.
2-(Furan-2-yl)-4-(thiophen-2-yl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-amine (5b), beige solid, (65% yield), m.p. 248 °C. IR (KBr, ν max cm−1): 3319, 3214 (NH), 2929, 2860 (CH), 1611 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.82 (s, 4H, 2CH2), 2.58 (t, 2H, CH2), 2.76 (t, 2H, CH2), 6.21 (s, 2H, NH2, D2O exchangeable), 6.74–8.26 (m, 7H, Ar-H); 13C-NMR (DMSO-d6, 100 MHz): δ = 22.75 (CH2), 22.99 (CH2), 23.86 (CH2), 33.09 (CH2), 110.81, 113.14, 113.23, 113.99, 116.60, 123.62, 127.59, 129.05, 132.53, 138.15, 140.54, 145.40, 147.25, 147.46, 152.67, 154.37, 163.17 (Ar–C); MS m/z (%) 403 (M+, 60), 402 (100), 387 (15), 374 (22), 360 (21), 347 (14), 253 (15), 225 (16), 198 (20). Anal. Calcd. For C22H17N3OS2 (403.52): C, 65.48; H, 4.25; N, 10.41%. Found: C, 65.14; H, 4.59; N, 10.12%.

3.1.6. Synthesis of Chloroacetamide Derivatives 6a,b

Chloroacetyl chloride (2.26 g, 20 mmol) in 1,4-dioxane (10 mL) was added to a cold solution of the amines 5a,b (10 mmol) in 1,4-dioxane (100 mL) containing a few drops of triethylamine at 5–10 °C, dropwise with stirring. After addition, the stirring was continued at room temperature for 8 h. The solvent was evaporated under vacuum and the residue was treated with boiled ethanol. The solid that formed was filtered off and recrystallized from DMF/H2O to give compounds 6a,b.
2-Chloro-N-(2-(furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)acetamide (6a), brown solid, (69% yield), m.p. 342–343 °C. IR (KBr, ν max cm−1): 3260 (NH), 3007, 2934, 2857 (CH), 1667 (C=O), 1601 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.80 (m, 4H, 2CH2), 2.76 (t, 4H, 2CH2), 3.87 (s, 3H, OCH3), 4.45 (s, 2H, CH2Cl), 6.74–7.79 (m, 8H, Ar-H), 10.51 (s, 1H, NH, D2O exchangeable); MS m/z (%) 506 (M++2, 16), 504 (M+, 50), 452 (9), 426 (100), 411 (18), 383 (17), 354 (49), 329 (11), 257 (10), 198 (33), 77 (54). Anal. Calcd. For C27H22ClN3O3S (504.00): C, 64.34; H, 4.40; N, 8.34%. Found: C, 64.11; H, 4.12; N, 8.58%.
2-Chloro-N-(2-(furan-2-yl)-4-(thiophen-2-yl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)acetamide (6b), brown solid, (71% yield), m.p. 338 °C. IR (KBr, ν max cm−1): 3247 (NH), 3011, 2938, 2855 (CH), 1659 (C=O), 1589 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.80–1.85 (m, 4H, 2CH2), 2.77 (s, 2H, CH2), 2.92 (s, 2H, CH2), 4.46 (s, 2H, CH2Cl), 6.73–8.14 (m, 7H, Ar-H), 10.54 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6, 100 MHz): δ = 22.21 (CH2), 22.71 (CH2), 24.71 (CH2), 32.92 (CH2), 43.13 (CH2Cl), 111.60, 113.29, 114.90, 122.34, 127.01, 129.43, 130.01, 134.44, 140.54, 145.84, 146.02, 147.82, 152.34,160.37, 162.49, 163.42 (Ar–C), 167.21 (C=O); MS m/z (%) 482 (M++2, 18), 480 (M+, 53), 482 (M++2, 18), 479 (100), 444 (12), 430 (4), 402 (59), 387 (28), 77(38). Anal. Calcd. For C24H18ClN3O2S2 (480.00): C, 60.06; H, 3.78; N, 8.75%. Found: C, 60.38; H, 4.09; N, 8.99%.

3.1.7. Synthesis of Hydrazinylacetamide Derivatives 7a,b

A mixture of chloroacetamide derivatives 6a,b (5 mmol) and hydrazine hydrate 99% (2 mL, excess) in absolute ethanol (100 mL) was refluxed for 12 h. The reaction mixture was then evaporated to dryness under reduced pressure and the residue was treated with cold water. The obtained solid was collected by filtration and recrystallized from EtOH/H2O to give compounds 7a,b.
N-(2-(furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)-2-hydrazinylacetamide (7a), pale yellow, (78% yield), m.p. 278 °C. IR (KBr, ν max cm−1): 3419, 3259 (NH), 2925, 2863 (CH), 1654 (C=O), 1599 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.74 (m, 4H, 2CH2), 2.19 (s, 2H, NH2, D2O exchangeable), 2.55–2.73 (m, 4H, 2CH2), 3.84 (s, 2H, CH2N), 3.87 (s, 3H, OCH3), 6.13 (s, 1H, NH, D2O exchangeable), 6.72–7.93 (m, 8H, Ar-H), 10.23 (s, 1H, NH, D2O exchangeable); MS m/z (%) 499 (M+, 24), 468 (14), 426 (100), 411 (16), 392 (17), 376 (15), 361 (17), 347 (14), 325 (11), 319 (10), 283 (13), 88 (10), 58 (12), 43 (11). Anal. Calcd. For C27H25N5O3S (499.59): C, 64.91; H, 5.04; N, 14.02%. Found: C, 64.67; H, 5.32; N, 13.78%.
N-(2-(furan-2-yl)-4-(thiophen-2-yl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)-2-hydrazinylacetamide (7b), pale yellow, (75% yield), m.p. 306 °C. IR (KBr, ν max cm−1):): 3422, 3262 (NH), 2935 (CH), 1656 (C=O), 1591 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.85 (m, 4H, 2CH2), 2.20 (s, 2H, NH2, D2O exchangeable), 2.78 (t, 2H, CH2), 2.91 (t, 2H, CH2), 3.67 (s, 2H, CH2N), 5.32 (s, 1H, NH, D2O exchangeable), 6.75–8.16 (m, 7H, Ar-H), 10.22 (s, 1H, NH, D2O exchangeable); MS m/z (%) 475 (M+, 28), 461 (16), 446 (100), 374 (10), 348 (12), 319 (14), 291 (13), 73 (9), 43 (10). Anal. Calcd. For C24H21N5O2S2 (475.59): C, 60.61; H, 4.45; N, 14.73%. Found: C, 60.88; H, 4.69; N, 14.98%.

3.1.8. Synthesis of Arylidene Derivatives 8a,b

A mixture of compounds 7a,b (1 mmol) and the appropriate aldehyde (1 mmol) in glacial acetic acid (20 mL) was refluxed for 8 h. After reaction completion, the reaction mixture was concentrated and poured into cold water. The formed solid was collected by filtration, washed with water, and recrystallized from ethanol to give 8a,b.
2-(2-(3,4-Dimethoxybenzylidene)hydrazinyl)-N-(2-(furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)acetamide (8a), brown solid, (66% yield), m.p. 187 °C. IR (KBr, ν max cm−1): 3429 (NH), 2922, 2856 (CH), 1647 (C=O), 1589 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.76–1.93 (m, 4H, 2CH2), 2.50–2.73 (m, 4H, 2CH2), 3.58 (s, 2H, NCH2), 3.85 (s, 9H, 3OCH3), 6.22 (br.s, 1H, NH, D2O exchangeable), 6.72–7.92 (m, 11H, Ar-H), 8.18 (s, 1H, CH=N), 10.11 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6, 100 MHz): δ = 22.32 (CH2), 22.73 (CH2), 23.80 (CH2), 32.92 (CH2), 47.54 (CH2N), 55.69, 55.95, 56.33 (3OCH3), 109.79, 111.70, 113.02, 113.29, 113.69, 114.20, 117.21, 117.37, 123.37, 126.63, 129.16, 129.51, 130.08, 132.32, 145.28, 145.58, 147.54, 147.84, 148.12, 149.61, 152.76, 152.94, 154.64, 160.19, 162.51 (Ar–C, CH=N), 168.86 (C=O); MS m/z (%) 647 (M+, 22), 591 (2), 574(100), 425(10), 355(2). Anal. Calcd. For C36H33N5O5S (647.75): C, 66.75; H, 5.14; N, 10.81%. Found: C, 66.45; H, 4.88; N, 11.12%.
N-(2-(furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)-2-(2-(4-methylbenzylidene)hydrazinyl)acetamide (8b), brown solid, (64% yield), m.p. 122 °C. IR (KBr, ν max cm−1): 3433 (NH), 2932, 2856 (CH), 1662 (C=O), 1608 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.78–1.94 (m, 4H, 2CH2), 2.37 (s, 3H, CH3), 2.51–2.75 (m, 4H, 2CH2), 3.64 (s, 2H, NCH2), 3.86 (s, 3H, OCH3), 4.45 (s, 1H, NH, D2O exchangeable), 6.78–8.04 (m, 12H, Ar-H), 8.67 (s, 1H, CH=N), 11.20 (s, 1H, NH, D2O exchangeable); MS m/z (%) 601 (M+, 8), 533 (12), 520 (12), 503 (41), 427 (17), 309 (100), 133 (10), 119(33), 68 (17). Anal. Calcd. For C35H31N5O3S (601.73): C, 69.86; H, 5.19; N, 11.64%. Found: C, 69.49; H, 4.88; N, 11.31%.

3.1.9. Synthesis of Amine Acetamide Derivatives 9a,b

A mixture of chloroacetamide derivative 6a (0.50 g, 1 mmol) and the appropriate amine (1 mmol) in N,N-dimethylformamide (10 mL) was refluxed for 12 h. After reaction completion, the reaction mixture was poured into an ice–water mixture. The obtained solid was collected by filtration and recrystallized from acetone to give compounds 9a,b.
N-(2-(furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)-2-morpholinoacetamide (9a), yellow solid, (69% yield), m.p. 143 °C. IR (KBr, ν max cm−1):): 3432 (NH), 2924(CH), 1651 (C=O), 1601 (C=N); 1H-NMR (CDCl3, 400 MHz): δ = 1.73–1.75 (m, 4H, 2CH2), 2.47–2.69 (m, 4H, 2CH2N), 2.80 (s, 2H, CH2), 2.87 (s, 2H, CH2), 3.31 (s, 2H, CH2N), 3.64 (s, 4H, 2CH2O), 3.93 (s, 3H, OCH3), 6.48–7.93 (m, 8H, Ar-H), 9.08 (s, H, NH, D2O exchangeable); 13C-NMR (CDCl3, 100 MHz): δ = 22.53 (CH2), 22.67 (CH2), 23.27 (CH2), 32.95 (CH2), 49.38 (CH2N), 55.43 (OCH3), 62.12 (2CH2N), 67.11 (2CH2O), 110.34, 111.62, 112.46, 112.86, 113.25, 114.12, 117.48, 118.27, 124.54, 129.42, 130.12, 131.56, 143.96, 144.53, 148.64, 153.19, 160.17, 160.97 (Ar–C); MS m/z (%) 554 (M+, 25), 498 (10), 454 (11), 426 (100), 380 (9), 100 (12), 86(14). Anal. Calcd. For C31H30N4O4S (554.67): C, 67.13; H, 5.45; N, 10.10%. Found: C, 67.42; H, 5.73; N, 9.88%.
N-(2-(furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)-2-(4-methylpiperazin-1-yl)acetamide (9b), yellow solid, (71% yield), m.p. 158-159°C. IR (KBr, ν max cm−1): 3422 (NH), 2923, 2851 (CH), 1654 (C=O), 1602 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.74 (s, 4H, 2CH2), 2.25 (s, 3H, NCH3), 2.51–2.56 (m, 8H, 4 NCH2), 2.73 (s, 2H, CH2), 2.88 (s, 2H, CH2), 3.28 (s, 2H, NCH2), 3.85 (s, 3H, OCH3), 6.71–7.95 (m, 8H, Ar-H), 9.65 (s, 1H, NH, D2O exchangeable); MS m/z (%) 567 (M+, 33), 430 (12), 427 (100), 393 (11), 379 (8), 365 (9). Anal. Calcd. For C32H33N5O3S (567.71): C, 67.70; H, 5.86; N, 12.34%. Found: C, 67.98; H, 5.54; N, 12.03%.

3.1.10. Synthesis of N-(2-(Furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno [3,2-b]quinolin-10-yl)benzenesulfonamide 10

A mixture of the amine 5a (0.43 g, 1 mmol) and benzenesulfonyl chloride (0.18 g, 1 mmol) in pyridine (10 mL) was refluxed for 12 h. After cooling, the reaction mixture was poured into an ice–water mixture. The precipitate formed was isolated by filtration, washed with water, dried, and recrystallized from acetone to give sulfonamide derivative 10, a grey solid, (74% yield), m.p. 303–304 °C. IR (KBr, ν max cm−1): 3424, (NH), 3088, 2927, 2852 (CH), 1600 (C=N), 1335, 1170 (SO2); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.80 (s, 4H, 2CH2), 2.71 (s, 2H, CH2), 3.09 (s, 2H, CH2), 3.90 (s, 3H, OCH3), 6.77–7.99 (m, 13H, Ar-H), 11.04 (s, 1H, NH, D2O exchangeable); MS m/z (%) 567 (M+, 15), 490 (6), 474 (14), 488 (5), 474 (14), 460 (21), 446 (47), 310 (100), 77 (4). Anal. Calcd. For C31H25N3O4S2 (567.68): C, 65.59; H, 4.44; N, 7.40%. Found: C, 65.33; H, 4.12; N, 7.12%.

3.1.11. Synthesis of 1-(2-(Furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno [3,2-b]quinolin-10-yl)-3-phenylurea 11

A mixture of amine 5a (0.43 g, 1 mmol) and phenyl isocyanate (0.12 g, 1 mmol) in absolute ethanol (20 mL) containing a few drops of glacial acetic acid was refluxed for 8 h. The reaction mixture was then evaporated to dryness under reduced pressure and the residue was treated with cold water. The formed solid was collected by filtration and recrystallized from 2-propanol to give phenylurea derivative 11, a pale yellow solid, (77% yield), m.p. 118–120 °C. IR (KBr, ν max cm−1): 3427, 3294 (NH), 2924, 2857 (CH), 1636 (C=O); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.78-1.79 (m, 4H, 2CH2), 2.55–2.62 (m, 4H, 2CH2), 3.86 (s, 3H, OCH3), 6.75–7.95 (m, 13H, Ar-H), 9.08, 10.15 (2s, 2H, 2NH, D2O exchangeable); MS m/z (%) 546 (M+, 31), 468 (12), 426 (45), 411 (12), 135 (10), 92 (40), 76 (40), 57 (100). Anal. Calcd. For C32H26N4O3S (546.65): C, 70.31; H, 4.79; N, 10.25%. Found: C, 70.60; H, 4.52; N, 10.69%.

3.1.12. Synthesis of Schiff Bases 12a,b

A mixture of amine 5a (0.43 g, 1 mmol) and the appropriate aldehyde (1 mmol) in glacial acetic acid (20 mL) was refluxed for 12 h. After reaction completion, the reaction mixture was poured into an ice–water mixture. The obtained solid was collected by filtration, washed with water, and recrystallized from ethanol to give compounds 12a,b.
4-(((2-(furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)imino)methyl)-N,N-dimethylaniline (12a), orange solid, (74% yield), m.p. 149°C. IR (KBr, ν max cm−1): 2921 (CH), 1604 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.75 (s, 4H, 2CH2), 2.57–2.84 (m, 4H, 2CH2), 2.93 (s, 6H, N(CH3)2), 3.84 (s, 3H, OCH3), 6.67–7.90 (m, 12H, Ar-H), 8.30 (s, 1H, CH=N)); 13C-NMR (DMSO-d6, 100 MHz): δ = 22.68 (CH2), 22.82 (CH2), 23.68 (CH2), 33.40 (CH2), 40.72 (2NCH3) 55.45 (OCH3), 109.50, 111.51, 112.19, 112.96, 113.12, 113.55, 114.21, 117.23, 125.21, 129.61, 130.94, 131.30, 131.69, 132.03, 147.73, 147.95, 149.71, 154.65, 157.65, 162.49, 162.62 (Ar–C, CH=N); MS m/z (%) 558 (M+, 29), 437(28), 425 (40), 407(19) 208 (100), 134 (23). Anal. Calcd. For C34H30N4O2S (558.21): C, 73.09; H, 5.41; N, 10.03%. Found: C, 73.38; H, 5.79; N, 9.68%.
N-(2-(furan-2-yl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)-1-(thiophen-2-yl)methanimine (12b), pale yellow solid, (76% yield), m.p. 114 °C. IR (KBr, ν max cm−1): 3095, 2921, 2854 (CH), 1611 (C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.72–1.78 (m, 4H, 2CH2), 2.61-2.73 (m, 4H, 2CH2), 3.86 (s, 3H, OCH3), 6.70-7.99 (m, 11H, Ar-H), 8.84 (s, 1H, CH=N)); MS m/z (%) 521 (M+, 84), 520 (100), 438 (4), 426 (68), 425 (11), 412 (5), 411(7), 398 (7), 110 (4), 96(7), 83 (5). Anal. Calcd. For C30H23N3O2S2 (521.65): C, 69.07; H, 4.44; N, 8.06%. Found: C, 69.35; H, 4.73; N, 7.74%.

3.1.13. Synthesis of Oxobutanamide Derivative 13

A mixture of amine 5b (0.40 g, 1 mmol) and ethyl acetoacetate (0.13 g,1 mmol) in dimethylsulfoxide (20 mL) containing anhydrous sodium carbonate (0.4 g) was heated at 80 °C with stirring for 8 h. The reaction mixture was poured into an ice–water mixture and left in the refrigerator overnight. The obtained solid was collected by filtration, washed with water and recrystallized from ethanol to give N-(2-(furan-2-yl)-4-(thiophen-2-yl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-yl)-3-oxobutanamide 13, a pale yellow solid, (76% yield), m.p. 217–218 °C. IR (KBr, ν max cm−1): 3427 (NH), 2921, 2852 (CH), 1716, 1643 (C=O); 1H-NMR (CDCl3, 400 MHz): δ = 1.90 (s, 7H, 2CH2, CH3), 2.62 (t, 2H, CH2), 2.91 (t, 2H, CH2), 4.27 (s, 2H, CH2C=O), 6.60 (s, 1H, Ar-H), 7.22 (s, 2H, Ar-H), 7.29 (s, 1H, NH, D2O exchangeable), 7.54 (s, d, 1H, J = 6 Hz, Ar-H), 7.62 (d, 1H, J =7.2 Hz, Ar-H), 7.90 (s, 1H, Ar-H), 8.19 (d, 1H, J = 3.6 Hz, Ar-H); MS m/z (%) 487 (M+, 22), 402 (100), 405 (13), 375 (25), 362 (8), 347 (4), 304 (11), 295 (4), 280 (5), 83 (4). Anal. Calcd. For C26H21N3O3S2 (487.59): C, 64.05; H, 4.34; N, 8.62%. Found: C, 64.33; H, 4.62; N, 8.94%.

3.1.14. Synthesis of N-(5-methyl-4H-pyrazol-3-yl)-10-amine Derivative 14

A mixture of 13 (0.49 g, 1 mmol) and hydrazine hydrate 99% (1 mL, excess) in N,N-dimethylformamide (15 mL) was refluxed for 12 h. After reaction completion, the reaction mixture was poured into an ice–water mixture. The obtained solid was collected by filtration, washed with water, and recrystallized from acetone to give 2-(furan-2-yl)-N-(5-methyl-4H-pyrazol-3-yl) -4-(thiophen-2-yl)-6,7,8,9-tetrahydropyrido[3′,2′:4,5]thieno[3,2-b]quinolin-10-amine 14, a brown solid, (68% yield), m.p. 274°C. IR (KBr, ν max cm−1): 3326 (NH), 2925 (CH), 1637, 1597(C=N); 1H-NMR (DMSO-d6, 400 MHz): δ = 1.79–1.81 (m, 4H, 2CH2), 2.51–2.77 (m, 4H, 2CH2), 2.89 (s, 3H, CH3), 3.00 (s, 2H, pyrazol-CH2), 6.73–8.26 (m, 7H, Ar-H), 8.53 (s, 1H, NH, D2O exchangeable); MS m/z (%) 483 (M+, 26), 447 (22), 403 (100), 401(45), 386 (57), 292 (25), 278 (42), 263 (13), 180 (37), 83 (33). Anal. Calcd. For C26H21N5OS2 (483.61): C, 64.57; H, 4.38; N, 14.48%. Found: C, 64.86; H, 4.66; N, 14.20%.

3.2. Antimicrobial Activity

3.2.1. Antibacterial Assay

The clinical control strains of Gram-negative bacteria (Escherichia coli 8739, Salmonella typhimurium 14028, Pseudomonas aeruginosa 27853) and Gram-positive bacteria (Bacillus subtilis 6633, Bacillus cereus 33018, Staphylococcus aureus 25923), were obtained from microbial inoculums by subculturing microorganisms into nutrient broth (NB) at 37 °C for 18 h, and were adjusted to 0.125 A° at 625 nm (equivalent to a McFarland 0.5) in 2-fold NB. A 100 μL measure of NB containing each test microorganism was added to the sterile Petri dishes. Antibacterial testing was carried out on all compounds (3a,b–14) using the agar disc-diffusion method [40]. Sterile nutrients were inoculated with 100 μL cell suspension of the chosen test microorganism and poured into Petri dishes (20 cm diameter). Filter paper discs (Whatman, No.3, diameter 5 mm) were loaded with 10 μL containing 100 μg of the tested compounds, as well as the standard drug. Composite compound disks were placed on the surface of inoculated agar plates and kept at low temperatures before incubation in order to support the swelling and diffusion of the microbial growth. The plates were incubated at 37 °C in the incubator. Experiments were performed in duplicate and the diameters of inhibition zones were measured after 24 h. A diameter of inhibition zone (DIZ) assay was performed to evaluate the antimicrobial potential of the compounds against the tested organisms compared with reference antibiotics. All measurements were done in DMSO as a solvent, which has zero inhibition activity [41], compared with amoxicillin trihydrate (inhibition zones against six strains = 20, 21, 18, 17, 19, and 19 mm, respectively) and gentamycin (inhibition zone against MRSA = 15 mm) as reference antibiotics. Minimum inhibition concentration (MIC) values of the active compounds and the standard drugs were determined using a 7-fold dilution (125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 μg/mL) procedure [42]. Tubes showing no growth of the test organism were counted and the minimum dilution of the sample which caused the inhibition of growth of the tested organism (MIC) was specified, compared with amoxicillin trihydrate as a positive control (MIC value for the six strains = 15.63 μg/mL), as shown in Table 1 and Table 2.

3.2.2. Escherichia coli DNA Gyrase Supercoiling Inhibition Assay

The assay for determining IC50 values (TopoGEN) was performed on black streptavidin-coated 96 well micro-titer plates (Thermo Scientific Pierce). The plate was hydrated with the wash buffer supplied (137 mM NaCl, 20 mM Tris-HCl (pH 7.6), 0.05% (v/v) Tween 20, 0.01% (w/v) BSA). Biotinylated oligonucleotide in wash buffer was immobilized onto the wells. The excess oligonucleotide was washed off and the enzyme assay was carried out in the wells (5 min). The final reaction volume was 30 µL in buffer (24 mM KCl; 35 mM TrisHCl (pH 7.5); 2 mM DTT; 4 mM MgCl2; 1.8 mM spermidine; 1 mM ATP; 0.1 mg/mL albumin; and 6.5% (w/v) glycerol), in addition to 1.5 U of DNA gyrase from E. coli, 0.008% Tween 20, 0.75 µg of relaxed pBR322 plasmid, and 3 µL of inhibitor solution in 10% DMSO. Reactions were incubated for 30 min at 37 °C and, after addition of the TF buffer (50 mM NaOAc (pH 5.0), 50 mM MgCl2 and 50 mM NaCl), which terminated the enzymatic reaction, for another 30 min at room temperature to allow triplex formation (biotin–oligonucleotide–plasmid). The unbound plasmid was washed off using TF buffer, and a solution of ethidium bromide stain in T10 buffer (10 mM Tris HCl (pH 8.0) and 1 mM EDTA) was added. After mixing, the fluorescence (excitation, 485 nm; emission, 535 nm) was read using a BioTek’s Synergy H4 microplate reader. Screening was investigated at inhibitor concentrations of 100 µM and 10 µM. For the most effective 10 compounds, IC50 was determined with 7 concentrations of the inhibitors. Calculation of IC50 values by using GraphPad Prism software and the concentration of inhibitor have been represented where the residual activity of the enzyme was 50% in three independent measurements; the final result is given as their average value. Novobiocin (IC50 = 4.17 µM for E. coli DNA gyrase) was used as a reference drug [43,44]. E. coli DNA gyrase supercoiling inhibition (IC50 µM) of the tested compounds (3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14) and reference antibiotics (novobiocin) are listed in Table 3.

3.2.3. Molecular Docking

The molecular docking simulation study was performed using Molecular Operating Environment (MOE®) 2008.10 software [45]. The crystal structures of E. coli topoisomerase II DNA gyrase B complexed with their ligands novobiocin (PDB code: 1AJ6) [37,38] was retrieved from the Protein Data Bank. Initially, the co-crystallized ligands were re-docked into the assigned active E. coli DNA gyrase B enzyme to evaluate a root-mean-square deviation value. The molecular docking procedure was done for the newly synthesized compounds (3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14) into the ATP-binding site of E. coli DNA gyrase B (PDB code: 1AJ6) following the previously reported method [45].

4. Conclusions

Novel bicyclic thieno[2,3-b]pyridines and tetracyclic pyridothienoquinolines (3a,b–14) were synthesized and evaluated for their in vitro antimicrobial activity against six bacterial strains. Whereas, compounds 3a,b, 4a, 5b, 6b, 7a, 9b, 12b, and 14 all showed significant activity, compounds 9a and 12b were the most active of them, with MIC = 15.63 µg /mL, equal to that of the reference for all tested organisms. Furthermore, the active synthesized compounds (4a, 7a, 9b, and 12b) which showed good in vitro growth inhibitory activity against S. aureus (MIC = 15.63 µg/mL) were selected to be evaluated for their inhibitory activity against the resistant bacteria (MRSA); the results of this preliminary test revealed that Schiff base 12b and the hydrazide derivative 7a had inhibition zones of 15 mm and 18 mm, while that of gentamicin was 15 mm. Subsequently, the most active compounds were also screened for their E. coli DNA gyrase inhibitory activity, with comparison with novobiocin as a reference DNA gyrase inhibitor. It was found that compounds 3a,b, 4a, 9b, and 12b displayed the highest inhibitory capacities (IC50 = 2.26–5.78 µM), with that of novobiocin being IC50 = 4.17 µM. In addition, docking studies were performed to estimate the mode of binding of the mostly active compounds to E. coli DNA gyrase B active binding site compared with binding mode of novobiocin. From the analysis of the docking data, compounds (3a,b, 4a, 9b, and 12b) adopted the best binding style with docking score = −6.83–−8.43 Kcal/mol, while novobiocin had a docking score = −6.30 Kcal/mol.
This study showed the importance of fused thienopyridine–quinolines which, with further optimization through structure-based design, could provide potent antimicrobial agents and DNA gyrase inhibitors through their new structural features able to deal with antimicrobial resistance.

Supplementary Materials

The following are available online, Figures S1–S53: NMR and MS spectra of compounds 2a–14.

Author Contributions

E.M.M.E.-D. conceptualization, investigated, and wrote original draft; E.A.A.E.-M. elucidated the structures, investigated, and wrote the final form, S.H. curated data, investigated, and reviewed manuscript, E.A.K. investigated the antimicrobial aspects and E.S.N. performed the software and docking study.

Funding

This research received no external funding.

Acknowledgments

The authors are grateful to National Research Centre for its support of this work.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 2a, 3a, 5a and 5b are available from the authors.
Molecules 24 03650 sch001 550
Scheme 1. Synthesis of target compounds 3a,b–5a,b.
Scheme 1. Synthesis of target compounds 3a,b–5a,b.
Molecules 24 03650 sch001
Molecules 24 03650 sch002 550
Scheme 2. Synthesis of fused thienopyridine-quinolines 6a,b–9a,b.
Scheme 2. Synthesis of fused thienopyridine-quinolines 6a,b–9a,b.
Molecules 24 03650 sch002
Molecules 24 03650 sch003 550
Scheme 3. Synthesis of fused thienopyridine-quinolines 10–14.
Scheme 3. Synthesis of fused thienopyridine-quinolines 10–14.
Molecules 24 03650 sch003
Molecules 24 03650 g001 550
Figure 1. Comparison of antibacterial activity (MIC in µg/mL) of compounds 3a,b–14 and reference antibiotic (amoxicillin trihydrate).
Figure 1. Comparison of antibacterial activity (MIC in µg/mL) of compounds 3a,b–14 and reference antibiotic (amoxicillin trihydrate).
Molecules 24 03650 g001
Molecules 24 03650 g002 550
Figure 2. 2D (a) and 3D (b) diagrams illustrating the binding patterns of the co-crystallized ligand novobiocin into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Figure 2. 2D (a) and 3D (b) diagrams illustrating the binding patterns of the co-crystallized ligand novobiocin into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Molecules 24 03650 g002
Molecules 24 03650 g003 550
Figure 3. 2D (a) and 3D (b) diagrams illustrating the binding patterns of compound 3a into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Figure 3. 2D (a) and 3D (b) diagrams illustrating the binding patterns of compound 3a into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Molecules 24 03650 g003
Molecules 24 03650 g004 550
Figure 4. 2D (a) and 3D (b) diagrams illustrating the binding patterns of compound 4a into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Figure 4. 2D (a) and 3D (b) diagrams illustrating the binding patterns of compound 4a into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Molecules 24 03650 g004
Molecules 24 03650 g005 550
Figure 5. 2D (a) and 3D (b) diagrams illustrating the binding patterns of compound 9b into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Figure 5. 2D (a) and 3D (b) diagrams illustrating the binding patterns of compound 9b into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Molecules 24 03650 g005
Molecules 24 03650 g006 550
Figure 6. 2D (a) and 3D (b) diagrams illustrating the binding patterns of compound 12b into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Figure 6. 2D (a) and 3D (b) diagrams illustrating the binding patterns of compound 12b into the ATP-active pocket of E. coli DNA gyrase B (PDB code: 1AJ6).
Molecules 24 03650 g006
Table
Table 1. The diameter of inhibition zones (mm) and minimum inhibitory concentration (MIC) values (µg/mL).
Table 1. The diameter of inhibition zones (mm) and minimum inhibitory concentration (MIC) values (µg/mL).
M.OE. coliS. typhimuriumP. aeruginosaB. subtilisB. cereusS. aureus
Compound
3a18
(15.63)		15
(15.63)		15
(15.63)		10
(31.25)		13
(31.25)		12
(31.25)
3b19
(15.63)		18
(15.63)		13
(31.25)		12
(31.25)		18
(15.63)		13
(31.25)
4a18
(15.63)		15
(15.63)		13
(31.25)		15
(15.63)		16
(15.63)		18
(15.63)
4b12
(31.25)		15
(15.63)		NA
10
(31.25)		NA
12
(31.25)
5a12
(31.25)		NA
12
(31.25)		10
(31.25)		13
(31.25)		NA
5b15
(15.63)		17
(15.63)		14
(15.63)		12
(31.25)		16
(15.63)		13
(31.25)
6a13
(31.25)		NA
13
(31.25)		10
(31.25)		13
(31.25)		12
(31.25)
6b16
(15.63)		12
(31.25)		12
(31.25)		15
(15.63)		15
(15.63)		NA
7a16
(15.63)		11
(31.25)		11
(31.25)		17
(15.63)		15
(15.63)		19
(15.63)
7bNA
10
(31.25)		12
(31.25)		8
(62.5)		11
(31.25)		10
(31.25)
8aNA
NA
NA
11
(31.25)		9
(31.25)		11
(31.25)
8bNA
NA
NA
9
(31.25)		9
(62.5)		8
(62.5)
9aNA
NA
11
(31.25)		14
(15.63)		16
(15.63)		12
(31.25)
9b20
(15.63)		19
(15.63)		18
(15.63)		15
(15.63)		18
(15.63)		16
(15.63)
10NA
NA
NA
8
(62.5)		9
(62.5)		9
(31.25)
11NA
NA
NA
9
(31.25)		8
(62.5)		8
(62.5)
12a11
(31.25)		8
(62.5)		9
(62.5)		13
(31.25)		11
(31.25)		10
(31.25)
12b18
(15.63)		16
(15.63)		16
(15.63)		15
(15.63)		15
(15.63)		16
(15.63)
138
(62.5)		7
(62.5)		11
(31.25)		15
(15.63)		16
(15.63)		12
(31.25)
1415
(15.63)		12
(31.25)		10
(31.25)		16
(15.63)		17
(15.63)		13
(31.25)
Amoxicillin20
(15.63)		21
(15.63)		18
(15.63)		17
(15.63)		19
(15.63)		19
(15.63)
M.O: microorganism; NA: no activity; Clear zone: mm; MIC ( ): µg/mL
Table
Table 2. The zones of inhibition (mm) against methicillin-resistant Staphylococcus aureus (MRSA).
Table 2. The zones of inhibition (mm) against methicillin-resistant Staphylococcus aureus (MRSA).
M.O.4a7a9b12bGentamicin
MRSA1318141515
M.O: microorganism.
Table
Table 3. E. coli DNA gyrase supercoiling inhibition (IC50 µM) of compounds 3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14 and reference antibiotic (novobiocin).
Table 3. E. coli DNA gyrase supercoiling inhibition (IC50 µM) of compounds 3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14 and reference antibiotic (novobiocin).
3a3b4a5b6a6b7a9b12b14Novobiocin
IC50 µM2.26 ± 0.194.50 ± 0.323.69 ± 0.1516.70 ± 0.8813.74 ± 0.655.95 ± 0.348.66 ± 0.615.78 ± 0.464.60 ± 0.2811.72 ± 0.934.17 ± 0.32
Table
Table 4. Docking results of the compounds (3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14) with E. coli DNA gyrase B kinase using MOE software version 2008.10.
Table 4. Docking results of the compounds (3a,b, 4a, 5b, 6a,b, 7a, 9b, 12b, and 14) with E. coli DNA gyrase B kinase using MOE software version 2008.10.
Compd. NO.Docking Score (Kcal/mol)Amino Acid Residues
(Bond Length A°)		Atoms of CompoundType of Bond
Novobiocin−6.30Asn46 (1.88);
Asp73 (1.89);
Arg76		H(OH)(oxan-4-yl);
H(OCONH2);
C6H2(coumarin) 		H–don
H–don
Arene–cation
3a−7.32Arg136 (2.64);
Thr165 (3.00)		O(CONH2);
N(pyridine)		H–acc
H–acc
3b−6.83Arg136 (2.52);
Thr165 (3.10)		O(CONH2);
N(pyridine)		H–acc
H–acc
4a−7.46Arg136 (2.67);
Thr165 (3.01)		O(CONH2);
N(pyridine)		H–acc
H–acc
5b−6.22Asn46 (3.29)N(pyridine)H–acc
6a−6.24Asn46 (3.00);
Val120 (3.2)		N(pyridine);
O(NHCOCH2)		H–acc
H–acc
6b−6.75Asn46 (2.25);
Val120 (2.72);
Arg76		N(pyridine);
O(NHCOCH2)
thiophene		H–acc
H–acc
Arene–cation
7a−6.25Asn46 (3.28);
Val120 (2.95)		N(pyridine);
O(NHCOCH2)		H–acc
H–acc
9b−8.43Asn46 (2.46);
Asn46 (2.79)		O(NHCOCH2);
N(pyridine)		H–acc
H–acc
12b−7.43Asn46 (2.98);
Arg76 (2.80)		N(N=CH);
O(OCH3)		H–acc
H–acc
14−5.45Asn46 (2.23);
Arg76		N(pyridine);
thiophene		H–acc
Arene–cation

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Mohi El-Deen, E.M.; Abd El-Meguid, E.A.; Hasabelnaby, S.; Karam, E.A.; Nossier, E.S. Synthesis, Docking Studies, and In Vitro Evaluation of Some Novel Thienopyridines and Fused Thienopyridine–Quinolines as Antibacterial Agents and DNA Gyrase Inhibitors. Molecules 2019, 24, 3650. https://doi.org/10.3390/molecules24203650

AMA Style

Mohi El-Deen EM, Abd El-Meguid EA, Hasabelnaby S, Karam EA, Nossier ES. Synthesis, Docking Studies, and In Vitro Evaluation of Some Novel Thienopyridines and Fused Thienopyridine–Quinolines as Antibacterial Agents and DNA Gyrase Inhibitors. Molecules. 2019; 24(20):3650. https://doi.org/10.3390/molecules24203650

Chicago/Turabian Style

Mohi El-Deen, Eman M., Eman A. Abd El-Meguid, Sherifa Hasabelnaby, Eman A. Karam, and Eman S. Nossier. 2019. "Synthesis, Docking Studies, and In Vitro Evaluation of Some Novel Thienopyridines and Fused Thienopyridine–Quinolines as Antibacterial Agents and DNA Gyrase Inhibitors" Molecules 24, no. 20: 3650. https://doi.org/10.3390/molecules24203650

APA Style

Mohi El-Deen, E. M., Abd El-Meguid, E. A., Hasabelnaby, S., Karam, E. A., & Nossier, E. S. (2019). Synthesis, Docking Studies, and In Vitro Evaluation of Some Novel Thienopyridines and Fused Thienopyridine–Quinolines as Antibacterial Agents and DNA Gyrase Inhibitors. Molecules, 24(20), 3650. https://doi.org/10.3390/molecules24203650

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