Synthesis, molecular modelling and docking studies of new thieno[2,3-b:4,5-b′] dipyridine compounds as antimicrobial agents

2.1 Materials and methods

Melting points were determined on an Electrothermal 9100 instrument. The IR spectra (KBr discs) were captured by a Thermo Scientific's Nicolet iS10 FTIR spectrometer. Using a spectrometer manufactured by JEOL, ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were recorded in DMSO-d₆. Mass analyses were recorded by a Quadrupole GC–MS (DSQII) mass spectrometer at a setting of 70 eV. Elemental analyses were determined using a Perkin-Elmer 2400 analyzer (C, H, and N).

2.2 Synthesis of 2-amino-4,7,9-trimethyl-3-substituted-thienodipyridines 2 and 3

A mixture of 2-acetyl-3-aminothienopyridine compound 1 (0.88 g, 4 mmol) and malononitrile (0.27 g, 4 mmol) or cyanoacetamide (0.34 g, 4 mmol) in sodium ethoxide solution (prepared from 0.14 g sodium metal and 30 mL absolute ethanol) was refluxed for 4 h. The mixture was poured into ice-cold water and neutralized by dilute HCl. The separated solid was collected and crystallized from ethanol to furnish the corresponding thieno-dipyridine compounds 2 and 3, respectively.

2.3 Synthesis of 2,4,7,9-tetramethyl-3-substituted-thienodipyridines 4 and 5

A mixture of 2-acetyl-3-aminothienopyridine compound 1 (0.88 g, 4 mmol) and acetylacetone (0.40 g, 4 mmol) or ethyl acetoacetate (0.52 g, 4 mmol) in sodium ethoxide solution (prepared from 0.14 g sodium metal and 30 mL absolute ethanol) was refluxed for 4 h. The mixture poured into ice-cold water and neutralized by dilute HCl. The solid that formed was collected and crystallized from ethanol to give the corresponding thieno-dipyridine compounds 4 and 5, respectively.

2.4 Synthesis of 4-hydroxy-7,9-dimethylthieno[2,3-b:4,5-b′]dipyridine compounds 6 and 8

To a solution of thienopyridine compound 1 and/or 7 (3 mmol) in 30 mL xylene, DMF-DMA (0.36 mL, 3 mmol) was added. The solution was subjected to reflux for 4 h. The solid that obtained upon cooling was collected and crystallized from EtOH to furnish the targeting 4-hydroxy-thieno[2,3-b:4,5-b′]dipyridine compounds 6 and 8, respectively.

2.5 Ethyl 4-hydroxy-7,9-dimethylthieno[2,3-b:4,5-b′]dipyridine-3-carboxylate (8)

Reddish brown powder; yield = 56%; m.p. = 302–303 °C. IR ( $\overline{v}$ /cm⁻¹): 3174 (O—H), 1667 (C⚌O), 1638 (C⚌N). ¹H NMR (δ/ppm): 1.29 (t, J = 7.00 Hz, 3H, —OCH₂CH₃), 2.55 (s, 3H, —CH₃), 2.71 (s, 3H, —CH₃), 4.23 (q, J = 7.00 Hz, 2H, —OCH₂CH₃), 6.89 (s, 1H, C5-pyridine), 8.73 (s, 1H, C2-pyridine), 14.62 (s, 1H, O—H). ¹³C NMR (δ/ppm): 14.27 (—OCH₂CH₃), 19.06 (—CH₃), 23.94 (—CH₃), 60.88 (—OCH₂CH₃), 111.85, 115.73, 122.72, 124.27, 144.47, 152.03, 157.29, 159.38, 160.15, 163.51, 165.64 (C⚌O). MS (EI, m/z): 302 (41.29%). Anal. Calcd. for C₁₅H₁₄N₂O₃S (302.07): C, 59.59; H, 4.67; N, 9.27%. Found: C, 59.76; H, 4.60; N, 9.38%.

2.6 Computational calculations

Geometrical optimization of the synthesized thieno-dipyridine derivatives was carried out by the Gaussian 09W program (Frisch et al., 2009) at DFT/B3LYP/6-311⁺⁺G(d,p) methodology (Becke, 1993, Lee et al., 1988, Perdew and Wang, 1992). The resulting electronic and frontier molecular orbitals of the optimized structures have been obtained using the GaussView program (Dennington et al., 2009). The B3LYP/DNP (version 3.5) method in the DMol3 module of Materials Studio software (BIOVIA, 2017) was applied in estimating the Fukui indices (Delley, 2006).

2.7 Antimicrobial assay

The antibacterial procedure that was utilized in the study of the synthesized thieno-dipyridines is the agar disc-diffusion assay (Azoro, 2002, Al-Anazi et al., 2019, Kaushal et al., 2018). Gram-positive and Gram-negative microorganisms “(Bacillus subtilis MTCC-5981, Staphylococcus aureus MTCC-740, Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa MTCC-424” are used in the testing. By sub-culturing microorganisms at 37 °C into microbial inoculums for 18 h, the bacteria were extracted. A 100 mL portion of every test microorganism was moved to the sterilised Petri dishes. The experimental findings are reported as a mean standard deviation because they were performed in triplicate (SD). After 24 h, the inhibition zone diameters (IZD) were evaluated in duplicate experiments. The antibacterial ability of the synthesized thieno-dipyridines toward the examined pathogens was assessed using the IZD assay in comparison to the reference antibiotic Ampicillin. In contrast, all experiments were carried out using the solvent DMSO, which has no inhibitory activity. Meanwhile, minimum inhibitory concentration (MIC) values of thieno[2,3-b:4,5-b′] dipyridine hybrids were specified using agar dilution bacterial cultures and mentioned in the supplementary file (Al-Anazi et al., 2019).

2.8 Molecular docking (MD)

M.O.E “2015.10” program was used to carry out the molecular docking exploration. The Protein Data Bank was used to locate the structure of “E. coli topoisomerase II DNA gyrase B” (PDB: 1AJ6) (Mohi El-Deen et al., 2019). To calculate a root-mean-square deviation value, the co-crystallized ligands were initially redocked inside the designated active “E. coli DNA gyrase B” enzyme, ignoring heteroatoms and water, docking ligand atoms at the proper sites using 10 poses, optimization, and the ligands were docked. Following a previously described process, the newly synthesized thieno-dipyridines (2, 3, 4, 5, 6, and 8) were molecularly docked inside the ATP-binding position of “E. coli DNA gyrase B” (PDB: 1AJ6).

3.1 Chemistry

The first goal of this study involves exploration the synthetic potentially of 2-acetyl-3-amino-4,6-dimethylthieno[2,3-b]pyridine (1) (Yassin, 2009) via its reaction with active methylene compounds. Thus, the thienopyridine compound 1 was reacted with activated nitriles (namely, malononitrile and cyanoacetamide) in refluxing ethanol/sodium ethoxide solution to give 3-(3-amino-4,6-dimethylthieno[2,3-b]pyridin-2-yl)but-2-enenitrile intermediate (A) formed via the loss of water molecule followed by intramolecular cyclization to afford 2-amino-4,7,9-trimethylthieno[2,3-b:4,5-b′]dipyridine compounds 2 and 3 as sole product in each case (Scheme 1). The structure of thieno-dipyridine compounds 2 and 3 were established based on the data of their spectral analyses. The IR spectrum of compound 2 lacked an absorption band of C⚌O group and displayed bands at 3360, 3294 and 2212 for —NH₂ and -C≡N groups, respectively. The ¹H NMR spectrum of 2 revealed singlet signals at δ 2.32, 2.42, and 2.71 for the protons of three methyl groups. The protons of pyridine-C5 and amino (—NH₂) were recorded as singlet signals at δ 7.04 and 7.12 ppm, respectively. The mass spectrum showed the expected molecular ion peak M⁺ at m/z 268 (13.32%) for the formula C₁₄H₁₂N₄S.

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Scheme 1

Synthesis of 2-aminothieno[2,3-b:4,5-b′]dipyridine derivatives 2 and 3.

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In addition, treatment of thienopyridine compound 1 with acetylacetone and/or ethyl acetoacetate in boiling sodium ethoxide solution was the synthetic route for the production of 2,4,7,9-tetramethylthieno[2,3-b:4,5-b′]dipyridine derivatives 4 and 5, respectively (Scheme 2). The IR spectrum of thieno-dipyridine analogue 4 exhibited the absorption of acetyl-carbonyl group at 1680 (C⚌O) cm⁻¹. The ¹H NMR spectrum of thieno-dipyridine analogue 4 showed five singlet signals at δ 2.19, 2.35, 2.51, 2.57 and 2.69 ppm for the protons of five methyl groups. The proton of pyridine-C5 was recorded as singlet δ 7.11 ppm. The mass analysis of thieno-dipyridine analogue 4 showed the molecular ion peak at m/z 284 (M⁺, 30.53%) referring to the formula C₁₆H₁₆N₂OS.

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Scheme 2

Synthesis of 2,4,7,9-tetramethylthieno[2,3-b:4,5-b′]dipyridine derivatives 4 and 5.

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The reaction of 2-acetyl-3-aminothieno[2,3-b]pyridine derivative 1 with DMF-DMA in boiling xylene did not stop at the intermediate (A) which undergoes intramolecular elimination of dimethylamine molecule. Aromaticity was the driving force behind intermediate (B) to furnish the final product, 4-hydroxythieno[2,3-b:4,5-b′]dipyridine compound 6 (Scheme 3) (Mohamed et al., 2018, Atta and Abdel-Latif, 2021). The IR spectrum exhibited absorptions at 3161 and 1641 cm⁻¹ that are ascribed to the stretching vibrations of hydroxyl (O—H) and imine (C⚌N) functions, respectively. The ¹H NMR spectrum displayed two singlet signals at δ 2.59 (pyridine-CH₃) and 2.78 ppm (pyridine-CH₃). The protons of pyridine ring (C3 and C2) were observed as two double signals at δ 6.70 and 8.17 ppm, respectively. The protons of pyridine-C5 and hydroxyl group were observed as singlet signals at δ 6.91 and 11.15 ppm, respectively. The mass analysis indicated a molecular ion peak at m/z 230 (M⁺, 65.37%) corresponding to the formula C₁₂H₁₀N₂OS.

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Scheme 3

Synthesis of 4-hydroxythieno[2,3-b:4,5-b′]dipyridine compound 6.

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The reaction of ethyl 3-(3-amino-4,6-dimethylthieno[2,3-b]pyridin-2-yl)-3-oxopropanoate (7) (Rodinovskaya and Shestopalov, 2000) with DMF-DMA in boiling xylene did not stop at the intermediate (C) which undergoes intramolecular elimination of dimethylamine molecule followed by tautomerization of the produced intermediate (D) into the tricyclic ring system, 4-hydroxythieno[2,3-b:4,5-b′]dipyridine compound 8 (Scheme 4). The IR spectrum showed the absorptions of hydroxyl (O—H) and ester-carbonyl (C⚌O) functions at 3174 and 1667 cm⁻¹. The ¹H NMR spectrum identified the protons of ethoxy group (COOCH₂CH₃) as triplet and quartet at δ 1.29 and 4.23 ppm. The protons of two methyl groups were observed as singlet signals at δ 2.55 and 2.17 ppm. The protons of pyridine ring systems were recorded at δ 6.89 and 8.73 ppm. The proton of hydroxyl group was recoded as singlet at 14.62 ppm.

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Scheme 4

Synthesis of ethyl 4-hydroxy-thieno[2,3-b:4,5-b′]dipyridine-3-carboxylate compound 8.

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3.2 Computational studies

The DFT calculations of the thieno[2,3-b:4,5-b′]dipyridine derivatives, 2–8, revealed that they have planar structures, with dihedral angles of 0.0° and/or 180.0° (Fig. 3). Whereas, in compound 3, the obtained dihedral angle disclosed that the amide and amino groups were lying out the plane of the thienodipyridine moiety where, for example, the CO_(amd)—C³_(tpy)—C²_(tpy)-N¹_(tpy) = 172.8°, C²_(tpy)—C³_(tpy)—CO_(amd)—OC_(amd) = 19.8°, C²_(tpy)—C³_(tpy)—CO_(amd)—NH_2(amd) = −157.8° and CO_(amd)—C³_(tpy)—C²_(tpy)—NH₂ = −7.1°. Likewise, in compound 4, the acetyl group has been departed the planarity as the CO_(Actl)—C³_(tpy)—C²_(tpy)-N¹_(tpy) = 177.1, C²_(tpy)—C³_(tpy)—CO_(Actl)—OC_(Actl) = 35.6 and C²_(tpy)—C³_(tpy)—CO_(Actl)-Me_(Actl) = −140.2 (Tables S1–S3).

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Fig. 3

Compounds 2–8 DFT Optimized structures.

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Moreover, the structural parameters, i.e., bond length and angle, displayed notable matching with the single crystal X-ray values of analogue derivatives (Klemm et al., 2000), where the lengths presented 0.16 Å maximum difference from the equivalent x-ray, RMSD 5.02–5.49 × 10⁻², while the differences of angles were 0.0–12.1°, RMSD = 4.36–4.86. Such differences might arise from the fact that no intermolecular columbic interactions take place in the quantum calculations because they involve a single gaseous molecule, whereas the actual information was gained from solid interacting molecules in crystal (Sajan et al., 2011) (Tables S1–S3).

The HOMO-LUMO shape and energy plays important role in empathizing the molecule’s electrons donation and acceptance abilities (Bulat et al., 2004). The molecule’s bioactivity may be influenced by HOMO-LUMO charge transfer, which becomes easier when the energy gap is reduced (Xavier et al., 2015, Makhlouf et al., 2018, Bouchoucha et al., 2018). The HOMO of the 2–8 derivatives was chiefly made of the π-orbitals and heteroatoms non-bonding lone pairs of electrons of the thienodipyridine ring along with those of the substituent groups except the ester group in 5 and 8 compounds, while, their LUMO were built principally of the whole molecule π*-orbitals (Fig. 4). The close configuration of HOMO-LUMO affected on the values of their energy, E_H and E_L (Table 1). For instance, the studied compounds offered E_H values from −6.43 eV for 8 to −5.67 eV for 3, as well as, the E_L values were from −3.68 to −2.91 eV and accordingly sorted as 6 < 3 < 5 < 2 < 8 < 4. Similarly, the considered compounds offered close and small gap (ΔE_H-L), 2.32–3.39 eV, and may be sorted as 3 < 2 < 4 < 8 < 5 < 6. As a result, the 2-amino derivatives revealed a lower gap than the methyl and hydroxyl compounds (Table 1).

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Fig. 4

The FMO plots of the compounds 2–8.

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As well, some chemical reactivity descriptors, such as electronegativity (χ), global hardness (η), softness (δ), electrophilicity (ω), electron-donating power (ω⁻) and electron-accepting power (ω⁺) were calculated using the E_H and E_L as follows (Xavier et al., 2015). $$\begin{array}{c}\chi =-\frac{1}{2}\left(E_{\mathit{HOMO}}+E_{\mathit{LUMO}}\right)\eta =-\frac{1}{2}\left(E_{\mathit{HOMO}}-E_{\mathit{LUMO}}\right)\delta =\frac{1}{\eta }\\ \omega =\frac{{\chi }^2}{8\eta }{\omega }^{-}=\frac{{\left(3I+A\right)}^2}{16(I-A)}{\omega }^{+}=\frac{{\left(I+3A\right)}^2}{16(I-A)}\end{array}$$

As shown in Table 1, the compound 3 was the most chemically reactive, lowest kinetically stable and softest derivative where it exhibited the lowest global hardness (η) and highest softness (δ) values. According to electrophilicity index (ω) which evaluate the stabilization energy of acquiring additional electronic charge from the environment, organic molecules with ω > 1.5 eV were ascribed as strong electrophile (Afolabi et al., 2022, Domingo et al., 2016). Thus, the studied compounds were strong electrophile as they exhibited ω index ranged from 1.56 to 2.34 eV following the order 6 < 5 < 3 < 2 < 8 < 4. Likewise, the electron donating (ω⁺) and acceptance (ω⁻) powers of the investigated derivatives, which demonstrated the capability to give and receive electrons, respectively, obeyed the pervious order but they exhibited more donating power, 4.16–7.02 eV, than acceptance one, 8.76–12.06 eV, where smaller values signify enhanced transaction (Afolabi et al., 2022, Domingo et al., 2016) (Table 1).

Moreover, the Mulliken’s atomic charges afforded a respectable interpretation of molecule’s electronegativity and charge transfer (Bhagyasree et al., 2013). The investigated compounds atomic charges revealed that the thienodipyridine nitrogen’s, N¹_(tpy) and N⁶_(tpy), had a positive charge, 0.022–0.376 and 0.013–0.158, that can be attached to their contribution in the fused rings resonance structure (Table 2). While, the negative charge of the sulfur atom, S⁵_(tpy), from −0.173 to −0.730, cleared that its lone pairs of electrons did not participate in resonance of the thienodipyridine moiety. In addition, the carbon atom, C²_(tpy), in the amino derivatives 2–3, has more negative charged than in the methyl ones 4–5, which could be ascribed to the amino group electron release effect, but it has positive charge in derivatives 6–8. Also, the presence of 3-cyano substituent in compound 2 resulted in negative charge, −0.130, on the carbon atom C³_(tpy) while it converted to be positive, 0.146–0.818, in the acetyl, amido and ester substituted derivatives 3–8. Correspondingly, the nitrogen and oxygen atoms of the substituents in all derivatives were negatively charged.

To explore the nucleophilic ( $f_k^{+}$ ) and electrophilic ( $f_k^{-}$ ) attacks susceptible sites, the Fukui’s indices have been estimated (Olasunkanmi et al., 2016, El Adnani et al., 2013, Mi et al., 2015, Messali et al., 2018). The inspected derivatives electrophilic attack indices ( $f_k^{-}$ ) showed that the thienodipyridine sulfur atom, S⁵_(tpy), has the maximum susceptibility while the amino nitrogen, NH₂, occupied the second position in compound 2 and 3. The hydroxy derivatives 6 and 8 presented coincided trend which is S⁵_(tpy) > C²_(tpy) > C²_(tpy) > OH while the methyl derivative 4 and 5 displayed different patterns (Table 3). Likewise, the radical attack indices ( $f_k^0$ ) data exhibited different patterns but the sulfur of thienodipyridine (S⁵_(tpy)) was the highest liable for radical attack in all compounds except in case of derivative 4 in which the first place was taken by acetyl oxygen atom (OC_(Actl)) and then the thienodipyridine sulfur atom. Otherwise, the Fukui’s indices ( $f_k^{+}$ ) demonstrated altered patterns where the S⁵_(tpy) was the most susceptible site in case of 3, 5 and 6 derivatives while it appeared in the second position in 2 and 8 after cyano nitrogen (NC) and carboxylate oxygen (O¹C_(Estr)) atoms, respectively. For instance, the amide derivative 3 displayed that S⁵_(tpy) > OC_(amd) > C⁷_(tpy) > C⁴_(tpy) while the derivative 5 exhibited another one, S⁵_(tpy) > O¹C_(Estr) > C⁷_(tpy) > C^9a_(tpy) (Table 3).

Occasionally, the Fukui’s indices offered inaccurate in estimation probable sites for attack, therefore, the local relative electrophilicity and nucleophilicity descriptors, $s_k^{-}/s_k^{+}$ and $s_k^{+}/s_k^{-}$ , respectively, were computed (Roy et al., 1998b, Roy et al., 1998a, Roy et al., 1999), where δ is global softness, $s_k^{+}=f_k^{+}\times \delta$ and $s_k^{-}=f_k^{-}\times \delta$ . The relative electrophilicity calculations, $s_k^{-}/s_k^{+}$ , presented entirely diverse patterns than the Fukui’s indices. For example, the amino nitrogen, NH₂, occupied the first position in case of 2–3 compounds, while in the other derivatives 4–8, the fused ring sulfur atom, S⁵_(tpy), was the top one followed by the carbon C²_(tpy) and C⁸_(tpy) atoms in 4–5 and 6–8 derivatives, respectively. Likewise, the relative nucleophilicity, $s_k^{+}/s_k^{-}$ , suggested other arrangements of the utmost vulnerable atoms. For instance, in compounds 2–4, although they have thienodipyridine carbon atom, C^9a_(tpy), as the most active site, their second place was captured differentially by cyano (CN), amide (CO_(amd)), and acetyl (CO_(Actl)) carbon atoms, respectively. Moreover, the other compounds 5 and 8 displayed close orders in which the carboxylate carbon presented on the top (CO_(Estr)) and then the carboxylate oxygen atoms, O²C_(Estr) and O¹C_(Estr), in the 2nd and 3rd rank, respectively (Table 3).

In addition, the polarizability (α_total), hyperpolarizabilities (β_total), and dipole moment (μ) are chief molecular parameters which evaluate the molecule’s softness and electron density that mainly effects on intermolecular interactions (Aziz et al., 2022), as well as, optical nonlinearity and response (Shi, 2001, Prasad and Williams, 1991, Williams, 1984, Khan et al., 2021). The dipole moment (μ), polarizability (α_total) and first-order hyperpolarizability (β_total) were described as (Sun et al., 2003, Abraham et al., 2008, Karamanis et al., 2008): $$\mathrm{\mu }=({\mathrm{\mu }}_{\mathrm{x}}^2+{\mathrm{\mu }}_{\mathrm{y}}^2+{\mathrm{\mu }}_{\mathrm{z}}^2){\mathrm{\alpha }}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}=\frac{({\mathrm{\alpha }}_{\mathrm{x}\mathrm{x}}+{\mathrm{\alpha }}_{\mathrm{y}\mathrm{y}}+{\mathrm{\alpha }}_{\mathrm{z}\mathrm{z}})}{3}$$ $${\mathrm{\beta }}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}=\sqrt{{\left({\mathrm{\beta }}_{\mathrm{x}\mathrm{x}\mathrm{x}}+{\mathrm{\beta }}_{\mathrm{x}\mathrm{y}\mathrm{y}}+{\mathrm{\beta }}_{\mathrm{x}\mathrm{z}\mathrm{z}}\right)}^2+{\left({\mathrm{\beta }}_{\mathrm{y}\mathrm{y}\mathrm{y}}+{\mathrm{\beta }}_{\mathrm{y}\mathrm{z}\mathrm{z}}+{\mathrm{\beta }}_{\mathrm{y}\mathrm{x}\mathrm{x}}\right)}^2+{\left({\mathrm{\beta }}_{\mathrm{z}\mathrm{z}\mathrm{z}}+{\mathrm{\beta }}_{\mathrm{z}\mathrm{x}\mathrm{x}}+{\mathrm{\beta }}_{\mathrm{z}\mathrm{y}\mathrm{y}}\right)}^2}$$

The examined compounds dipole moment (μ) was from 4.04 D, for compound 2, to 0.69 D, for compound 5. On comparison with urea (μ = 1.3732 Debye (Ahmed et al., 2008)), the derivatives 3 and 5 have lower dipole moment, 0.92 and 0.50 times, while other exhibited higher values, 1.21–2.94 times, which may be resulted from an overall charge inequality (Table 4). Moreover, the polarizability (α_total) data of the investigated derivatives displayed close values, where the derivative 5 and 6 exhibited the highest and lowest values, 1.94 × 10⁻²³ and 1.37 × 10⁻²³ esu, respectively, and can be arranged as 6 < 2 < 3 < 8 < 4 < 5. Whereas, the first-order hyperpolarizability data of the studied compounds disclosed that the compound 2 has the higher value, β_total = 1.95 × 10⁻³⁰ esu, while 3 has the lowest, β_total = 3.62 × 10⁻³¹ esu. (Table 4). On comparison with urea hyperpolarizability as reference material (β = 3.7389 × 10⁻³¹ esu (Ahmed et al., 2008)), the data indicated that compound 3 and 2 were almost equal and greater than urea, 0.97 and 5.22 times, respectively.

3.3 Biological evaluation

3.3.1

3.3.1 Antimicrobial activity

Using IZD and MIC techniques, the newly synthesized thieno-dipyridine derivatives were examined toward antibacterial effectiveness across Gram-positive and Gram-negative strains. Fig. 5 established the antimicrobial behaviours of the synthesized analogues derivatives toward the antibacterial action because thienopyrimidines have the capability to block the construction of folic acid through both of the two bacterial strains (Wilding and Klempier, 2017). Over the investigated derivatives, they exhibited more reliable results against both “S. aureus and B. subtilis” Gram-positive and “S. typhimurium and E. coli” Gram-negative bacterial strains. Wherever, thieno-dipyridine analogues 2, 3, 5 and 8 nominated good activity as a general, especially toward Gram-positive bacteria rather than Gram-negative bacteria comparative to the results of ampicillin drug reference. Meanwhile, thieno-dipyridine derivative 2 have amino and nitrile groups displayed higher inhibition zone of (IZD = 26 mm, MIC = 18.52 µg/mL) against S. aureus, more than the reference ampicillin (IZD = 24 mm, MIC = 18.52 µg/mL). Likewise, thieno[2,3-b:4,5-b′]dipyridine derivative 3 have amino and carboxamide groups revealed (IZD = 21 mm, MIC = 18.52 µg/mL) against B. subtilis (Gram-positive) in comparison to the reference IZD = 22 mm, MIC = 18.52 µg/mL). Also, thieno-dipyridine derivative 5 have ethyl carboxylate ester moiety shown amazing inhibition against S. aureus with (IZD = 23 mm, MIC = 18.52 µg/mL). Whilst thieno-dipyridine derivative 8 has hydroxyl and ester groups exposed moderate inhibition against S. aureus with (IZD = 22 mm, MIC = 18.52 µg/mL).

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Fig. 5

MIC of the examined derivatives toward both of Gram’s positive and Gram’s negative bacteria.

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Moreover, the results of hybrids 2, 3, 5 and 8 toward the Gram-negative bacteria “S. typhimurium and E. coli” were changed in their reactivity order. Derivatives 5 presented moral inhibition (IZD = 25 mm, MIC = 18.52 µg/mL) against E.coli more than the Ampicillin reference (IZD = 24 mm, MIC = 18.52 µg/mL). Whereas, derivatives 8 displayed moral inhibition (IZD = 23 mm, MIC = 18.52 µg/mL) against S. typhimurium equal to the reference (IZD = 23 mm, MIC = 18.52 µg/mL). Though, hybrid 2 revealed good inhibition (IZD = 21 mm, MIC = 18.52 µg/mL) against E.coli. Moreover, derivative 3 revealed (IZD = 20 mm, MIC = 18.52 µg/mL) against S. typhimurium (Table 5).

Table 5 Inhibition zone and diameter (IZD mm) and minimum inhibition concentrations (MIC µg/mL) of the synthesized thieno[2,3-b:4,5-b′] dipyridine derivatives.

Organism	Gram-positive bacteria		Gram-negative bacteria
	S. aureus	B. subtilis	S. typhimurium	E. coli
2	26 (18.52)	18(18.52)	16(21.43)	21(18.52)
3	20(18.52)	21(18.52)	20(21.43)	18(18.52)
4	NA	17(18.52)	15(21.43)	18(18.52)
5	23(18.52)	19(18.52)	19(21.43)	25(18.52)
6	16(21.43)	14(21.43)	NA	16(21.43)
8	18(18.52)	16(18.52)	23(18.52)	20(18.52)
Ampicillin	25(18.52)	22(18.52)	23(18.52)	24(18.52)

Notes: (IZD) inhibition zone diameter (mm), (MIC) minimum inhibition concentration (µg/mL), (NA) no activity, and Ampicillin is a standard antibiotic in case of Gram-positive bacteria, Cephalothin is a standard antibiotic in case of Gram-negative bacteria.

3.4 Molecular docking (MD)

Molecular docking is one of the most crucial techniques in structure-based drug design as it may provide an understanding of the novel molecules' modes of binding in the appropriate target's binding site, which is an essential stage in drug design (Mohi El-Deen et al., 2019; Nakamura et al., 2017). As the literary survey and PDB database were displayed the binding interactions with the thieno-pyridine derivatives for “E. coli DNA gyrase B” active site (PDB: 1AJ6), it was encouraged to inspect the binding affinity of the newly synthesized thieno[2,3-b:4,5-b′]dipyridine analogues towards (PDB code: 1AJ6) (Mohi El-Deen et al., 2019). In this study, MD studies were carried out to reveal the mechanism of binding between the novel bioactive chemical and the enzyme active binding site as well as any potential binding and the interaction score. Through the thieno-dipyridine system indicating their potential interactions inside “E. coli DNA gyrase B”, docking modeling was used to connect the experiential potencies and structural activity relationships (SAR) of newly created analogues. Table 6 contains the docking outcomes for the examined variants. Meanwhile, Derivative 2 was displayed two π-H bonds among Ile 78 and both of pyridine and thiophene rings through two bonds with length 4.50 and 3.86 Å, respectively. In addition, one H-acceptor between N19 of nitrile moiety with Gly 77 over docking score −5.5039 Kcal/mol and root-main square (RMSD) 0.8837 (Fig. S1). However, derivative 3 was revealed interaction between N3 of pyridine ring with His 99 amino-acid over H-acceptor, docking score −5.2587 Kcal/mol, RMSD = 1.3778, and bond length 3.54 Å (Fig. S2). Moreover, derivative 4 was exposed to interaction between S7 of thiophene ring with Asn 46 amino-acid over H-donor, RMSD = 1.0430, docking score −5.0497 Kcal/mol, and bond length 3.18 Å (Fig. S3). Though, derivative 5 was exhibited π-H interaction between pyridine ring with Val 120 of 1AJ6 amino-acid, highest docking score −5.6385 Kcal/mol, RMSD = 0.9995, and bond length 4.57 Å (Fig. 6).

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Fig. 6

Docking interactions over analogue 5 with 1AJ6.

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Likewise, derivative 6 was H-donor interaction between S7 of thiophene ring with Ala 100 of amino-acid, lowest docking score −4.9306 Kcal/mol, RMSD = 1.1946, and bond length 3.37 Å (Fig. S4). Furthermore, derivative 8 was presented π-H interaction between pyridine ring with Val 120 of 1AJ6 amino-acid, acceptable docking score −5.5244 Kcal/mol, RMSD = 1.3913, and bond length 4.53 Å (Fig. 7).

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Fig. 7

Docking interactions over analogue 8 with 1AJ6.

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Additionally, ampicillin was afforded H-donor between O 10 of carboxylic acid with Asn 46. H-acceptor bond among O 16 of urea moiety and Val 120, and π-H among benzene moiety with Hiss 99 over bond length 2.92, 3.22, and 3.57 Å, respectively, with score −6.2245 Kcal/mol, RMSD = 1.3393, (Fig. S5).

Finally, MD stimulation was pragmatic to endorse how far the synthesized thieno-dipyridine analogues interact as they bonded with many 1AJ6 amino-acids. Each of the six synthesized thieno-dipyridine analogues was reinforced by a network of π-H and H-bonds (H-doner, H-acceptor) with the N-atom and S-atom of both of pyridine and thiophene rings and chemical aromatic structures of thieno-dipyridines cooperate straight with 1AJ6 amino acids. The most of the synthesized thieno-dipyridine analogues had the 1AJ6 pocket, which were appeared as fork over polar and nonpolar residues of 1AJ6 amino acids (Gly 77, Ile 78, His 99, Asn 46, Val 120, and Ala 100). As well, the plurality of the thieno-dipyridine derivatives have the same amino acids, which is viewed as strong evidence for the effectiveness of the docking procedure.