Introduction

Fused heterocyclic compounds containing pyridine moiety represent a variety of biological activities in different areas [1]. Pyridine scaffold is an essential core in the chemical structure of a variety of approved drugs in the pharmaceutical market (Fig. 1) [1,2,3,4,5,6]. Numerous pyridine derivatives have been studied for their biological and pharmacological activities; some of these compounds have been shown to have antibacterial properties such as Sulfapyridine (Fig. 1). On the other hand, one of the most common pyridine derivatives is known as thienopyridine compounds. These scaffolds are employed as precursors in the synthesis of target agrochemicals and pharmaceuticals (Fig. 1). Fused thienopyridine derivatives have been reported associated with different medicinal and biological properties including anti-inflammatory [7], antibacterial [8], antifungal [9], anticancer [10], and antimicrobial agents [11]. Rateb et al. [12]. reported a new series thienopyridine derivatives with potent antifungal activity (compounds I, Fig. 1), likewise, Mohi El-Deen et al. [13]. reported a novel series of thienopyridine derivatives with high antibacterial properties (compounds II, Fig. 1). Moreover, some drugs in the pharmaceutical market contain thienopyridine moiety in their structures such as Clopidogrel [14], Ticlopidine [15], and Prasugrel [16] (Fig. 1).

Fig. 1
figure 1

Pyridine and thienopyridine scaffold-bearing compounds in therapeutic applications

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Antibiotics were arguably the most successful chemotherapeutic agents developed in the 20th century. After many decades from their discovery and introduction to the market, antibiotics have saved countless lives [17]. Modern medicine depends on antibiotics’ capacity to both cure and prevent a wide range of illnesses. It is well known that bacteria may become resistant to antibiotics [18]. Antibiotics were often taken for granted since, in the beginning, there were enough new ones available to battle the resistance that was being shown [19]. On the other hand, the level of bacterial resistance has continued to rise dramatically as the availability of new antibiotics has decreased. Overuse of antibiotics combined with the ongoing spread and development of mobile genetic resistance elements over the past 20 years has led to an increase in the prevalence of multidrug resistance [17]. Contrasting sharply with the current level of investment in antimicrobial development is the growing need for new drugs to treat potentially fatal infections brought on by the global spread of multidrug-resistant bacterial pathogens. This is especially true for small molecules derived from natural products and synthetic compounds. To combat the threat that antimicrobial resistance poses to public health, novel agents with inventive chemistry and modes of action are sorely needed on a global scale [20].

Therefore, due to these facts and in continuation of our previous work on the synthesis of new organic compounds with evaluation of their biological activities [21,22,23,24,25,26,27,28,29,30]. We report herein reliable synthetic approaches for a new series of pyridine and thienopyridine derivatives and evaluation of their biological activities as antimicrobial agents.

Results and discussion

Chemistry

According to the literature [31, 32], 4-bromo acetophenone (1) and vetraldehyde (3,4-dimethoxy benzaldehyde) (2) were reacted to produce 3-(3,4-dimethoxyphenyl)-1-(4-bromophenyl)prop-2-en-1-one (3) in the presence of piperidine or NaOH. Compound 3 reacted with malononitrile in methanol or ethanol as a suitable solvent to yield the unexpected alkoxypyridine compounds 4 and 5, respectively (rather than 4-H-cyanopyran 6) (Scheme 1). The lone pairs of the hydroxy groups in the methanol or ethanol solvent, as shown in Scheme 2, are essential to the suggested reaction mechanism and the end products 4 and 5 (Scheme 2) [33]. The chemical structures of the synthesized compounds 4 and 5 were elucidated by spectral data and elemental analysis. The 1H NMR spectrum of the methoxypyridine derivative 4 was characterized by the presence of a singlet signal at δ 4.21 (ppm) for the OCH3 group, while the 1H NMR spectrum of compound 5 showed a triplet signal (CH3 group) at δ 1.45 (ppm) (J = 7.2 Hz) and a quartet signal (CH2 group) at δ 4.63 (ppm) (J = 7.2 Hz) due to the ethyl group. The mass spectrometry of compounds 4 and 5 showed two peaks at m/z = 425 and m/z = 438 for both 4 and 5, respectively.

Scheme 1
scheme 1

Synthesis of chalcone 3

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Scheme 2
scheme 2

Synthesis of compounds 4 and 5 with the expected reaction mechanism (in case of using KOH as a catalyst)

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The intermediate (a) was produced by a Michael addition reaction between compound 3 and malononitrile. The expected intermediate (c) was then produced by the nucleophilic attack of the alkoxide anion (RO-) from the solvent (methanol in case of (4) or ethanol in case of (5)) over the ketone-imine (b). The expected intermediate (d) was then formed by intramolecular N-cyclization of the intermediate (c), followed by the elimination of the water molecule, and then oxidative aromatization to afford the corresponding products 4 or 5. It is important to note that, the expected reaction mechanism in Scheme 2 is could be considered only by using KOH as a catalyst.

Treatment of compound 3 with 2-cyanothioacetamide in ethanol in the presence of trimethylamine afforded orang precipitate, of the corresponding cynopyridinethione derivative (7) with excellent yield (94%, Scheme 3). In the same context, cynopyridinethione (7) could be obtained as one pot step via the reaction of 4-bromoacetophenone (1), vetraldehyde (2) and 2-cyanothioacetamide in a molar ratio of 1:1:1 in ethanol under reflux for 4 hours as shown in Scheme 3. 1H NMR spectrum of pyridinethione (7), showed a multiplet signal at chemical shist δ 3.82–3.98 (ppm) assigned to the protons of the two methoxy groups, while the aromatic protons were observed in the aromatic zone at δ 6.88–7.16 and 7.26–7.73 (ppm). The CN group is observed with a band at 2226 cm−1 in the infrared spectrum, and the molecular ion peak of 7 is identified by a peak (M+) at m/z (EI) = 426 in the mass spectrum.

Scheme 3
scheme 3

Synthesis of 6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (7)

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The cynopyridinethione (7) and 2-chloro-N-arylacetamide derivatives (8a–c) were refluxed in ethanol and with a few drops of Et3N to yield the corresponding compounds 9a–c, respectively. The CH2 group in 1H NMR spectra of each compound was reported as a singlet signal at δ 4.33, 4.32, and 4.31 (ppm), respectively. It is noteworthy to mention that, the appearance of these signals (of the CH2 groups) confirms the open structure form as visualized in Scheme 4. As a result, compounds 9a–c were cyclized by refluxing in sodium-ethoxide solution to yield the corresponding thienopyridine derivatives (10a–c). Compounds 10a–c, on the other hand, were obtained in a single step by treating 7 with 8a–c in a refluxing sodium-ethoxide solution (Scheme 4). Elemental analysis and spectral data were used to prove the chemical structure of compounds 10a–c (Scheme 4). In this regard, the 1H-NMR spectra of compounds 10a–c confirmed that the singlet signals of the CH2 group were missed and new signals for the NH2 groups appeared at 6.01, 5.91, and 5.90 (ppm), respectively (see experimental part).

Scheme 4
scheme 4

Synthetic pathway for the preparation of compounds 9a–c and 10a–c

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Moreover, the reaction of 7 with some α-halo ketone derivatives such as chloroacetone, phenacyl chloride and ethyl chloroacetate gave the corresponding S-alkyl derivatives (11a–c), respectively (Scheme 5). Cyclization of 11a–c was carried out by refluxing these compounds in freshly prepared sodium ethoxide solution to give the corresponding thieno[2,3-b]pyridine derivatives (12a–c), respectively. The chemical structure of both S-alkyl derivatives (11a–c) and thieno[2,3-b] pyridine derivatives (12a–c) was confirmed by the correct elemental analyses and spectral data. On the other hand, the thieno[2,3-b]pyridine derivatives (12a–c) could be prepared directly as a one-pot reaction by the treatment of compound 7 with α-halo carbonyl compounds in the presence of sodium ethoxide (Scheme 5). Similarly, compound 7 was reacted with methyl iodide to give the corresponding S-methyl derivative 13 (Scheme 5). The CN group in the IR spectrum of 13 showed a band at 2211 cm−1. Also, the 1H-NMR spectrum of 13 revealed the methyl group at δ 2.72 (ppm) as a singlet, while the methoxy groups were assigned at δ 3.81 and 3.83 (ppm). In the mass spectrometry, the highest recorded peak of 13 showed at m/z (EI) = 441 (56%) in mass spectrometry.

Scheme 5
scheme 5

Synthesis of compounds 11a–c, 12a–c and 13

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Furthermore, compound 7 was reacted with chloroacetonitrile to give the S-alkyl derivative (14) which was cyclized in refluxed sodium ethoxide solution to afford compound 15 as shown in Scheme 6. The formation of compound 15 rather than 16 (Scheme 6) could be attributed to the hydrolysis of the CN group to the amide group (Scheme 5). The IR spectrum of compound 15 showed a disappearance of the cyano group and a new absorption band at 1641 cm−1 for the C = O group. Moreover, in the 1H NMR spectrum, two singlet signals at δ 5.89 and 7.18 (ppm) could be attributed to the presence of two NH2 groups. The 13C NMR spectrum of compound 15 showed a signal at δ 166.9 (ppm) owing to the presence of the C = O group. The mass spectrometry (EI) showed a peak at m/z = 483 corresponding to the molecular ion of 15.

Scheme 6
scheme 6

Synthetic pathway for the newly synthesized compounds 14 and 15

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Antimicrobial activity

To test the antibacterial potency of the produced compounds, E. coli, B. mycoides, and C. albicans were used as representations for Gram-negative, Gram-positive bacterial strains, and a non-filamentous fungal strain, respectively. These three typical microorganisms were used in the well diffusion experiment for the produced compounds. In general, the compositions and functional groups included in each synthetic chemical have a significant impact on the resistance of microbial strains to such compounds [34,35,36].

The results, which show how various tested microorganisms react differently to the synthesized compounds 4–15, are listed in Table 1 and shown in Fig. 2. Compounds 12a and 15 showed the greatest inhibition zone against the tested microbial strains, with maximum antibacterial activity against B. mycoides (33 mm) and C. albicans (29 mm), respectively, which are higher than the activity exerted by the applied control antibiotics (Ampicillin 10 µg, Gentamicin 10 µg, and Tobramycin 10 µg). Contrarily, C. albicans showed the least reaction (inhibition zone of 16 mm) to compounds 9a, 9c and 10a among the studied microbial strains (Table 1). However, the majority of the investigated compounds exhibited modest antibacterial efficacy against the described microbes. Therefore, the highest two compounds 12a and 15 were chosen to calculate their MIC values for the three investigated microbes.

Table 1 Assessment of antimicrobial properties of compounds 4–15 using the agar diffusion technique
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Fig. 2
figure 2

Assessment of synthetic chemicals’ antimicrobial properties using the agar diffusion technique. Arrows indicate the selected compounds 12a (a, c, e) and 15 (b, d, f) with the highest microbial inhibition activity against E coli (a, b), B. mycoides (c, d), and C. albicans (e, f)

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Different concentrations of each compound were examined for their antibacterial activity during the 18-h incubation period to assess the antimicrobial activity durability of both 12a and 15 compounds against the indicated pathogens. Figures 3, S1, S2, and Table S1 (supplementary data) show the results obtained and demonstrate that the synthesized compounds have the ability to inhibit the tested microbes even at low concentrations. This behavior was also demonstrated by the measured MIC values (Table 2 and Fig. 3), which demonstrate that compound 12a has lower MIC values than compound 15 (which has a greater capacity to inhibit microbial growth), with the exception of E. coli, which was inhibited by a lower concentration of compound 15. B. mycoides and C. albicans’ growth was suppressed by compound 12a at MIC levels lower than 0.0048 mg/mL, whereas compound 15 was successful in preventing their visible development at MIC levels of 0.0098 and 0.039 mg/mL, respectively (Table 2).

Fig. 3
figure 3

Compounds 12a (a) and 15 (b) antimicrobial activity at various doses

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Table 2 Minimum Inhibitory Concentration (MIC) values of compounds 12a and 15 against represented microorganisms
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Structure-activity relationship (SAR)

Structure-activity relationship will be discussed to correlate the relation between the chemical structure of the synthesized pyridine and thienopyridine compounds and their antimicrobial properties as visualized in Fig. 4. In general, most of the newly synthesized pyridine and thienopyridine derivatives (4–15) exhibited well to strong antimicrobial activity against pathogens E. coli, B. mycoides and C. albicans. Thienopyridine derivatives (10, 12 and 15) showed strong antimicrobial activity with the highest inhibition zone than that observed in the case of pyridine derivatives (4, 5, 7, 9, 11, 13 and 14). Compound 12a (R = COOEt) prevented the growth of E. coli, at MIC level of 0.0195 mg/mL, and B. mycoides and C. albicans at MIC level below than 0.0048 mg/mL, respectively, while compound 15 (R = CONH2) prevented the visible growth of E. coli, B. mycoides, and C. albicans at MIC values of >0.0048, 0.0098, and 0.039 mg/mL, respectively.

Fig. 4
figure 4

Structure-activity relationship study of the synthesized pyridine and thienopyridine (4–15) compounds

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Conclusions

In summary, an efficient and convenient route to the synthesis of a new series of substituted pyridines. The newly synthesized pyridine derivatives were used for the preparation of a novel series of fused thienopyridine derivatives. All the newly synthesized pyridine and thienopyridine derivatives were tested for their antimicrobial activity against E. coli, B. mycoides, and C. albicans as representatives for Gram-negative, Gram-positive bacterial strains, and a non-filamentous fungal strain, respectively. The findings of the study indicated that the selected synthesized derivatives with the highest antimicrobial activity can be implemented in future studies to synthesize new antimicrobial agents.

Materials and methods

General

All melting points were measured on a Gallenkamp melting point apparatus (Weiss Gallenkamp, London, UK). The infrared spectra were recorded in potassium bromide disks on Pye Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometers (Pye Unicam Ltd. Cambridge, England and Shimadzu, Tokyo, Japan, respectively). The NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer (Varian, Palo Alto, CA, USA). 1H NMR spectra were run at 300 MHz or (Joel, Japan) 500 MHz and 13C NMR spectra were run at 75.46 MHz or 100.06 MHz in deuterated chloroform (CDCl3) or dimethyl sulfoxide (DMSO-d6). Chemical shifts are given in parts per million and are related to that of the solvent. Mass spectra were recorded with a Shimadzu GCMS–QP–1000 EX mass spectrometer in EI (70 eV) model. Elemental analyses were carried out at the Micro analytical Centre of Cairo University, Giza, Egypt and recorded on Elementar-Vario EL (Germany) automatic analyzer. 3-(4-Bromophenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (3) was synthesized by Claisen-Schmidt condensation according to literature [31, 32]. 4-bromo acetophenone and veratraldehyde (3,4-dimethoxy benzaldehyde) in a 1:1 molar ratio were dissolved in ethanol (10 mL). Aqueous NaOH solution (10%, 20 mL) was added to the reaction mixture. The reaction mixture was monitored by TLC using chloroform: methanol (4.8:0.2) as a solvent system. When at least one of the starting compounds finished, reactions were stopped. The reaction content was poured on ice water and neutralized by HCl (10%). The precipitate obtained was washed with cold water and ethanol, filtered, and dried. Chalcone 3 was used for the synthesis of pyridine and thienopyridine derivatives without further purification [37].

Synthesis of compounds 4 and 5

Method A

Sodium hydroxide (20%) was added to a mixture of malononitrile (10 mmol) and 3-(4-bromophenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (3) (10 mmol) in refluxed methanol (10 ml) or ethanol (10 ml) for 3 h. Compounds 4 or 5 were obtained by cooling the reaction, collecting the precipitate through filtration, washing it with ice water, drying and recrystallizing it from methanol.

Method B

In a 250 ml round flask, At room temperature, drops of piperidine were added to a mixture of 10-(4-bromophenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (3) (10 mmol) and malononitrile (10 mmol) in methanol (10 ml) or ethanol (10 ml). After the reaction had advanced for four hours, as verified by TLC, compounds 4 or 5 were obtained by filtering off, drying, and recrystallizing the precipitate from methanol.

6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-2-methoxynicotinonitrile (4)

Yellow solid, (method A, 265 mg, 62%); (method B, 350 mg, 79%); mp: 190–192 °C. IR (KBr) v (cm−1) 3447, 3079, 2978, 2943, 2838, 2219, 1581, 1541, 1521, 1449, 1369, 1327, 1253, 1024, 865, 771, 599. 1H NMR (300 MHz, CDCl3) δ (ppm) 3.97 (3H, s, OCH3), 3.99 (3H, s, OCH3), 4.21 (3H, s, OCH3), 6.98 (1H, d, J = 9 Hz, Ar), 7.27–7.37 (3H, m, Ar), 7.52 (1H, d, J = 8.7 Hz, Ar), 7.67–7.69 (3H, m, Ar + CH-pyridine). MS (EI, 70 eV) m/z (%): 425 [M+] (25), 410 [M+-CH3] (25), 394 [M+-OCH3] (10), 257 [M+-C9H12O3](14). Anal. Calcd. for C21H17BrN2O3 (425.28) (%): C, 59.31; H, 4.03; N, 6.59. Found (%): C, 59.37; H, 4.09; N, 6.56.

6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-2-ethoxynicotinonitrile (5)

Yellow solid; (method A, 279 mg, 65%), (method B, 350 mg, 75%); mp: 190–192 °C. IR (KBr) v (cm−1) 3447, 2956, 2833, 2221, 1593, 1578, 1540, 1519, 1421, 1343, 1251, 1142, 1039, 1021, 1009, 822, 804, 615. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.45 (3H, t, J = 7.2 Hz, CH3), 3.84 (3H, s, OCH3), 3.86 (3H, s, OCH3), 4.63 (2H, q, J = 7.2 Hz, CH2), 7.10 (1H, d, J = 8.7 Hz, Ar), 7.66–7.89 (7H, m, Ar + CH-pyridine). 13C NMR (DMSO-d6, 75.5 MHz): δ (ppm) 165.1, 157.8, 155.1, 151.3, 149.2, 136.1, 132.0, 129.8, 128.5, 124.4, 120.5, 112.1, 111.1, 110.5, 106.1, 92.1, 63.26, 61.63, 56.0, 55.9, 14.6, 14.3. MS (EI, 70 eV) m/z (%): 438 [M+] (100), 423 [M+-CH3] (27.65), 410 [M+-CH2CH3] (13.2), 394 [M+-OCH2CH3] (10), 257 [M+-C9H12O3] (9). Anal. calcd. for C22H19BrN2O3 (439.31) (%): C, 60.15; H, 4.36; N, 6.38. Found (%): C, 60.21; H, 4.40; N, 6.43.

Synthesis of 6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (7)

Drops of trimethylamine were added under refluxing temperature to a solution of 3-(4-bromophenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (3) (10 mmol) and cyanothioacetamide (10 mmol) in absolute ethanol (15 mL). The reaction was given time to cool and after a one-hour observation, the orange precipitate was collected by filteration, and the resulting precipitate was dried, and recrystallized from methanol to yield the cyanothiopyridine derivative 7.

Orange solid; (4 g, 94%); mp: 240–245 °C. IR (KBr) v (cm−1) 3447, 2050, 2933, 2226 (CN), 1597, 1549, 1518 (C = N), 1489, 1350, 1261, 1144, 1068, 1019, 795. 1H NMR (300 MHz, CDCl3) δ (ppm): 3.82–3.98 (6H, m, OCH3), 6.88–7.16 (2H, m, Ar), 7.26–7.73 (6H, m, Ar + CH-pyridine). MS (EI, 70 eV) m/z (%): 426 [M + ] (96), 411[M+-NH] (11), 368[M+-CSNH] (5). Anal. Calcd. for C20H15BrN2O2S (427.31) (%): C, 56.22; H, 3.54; N, 6.56. Found (%): C, 56.30; H, 3.62; N, 6.61.

Synthesis of compounds 9a–c

Compound 7 (10 mmol) was added to the derivatives of 2-chloro-N-phenylacetamide (10 mmol) in 15 ml of refluxing ethanol. Drops of triethylamine were then added, and the reflux was left for two hours. The corresponding S-alkyl pyridine derivatives (9a–c) were obtained by filtering off, drying, and recrystallizing the precipitate that was formed after the reaction was allowed to cool.

2-((6-(4-bromophenyl)-3-cyano-4-(3,4-dimethoxyphenyl)pyridin-2-yl)thio)-N-phenylacetamide (9a)

Yellow solid; (55 mg, 91%); mp: 230–235 °C. IR (KBr) v (cm−1) 3313 (NH), 3060, 2967, 2927, 2830, 2213 (CN), 1658 (C = O), 1598 (C = N), 1519, 1375, 1257, 1170, 1133, 821, 757, 964, 495. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.76 (3H, s, OCH3), 3.79 (3H, s, OCH3), 4.33 (2H, s, CH2), 6.84 (1H, d, J = 8.4 Hz, Ar), 7.05 (1H, t, J = 8.0 Hz, Ar), 7.30 (2H, t, J = 7.5 Hz, Ar), 7.57–7.87 (9H, m, Ar + CH-pyridine), 10.30 (1H, s, NH). MS (EI, 70 eV) m/z (%): 559 [M+] (40), 469 [M+-C6H5NH] (46), 439 [M+-C6H5NHCO] (95), 425 [M+-C6H5NHCOC] (18). Anal. Calcd. for C28H22BrN3O3S (560.46) (%): C, 60.01; H, 3.96; N, 7.50. Found (%): C, 60.11; H, 4.03; N, 7.56.

2-((6-(4-bromophenyl)-3-cyano-4-(3,4-dimethoxyphenyl)pyridin-2-yl)thio)-N-(p-tolyl)acetamide (9b)

Yellow solid; (550 mg, 92%); mp: 250–252 °C. IR (KBr) v (cm−1) 3241 (NH), 3048, 2931, 2834, 2215 (CN), 1652 (C = O), 1598 (C = N), 1517, 1376, 1259, 1137, 1020, 819, 503. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 2.25 (3H, s, CH3), 3.77 (3H, s, OCH3), 3.79 (3H, s, OCH3), 4.31 (2H, s, CH2), 6.85 (1H, d, J = 8.7 Hz, Ar), 7.10 (2H, d, J = 8.1 Hz, Ar), 7.46 (2H, d, J = 8.4 Hz, Ar), 7.69 (2H, d, J = 8.4 Hz, Ar), 7.67–7.87 (5H, m, Ar + CH-pyridine), 10.19 (1H, s, NH). MS (EI, 70 eV) m/z (%): 573 [M+] (30), 467 [M+-CH3C6H5NH] (38), 438 [M+-CH3C6H5NHCO] (86), 425 [M+-CH3C6H5NHCOC] (16), 393 [M+-FC6H5NHCOCS] (2.5). Anal. Calcd. for C29H24BrN3O3S (574.49) (%): C, 60.63; H, 4.21; N, 7.31. Found (%): C, 60.68; H, 4.25; N, 7.36.

2-((6-(4-bromophenyl)-3-cyano-4-(3,4-dimethoxyphenyl)pyridin-2-yl)thio)-N-(4-fluorophenyl)acetamide (9c)

Yellow solid; (510 mg, 87%); mp: 259–262 °C. IR (KBr) v (cm−1) 3434, 3257 (NH), 3056, 2935, 2836, 2215 (CN), 1654 (C = O), 1598 (C = N), 1513, 1376, 1259, 1218, 1133, 1074, 821, 511. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 3.77 (3H, s, OCH3), 3.80 (3H, s, OCH3), 4.32 (2H, s, CH2), 6.88 (1H, d, J = 8.4 Hz, Ar), 7.14 (2H, t, J = 8.7 Hz, Ar), 7.56–7.61 (2H, m, Ar), 7.65–7.71 (2H, m, Ar), 7.77–7.88 (5H, m, Ar + CH-pyridine), 10.35 (1H, s, NH). 13C NMR (100 MHz, DMSO-d6) δ (ppm) 166.2, 164.6, 162.4, 158.7, 157.6, 155.0, 153.6, 153.4, 151.9, 149.5, 147.3, 135.6, 132.4, 131.3, 129.6, 124.3, 121.9, 121.5, 121.4, 121.3, 116.0, 115.9, 115.7, 112.0, 111.4, 56.3, 56.1, 35.4. MS (EI, 70 eV) m/z (%): 579 [M+] (50), 467 [M+ -FC6H5NH] (50.8), 439 [M+-FC6H5NHCO] (91), 425 [M+-FC6H5NHCOC] (16), 393 [M+-FC6H5NHCOCS] (16). Anal. Calcd. for C28H21BrFN3O3S (578.46) (%):C, 58.14; H, 3.66; N, 7.26. Found (%): C, 58.14; H, 3.66; N, 7.32.

Synthesis of the thienopyridine derivatives (10a–c)

Method A

To a round flask containing sodium ethoxide solution, for three hours, compounds 9a–c (10 mmol) were refluxed. In all cases, a yellow precipitate was seen. After allowing the reactions to cool, the precipitates were removed by filtering, drying, and recrystallizing in the appropriate solvent to yield the corresponding thieno[2,3-b] derivatives of pyridine-2-carboxamide (10a–c).

Method B

Compound 7 (10 mmol) was refluxed in sodium ethoxide solution for three hours along with the appropriate derivative of 2-chloro-N-phenylacetamide (10 mmol). A yellow precipitate was observed and allowed to cool. The precipitate was then filtered off, dried, and crystallized from the appropriate solvent to yield the corresponding thieno[2,3-b]pyridine-2-carboxamide derivatives (10a–c).

3-amino-6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-N-phenylthieno[2,3-b]pyridine-2-carboxamide (10a)

Yellow solid; (method A, 45 mg, 83%), (method B, 40 mg, 71%); mp: 230–232 °C. IR (KBr) v (cm−1) 3477–3397 (NH2), 3318 (NH), 3081, 3000, 2927, 2829, 1631 (C = O), 1594 (C = N), 1517, 1482, 1432, 1317, 1238, 1027, 802, 752, 690, 499, 422. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 3.82 (3H, s, OCH3), 3.87 (3H, s, OCH3), 6.01 (2H, br, NH2), 7.02–7.10 (2H, m, Ar), 7.31 (2H, t, J = 7.8 Hz, Ar), 7.53 (2H, d, J = 8.1 Hz, Ar), 7.68–7.78 (7H, m, Ar + CH-pyridine), 9.41 (1H, br, NH). 13C NMR (DMSO-d6, 75.5 MHz) δ (ppm) 164.1, 160.1, 155.9, 150.8, 149.2, 146.8, 146.2, 138.9, 135.9, 131.7, 131.0, 130.2, 128.5, 123.7, 123.5, 122.9, 121.6, 120.5, 120.3, 120.2, 117.9, 111.9, 110.6, 98.4, 55.9, 55.7. MS (EI, 70 eV) m/z (%): 561 [M+] (81), 467 [M+-C6H5NH] (100), 439 [M+-C6H5NHCO] (3.2). Anal. Calcd. for C28H22BrN3O3S (560.46) (%): C, 60.01; H, 3.96; N, 7.50. Found (%): C, 60.08; H, 4.02; N, 7.55.

3-Amino-6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-N-(p-tolyl)thieno[2,3-b]pyridine-2-carboxamide (10b)

Yellow solid; (method A, 540 mg, 94%), (method B, 45 mg, 76%); mp: 140–142 °C. IR (KBr) v (cm−1) 3473 (NH2), 3316 (NH), 3079, 2996, 2829, 1633 (C = O), 1592 (C = N), 1513, 1398, 1315, 1238, 1027, 806, 505. 1H NMR (300 MHz, CDCl3) δ (ppm): 2.32 (3H, s, CH3), 3.93 (3H, s, OCH3), 3.99 (3H, s, OCH3), 5.82 (2H, s, NH2), 6.92 (1H, d, J = 8.4 Hz, Ar), 7.12 (2H, d, J = 8.1 Hz, Ar), 7.37–7.43 (6H, m, Ar), 7.57–7.75 (4H, m, Ar + CH-pyridine + NH). 13C NMR (DMSO-d6, 75.5 MHz) δ (ppm) 163.7, 159.8, 155.5, 150.5, 148.9, 146.1, 145.8, 136.5, 135.8, 132.2, 131.4, 130.8, 130.0, 128.6, 122.6, 121.5, 120.4, 120.1, 117.6, 111.7, 110.4, 98.8, 55.6, 55.4, 20.3. MS (EI, 70 eV) m/z (%): 573 [M+] (98), 467 [M+ -C7H10N] (100), 439 [M+-C8H8NO] (8), 367 [M+-C10H10N2OS] (3). Anal. Calcd. for C29H24BrN3O3S (574.49) (%): C, 60.63; H, 4.21; N, 7.31. Found (%): C, 60.65; H, 4.27; N, 7.38.

3-amino-6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-N-(4-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide (10c)

Yellow solid; [A] (530 mg, 90%). [B] (50 mg, 75%); mp: 205–207 °C. IR (KBr) v (cm−1) 3531–3484 (NH2), 3324 (NH), 3077, 2965, 2836, 1635 (C = O), 1596 (C = N), 1509, 1400, 1315, 1240, 1012, 829, 809, 636, 514, 493. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 3.95 (3H, s, OCH3), 4.01 (3H, s, OCH3), 5.90 (2H, s, NH2), 6.94–7.07 (3H, m, Ar), 7.21–7.27 (1H, m, Ar), 7.39–7.52 (5H, m, Ar), 7.60–7.76 (4H, m, Ar + CH-pyridine + NH). MS (EI, 70 eV) m/z (%): 579 [M+] (76), 467 [M+-FC6H5NH] (100), 439 [M+-FC6H5NHCO] (3), 367 [M-C9H7FN2OS] (3). Anal. Calcd. for C28H21BrFN3O3S (578.45) (%): C, 58.14; H, 3.66; N, 7.26. Found (%): C, 58.1; H, 3.61; N, 7.55.

Synthesis of compounds 11a–c

Method A

A solution of 7 (10 mmol) and the suitable ɑ-chloroketones ethyl chloroacetate, phenacyl chloride, or chloroacetone (10 mmol) in absolute ethanol (15 mL) was prepared by adding drops of triethylamine. The mixture was refluxed for 4 h and then allowed to cool. The precipitate that was formed was filtered off, dried, and crystallized from the appropriate solvent to yield the corresponding derivatives of compounds 11a–c, respectively.

Method B

After 10 h of vigorous stirring in DMF (15 mL) containing 7 (10 mmol), the appropriate ɑ-chloroketones (10 mmol), and potassium hydroxide (10 mmol), the pale yellow product in each case was filtered off, dried, and crystallized from methanol to yield the S-alkylated pyridine derivatives (11a–c).

Ethyl 2-((6-(4-bromophenyl)-3-cyano-4-(3,4-dimethoxyphenyl)pyridin-2-yl)thio)acetate (11a)

Yellow solid; (method A, 45 mg, 90%), (method B, 38 mg, 67%); mp: 165–167 °C. IR (KBr) v (cm−1) 3447, 3081, 2970, 2935, 2677, 2207 (CN), 1735 (C = O), 1569 (C = N), 1569, 1517, 1413, 1267, 1148, 1023, 815. 1H NMR (300 MHz, DMSO-d6) δ (ppm) 1.11 (3H, t, J = 6.9 Hz, CH3), 3.85 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.08 (2H, q, J = 6.9 Hz, CH2), 4.28 (2H, s, CH2), 7.09 (1H, d, J = 8.4 Hz, Ar), 7.68–7.91 (6H, m, Ar), 8.06 (1H, s, CH-pyridine). MS (EI, 70 eV) m/z (%): 514.22 [M+] (81), 484[M+-CH2CH3] (7), 467 [M+-CH2CH3O] (100), 439 [M+-C3H5O2] (3), 394 [M+-C4H7O2S] (4). Anal. Calcd. for C24H21BrN2O4S (513.41) (%): C, 56.15; H, 4.12; N, 5.46. Found (%): C, 56.21; H, 4.11; N, 5.55.

6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-2-((2-oxo-2-phenylethyl)thio)-nicotinonitrile (11b)

Yellow solid; (method A, 55 mg, 94%), (method B, 50 mg, 89%); mp: 201–202 °C. IR (KBr) v (cm−1) 3064, 2998, 2937, 2908, 2838, 2211 (CN), 1675 (C = O), 1596 (C = N), 1565, 1517, 1411, 1265, 1143, 1016, 829, 811, 757, 692, 644, 570. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 3.57 (3H, s, OCH3), 3.91 (3H, s, OCH3), 4.86 (2H, s, CH2), 6.80 (1H, d, J = 8.4 Hz, Ar), 7.41–7.54 (7H, m, Ar + CH-pyridine), 7.62–7.70 (3H, m, Ar + CH-pyridine), 8.05 (2H, d, J = 8.4 Hz, Ar). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 193.1, 162.1, 158.4, 153.6, 151.8, 149.4, 136.3, 135.5, 134.2, 132.4, 131.4, 129.4, 128.9, 127.0, 124.3, 121.5, 116.3, 115.9, 114.2, 111.95, 110.9, 110.8, 56.1, 55.6, 38.7. MS (EI, 70 eV) m/z (%): 544 [M+] (25), 439 [M+-C7H5O] (55), 393 [M+-C8H7OS] (2), 105 [M+-C21H16BrN2O2S] (100). Anal. Calcd. for C28H21BrN2O3S (545.45) (%): C, 61.66; H, 3.88; N, 5.14. Found (%): C, 61.71; H, 3.95; N, 5.16.

6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-2-((2-oxopropyl)thio)nicotinonitrile (11c)

Yellow solid; (method A, 45 mg, 91%), (method B, 39 mg, 80%); mp: 185–187 °C. IR (KBr) v (cm−1) 3430, 3085, 3002, 2938, 2210 (CN), 1718 (C = O), 1596 (C = N), 1569, 1521, 1375, 1359, 1257, 1137, 1018, 819, 578, 505. 1H NMR (300 MHz, CDCl3): δ (ppm) 2.36 (3H, s, CH3), 3.97 (3H, s, OCH3), 4.05 (2H, s, CH3), 4.06 (3H, s, OCH3), 6.97 (1H, d, J = 8.4 Hz, Ar), 7.47–7.71 (7H, m, Ar + CH-pyridine). MS (EI, 70 eV) m/z (%): 484 [M+] (45), 467 [M+-CH3] (7), 439 [M+-C2H3O] (100), 425 [M+-C3H5O] (13), 393 [M+-C3H5OS] (2). Anal. Calcd. for C23H19BrN2O3S (483.38) (%): C, 57.15; H, 3.96; N, 5.80. Found (%): C, 57.22; H, 4.01; N, 5.87.

Synthesis of compounds 12a–c

Method A

For three to six hours, compounds 11a–c (10 mmol) were refluxed in a sodium ethoxide solution. In each case, the mixtures were cooled to precipitate a yellow solid, which was subsequently filtered off, dried, and crystallized from the appropriate solvent to yield the corresponding thieno[2,3-b]pyridine derivatives (12a–c).

Method B

Compound 7 (10 mmol) was refluxed in sodium ethoxide solution for three hours along with the appropriate ɑ-haloketone derivatives, including ethyl chloroacetate, phenacyl chloride, or chloroacetone (10 mmol). The corresponding thieno[2,3-b]pyridine derivatives (12a–c) were then obtained by filtering off, drying, and recrystallizing the precipitate that had formed after the reaction was allowed to cool (12a–c).

Ethyl 3-amino-6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)thieno[2,3-b]pyridine-2-carboxylate (12a)

Yellow solid; (method A, 45 mg, 90%), (method B, 40 mg, 76%); mp: 218–222 °C. IR (KBr) v (cm−1) 3487–3354 (NH2), 3078, 2994, 2829, 1660 (C = O), 1599 (C = N), 1516, 1484, 1402, 1351, 1248, 1141, 1011, 800, 767, 638. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 1.29 (3H, t, J = 6.9 Hz, CH3), 3.83 (3H, s, OCH3), 3.88 (3H, s, OCH3), 4.27 (2H, q, J = 6.9 Hz, CH2), 5.48 (2H, s, NH2), 7.04 (1H, d, J = 9.3 Hz, Ar), 7.49–7.81 (7H, m, Ar + CH-pyridine). MS (EI, 70 eV) m/z (%): 514 [M+] (91), 484[M+-CH2CH3] (7), 467 [M+-CH2CH3O] (41), 439 [M+-C3H5O2] (3), 397 [M+-C5H7NO2] (4). Anal. Calcd. for C24H21BrN2O4S (513.40) (%): C, 56.15; H, 4.12; N, 5.46. Found (%): C, 56.21; H, 4.15; N, 5.52.

(3-amino-6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)thieno[2,3-b]pyridin-2-yl)(phenyl)methanone (12b)

Yellow solid; (method A, 55 mg, 95%), (method B, 35 mg, 65%); mp: 275–278 °C. IR (KBr) v (cm−1) 3473–3286 (NH2), 3083, 2948, 2927, 2825, 1596 (C = N), 1535, 1513, 1457, 1428, 1324, 1255, 1022, 732, 701, 653, 593, 404. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.81 (3H, s, OCH3), 3.84 (3H, s, OCH3), 6.93–7.00 (3H, m, Ar), 7.53 (4H, d, J = 7.8 Hz, Ar), 7.72–7.78 (8H, m, Ar + CH-pyridine + NH2). 13C NMR (DMSO-d6, 75.5 MHz): δ (ppm) 189.0, 162.4, 157.2, 151.1, 150.4, 149.2, 147.4, 140.9, 135.6, 132.0, 131.2, 130.9, 129.8, 128.5, 127.5, 123.2, 121.4, 120.8, 120.7, 119.1, 117.9, 111.9, 110.6, 103.7, 55.8. MS (EI, 70 eV) m/z (%): 546 [M+] (97), 529 [M+-NH2] (10), 105 [M+-C21H16BrN2O2S] (12). Anal. Calcd. for C28H21BrN2O3S (545.45) (%): C, 61.66; H, 3.88; N, 5.14. Found (%): C, 61.65; H, 3.98; N, 5.16.

1-(3-amino-6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)thieno[2,3-b]pyridin-2-yl)ethan-1-one (12c)

Yellow solid; (method A, 43 mg, 90%), (method B, 35 mg, 73%); mp: 255–257 °C. IR (KBr) v (cm−1) 3480–3311 (NH2), 3085, 2996, 2931, 2830, 1614 (C = O), 1531 (C = N), 1463, 1247, 1139, 1024, 673, 572, 501, 414. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 2.37 (3H, s, CH3), 3.83 (3H, s, OCH3), 3.87 (3H, s, OCH3), 6.56 (2H, br, NH2), 7.08 (1H, d, J = 8.4 Hz, Ar), 7.53–7.60 (2H, m, Ar), 7.78–7.85 (5H, m, Ar + CH-pyridine). MS (EI, 70 eV) m/z (%): 484 [M+] (92), 467 [M+-CH3] (7), 439 [M+-CH3CO] (3). Anal. Calcd. for C23H19BrN2O3S (483.38) (%): C, 57.15; H, 3.96; N, 5.80. Found (%): C, 57.22; H, 4.04; N, 5.85.

Synthesis of compounds 13 and 14

To a round flask, Triethylamine drops were added to a mixture of 10 mmol of compound 7, 10 mmol of methyl iodide or 10 mmol of chloro acetonitrile in 15 mL of absolute ethanol. After three hours of reflux, the mixture was allowed to cool. Compounds 13 and 14 were obtained by filtering off, drying, and crystallizing the precipitate that had formed in each case using the appropriate solvent.

6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)-2-(methylthio)nicotinonitrile (13)

Yellow solid; (40 mg, 91%); mp: 198–201 °C. IR (KBr) v (cm−1) 3068, 2999, 2969, 2918, 2819, 2208 (CN), 1590 (C = N), 1568, 1519, 1414, 1358, 1272, 1172, 1150, 1023, 808, 584, 572, 474, 458. 1H NMR (300 MHz, CDCl3): δ (ppm) 2.72 (3H, s, CH3), 3.81 (3H, s, OCH3), 3.83 (3H, s, OCH3), 6.98 (1H, d, J = 9.5 Hz, Ar), 7.42 (1H, s, Ar), 7.51 (2H, d, J = 9.1 Hz, Ar), 7.66–7.75 (4H, m, Ar + CH-pyridine). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 181.9, 163.5, 153.3, 152.7, 151.9, 149.6, 149.6, 135.6, 132.4, 131.9, 131.4, 130.8, 129.7, 124.2, 121.6, 115.4, 112.4, 111.2, 102.3, 56.2, 13.6. MS (EI, 70 eV) m/z (%): 442 [M+] (56), 425 [M+-CH3] (16), 393 [M+-CH3S] (3). Anal. Calcd. for C21H17BrN2O2S (441.34) (%): C, 57.15; H, 3.88; N, 6.35. Found (%): C, 57.22; H, 3.92; N, 6.43.

6-(4-bromophenyl)-2-((cyanomethyl)thio)-4-(3,4-dimethoxyphenyl)nicotinonitrile (14)

Yellow solid; (40 mg, 88%); mp: 165–167 °C. IR (KBr) v (cm−1) 3432, 3089, 2991, 2931, 2211 (CN), 1592 (C = N), 1571, 1517, 1415, 1270, 1145, 1020, 817, 572, 470. 1H NMR (300 MHz, CDCl3): δ (ppm) 3.98 (3H, s, OCH3), 4.02 (3H, s, 2OCH3), 4.07 (2H, s, CH2), 7.00 (1H, d, J = 8.4 Hz, Ar), 7.50–7.55 (3H, m, Ar), 7.62–7.73 (3H, m, Ar+ CH-pyridine), 7.86 (1H, s, Ar). MS (EI, 70 eV) m/z (%): 467 [M+] (52), 450 [M+-CH3] (23), 386 [M+-Br] (15). Anal. Calcd. for C22H16BrN3O2S (466.35) (%): C, 56.66; H, 3.46; N, 9. 01. Found (%): C, 56.74; H, 3.51; N, 9.09.

Preparation of 3-amino-6-(4-bromophenyl)-4-(3,4-dimethoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (15)

To obtain compound 15, compound 14 (10 mmol) was refluxed in sodium ethoxide solution for three hours. The reaction was then allowed to cool, and the precipitate that was formed was filtered off, dried, and crystallized from methanol.

Yellow solid; (35 mg, 80%); mp: 235–238 °C. IR (KBr) v (cm−1) 3482, 3380, 3087 (2NH2), 2829, 1641 (C = O), 1585 (C = N), 1403, 1353, 1251, 1025, 580, 759, 647, 559, 449, 416. 1H NMR (300 MHz, DMSO-d6): δ (ppm) 3.82 (3H, s, OCH3), 3.87 (3H, s, OCH3), 5.90 (2H, s, NH2), 7.03 (1H, d, J = 8.4 Hz, Ar), 7.18 (2H, s, NH2), 7.51 (2H, d, J = 7.5 Hz, Ar), 7.72–7.76 (5H, m, Ar + CH-pyridine). 13C NMR (DMSO-d6, 75.5 MHz): δ (ppm) 166.9, 159.5, 155.3, 150.5, 148.9, 145.9, 145.5, 135.8, 131.4, 130.8, 130.0, 122.5, 120.3, 120.1, 117.5, 111.7, 110.3, 98.4, 55.6, 55.5. MS (EI, 70 eV) m/z (%): 485 [M+] (95), 467 [M+-NH2] (78), 439 [M+-CONH2] (14). Anal. Calcd. for C22H18BrN3O3S (484.37) (%): C, 54.55; H, 3.75; N, 8.68. Found (%): C, 54.58; H, 3.80; N, 8.71.

Antimicrobial activity

Media and microorganisms

Modified nutrient agar medium slants (g/L) at pH 7.0 were used to sustain Escherichia coli, Bacillus mycoides, and Candida albicans: Peptone: 3, Yeast extract: 1.5, Meat extract: 1.5, Glucose: 0.5, NaCl: 0.25, and Agar: 20.0. The microbial strains were seeded and grown at 37 °C on nutrient agar medium (70148 Nutrient agar, Fluka, Spain) with the following components (g/L): peptone, 5.0; yeast extract, 2.0; meat extract, 1.0; sodium chloride, 5.0; and agar, 15.0. This was done to conduct the agar diffusion method for antimicrobial assessment, where 28 g of the ready medium was dissolved in 1.0 L of distilled water to create the nutrient agar medium at pH 7.0. Autoclaving was used to sterilize the aforementioned media for 20 min at 121 degrees Celsius while using 1.5 atmospheres.

Assessment of antimicrobial activity

Utilizing the agar diffusion technique, the effectiveness of the produced compounds as antibacterial agents was evaluated against three representative microorganisms [34]. Using 100 μl (1.5 × 105 CFU/100 μl) of re-suspended overnight culture at 37 °C, the microbial cultures were seeded in the nutrient agar medium (70148 Nutrient agar, Fluka) as follows: The specified microbial inoculums were added to the melting medium at around 45 °C before the nutrient agar media was poured onto a series of Petri dishes. At room temperature, the plates were allowed to harden before 200 μl of dissolved substances (10 mg/mL in DMSO) were administered to 15 mm holes made in the infected agar plates. Standard antibiotic disks (7 mm) (Ampicillin 10 µg, Gentamicin 10 µg, and Tobramycin 10 µg) from Bioanalyse® were also used for comparison with the tested compounds. The test samples were serially diluted in DMSO to achieve concentrations ranging from 5 to 0.0048 mg/mL in order to establish the minimal inhibitory concentration (MIC). The MIC was established as the lowest concentration that could prevent observable microbial growth [27, 38, 39]. Following a 24-h incubation period at 37 °C, the established inhibitory zones on culture plates were examined [40].