In continuation of research on the synthesis of compounds with antibacterial activity [1], a series of 22 3-pyrrol- 1-ylthieno[2,3-b]pyridine-2-carboxylic acid [(phenyl-, 1,3-benzodioxol-5-yl)methylene]hydrazides (Ia – v) were synthesized and their antibacterial activity was studied. The starting materials for preparing compounds I were 3-(1-pyrrolyl)- thieno[2,3-b]pyridine-2-carbohydrazides (IIa and -b) [2] and several aldehydes (IIIa – l) containing pharmacophores from the chemical-block.com database.

Target compounds Ia – v were synthesized by refluxing 3-(1-pyrrolyl)thieno[2,3-b]pyridine-2-carbohydrazides II and aldehydes III in EtOH:DMF (1:1, v/v) with p-toluenesulfonic acid catalyst. The mixture with DMF was used because the hydrazides were poorly soluble in EtOH. The reaction time depended on the nature of the aldehyde and varied from 10 – 15 min to several hours.

The yields of target compounds Ia – v reached 98% (Table 1). Compounds Ia – v were crystalline solids ranging from colorless (Ia, b, o, p) to bright yellow (If – h, j – m, q – s) with melting points exceeding those of the corresponding starting hydrazides II.

Table 1 Characteristics of Synthesized Compounds
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The structures of Ia – v were confirmed by IR and PMR spectra (Tables 2 and 3). PMR spectra of hydrazones Ia – v were missing the NH2 singlet at 4.23 or 4.24 ppm that appeared in spectra of the corresponding starting hydrazones II [2]. Instead of it, resonances for protons of the added CH–R2 group appeared. The CH group resonated at 7.70 – 8.98 ppm. Doubling of this resonance in addition to resonances for CONH and the á-, and â-protons of the pyrrole ring etc. indicated that the solution contained two conformers of target products Ia – v, i.e., syn and anti. Another feature of the PMR spectra of the examined compounds was a singlet for the pyrrole protons of both the synand anti-conformers.

Table 2 PMR Spectra of Synthesized Compounds
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Table 3 IR Spectra of Ia, b, i, j, l, n, q
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figure a

IR spectra of products I had only one íN–H stretching band at 3295 – 3260 cm–1 (Table 3), in contrast with those of starting hydrazides IIa and -b [2]. Stretching bands of C–HAr and C=N groups appeared at 3110 – 3080 and 1580 – 1560 cm–1, respectively. Carbonyl stretching bands shifted from 1620 – 1615 to 1660 – 1640 cm–1.

Experimental Chemical Part

IR spectra were taken in mineral oil on a Specord IR-75 spectrophotometer at room temperature. PMR spectra were recorded in DMSO-d6 with TMS internal standard on a Bruker DRX-500 spectrometer at operating frequency 500.13 MHz. TLC was performed on Sorbfil plates (PTS-AF-A).

6-Methyl-4-methoxymethyl-3-pyrrol-1-ylthieno[2,3-b ]pyridine-2-carboxylic acid (3,4-dichlorobenzylidene)- hydrazide (Ia). A mixture of 6-methyl-4-methoxymethyl-3- pyrrol-1-ylthieno[2,3-b]pyridine-2-carbohydrazide (IIa, 1.0 g, 3.16 mmol) and 3,4-dichlorobenzaldehyde (IIIa, 0.6 g, 3.5 mmol) was dissolved with heating in EtOH:DMF (30 mL, 1:1, v/v), treated with a catalytic amount of p-TsOH, and refluxed. The end of the reaction was determined by chromatography (toluene:EtOH eluent, 2:1). The reaction time was 15 min. The mixture was cooled to room temperature. The precipitate was filtered off, rinsed with H2O, dried in air, and recrystallized from i-PrOH:DMF (1:2, v/v). Yield 1.4 g (96%).

Carbohydrazides Ib – v were synthesized analogously. The only differences were that the reaction time for Ig – q was 1 h; for Ir – v, 3 h.

Experimental Biological Part

The antibacterial properties of the new compounds were screened microbiologically using a qualitative method (diffusion in AGV solid growth medium) and a quantitative method (serial dilutions in growth broth) [3]. The test organisms were Staphylococcus aureus and Escherichia coli. The tested compounds were dissolved in DMSO. Preliminary screening used compound concentrations of 12 mg/mL (300 μg/25 μL). Antibacterial activity was estimated from the diameter of the growth inhibition zone (DGIZ, mm) and the minimum inhibiting concentration (MIC, μg/mL). These were measured in three repetitions followed by statistical processing using Microsoft Excel software to calculate the mean (M), standard deviation of the mean (m), and statistical significance (p) of the differences from the control. The control was the known broad-spectrum antibacterial drug furazolidone (drug substance, Zhehem Pharmachem Co., P. R. China).

The results showed that several of the synthesized hydrazones I and hydrazides IIa and -b suppressed the growth of the test bacteria (Table 4).

Table 4 Biological Test Results of Synthesized Compounds
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It was found that 3-pyrrol-1-ylthieno[2,3-b]pyridine-2- carbohydrazides IIa and -b exhibited strong antibacterial activity against E. coli. Hydrazones I that were prepared from them showed different properties depending on the nature of the introduced radical R2.

Only 11 (Ia, -c, -d, -e, -g, -k, -m, -o, -p, -r, -t) of the 22 synthesized hydrazones (Ia – v) displayed antibacterial properties that were characterized as inhibiting growth of the test strains with DGIZ of 10 – 20 mm. The DGIZ for the control was 20 – 26 mm. Compounds Ia, -c, and -m inhibited the growth of only S. aureus; Id and –k, only E. coli.

Quantitative assay of the antibacterial activity of Ia, -c, -d, -e, -g, -k, -m, -o, -p, -r, and -t found (Table 4) that their MIC values fell in the range 62.5 – 1,000 μg/mL. The most active against S. aureus were Ic, -g, -o, -r, and -t with R2 radicals 2-hydroxy-3,5-dichlorophenyl (Ic), 2-hydroxynaphthalene (Ig), 4,7-dimethoxy-6-propylbenzo[1, 3]dioxol-5-yl (Io), 4,7-dimethoxy-6-[3-(4-methoxyphenyl)-4,5-dihydroisoxazol- 5-ylmethyl]benzo[1, 3]dioxol-5-yl (Ir), and 6-[3-(4-fluorophenyl)- 4,5-dihydroisoxazol-5-ylmethyl]-4,7-dimethoxyben zo[1, 3]dioxol-5-yl (It).

The most active antibacterial compound was Ic, the MIC of which against S. aureus was (62.5 ± 30.62) μg/mL; against E. coli, (500.0 ± 244.95) μg/mL. The most active compound that inhibited growth of only S. aureus was Io, the MIC of which was (62.5 ± 30.62) μg/mL; of only E. coli, Ik, the MIC of which was (250.0 ± 176.77) μg/mL.

Compounds Ia, -c, -d, -k, and –m were active against E. coli. The strongest antibacterial derivatives were Ia, -k, and -m with R2 radicals 3,4-dichlorophenyl, 4,7-dimethoxybenzo- [1, 3]dioxol-5-yl, and 6,7-dimethoxybenzo[1, 3]dioxol-5-yl, respectively. Introducing a substituent in the 6-benzo- [1, 3]dioxole position could have a drastic effect on the antibacterial activity. Thus, a propyl (Io) or 4,5-dihydroisoxazol- 5-ylmethyl (Ip – v) substituent destroyed the activity of the tested compounds against E. coli.

The nature of the radical in the pyridine 4-position affected considerably the antibacterial properties of the synthesized compounds. Adding a methoxymethyl group to the 4-position increased the activity against S. aureus (Ia, -c, -e, -g, -m, -o, -p, -r, -t) and E. coli (Ia, -c, -k, -m). Moreover, replacing the methoxymethyl by methyl (Ib, -d, -f, -h, -j, -l, -n, -q, -s, -v) caused the antibacterial activity to disappear. The exception was Id, which exhibited moderate antibacterial properties against E. coli.

Thus, 11 of the 22 new compounds possessed antibacterial activity; 3 of them, against both S. aureus (Gram-positive) and E. coli (Gram-negative). This characterized them as broad-spectrum compounds. The most promising compounds for further research were Ic and Io.