Introduction

Multi-component reactions (MCRs) are of increasing importance in organic and medicinal chemistry [1, 2]. MCRs are reactions in which three or more reagents react in a one-pot procedure to give a single product [3]. These reactions have become increasingly popular during the last decade [4–6]. MCRs offer a wide range of advantages over single-component reactions such as high degree of atom economy, convergence, ease of execution, and a quick access to complex molecules [7].

Indole fragment is featured widely in broad ranges of pharmacologically and biologically active compounds [8–13]. Therefore, the synthesis and selective functionalization of indoles have been the focus of active research over the years [14–17]. In particular, 3-substituted indoles are privileged scaffolds in medicinal chemistry, which have been found in a fascinating array of bioactive natural products and pharmaceutical compounds [18–25]. These scaffolds are found in a number of biologically active compounds, especially with anti-cancer [26–29], anti-tumor [30], hypoglycemic [31], anti-inflammatory [32], analgesic [33, 34], and anti-pyretic activities [35].

Kojic acid is a natural product that has been isolated from various strains of microorganisms such as Penicillium, Aspergillus, and Gluconacetobacter [36]. Kojic acid and its derivatives have shown to possess various bioactivities such as anti-microbial [37, 38], cosmetic skin-whitening [39], anti-speck [40], pesticide and insecticide [41], anti-tumor [42], anti-diabetic [43], and anti-proliferative activities [44].

Therefore, in continuation of our previous work on the synthesis of novel biologically active heterocyclic compounds [45–47], we wish to report herein a simple protocol for the synthesis of novel 3-substituted indole and kojic acid derivatives 4a–4j by the three-component condensation of 3-(cyanoacetyl)indole (1), benzaldehyde derivatives 2, and kojic acid (3a) in the presence of ammonium acetate as catalyst (Scheme 1).

scheme 1

Also, antibacterial activities of 3-substituted indole derivatives 4a–4j were investigated against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis.

Results and discussion

Chemistry

In order to optimize the reaction conditions, the three-component reaction of 3-(cyanoacetyl)indole (1) with benzaldehyde (2a) and kojic acid (3a) was investigated as model reaction. Initially, the reaction was carried out without any catalysts, but did not give any product (Table 1, entry 1). Also, when piperidine was used as the catalyst, the reaction did not proceed (Table 1, entry 2). The best result was obtained when the reaction was performed in the presence of ammonium acetate as catalyst. We also evaluated the amount of catalyst required for this transformation. It was found that when the amount of ammonium acetate increased from 10 to 20 mol% and 30 mol%, the yields increase from 20 to 55 % and 95 %, respectively. Using 30 mol% of ammonium acetate is sufficient to promote the reaction. Additional amounts of the catalyst did not improve the product yields (Table 1, entry 7).

Table 1 Optimization of catalysts
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To select the optimal solvent and temperature for this reaction, we performed the model reaction in different solvents, and at room temperature and at reflux conditions. It was observed that when using EtOH as the solvent at reflux condition, shorter reaction time and higher yield resulted (Table 2).

Table 2 Optimization of solvent and temperature
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To study the scope the reaction, a series of substituted aldehydes 2a–2l and kojic acid (3a) or some example of other compounds containing an enol group 3b–3d were reacted with 3-(cyanoacetyl)indole (1) using ammonium acetate (30 mol% in boiling EtOH. Under the optimized reaction conditions, a series of 3-(cyanoacetyl)indole derivatives 4a–4j were synthesized in excellent yields (Table 3). As presented in Table 3, the reaction of aldehydes with electron accepting groups leads to the highest yields and the shortest reaction times (entries 7 and 10), whereas the reaction of aldehydes 2k, 2l with strong electron releasing group (e.g., NMe2) and the other OH acids 3b–3d (entries 13–15) did not proceed, therefore no products could be isolated. The expected structures of the products 4m–4o are depicted in Fig. 1.

Table 3 Synthesis of three-substituted indole derivatives 4a–4j
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Fig. 1
figure 1

The expected structures of the products 4m–4o

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As shown in Fig. 2, compounds 4a–4j possess two chiral centers and therefore, they can exist as two diastereomeric pairs. For example, compound 4a exists as two diastereomeric pairs, RR or its enantiomer SS, and RS or its enantiomer SR.

Fig. 2
figure 2

Structures of two diastereomeric pairs of 4a (I (RR/SS or RS/SR) and II (RR/SS or RS/SR))

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The 1H NMR spectrum of 4a shows existence of both two diastereomeric pairs. Unfortunately, all our attempts to separate the two diastereomeric pairs including recrystallization, flash and column chromatography by using different ratios of various solvents have failed and we could only isolate the mixture of two diastereomers.

The structures and the purities of the products 4a–4j were determined on the basis of their elemental analyses, mass spectra, 1H and 13C NMR and IR spectroscopic data. The 1H NMR spectrum of 4a in DMSO-d 6 for the first diastereomeric pair (RR/SS or RS/SR) exhibited two doublet of doublets at 4.07 and 4.25 ppm (2 J HH = 16.0 and 3 J HH = 6.0 Hz) for CH2 group (AB system), two doublets at 5.00 ppm for Hb and 5.74 ppm for Ha (3 J HH = 11.6 Hz), a triplet at 5.64 ppm (3 J HH = 6.4 Hz) for OH group and a singlet at 6.10 ppm for vinyl proton of kojic acid ring. The other signals at 7.16–7.50, 7.61, and 8.11 ppm consisted of characteristic resonances in the appropriate regions for nine aromatic protons along with three singlets at 8.91, 9.28, and 12.43 ppm for vinyl proton of indole, OH and NH groups, respectively. The proton decoupled 13C NMR spectrum of 4a showed 22 signals, which is in agreement with the proposed structure.

The 1H NMR spectrum of 4a in DMSO-d 6 for the second diastereomeric pair (RR/SS or RS/SR) exhibited two doublet of doublets at 4.44 and 4.48 ppm (2 J HH = 16.0 and 3 J HH = 6.4 Hz) for CH2 group (AB system), two doublets at 5.18 ppm (3 J HH = 10.8 Hz) and 5.61 ppm (3 J HH = 11.2 Hz) for Hb and Ha, a triplet at 5.79 ppm (3 J HH = 6.4 Hz) for OH group and a singlet at 6.36 ppm for vinyl proton of kojic acid ring. The other signals at 7.16–7.50, 7.52, and 8.05 consisted of characteristic resonances in the appropriate regions for aromatic protons along with three singlets at 8.75, 9.43, and 12.38 ppm for vinyl proton of indole, OH and NH groups, respectively. The proton decoupled 13C NMR spectrum of 4a showed 22 signals, which is in agreement with the proposed structure. The 1H and 13C NMR spectra of 4b–4j were similar to those of 4a, except to the substituted phenyl moiety that exhibited characteristic resonances in the appropriate regions of the spectra.

A plausible mechanism for the formation of products 4a–4j is given in Scheme 2. The formation of these products can be rationalized by initial formation of intermediate 5 by Knoevenagel condensation of the aldehyde 2 and 3-(cyanoacetyl)indole. As shown in Scheme 2, the anion of kojic acid can attack to intermediate 5 in four routes: Michael-type addition (route a), direct addition (route b), C-alkylation (route c), or O-alkylation (route d). Among these four routes, the reaction proceeded regioselectively via route a, and led to intermediate 7. Then, enolization and protonation of 7 in the reaction conditions result in final product 4.

scheme 2

Antibacterial activity

The synthesized compounds were evaluated for their antibacterial activity against two Gram-positive S. aureus and B. subtilis and two Gram-negative bacterial strains E. coli and P. aeruginosa. Standard antibacterial drug gentamicin and chloramphenicol were also tested under similar conditions against these organisms (Table 4). Compound 4e (p-chlorobenzaldehyde derivative) demonstrated excellent activity against S. aureus and B. subtilis, even more than standard compounds. Except compound 4f (m-chlorobenzaldehyde derivative), none of the compounds did show antibacterial activities against P. aeruginosa. Also, compounds 4e, 4f, 4h, 4i, and 4j (with Cl, Br, or NO2 substituent) exhibited good activity against E. coli.

Table 4 Antibacterial activity of the complex using Kirby–Bauer technique (zone of growth inhibition/mm)
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Experimental

All chemicals and reagents were purchased from Fluka and Merck and used without further purification. Melting points were measured on an Electrothermal 9100 apparatus. NMR spectra were recorded on a Bruker DRX-400 AVANCE instrument (400.1 MHz for 1H, 100.6 MHz for 13C) with DMSO-d 6 as solvent. Chemical shifts (δ) are given in parts per million (ppm) relative to TMS, and coupling constants (J) are reported in hertz (Hz). IR spectra were recorded on an FT-IR Bruker vector 22 spectrometer. Mass spectra were recorded on a Finnigan-Matt 8430 mass spectrometer operating at an ionization potential of 70 eV. Elemental analyses were carried on a Perkin-Elmer 2400II CHNS/O Elemental Analyzer. 3-(Cyanoacetyl)indole was prepared as reported in the literature [48].

General procedure for the synthesis of compounds 4a – 4j

A mixture of 3-(cyanoacetyl)indole (1 mmol), benzaldehyde derivatives (1 mmol), kojic acid (1 mmol), and ammonium acetate (30 mol%) in 5 cm3 ethanol was refluxed for appropriate time as indicated in Table 2. The completion of the reaction was indicated by the disappearance of the starting material in thin layer chromatography (EtOAc/n-hexane 18:2). After completion of the reaction, the product was recrystallized from methanol and washed with water.

3-[3-Hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)-3-phenylpropanenitrile (4a, C24H18N2O5)

White powder, 0.39 g (95 %); m.p.: 236-238 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4a-I (RR/SS or RS/SR): yield 56 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.07 and 4.25 (2dd, AB system, 2H, 2 J HH = 16.0 Hz, 3 J HH = 6.0 Hz, CH2), 5.00 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.64 (t, 1H, 3 J HH = 6.4 Hz, OH), 5.74 (d, 1H, 3 J HH = 11.6 Hz, CH), 6.10 (s, 1H, CHkojic), 7.16–7.50 (m, 7H, 7CHAr), 7.61 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.11 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.91 (s, 1H, CH–NH), 9.28 (s, 1H, OH), 12.43 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.47, 43.35 (2CH), 59.67 (CH2), 109.26 (CHkojic), 113.05 (CHindole), 114.73, 118.10 (2Cindole), 121.63, 123.17, 124.19 (3CHindole), 125.65 (Cindole), 128.29, 128.58, 129.19, 136.89 (5CH, CAr), 137.29 (CHindole), 137.45 (CN), 142.14, 147.42, 167.30 (3Ckojic), 173.68, 182.66 (2CO) ppm.

4a-II (RR/SS or RS/SR): yield 39 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.44 and 4.48 (2dd, AB system, 2H, 2 J HH = 16.0 Hz, 3 J HH = 6.4 Hz, CH2), 5.18 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.61 (d, 1H, 3 J HH = 11.2 Hz, CH), 5.79 (t, 1H, 3 J HH = 6.4 Hz, OH), 6.36 (s, 1H, CHkojic), 7.16–7.50 (m, 7H, 7CHAr), 7.52 (d, 1H, 3 J HH = 7.6 Hz, CHAr), 8.05 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.75 (s, 1H, CH–NH), 9.43 (s, 1H, OH), 12.38 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 42.04, 44.42 (2CH), 59.91 (CH2), 109.38 (CHkojic), 113.06 (CHindole), 115.30, 118.33 (2Cindole), 121.68, 123.18, 124.20 (3CHindole), 125.73 (Cindole), 128.30, 128.97, 129.36, 137.20 (5CH, CAr), 137.36 (CHindole), 137.66 (CN), 142.45, 148.21, 168.17 (3Ckojic), 174.00, 183.55 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,425 (OH), 3,247 (NH), 2,250 (CN), 1,630 (C=O), 1,599 (C=C), 1,097 (C–O) cm–1; MS (70 eV): m/z (%) = 414 (M+., 8), 270 (M+-C9H6NO, 29), 144 (M+-C15H12NO4, 100), 116 (M+-C16H12NO5, 66).

3-[3-Hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)-3-(4-methoxyphenyl)propanenitrile (4b, C25H20N2O6)

White powder, 0.40 g (91 %); m.p.: 232–234 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4b-I (RR/SS or RS/SR): yield 53 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 3.77 (s, 3H, CH3), 4.04–4.26 (m, 2H, CH2), 4.94 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.55 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.67–5.71 (m, 1H, OH), 6.09 (s, 1H, CHkojic), 6.99 (d, 1H, 3 J HH = 8.8 Hz, CHAr), 7.19–7.53 (m, 6H, 6CHAr), 8.09 (d, 1H, 3 J HH = 7.6 Hz, CHAr), 8.89 (s, 1H, CH–NH), 9.29 (s, 1H, OH), 12.42 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.54, 42.66 (2CH), 55.42 (OCH3), 59.69 (CH2), 109.23 (CHkojic), 113.04 (CHindole), 114.53, 115.41 (2Cindole), 121.69, 123.17, 124.22 (3CHindole), 125.66 (Cindole), 128.68, 129.59, 129.75, 130.11 (4CH, 2CAr), 137.19 (CHindole), 141.90 (CN), 148.57, 159.12, 167.20 (3Ckojic), 173.70, 182.78 (2CO) ppm.

4b-II (RR/SS or RS/SR): yield 38 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 3.62 (s, 3H, CH3), 4.39-4.50 (m, 2H, CH2), 5.12 (d, 1H, 3 J HH = 11.2 Hz, CH), 5.70 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.82 (t, 1H, 3 J HH = 6.8 Hz, OH), 6.35 (s, 1H, CHkojic), 6.79 (d, 1H, 3 J HH = 8.8 Hz, CHAr), 7.19–7.53 (m, 6H, 6CHAr), 8.06 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.74 (s, 1H, CH–NH), 9.30 (s, 1H, OH), 12.41 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 42.30, 43.66 (2CH), 55.59 (OCH3), 59.92 (CH2), 109.34 (CHkojic), 113.05 (CHindole), 114.68, 118.14 (2Cindole), 121.70, 123.18, 124.23 (3CHindole), 125.67 (Cindole), 128.69, 129.60, 129.76, 130.12 (4CH, 2CAr), 137.31 (CHindole), 142.18 (CN), 148.58, 159.44, 168.08 (3Ckojic), 174.02, 182.79 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,427 (OH), 3,233 (NH), 2,259 (CN), 1,632 (C=O), 1,515 (C=C), 1,033 (C–O) cm−1; MS (70 eV): m/z (%) = 444 (M+., 5), 300 (M+-C9H6NO, 27), 261 (M+-C11H7N2O, 25), 144 (M+-C16H14NO5, 100), 116 (M+-C17H14NO6, 20).

3-[3-Hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)-3-(3-methoxyphenyl)propanenitrile (4c, C25H20N2O6)

White powder, 0.35 g (80 %); m.p.: 211–213 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4c-I (RR/SS or RS/SR): yield 47 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 3.81 (s, 3H, CH3), 4.08 and 4.24 (2dd, 2H, 2 J HH = 16.0 Hz, 3 J HH = 5.6 Hz, CH2), 4.98 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.64 (br s, 1H, OH), 5.74 (d, 1H, 3 J HH = 12.0 Hz, CH), 6.10 (s, 1H, CHkojic), 6.73–7.29 (m, 6H, 6CHAr), 7.51 (t, 1H, 3 J HH = 8.4 Hz, CHAr), 8.11 (d, 1H, 3 J HH = 8.0 Hz, CHAr), 8.92 (s, 1H, CH–NH), 9.28 (s, 1H, OH), 12.43 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.35, 43.28 (2CH), 55.46 (CH3), 59.71 (CH2), 109.30 (CHkojic), 113.06 (CHindole), 113.45, 114.31 (2Cindole), 114.93, 118.07, 120.94 (3CHindole), 121.64 (Cindole), 123.19, 124.20, 125.66, 130.26, 137.25, 137.36 (4CH, 2CAr), 138.23 (CHindole), 139.00 (CN), 147.28, 159.69, 167.24 (3Ckojic), 173.68, 182.66 (2CO) ppm.

4c-II (RR/SS or RS/SR): yield 33 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 3.66 (s, 3H, CH3), 4.40–4.51 (m, 2H, CH2), 5.16 (d, 1H, 3 J HH = 11.2 Hz, CH), 5.61 (d, 1H, 3 J HH = 11.2 Hz, CH), 5.79 (t, 1H, 3 J HH = 6.4 Hz, OH), 6.37 (s, 1H, CHkojic), 6.73–7.29 (m, 6H, 6CHAr), 7.36 (t, 1H, 3 J HH = 7.6 Hz, CHAr), 8.07 (d, 1H, 3 J HH = 8.0 Hz, CHAr), 8.77 (s, 1H, CH–NH), 9.46 (s, 1H, OH), 12.40 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.93, 44.33 (2CH), 55.60 (CH3), 59.94 (CH2), 109.43 (CHkojic), 113.07 (CHindole), 113.54, 114.73 (2Cindole), 115.33, 118.34, 121.26 (3CHindole), 121.68 (Cindole), 123.20, 124.24, 125.72, 130.43, 137.29, 137.48 (4CH, 2CAr), 138.24 (CHindole), 139.01 (CN), 148.11, 159.95, 168.09 (3Ckojic), 174.01, 183.53 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,421 (OH), 3,224 (NH), 2,261 (CN), 1,633 (C=O), 1,596 (C=C), 1,042 (C–O) cm−1; MS (70 eV): m/z (%) = 444 (M+., 2), 262 (M+-C11H6N2O, 96), 144 (M+-C16H14NO5, 100), 116 (M+-C17H14NO6, 34).

3-[3-Hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)-3-(4-methylphenyl)propanenitrile (4d, C25H20N2O5)

White powder, 0.39 g (92 %); m.p.: 233–235 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4d-I (RR/SS or RS/SR): yield 55 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 2.32 (s, 3H, CH3), 4.03–4.27 (m, 2H, CH2), 4.96 (d, 1H, 3 J HH = 11.2 Hz, CH), 5.59 (d, 1H, 3 J HH = 11.2 Hz, CH), 5.67 (t, 1H, 3 J HH = 6.4 Hz, OH), 6.09 (s, 1H, CHkojic), 7.04–7.53 (m, 7H, 7CHAr), 8.10 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.90 (s, 1H, CH–NH), 9.27 (s, 1H, OH), 12.43 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 20.97 (CH3), 42.03, 44.00 (2CH), 59.65 (CH2), 109.21 (CHkojic), 113.04 (CHindole), 114.72, 118.13 (2Cindole), 121.67, 123.21, 124.27 (3CHindole), 125.62 (Cindole), 128.44, 129.75, 133.78, 134.60 (4CH, 2CAr), 137.28 (CHindole), 137.60 (CN), 141.93, 147.61, 167.27 (3Ckojic), 173.66, 182.66 (2CO) ppm.

4d-II (RR/SS or RS/SR): yield 37 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 2.14 (s, 3H, CH3), 4.41 and 4.47 (2dd, AB system, 2H, 2 J HH = 16.0 Hz, 3 J HH = 6.0 Hz, CH2), 5.15 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.71 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.82 (t, 1H, 3 J HH = 6.8 Hz, OH), 6.35 (s, 1H, CHkojic), 7.04–7.53 (m, 7H, 7CHAr), 8.05 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.77 (s, 1H, CH–NH), 9.28 (s, 1H, OH), 12.42 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 21.16 (CH3), 42.95, 44.07 (2CH), 59.89 (CH2), 109.33 (CHkojic), 113.05 (CHindole), 115.37, 118.38 (2Cindole), 121.68, 123.22, 124.28 (3CHindole), 125.69 (Cindole), 128.81, 129.91, 133.79, 134.61 (4CH, 2CAr), 137.29 (CHindole), 137.90 (CN), 141.94, 148.41, 168.14 (3Ckojic), 174.00, 183.65 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,425 (OH), 3,248 (NH), 2,260 (CN), 1,632 (C=O), 1,519 (C=C), 1,036 (C–O) cm−1; MS: m/z (%) = 428 (M+., 8), 284 (M+-C9H6NO, 43), 245 (M+-C11H7N2O, 19), 144 (M+-C16H14NO4, 100), 116 (M+-C17H14NO5, 48).

3-(4-Chlorophenyl)-3-[3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)propanenitrile (4e, C27H17 ClN2O5)

White powder, 0.41 g (92 %); m.p.: 217–219 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4e-I (RR/SS or RS/SR): yield 55 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.05–4.28 (m, 2H, CH2), 5.04 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.77 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.69 (br s, 1H, OH), 6.13 (s, 1H, CHkojic), 7.21–7.67 (m, 7H, 7CHAr), 8.11 (d, 1H, 3 J HH = 7.6 Hz, CHAr), 8.89 (s, 1H, CH–NH), 9.39 (s, 1H, OH), 12.45 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.42, 42.70 (2CH), 59.66 (CH2), 109.31 (CHkojic), 113.08 (CHindole), 114.69, 117.94 (2Cindole), 121.62, 123.23, 124.25 (3CHindole), 125.61 (Cindole), 129.23, 130.46, 130.87, 133.08 (4CH, 2CAr), 135.81 (CHindole), 137.36 (CN), 142.23, 146.92, 167.42 (3Ckojic), 173.70, 182.44 (2CO) ppm.

4e-II (RR/SS or RS/SR): yield 37 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.41–4.51 (m, 2H, CH2), 5.20 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.63 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.84 (br s, 1H, OH), 6.39 (s, 1H, CHkojic), 7.21–7.67 (m, 7H, 7CHAr), 8.07 (d, 1H, 3 J HH = 7.6 Hz, CHAr), 8.76 (s, 1H, CH–NH), 9.55 (s, 1H, OH), 12.44 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.88, 43.76 (2CH), 59.89 (CH2), 109.44 (CHkojic), 113.09 (CHindole), 115.22, 118.23 (2Cindole), 121.67, 123.28, 124.32 (3CHindole), 125.69 (Cindole), 129.37, 130.47, 130.88, 133.40 (4CH, 2CAr), 136.57 (CHindole), 137.43 (CN), 142.54, 147.65, 168.27 (3Ckojic), 174.03, 183.27 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,417 (OH), 3,239 (NH), 2,259 (CN), 1,632 (C=O), 1,594 (C=C), 1,035 (C–O) cm−1; MS (70 eV): m/z (%) = 448 (M+., 9), 450 (M+.+2, 3), 304 (M+-C9H6NO, 26), 306 (M++2-C9H6NO, 14), 265 (M+-C11H7N2O, 14), 267 (M++2-C11H7N2O, 6), 144 (M+-C15H11ClNO4, 100), 116 (M+-C16H11ClNO5, 33).

3-(3-Chlorophenyl)-3-[3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)propanenitrile (4f, C27H17 ClN2O5)

White powder, 0.40 g (91 %); m.p.: 210–212 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4f-I (RR/SS or RS/SR): yield 58 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.07 and 4.25 (2dd, AB system, 2H, 2 J HH = 16.0 Hz, 3 J HH = 6.0 Hz, CH2), 5.04 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.65 (t, 1H, 3 J HH = 6.4 Hz, OH), 5.81 (d, 1H, 3 J HH = 11.6 Hz, CH), 6.13 (s, 1H, CHkojic), 7.20–7.76 (m, 7H, 7CHAr), 8.11 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.94 (d, 1H, 3 J HH = 2.8 Hz, CH–NH), 9.42 (s, 1H, OH), 12.46 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.36, 42.86 (2CH), 59.68 (CH2), 109.37 (CHkojic), 113.10 (CHindole), 114.70, 117.90 (2Cindole), 121.62, 123.22, 124.24 (3CHindole), 125.64 (Cindole), 127.45, 128.33, 128.58, 131.12, 133.72, 137.31 (4CH, 2CAr), 137.55 (CHindole), 139.22 (CN), 142.36, 146.70, 167.40 (3Ckojic), 173.69, 182.34 (2CO) ppm.

4f-II (RR/SS or RS/SR): yield 33 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.45–4.52 (m, 2H, CH2), 5.20 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.67 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.80 (br s, 1H, OH), 6.39 (s, 1H, CHkojic), 7.20–7.76 (m, 7H, 7CHAr), 8.07 (d, 1H, 3 J HH = 7.6 Hz, CHAr), 8.79 (d, 1H, 3 J HH = 2.8 Hz, CH–NH), 9.59 (s, 1H, OH), 12.44 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.65, 44.00 (2CH), 59.91 (CH2), 109.51 (CHkojic), 113.11 (CHindole), 115.14, 118.21 (2Cindole), 121.67, 123.27, 124.30 (3CHindole), 125.71 (Cindole), 128.00, 128.45, 128.73, 131.26, 133.97, 137.37 (4CH, 2CAr), 137.56 (CHindole), 139.94 (CN), 142.68, 147.42, 168.24 (3Ckojic), 174.00, 183.15 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,481 (OH), 3,293 (NH), 2,248 (CN), 1,633 (C=O), 1,579 (C=C), 1,088 (C–O) cm−1; MS: m/z (%) = 448 (M+., 3), 450 (M+.+2, 1), 304 (M+.-C9H6NO, 8), 306 (M+.+2-C9H6NO, 9), 144 (M+-C15H11ClNO4, 100), 116 (M+-C16H11ClNO5, 30).

3-[3-Hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)-3-(4-isocyanophenyl)propanenitrile (4g, C25H17 N3O5)

White powder, 0.42 g (97 %); m.p.: 235–237 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4g-I (RR/SS or RS/SR): yield 60 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.06 and 4.24 (2dd, AB system, 2H, 2 J HH = 16.0 Hz, 3 J HH = 6.4 Hz, CH2), 5.11 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.64 (t, 1H, 3 J HH = 6.4 Hz, OH), 5.82 (d, 1H, 3 J HH = 11.6 Hz, CH), 6.12 (s, 1H, CHkojic), 7.20–7.30 (m, 2H, 2CHAr), 7.50–7.55 (m, 1H, CHAr), 7.84 (d, 2H, 3 J HH = 8.4 Hz, 2CHAr), 7.95 (d, 2H, 3 J HH = 8.4 Hz, 2CHAr), 8.10 (d, 1H, 3 J HH = 8.0 Hz, CHAr), 8.90 (s, 1H, CH–NH), 9.47 (s, 1H, OH), 12.45 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.23, 43.08 (2CH), 59.63 (CH2), 109.37 (CHkojic), 111.29 (CHindole), 113.11, 114.64 (2Cindole), 117.78, 118.77, 121.59 (3CHindole), 123.25 (Cindole), 124.27, 125.61, 129.62, 133.20 (4CH, 2CAr), 137.30 (CHindole), 137.51, 142.33 (2 CN), 142.87, 146.21, 167.51 (3Ckojic), 173.66, 182.13 (2CO) ppm.

4g-II (RR/SS or RS/SR): yield 37 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.43–4.46 (m, 2H, CH2), 5.27 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.69 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.79 (t, 1H, 3 J HH = 6.4 Hz, OH), 6.37 (s, 1H, CHkojic), 7.20–7.30 (m, 2H, 2CHAr), 7.50–7.55 (m, 1H, CHAr), 7.64 (d, 2H, 3 J HH = 8.4 Hz, 2CHAr), 7.76 (d, 2H, 3 J HH = 8.4 Hz, 2CHAr), 8.04 (d, 1H, 3 J HH = 8.0 Hz, CHAr), 8.77 (s, 1H, CH–NH), 9.63 (s, 1H, OH), 12.44 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.44, 44.25 (2CH), 59.86 (CH2), 109.49 (CHkojic), 111.57 (CHindole), 113.12, 115.00 (2Cindole), 118.06, 118.99, 121.65 (3CHindole), 123.29 (Cindole), 124.33, 125.70, 130.10, 133.34 (4CH, 2CAr), 137.37 (CHindole), 137.52, 142.54 (2CN), 142.96, 146.90, 168.35 (3Ckojic), 173.97, 182.92 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,221 (OH, NH), 2,231 (CN), 1,656, 1,626 (C=O), 1,585 (C=C), 1,042 (C–O) cm−1; MS: m/z (%) = 439 (M+., 3), 295 (M+-C9H6NO, 7), 144 (M+-C16H11N2O4, 100), 116 (M+-C17H11N2O5, 21).

3-(4-Bromophenyl)-3-[3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)propanenitrile (4h, C24H17Br N2O5)

White powder, 0.45 g (93 %); m.p.: 223–225 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4h-I (RR/SS or RS/SR): yield 56 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.06 and 4.25 (2d, AB system, 2H, 2 J HH = 15.2 Hz, CH2), 5.01 (d, 1H, 3 J HH = 9.2 Hz, CH), 5.78 (br s, 2H, 1CH, OH), 6.11 (s, 1H, CHkojic), 7.21–7.69 (m, 7H, 7CHAr), 8.10 (d, 1H, 3 J HH = 7.6 Hz, CHAr), 8.89 (s, 1H, CH–NH), 9.38 (s, 1H, OH), 12.44 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.30, 42.69 (2CH), 59.65 (CH2), 109.31 (CHkojic), 113.09 (CHindole), 114.69, 117.92 (2Cindole), 121.65, 123.25, 124.30 (3CHindole), 125.63 (Cindole), 130.78, 131.20, 132.15, 136.27 (4CH, 2CAr), 137.03 (CHindole), 137.30 (CN), 142.25, 146.84, 167.39 (3Ckojic), 173.68, 182.39 (2CO) ppm.

4h-II (RR/SS or RS/SR): yield 37 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.38–4.53 (m, 2H, CH2), 5.18 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.62 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.60–5.63 (m, 1H, OH), 6.37 (s, 1H, CHkojic), 7.21–7.69 (m, 7H, 7CHAr), 8.06 (d, 1H, 3 J HH = 7.6 Hz, CHAr), 8.77 (s, 1H, CH–NH), 9.39 (s, 1H, OH), 12.43 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.76, 43.81 (2CH), 59.88 (CH2), 109.42 (CHkojic), 113.10 (CHindole), 115.19, 118.23 (2Cindole), 121.96, 123.26, 124.31 (3CHindole), 125.72 (Cindole), 130.79, 131.21, 132.28, 136.28 (4CH, 2CAr), 137.04 (CHindole), 137.31 (CN), 142.56, 147.54, 168.26 (3Ckojic), 174.00, 183.22 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,414 (OH), 3,239 (NH), 2,258 (CN), 1,632 (C=O), 1,593 (C=C), 1,034 (C–O) cm−1; MS: m/z (%) = 492 (M+., 1), 494 (M+.+2, 1), 308 (M+-C11H7 N2O, 15), 310 (M++2-C11H7 N2O, 19), 144 (M+-C15H11 BrNO4, 100), 116 (M+-C16H11 BrNO5, 23).

3-(2-Bromophenyl)-3-[3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)propanenitrile (4i, C24H17Br N2O5)

White powder, 0.44 g (90 %); m.p.: 219–221 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4i-I (RR/SS or RS/SR): yield 58 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.05 and 4.25 (2dd, AB system, 2H, 2 J HH = 16.0 Hz, 3 J HH = 6.0 Hz, CH2), 5.63–5.77 (m, 3H, 2CH, OH), 6.12 (s, 1H, CHkojic), 7.12–8.28 (m, 8H, 8CHAr), 8.86 (s, 1H, CH–NH), 9.29 (s, 1H, OH), 12.42 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 42.03, 42.28 (2CH), 59.65 (CH2), 109.23 (CHkojic), 113.07 (CHindole), 114.68, 117.66 (2Cindole), 121.68, 123.13, 124.22 (3CHindole), 125.69 (Cindole), 128.42, 129.90, 130.48, 133.19, 133.55, 136.02 (4CH, 2CAr), 137.03 (CHindole), 137.28 (CN), 142.78, 146.34, 167.52 (3Ckojic), 173.71, 182.29 (2CO) ppm.

4i-II (RR/SS or RS/SR): yield 32 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.35 and 4.43 (2dd, AB system, 2H, 2 J HH = 16.0 Hz, 3 J HH = 6.0 Hz, CH2), 5.63–5.77 (m, 3H, 2CH, OH), 6.36 (s, 1H, CHkojic), 7.12–8.28 (m, 8H, 8CHAr), 8.73 (s, 1H, CH–NH), 9.43 (s, 1H, OH), 12.37 (s, 1H, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 42.21, 42.85 (2CH), 59.68 (CH2), 109.24 (CHkojic), 113.11 (CHindole), 114.98, 117.86 (2Cindole), 121.84, 123.19, 124.28 (3CHindole), 125.73 (Cindole), 128.74, 130.18, 130.76, 133.50, 133.65, 136.89 (4CH, 2CAr), 137.22 (CHindole), 137.35 (CN), 143.14, 146.37, 168.40 (3Ckojic), 174.08, 183.07 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,426 (OH), 3,242 (NH), 2,257 (CN), 1,655, 1,626 (C=O), 1,599 (C=C), 1,036 (C–O) cm−1; MS: m/z (%) = 492 (M+, 4), 494 (M+.+2, 4), 347 (M+-C9H6NO, 12), 349 (M++2-C9H6NO, 14), 144 (M+-C15H11 BrNO4, 100), 116 (M+-C16H11 BrNO5, 10).

3-[3-Hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl]-2-(1H-indol-3-ylcarbonyl)-3-(4-nitrophenyl)propanenitrile (4j, C24H17 N3O7)

White powder, 0.44 g (97 %); m.p.: 263–265 °C; R f = 0.4 (n-hexane/ethyl acetate 1:9 v/v).

4j-I (RR/SS or RS/SR): yield 57 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.06 and 4.26 (2dd, 2H, 2 J HH = 16.0 Hz, 3 J HH = 6.0 Hz, CH2), 5.19 (d, 1H, 3 J HH = 11.6 Hz, CH), 5.64 (t, 1H, 3 J HH = 6.4 Hz, OH), 5.87 (d, 1H, 3 J HH = 11.6 Hz, CH), 6.13 (s, 1H, CHkojic), 7.26–7.31 (m, 2H, 2CHAr), 7.54 (d, 1H, 3 J HH = 6.4 Hz, CHAr), 7.93 (d, 2H, 3 J HH = 8.8 Hz, 2CHAr), 8.11 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.32 (d, 1H, 3 J HH = 8.8 Hz, CHAr), 8.91 (d, 1H, 3 J HH = 3.2 Hz, CH–NH), 9.50 (s, 1H, OH), 12.46 (d, 1H, 3 J HH = 3.2 Hz, NH) ppm; 13C NMR (100.6 MHz, DMSO-d 6 ): δ = 41.24, 42.81 (2CH), 59.63 (CH2), 109.36 (CHkojic), 113.12 (CHindole), 114.63, 117.73 (2Cindole), 121.59, 123.25, 124.37 (3CHindole), 125.62 (Cindole), 129.96, 130.46, 137.20, 137.38 (4CH, 2CAr), 142.62 (CHindole), 144.26 (CN), 146.00, 147.49, 167.57 (3Ckojic), 173.65, 182.03 (2CO) ppm.

4j-II (RR/SS or RS/SR): yield 40 %; 1H NMR (400.13 MHz, DMSO-d 6 ): δ = 4.44–4.50 (m, 2H, CH2), 5.34 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.74 (d, 1H, 3 J HH = 10.8 Hz, CH), 5.79 (t, 1H, 3 J HH = 6.8 Hz, OH), 6.38 (s, 1H, CHkojic), 7.26–7.31 (m, 2H, 2CHAr), 7.51 (d, 1H, 3 J HH = 7.6 Hz, CHAr), 7.74 (d, 2H, 3 J HH = 8.8 Hz, 2CHAr), 8.04 (d, 1H, 3 J HH = 7.2 Hz, CHAr), 8.14 (d, 2H, 3 J HH = 8.8 Hz, 2CHAr), 8.80 (d, 1H, 3 J HH = 3.2, CH–NH), 9.67 (s, 1H, OH), 12.44 (d, 1H, 3 J HH = 2.8 Hz, NH) ppm; 13C NMR (100 MHz, CDCl3): δ = 41.43, 44.01 (2CH), 59.85 (CH2), 109.48 (CHkojic), 113.13 (CHindole), 114.98, 118.03 (2Cindole), 121.65, 124.27, 124.49 (3CHindole), 125.71 (Cindole), 129.97, 130.47, 137.31, 137.52 (4CH, 2CAr), 142.94 (CHindole), 144.87 (CN), 146.69, 147.82, 168.42 (3Ckojic), 173.96, 182.85 (2CO) ppm; IR (KBr): \( \overline{\nu } \) = 3,222 (NH, OH), 2,252 (CN), 1,656, 1,626 (C=O), 1,586 (C=C), 1,044 (C–O) cm−1; MS: m/z (%) = 459 (M+., 1), 315 (M+.-C9H6NO, 3), 144 (M+-C15H11N2O6, 100), 116 (M+-C16H11N2O7, 21).

General procedure for evaluation of antibacterial activity

Antibacterial activities of the synthesized 3-(cyanoacetyl)indole derivatives 4a–j were assayed using Kirby–Bauer disc diffusion method, where a filter disc was impregnated with the compounds and placed on the surface of inoculated agar plates [49]. The synthesized compounds were dissolved into DMSO to achieve 20 mg cm−3 solution then filter sterilized using a 0.22-μm Ministart (Sartorius).

The antibacterial activities of the synthesized compounds were investigated against four bacterial species. Test organisms included E. coli PTCC 1330, P. aeruginosa PTCC 1074, S. aureus ATCC 35923, and B. subtilis PTCC 102 [50]. Late exponential phase of the bacteria was prepared by inoculating 1 % (v/v) of the cultures into the fresh Muller–Hinton broth (Merck) and incubating in an orbital shaker at 37 °C and 100 rpm overnight. Before using the cultures, they were standardized with a final cell density of approximately 108 cfu cm−3. Muller–Hinton agar (Merck) was prepared and inoculated with the standardized cultures of the test organisms and then spread as uniformly as possible throughout the entire media. Sterile paper discs (6 mm diameter, Padtan, Iran) were impregnated with 20 mm3 of the compound solution then allowed to dry. The impregnated disc was introduced on the upper layer of the seeded agar plate and incubated at 37 °C for 24 h. The antibacterial activities of the synthesized compounds were compared with known antibiotic gentamicin (10 µg/disc) and chloramphenicol (30 µg/disc) as positive control and DMSO (20 mm3/disc) as negative control. Antibacterial activity was evaluated by measuring the diameter of inhibition zone (mm) on the surface of plates, and the results were reported as mean ± SD after three repeats.