Introduction

The potentiality of biologically active compound can be greatly altered by its minor structural modifications. This implies that a major line of approach is served by promising lead compounds substantiating molecular manipulation which involves an effort to combine separate pharmacophoric groups into one compound to achieve more potent molecule.

It is well known that the indole moiety which is probably the most widely spread nitrogen heterocycle in nature is very important for its medicinal and biological aspects, thus attracting a lot of scientific attention. It has been found to possess pharmacological and chemotherapeutic properties such as anticancer [1], antidiabetic [2], antinflammatory [3], antimalarial [4], antibacterial [5–7], antifungal [8], antiviral [9, 10], and so forth.

A large number of fused pyrimidines have been the focus of great importance and have reflected significant biological activities with appreciably wider spectrum like antiplatelet activity [11], anticancer [12], antimicrobial [13], Escherichia coli and Staphylococcus aureus SecA inhibitors [14], c-Met kinase inhibitors [15], and serotonin 5-HT6 receptor antagonists [16]. The versatile applicability of these heterocycles has helped long to plan, organize, and implement new approaches towards discovery of novel drugs.

Hence, the synthesis of triazolopyrimidine derivatives has become a topic of particular interest because of their broad spectrum of biological properties. Some of these derivatives have shown remarkable biological properties such as antiviral, antitumor [17], adenosine A2A receptor [18], analgesic, anticonvulsant, anti-inflammatory [19], antineoplastic [20], anticancer [21], and herbicidal [22]. Thus, fusion of pyrimidine moiety with different heterocyclic scaffolds gives rise to a new class of hybrid heterocycles possessing improved activities.

Owing to the important role played by indole and evaluating the pharmacological profile of pyrimidine we have designed the program for the synthesis of indole fused to triazolopyrimidine so as to get biologically more potent molecules.

Results and discussion

Chemistry

The synthetic strategy to synthesize the target indolyl-triazolopyrimidine compounds is depicted in Scheme 1. The starting material 1-benzyl-5-amino-1H-1,2,3-triazole-4-carboxamide (1) was prepared by the 1,3-dipolar cycloaddition of benzyl azide and cyanoacetamide as previously reported method [23]. Reaction of 1 with ethyl 5-substituted-3-phenylindole-2-carboxylates 2a–2c afforded the annulation product 3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-3H-[1,2,3] triazolo[4,5-d]pyrimidin-7-ols 3a–3c. The triazolopyrimidine compounds 3a–3c when subjected to chlorination with POCl3 gave 3-benzyl-7-chloro-5-(5-substituted-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidines 4a–4c which on reaction with hydrazine hydrate in refluxing ethanol afforded the key intermediate 3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-7-hydrazinyl-3H-[1,2,3] triazolo[4,5-d]pyrimidines 5a–5c. The hydrazines 5a–5c on cyclocondensation with acetyl acetone, dibenzoyl methane, and ethyl acetoacetate in refluxing methanol gave the respective pyrazole derivatives 6a–6c, 7a–7c, and 8a–8c. Similarly, the hydrazines 5a–5c on cyclization with benzoyl chloride and acetic acid under reflux conditions yielded the respective cyclization products 9a–9c and 10a–10c. Further the hydrazines 5a–5c when reacted with phthalic anhydride in dimethyl formamide at reflux temperature gave the compounds 2-[3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-2,3-dihydrophthalazine-1,4-diones 11a–11c. The structures of all the new compounds were confirmed by IR, 1H NMR, 13C NMR, and mass spectral studies and elemental analyses.

scheme 1

In the IR spectrum of 3a, absorption bands at 3351 and 3318 cm−1 were observed due to OH and indole NH groups, respectively. Furthermore, its 1H NMR spectrum exhibited characteristic signals at δ  = 11.86, 5.64, and 5.35 ppm due to the protons of indole NH, CH2 of benzyl moiety, and OH, respectively. Formation of 4a was confirmed by the disappearance of OH stretching frequency at 3351 cm−1 and the appearance of new C–Cl stretching frequency at 761 cm−1 in its IR spectrum. The absence of the signal due to OH proton at 5.35 ppm in its 1H NMR spectrum is the additional evidence for the formation of 4a. The IR spectrum of 5a exhibited absorption bands at 3407/3261 cm−1 and in its 1H NMR spectrum, signals at 8.93 and 4.50 ppm corresponding to NH/NH2, respectively. In the IR spectrum of compounds 6a, 7a, and 8a the absorption bands due to NH/NH2 groups at 3407/3261 cm−1 were missing. In its 1H NMR spectrum, compound 6a revealed signals at 5.93, 2.40, and 2.01 ppm due to the protons of pyrazole CH and the two CH3 groups. While in the 1H NMR spectrum of 7a, signal at 6.75 ppm resonated due to pyrazole CH. In the IR spectrum of 8a, characteristic absorption band at 1732 cm−1 was observed due to C=O group and its 1H NMR spectrum exhibited signals at 4.10 and 3.10 ppm due to pyrazole CH2 and CH3, respectively. In the IR spectra of compounds 9a and 10a, the absorption bands at 3407/3261 cm−1 due to NH/NH2 groups were missing. The 1H NMR spectrum of 10a showed signal at 1.25 ppm which was attributed to the protons of CH3. Compound 11a in its IR spectrum exhibited absorption band at 3363 cm−1 corresponding to the NH group and in its 1H NMR spectrum a signal at 10.88 ppm appeared due to NH. The structures of all the compounds were further established by 13C NMR and mass spectral studies and elemental analyses.

In vitro antimicrobial activity

The newly synthesized compounds were screened for their in vitro antimicrobial activity by cup-plate method (Indian Pharmacopoeia, 1985). This method depends on the diffusion of antibiotic from a cavity through the solidified agar layer in a petri-dish to an extent such that the growth of the added microorganism is prevented in a circular zone around the cavity containing a solution of antibiotic. For antibacterial activity S. aureus (ATCC-29513), E. coli (MTCC-723), and Pseudomonas aeruginosa (MTCC-1688) were used as pathogenic representatives, whereas Aspergillus niger (MTCC-281), Aspergillus flavus (MTCC-1973), and Aspergillus oryzae (MTCC-3567T) were used for antifungal activity. Streptomycin and fluconazole were used as standards for antibacterial and antifungal activities, respectively.

The investigation of antibacterial screening (Table 1) revealed that all tested compounds have moderate to high antibacterial activity as compared to the standard drug streptomycin. Compounds 4a, 8a, and 11a showed excellent antibacterial activity against the tested microorganism S. aureus. Compounds 4c, 7a, and 10c exhibited good activity against E. coli. While the compounds 5a, 8c, and 11a displayed good activity against P. aeruginosa.

Table 1 In vitro antibacterial activity of compounds 3–11
Full size table

On the other hand, the antifungal activity results (Table 2) suggested that the compounds 5a, 6c, and 8a revealed profound activity against A. niger. Compound 3c showed good activity against A. flavus. Whereas the compounds 3a and 11c exhibited good activity against A. oryzae. Rest of the compounds was either moderately active or inactive against the bacterial and fungal strains.

Table 2 In vitro antifungal activity of compounds 3–11
Full size table

Antioxidant activities

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (RSA)

1,1-Diphenyl-2-picrylhydrazyl, a stable free radical, has an odd electron and thus a strong absorption at 517 nm. When this electron becomes paired off, the absorption decreases stoichiometrically with respect to the number of electrons or hydrogen atoms taken up. Such a change in absorption by this reaction has been extensively adopted to test the capacity of several molecules to act as free radical scavengers. The scavenging effects of the synthesized compounds on the DPPH radical were evaluated. The results were compared with the standards 2-tert-butyl-4-methoxyphenol (butylated hydroxyanisole, BHA), 2-(1,1-dimethylethyl)-1,4-benzenediol (2-tert-butyl hydroquinone, TBHQ), and ascorbic acid (AA). The antioxidant capacity was expressed as the 50% inhibitory concentration (IC50) based on the amount of compound required for a 50% decrease of the initial DPPH radical concentration. The values of IC50, the effective concentration at which 50% of the radicals scavenged, were calculated to evaluate the antioxidant activity. The compounds 3–11 showed minimum IC50 value, i.e., less than the 25 µg cm−3 concentration. The results, as depicted in Table 3 (Figs. 1, 2, 3, 4, 5) suggested that compounds 5a and 8a showed promising RSA at all concentrations. Compound 8c was found to enhance the RSA 72.17, 76.95, and 77.82% at 50, 75, and 100 µg cm−3, respectively. Compound 11a showed good activity, i.e., 71.73 and 72.17 at 25 and 100 µg cm−3, respectively. While compound 3c showed good activity, i.e., 72.54% at 25 µg cm−3 and 5c showed 76.95% activity at 75 µg cm−3. Rest of the compounds was found to augment the RSA to a lesser extent.

Table 3 In vitro antibacterial activity with IC 50% values of compounds 3–11
Full size table
Fig. 1
figure 1

Graphical representation for DPPH radical scavenging activity of compounds 3a–3c and 4a–4c

Full size image
Fig. 2
figure 2

Graphical representation for DPPH radical scavenging activity of compounds 5a–5c and 6a–6c

Full size image
Fig. 3
figure 3

Graphical representation for DPPH radical scavenging activity of compounds 7a–7c and 8a–8c

Full size image
Fig. 4
figure 4

Graphical representation for DPPH radical scavenging activity of compounds 9a–9c and 10a–10c

Full size image
Fig. 5
figure 5

Graphical representation for DPPH radical scavenging activity of compounds 11a–11c

Full size image

Ferric ion (Fe3+) reducing antioxidant power (FRAP)

The reductive ability of the synthesized compounds was assessed by the extent of conversion of Fe3+/ferricyanide complex to the Fe2+/ferrous form, at different concentrations (25, 50, 75, 100 µg cm−3) and the results were compared with standards BHA, TBHQ, and AA. Reductive ability results (Figs. 6, 7, 8, 9 10) suggested that compounds 6a, 7a, 7c, and 8a reduced metal ion complex to its lower oxidation state or took part in electron transfer reaction to a quite good extent. Rest of the compounds showed comparatively lesser reducing ability. In other words, these compounds showed the ability of electron donor to scavenge free radicals. The reducing ability of the synthesized compounds indicated that increase in the concentration of samples increases the reductive ability. Thus, higher the absorbance of compounds, greater is the reducing power.

Fig. 6
figure 6

Graphical representation for ferric ion (Fe3+) reducing antioxidant power (FRAP) of compounds 3a–3c and 4a–4c

Full size image
Fig. 7
figure 7

Graphical representation for ferric ion (Fe3+) reducing antioxidant power (FRAP) of compounds 5a–5c and 6a–6c

Full size image
Fig. 8
figure 8

Graphical representation for ferric ion (Fe3+) reducing antioxidant power (FRAP) of compounds 7a–7c and 8a–8c

Full size image
Fig. 9
figure 9

Graphical representation for ferric ion (Fe3+) reducing antioxidant power (FRAP) of compounds 9a–9c and 10a–10c

Full size image
Fig. 10
figure 10

Graphical representation for ferric ion (Fe3+) reducing antioxidant power (FRAP) of compounds 11a–11c

Full size image

Ferrous (Fe2+) ion metal chelating activity

Ferrous ion is the prooxidant among the various species of metal ion. Minimizing ferrous ions afford protection of reactive oxygen species and lipid production. The effective ferrous ion chelators protects by removing ferrous ion (Fe2+) that may otherwise participate in the generation of hydroxyl radical. The chelating effect of ferrous ion with test compounds was determined and results were compared with the standards BHA, TBHQ, and AA. Ferrozine can quantitatively form complex with ferrous ion in this method. In the presence of chelating agents the complex formation is disrupted resulting in a decrease in red color of the complex. Measurement of color reduction, therefore, allows estimating the metal chelating activity of the coexisting chelators. Lower absorption indicates higher metal chelating activity. In this assay, synthesized compounds interfere with the formation of ferrous and ferrozine complex. These results (Figs. 11, 12, 13, 14, 15) suggested that the compounds 6a, 7a, and 7b exhibited enhanced activity at all concentrations. Compound 8a displayed good activity, i.e., 71.46 and 72.82% at 50 and 75 µg cm−3. Compound 4a showed good activity, i.e., 76.34 and 79.03% at 75 and 100 µg cm−3, respectively. While compounds 3a and 3c exhibited good activity, i.e., 75.26 and 78.76%, respectively, at 100 µg cm−3, compound 6c showed 73.09% activity at 50 µg cm−3. Of the remaining, some compounds were moderately active and some were less active.

Fig. 11
figure 11

Graphical representation for ferrous (Fe2+) ion metal chelating activity of compounds 3a–3c and 4a–4c

Full size image
Fig. 12
figure 12

Graphical representation for ferrous (Fe2+) ion metal chelating activity of compounds 5a–5c and 6a–6c

Full size image
Fig. 13
figure 13

Graphical representation for ferrous (Fe2+) ion metal chelating activity of compounds 7a–7c and 8a–8c

Full size image
Fig. 14
figure 14

Graphical representation for ferrous (Fe2+) ion metal chelating activity of compounds 9a–9c and 10a–10c

Full size image
Fig. 15
figure 15

Graphical representation for ferrous (Fe2+) ion metal chelating activity of compounds 11a–11c

Full size image

Conclusion

The objective of the present study was to synthesize the entitled compounds, i.e., indole derivatives incorporated with triazolopyrimidine and evaluate their antimicrobial and antioxidant activities which were done efficiently and the structures for all of which have been established on the basis of their spectral studies. From the SAR study, we can conclude that the antimicrobial and antioxidant activities may be due to:

  • The presence of chloro and methoxy substitutions; evidently by the presence of which enhanced the activity in contrast to the same devoid of any substitution.

  • The triazolopyrimidine generally augmenting the activity.

  • The indole moiety proving essential for activity.

Furthermore, by the integration of triazolopyrimidine with the parent indole moiety, we have added a new class of compounds to the unit of bioactive molecules.

Experimental

All the reagents were obtained commercially and used by further purification. Melting points were determined by an open capillary method. Purity of the compounds was checked by thin layer chromatography using silica gel-G coated aluminium plates (Merck). The IR spectra were recorded on Thermo Fischer (id S-5) FT-IR spectrometer. The 1H NMR spectra were recorded with a BRUKER NMR (400 or 500 MHz), 13C NMR spectra were recorded by BRUKER AVANCE II (100 MHz) spectrometer and the mass spectral data were recorded by electron impact method on JEOL GCMATE II GC–MS mass spectrometer. Elemental analysis was carried out using Flash EA 1112 series elemental analyzer.

1-Benzyl-5-amino-1H-1,2,3-triazole-4-carboxamide (1) was synthesized by the reported literature method [23]. Ethyl 5-substituted-3-phenylindol-2-carboxylates 2a–2c were prepared as per the reported literature procedure [24].

General procedure for the synthesis of 3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-ols 3a–3c

To a solution of compound 1 (1 mmol) in anhydrous ethyl alcohol ethyl-5-substituted-3-phenylindol-2-carboxylates 2a–2c (2 mmol) was added and refluxed for 40 h. Further the excess of solvent was removed under reduced pressure and the residue was decomposed in crushed ice followed by neutralization with acetic acid to get the product which was collected by filtration, washed with water, dried, and recrystallized from ethanol.

3-Benzyl-5-(5-chloro-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-ol (3a, C25H17ClN6O)

Pale green crystals; 0.36 g (72%); m.p.: 195 °C; R f = 0.72 (benzene/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.86 (s, 1H, indole NH), 7.18-7.80 (m, 13H, Ar–H), 5.64 (s, 2H, CH2), 5.35 (s, 1H, OH) ppm; 13C NMR (100 MHz, CDCl3): δ = 165.13, 159.19, 154.18, 152.15, 139.13, 132.88, 132.44, 132.36, 131.46, 131.12, 130.85, 130.00, 129.49 128.93, 128.55, 126.25, 125.14, 122.72, 121.84, 113.14, 46.55 ppm; IR (thin film): \({\bar{\nu }}\) = 3351 (OH), 3318 (indole NH), 1666 (C=N) cm−1; MS (70 eV): m/z = 452 (M+), 454 ([M + 2]+).

3-Benzyl-5-(5-methyl-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-ol (3b, C26H20N6O)

Pale green crystals; 0.32 g (65%); m.p.: 120 °C; R f = 0.70 (benzene/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.62 (s, 1H, indole NH), 7.15–7.79 (m, 13H, Ar–H), 5.61 (s, 2H, CH2), 5.26 (s, 1H, OH), 2.8 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 164.55, 160.05, 154.25, 152.55, 140.02, 133.01, 132.54, 132.39, 131.52, 131.09, 130.94, 130.09, 129.68 129.03, 128.35, 126.75, 125.38, 122.58, 121.66, 113.16, 46.98, 27.89 ppm; IR (thin film): \({\bar{\nu }}\) = 3345 (OH), 3310 (indole NH), 1658 (C=N) cm−1.

3-Benzyl-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-ol (3c, C26H20N6O2)

Green crystals; 0.33 g (67.7%); m.p.: 177 °C; R f = 0.69 (benzene/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.89 (s, 1H, indole NH), 7.20–7.88 (m, 13H, Ar–H), 5.68 (s, 2H, CH2), 5.40 (s, 1H, OH), 3.82 (s, 3H, OCH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 164.98, 159.99, 154.15, 152.98, 140.42, 133.25, 132.64, 132.40, 131.48, 131.19, 130.86, 130.10, 129.71 129.26, 128.35, 126.58, 125.42, 122.60, 121.70, 113.20, 50.55, 49.42 ppm; IR (thin film): \({\bar{\nu }}\) = 3352 (OH), 3320 (indole NH), 1664 (C=N) cm−1.

General procedure for the synthesis of 3-benzyl-7-chloro-5-(5-substituted-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidines 4a–4c

Compound 3 (1 mmol) in 5 cm3 POCl3 was heated under reflux for 8 h. The reaction mixture was cooled to room temperature and decomposed in crushed ice. The product separated was filtered, washed with water, dried, and recrystallized from benzene.

3-Benzyl-7-chloro-5-(5-chloro-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (4a, C25H16Cl2N6)

Pale yellow crystals; 0.36 g (73%); m.p.: 230 °C; R f = 0.54 (chloroform/ethyl acetate 7:3 v/v);1H NMR (500 MHz, DMSO-d 6 ): δ = 12.04 (s, 1H, indole NH), 7.25–7.70 (m, 13H, Ar–H), 5.68 (s, 2H, CH2) ppm; 13C NMR (100 MHz, CDCl3): δ = 167.25, 160.39, 156.25, 153.18, 140.09, 134.55, 132.46, 130.41, 130.09, 129.70, 129.43, 129.19, 128.85, 128.55, 128.30, 127.18, 126.86, 125.14, 122.12, 113.14, 48.11 ppm; IR (thin film): \({\bar{\nu }}\) = 3319 (indole NH), 1678 (C=N), 761 (C–Cl) cm−1; MS (70 eV): m/z = 470 (M+), 472 ([M + 2]+), 474 ([M + 4]+).

3-Benzyl-7-chloro-5-(5-methyl-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (4b, C26H19ClN6)

Pale yellow crystals; 0.31 g (63%); m.p.: 220 °C; R f = 0.55 (chloroform/ethyl acetate 7:3 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 12.01 (s, 1H, indole NH), 7.25–7.69 (m, 13H, Ar–H), 5.65 (s, 2H, CH2), 2.52 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 167.48, 160.42, 156.30, 153.10, 140.15, 134.68, 132.49, 130.57, 130.21, 129.85, 129.44, 129.21, 128.96, 128.32, 128.29, 127.15, 126.46, 125.11, 122.42, 113.23, 50.56, 29.28 ppm; IR (thin film): \({\bar{\nu }}\) = 3315 (indole NH), 1672 (C=N), 758 (C–Cl) cm−1.

3-Benzyl-7-chloro-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (4c, C26H19ClN6O)

Pale yellow crystals; 0.34 g (69%); m.p.: 196 °C; R f = 0.54 (chloroform/ethyl acetate 7:3 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 12.09 (s, 1H, indole NH), 7.35–7.72 (m, 13H, Ar–H), 5.71 (s, 2H, CH2), 3.53 (s, 3H, OCH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 167.05, 160.15, 156.10, 153.05, 140.02, 134.48, 132.39, 130.38, 130.00, 129.60, 129.28, 129.14, 128.67, 128.35, 128.10, 127.08, 126.14, 125.02, 122.01, 113.41, 51.58, 49.99 ppm; IR (thin film): \({\bar{\nu }}\) = 3322 (indole NH), 1680 (C=N), 765 (C–Cl) cm−1.

General procedure for the synthesis of 3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-7-hydrazinyl-3H-[1,2,3]triazolo[4,5-d]pyrimidines 5a–5c

Compounds 4a–4c (10 mmol) and 5 cm3 of hydrazine hydrate (98%) were refluxed in absolute alcohol for 9 h. The hydrazine derivative separated after cooling was filtered, washed with alcohol, dried, and crystallized using benzene to afford the compounds 5a–5c.

3-Benzyl-5-(5-chloro-3-phenyl-1H-indol-2-yl)-7-hydrazinyl-3H-[1,2,3]triazolo[4,5-d]pyrimidine (5a, C25H19ClN8)

Brown crystals; 0.41 g (82%); m.p.: 170 °C; R f = 0.54 (chloroform/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.95 (s, 1H, indole NH), 8.93 (s, 1H, NH), 7.22–7.47 (m, 13H, Ar–H), 5.49 (s, 2H, CH2), 4.50 (s, 2H, NH2) ppm; 13C NMR (100 MHz, CDCl3): δ = 169.54, 161.22, 158.35, 150.06, 133.08, 133.00, 132.90, 130.68, 129.91, 129.84, 129.24, 128.86, 128.35, 126.69, 126.68, 126.45, 125.92, 120.06, 119.59, 113.33, 49.28 ppm; IR (thin film): \({\bar{\nu }}\) = 3407/3261 (NH/NH2), 3056 (indole NH), 1602 (C=N) cm−1; MS (70 eV): m/z = 466 (M+), 468 ([M + 2]+).

3-Benzyl-5-(5-methyl-3-phenyl-1H-indol-2-yl)-7-hydrazinyl-3H-[1,2,3]triazolo[4,5-d]pyrimidine (5b, C26H22N8)

Pale brown crystals; 0.35 g (71%); m.p.: 214 °C; R f = 0.55 (chloroform/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.92 (s, 1H, indole NH), 8.87 (s, 1H, NH), 7.24–7.45 (m, 13H, Ar–H), 5.45 (s, 2H, CH2), 4.48 (s, 2H, NH2), 2.74 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 169.89, 161.55, 158.25, 150.04, 133.10, 133.02, 132.70, 130.55, 129.84, 129.56, 129.14, 128.75, 128.28, 126.70, 126.48, 126.35, 125.81, 120.02, 119.60, 113.35, 51.52, 29.88 ppm; IR (thin film): \({\bar{\nu }}\) = 3405/3260 (NH/NH2), 3052 (indole NH), 1589 (C=N) cm−1.

3-Benzyl-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-7-hydrazinyl-3H-[1,2,3]triazolo[4,5-d]pyrimidine (5c, C26H22N8O)

Brown crystals; 0.31 g (63%); m.p.: 156 °C; R f = 0.55 (chloroform/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.98 (s, 1H, indole NH), 8.95 (s, 1H, NH), 7.50–7.72 (m, 13H, Ar–H), 5.52 (s, 2H, CH2), 4.51 (s, 2H, NH2), 3.98 (s, 3H, OCH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 170.01, 161.85, 158.55, 150.45, 133.45, 133.34, 132.89, 130.65, 129.98, 129.65, 129.34, 128.91 128.48, 126.82, 126.51, 126.41, 125.95, 120.21, 119.55, 113.48, 51.98, 49.98 ppm; IR (thin film): \({\bar{\nu }}\) = 3410/3264 (NH/NH2), 3059 (indole NH), 1610 (C=N) cm−1.

General procedure for the synthesis of 3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-7-(3,5-dimethyl-1H-pyrazol-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidines 6a–6c, 3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-7-(3,5-diphenyl-1H-pyrazol-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidines 7a–7c, and 1-[3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-3-methyl-1H-pyrazol-5(4H)-ones 8a–8c

A mixture of hydrazine derivatives 5a–5c (1 mmol), acetylacetone (or dibenzoylmethane or ethyl acetoacetate) (1 mmol) in 20 cm3 of dry methanol containing 4–5 drops of conc. HCl was refluxed for 4 h on steam bath. The excess of methanol was removed under vacuum. The reaction mixture was cooled to room temperature. The separated solid was filtered off, washed with little methanol, dried, and recrystallized using ethanol to furnish compounds 6a–6c, 7a–7c, and 8a–8c.

3-Benzyl-5-(5-chloro-3-phenyl-1H-indol-2-yl)-7-(3,5-dimethyl-1H-pyrazol-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (6a, C30H23ClN8)

Grey crystals; 0.33 g (66%); m.p.: 118 °C; R f = 0.62 (toluene/ethyl acetate 6:4 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.50 (s, 1H, indole NH), 7.26–7.75 (m, 13H, Ar–H), 5.93 (s, 1H, pyrazole CH), 5.24 (s, 2H, CH2), 2.40 (s, 3H, CH3), 2.01 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 162.16, 158.81, 152.39, 146.46, 134.41, 134.28, 133.96, 133.81, 133.76, 132.93, 131.30, 130.96, 130.69, 129.47, 128.75, 128.62, 127.93, 126.92, 126.25, 125.27, 123.19, 113.11, 111.41, 60.06, 15.09, 14.19 ppm; IR (thin film): \({\bar{\nu }}\) = 3289 (indole NH), 1603 (C=N) cm−1; MS (70 eV): m/z = 531 (M+), 533 ([M + 2]+).

3-Benzyl-5-(5-methyl-3-phenyl-1H-indol-2-yl)-7-(3,5-dimethyl-1H-pyrazol-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (6b, C31H26N8)

Dark blue crystals; 0.29 g (58%); m.p.: 98–100 °C; R f = 0.62 (toluene/ethyl acetate 6:4 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.49 (s, 1H, indole NH), 7.25-7.73 (m, 13H, Ar–H), 5.90 (s, 1H, pyrazole CH), 5.24 (s, 2H, CH2), 2.62 (s, 3H, CH3), 2.40 (s, 3H, CH3), 2.01 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 162.99, 158.75, 152.30, 146.45, 134.50, 134.41, 133.85, 133.75, 133.68, 132.89, 131.35, 130.85, 130.71, 129.52, 128.68, 128.51, 127.89, 126.85, 126.34, 125.30, 123.51, 113.18, 111.38, 60.15, 29.98, 15.55, 14.86 ppm; IR (thin film): \({\bar{\nu }}\) = 3285 (indole NH), 1598 (C=N) cm−1.

3-Benzyl-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-7-(3,5-dimethyl-1H-pyrazol-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (6c, C31H26N8O)

Dark blue crystals; 0.34 g (69%); m.p.: 135 °C; R f = 0.66 (toluene/ethyl acetate 6:4 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 11.52 (s, 1H, indole NH), 7.25-7.75 (m, 13H, Ar–H), 5.96 (s, 1H, pyrazole CH), 5.25 (s, 2H, CH2), 3.42 (s, 3H, OCH3), 2.43 (s, 3H, CH3), 2.04 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 162.88, 158.77, 152.40, 146.51, 134.45, 134.30, 133.85, 133.99, 133.80, 132.95, 131.42, 131.05, 130.95, 129.51, 128.84, 128.72, 127.95, 126.95, 126.45, 125.44, 123.33, 113.18, 111.84, 60.58, 50.55 15.78, 14.55 ppm; IR (thin film): \({\bar{\nu }}\) = 3291 (indole NH), 1609 (C=N) cm−1.

3-Benzyl-5-(5-chloro-3-phenyl-1H-indol-2-yl)-7-(3,5-diphenyl-1H-pyrazol-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (7a, C40H27ClN8)

Yellow crystals; 0.44 g (88%); m.p.: 154 °C; R f = 0.68 (toluene/ethyl acetate 6:4 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.21 (s, 1H, indole NH), 7.20-7.92 (m, 23H, Ar–H), 6.75 (s, 1H, pyrazole CH), 4.54 (s, 2H, CH2) ppm; 13C NMR (100 MHz, CDCl3): δ = 168.10, 167.87, 153.96, 152.09, 150.52, 142.39, 137.86, 136.09, 135.29, 133.76, 133.29, 132.96, 131.49, 130.95, 130.66, 130.55, 130.11, 129.31, 128.91, 128.82, 128.78, 128.51, 128.10, 127.86, 127.77, 127.50, 127.38, 126.15, 111.44, 105.55, 55.95 ppm; IR (thin film): \({\bar{\nu }}\) = 3061 (indole NH), 1597 (C=N) cm−1; MS (70 eV): m/z = 654 (M+), 656 ([M + 2]+).

3-Benzyl-5-(5-methyl-3-phenyl-1H-indol-2-yl)-7-(3,5-diphenyl-1H-pyrazol-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (7b, C41H30N8)

Yellow crystals; 0.34 g (69%); m.p.: 261 °C; R f = 0.78 (toluene/ethyl acetate 6:4 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.18 (s, 1H, indole NH), 7.20–7.90 (m, 23H, Ar–H), 6.74 (s, 1H, pyrazole CH), 4.52 (s, 2H, CH2), 2.35 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 168.08, 167.84, 153.89, 152.06, 150.48, 142.25, 137.75, 136.05, 135.18, 133.69, 133.19, 132.88, 131.98, 130.88, 130.51, 130.48, 130.05, 129.25, 128.84, 128.78, 128.69, 128.49, 128.05, 127.77, 127.68, 127.41, 127.29, 126.09, 111.35, 105.45, 55.85, 30.05 ppm; IR (thin film): \({\bar{\nu }}\) = 3059 (indole NH), 1595 (C=N) cm−1.

3-Benzyl-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-7-(3,5-diphenyl-1H-pyrazol-1-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidine (7c, C41H30N8O)

White crystals; 0.37 g (74%); m.p.: >300 °C; R f = 0.62 (toluene/ethyl acetate 6:4 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.25 (s, 1H, indole NH), 7.22–7.94 (m, 23H, Ar–H), 6.76 (s, 1H, pyrazole CH), 4.56 (s, 2H, CH2), 3.27 (s, 3H, OCH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 168.25, 167.99, 154.06, 152.51, 150.62, 142.45, 137.91, 136.25, 135.34, 133.95, 133.40, 132.99, 131.65, 130.98, 130.75, 130.60, 130.15, 129.45, 128.89, 128.79, 128.68, 128.60, 128.20, 127.95, 127.85, 127.65, 127.43, 126.25, 111.48, 105.60, 56.10, 50.94 ppm; IR (thin film): \({\bar{\nu }}\) = 3063 (indole NH), 1602 (C=N) cm−1.

1-[3-Benzyl-5-(5-chloro-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-3-methyl-1H-pyrazol-5(4H)-one (8a, C29H21ClN8O)

Purple crystals; 0.37 g (75%); m.p.: 100 °C; R f = 0.62 (n-hexane/ethyl acetate 8:2 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.91 (s, 1H, indole NH), 7.21–7.51 (m, 13H, Ar–H), 5.36 (s, 2H, CH2), 4.10 (s, 2H, pyrazole CH2), 3.10 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 165.73, 162.72, 159.19, 154.10, 152.75, 139.41, 134.32, 132.46, 130.47, 130.09, 129.70, 129.43, 129.19, 128.85, 128.55, 128.38, 128.17, 127.78, 126.25, 125.14, 122.72, 116.10, 46.55, 40.09, 15.70 ppm; IR (thin film): \({\bar{\nu }}\) = 3233 (indole NH), 1732 (C=O), 1660 (C=N) cm−1; MS (70 eV): m/z = 532 (M+), 534 ([M + 2]+).

1-[3-Benzyl-5-(5-methyl-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-3-methyl-1H-pyrazol-5(4H)-one (8b, C30H24N8O)

Blue crystals; 0.34 g (68%); m.p.: 274 °C; R f = 0.55 (n-hexane/ethyl acetate 8:2 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.88 (s, 1H, indole NH), 7.19–7.50 (m, 13H, Ar–H), 5.33 (s, 2H, CH2), 3.97 (s, 2H, pyrazole CH2), 3.08 (s, 3H, CH3), 2.56 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 165.65, 162.61, 159.15, 154.05, 152.68, 139.35, 134.28, 132.35, 130.39, 130.01, 129.65, 129.35, 129.11, 128.77, 128.50, 128.26, 128.06, 127.66, 126.15, 125.11, 122.68, 116.01, 46.59, 40.95, 30.11, 15.68 ppm; IR (thin film): \({\bar{\nu }}\) = 3231 (indole NH), 1730 (C=O), 1658 (C=N) cm−1.

1-[3-Benzyl-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-3-methyl-1H-pyrazol-5(4H)-one (8c, C30H24N8O2)

Blue crystals; 0.34 g (69%); m.p.: 140 °C; R f = 0.60 (n-hexane/ethyl acetate 8:2 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.97 (s, 1H, indole NH), 7.28-7.56 (m, 13H, Ar–H), 5.39 (s, 2H, CH2), 4.12 (s, 2H, pyrazole CH2), 3.62 (s, 3H, OCH3), 3.12 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 165.95, 162.88, 159.48, 154.32, 152.98, 139.65, 134.41, 132.64, 130.71, 130.25, 129.84, 129.51, 129.28, 128.92, 128.63, 128.55, 128.27, 127.95, 126.51, 125.29, 122.84, 116.22, 51.05, 47.05, 40.98, 15.95 ppm; IR (thin film): \({\bar{\nu }}\) = 3235 (indole NH), 1736 (C=O), 1664 (C=N) cm−1.

General procedure for the synthesis of 3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-7-phenyl-3H-[1,2,3]triazolo[4,5-e][1,2,4] triazolo[4,5-c]pyrimidines 9a–9c

A mixture of compounds 5a–5c (2 mmol) and 20 cm3 of benzoyl chloride was heated under reflux for 8 h. Excess of acid chloride was removed under reduced pressure. After cooling the product separated was filtered, washed with hot petroleum ether (40–60 °C), dried, and recrystallized from acetic acid to obtain 9a–9c.

3-Benzyl-5-(5-chloro-3-phenyl-1H-indol-2-yl)-7-phenyl-3H-[1,2,3]triazolo[4,5-e][1,2,4]triazolo[4,5-c]pyrimidine (9a, C32H21ClN8)

Dark green crystals; 0.34 g (68%); m.p.: 120 °C; R f = 0.82 (benzene/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 12.99 (s, 1H, indole NH), 7.49–7.97 (m, 18H, Ar–H), 5.24 (s, 2H, CH2) ppm; 13C NMR (100 MHz, CDCl3): δ = 167.05, 154.02, 152.5, 152.3, 141.12, 140.03, 133.91, 133.25, 132.44, 132.00, 132.36, 131.34, 131.12, 130.94, 130.20, 129.95, 129.83, 128.98, 128.82, 128.61, 128.35, 128.19, 127.96, 126.65, 114.00, 54.02 ppm; IR (thin film): \({\bar{\nu }}\) = 3071 (indole NH), 1601 (C=N) cm−1; MS (70 eV): m/z = 553 (M+), 555 ([M + 2]+).

3-Benzyl-5-(5-methyl-3-phenyl-1H-indol-2-yl)-7-phenyl-3H- [1,2,3]triazolo[4,5-e][1,2,4]triazolo[4,5-c]pyrimidine (9b, C33H24N8)

Green crystals; 0.26 g (53%); m.p.: 220 °C; R f = 0.82 (benzene/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 12.91 (s, 1H, indole NH), 7.49–7.95 (m, 18H, Ar–H), 5.23 (s, 2H, CH2), 2.41 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 167.02, 154.00, 152.48, 152.28, 141.09, 140.00, 133.85, 133.04, 132.25, 132.11, 132.28, 131.19, 131.02, 130.88, 130.15, 129.86, 129.77, 128.78, 128.78, 128.55, 128.28, 128.05, 127.88, 126.59, 114.05, 54.18, 29.55 ppm; IR (thin film): \({\bar{\nu }}\) = 3067 (indole NH), 1595 (C=N) cm−1.

3-Benzyl-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-7-phenyl-3H-[1,2,3]triazolo[4,5-e][1,2,4]triazolo[4,5-c]pyrimidine (9c, C33H24N8O)

Pale green crystals; 0.38 g (77%); m.p.: 130 °C; R f = 0.87 (benzene/ethyl acetate 8:2 v/v); 1H NMR (500 MHz, DMSO-d 6 ): δ = 13.01 (s, 1H, indole NH), 7.52–7.99 (m, 18H, Ar–H), 5.26 (s, 2H, CH2), 3.02 (s, 3H, OCH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 167.35, 154.22, 152.45, 152.13, 141.25, 140.43, 134.01, 133.55, 132.61, 132.19, 132.01, 131.54, 131.22, 130.99, 130.40, 129.88, 129.79, 128.99, 128.90, 128.75, 128.41, 128.25, 127.98, 126.81, 114.24, 54.41, 51.56 ppm; IR (thin film): \({\bar{\nu }}\) = 3077 (indole NH), 1605 (C=N) cm−1.

General procedure for the synthesis of 3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-7-methyl-3H-[1,2,3]triazolo[4,5-e][1,2,4] triazolo[4,5-c]pyrimidines 10a–10c

A mixture of hydrazines 5a–5c (10 mmol) and 30 cm3 of acetic acid was heated under reflux for 10 h, cooled to room temperature, and poured into crushed ice. The product obtained was filtered, washed with water, dried, and recrystallized from ethanol to yield compounds 10a–10c.

3-Benzyl-5-(5-chloro-3-phenyl-1H-indol-2-yl)-7-methyl-3H-[1,2,3]triazolo[4,5-e][1,2,4]triazolo[4,5-c]pyrimidine (10a, C27H19ClN8)

Black crystals; 0.40 g (80%); m.p.: 164 °C; R f = 0.79 (benzene/ethyl acetate 6:4 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 10.19 (s, 1H, indole NH), 7.35–7.53 (m, 13H, Ar–H), 5.36 (s, 2H, CH2), 1.25 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 162.88, 154.76, 152.36, 148.93, 139.49, 133,76, 133.25, 132.88, 132.52, 130.85, 130.00, 129.62, 129.13, 128.69, 128.19, 127.34, 126.48, 126.40, 123.96, 123.19, 113.96, 52.00, 12.96 ppm; IR (thin film): \({\bar{\nu }}\) = 3213 (indole NH), 1635 (C=N) cm−1; MS (70 eV): m/z = 490 (M+), 492 ([M + 2]+).

3-Benzyl-5-(5-methyl-3-phenyl-1H-indol-2-yl)-7-methyl-3H-[1,2,3]triazolo[4,5-e][1,2,4]triazolo[4,5-c]pyrimidine (10b, C28H22N8)

Brown crystals; 0.29 g (59%); m.p.: 86 °C; R f = 0.83 (benzene/ethyl acetate 6:4 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 10.15 (s, 1H, indole NH), 7.32-7.53 (m, 13H, Ar–H), 5.34 (s, 2H, CH2), 2.23 (s, 3H, CH3), 1.23 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 162.75, 154.69, 152.28, 148.85, 139.35, 133,69, 133.15, 132.75, 132.48, 130.77, 130.05, 129.55, 129.11, 128.55, 128.15, 127.28, 126.35, 126.20, 123.85, 123.09, 113.85, 52.55, 28.98, 12.89 ppm; IR (thin film): \({\bar{\nu }}\) = 3210 (indole NH), 1631 (C=N) cm−1.

3-Benzyl-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-7-methyl-3H-[1,2,3] triazolo[4,5-e][1,2,4]triazolo[4,5-c]pyrimidine (10c, C28H22N8O)

Pale brown crystals; 0.38 g (76%); m.p.: 120 °C; R f = 0.66 (benzene/ethyl acetate 6:4 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 10.23 (s, 1H, indole NH), 7.38–7.54 (m, 13H, Ar–H), 5.39 (s, 2H, CH2), 3.41 (s, 3H, OCH3), 1.26 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 162.92, 154.85, 152.42, 148.99, 139.55, 133,84, 133.44, 132.91, 132.65, 130.92, 130.18, 129.65, 129.25, 128.71, 128.21, 127.42, 126.55, 126.52, 123.89, 123.25, 114.00, 52.58, 51.55, 12.88 ppm; IR (thin film): \({\bar{\nu }}\) = 3215 (indole NH), 1639 (C=N) cm−1.

General procedure for the synthesis of 2-(3-benzyl-5-(5-substituted-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl)-2,3-dihydrophthalazine-1,4-diones 11a–11c

A mixture of compounds 5a–5c (1 mmol) and phthalic anhydride (1 mmol) was refluxed in dimethyl formamide for 12 h. After completion of the reaction, the reaction mixture was cooled to room temperature and poured into crushed ice. The solid obtained was filtered, washed with water, dried, and recrystallized from methanol to yield compounds 11a–11c.

2-[3-Benzyl-5-(5-chloro-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-2,3-dihydrophthalazine-1,4-dione (11a, C33H21ClN8O2)

Pale yellow crystals; 0.42 g (85%); m.p.: 154 °C; R f = 0.66 (benzene/ethyl acetate 6:4 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.50 (s, 1H, indole NH), 10.88 (NH), 7.24–7.48 (m, 17H, Ar–H), 4.88 (s, 2H, CH2) ppm; 13C NMR (100 MHz, CDCl3): δ = 171.67, 167.85, 164.44, 160.19, 154.35, 154.10, 140.03, 135.12, 134.24, 133.91, 132.88, 131.46, 130.94, 130.00, 129.62, 128.98, 128.61, 128.19, 127.34, 126.65, 124.44, 123.96, 123.49, 123.36, 113.88, 48.10 ppm; IR (thin film): \({\bar{\nu }}\) = 3363 (NH), 3291 (indole NH), 1742, 1702 (C=O), 1660 (C=N) cm−1; MS (70 eV): m/z = 597 (M+), 599 ([M + 2]+).

2-[3-Benzyl-5-(5-methyl-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-2,3-dihydrophthalazine-1,4-dione (11b, C34H24N8O2)

Yellow crystals; 0.39 g (79%); m.p.: 76 °C; R f = 0.74 (benzene/ethyl acetate 8:2 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.47 (s, 1H, indole NH), 10.85 (NH), 7.23–7.48 (m, 17H, Ar–H), 4.87 (s, 2H, CH2), 2.51 (s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 171.55, 167.78, 164.35, 160.10, 154.28, 154.05, 140.00, 135.03, 134.12, 133.85, 132.77, 131.41, 130.86, 130.05, 129.59, 128.88, 128.58, 128.09, 127.24, 126.53, 124.39, 123.88, 123.35, 123.28, 113.79, 48.56, 29.96 ppm; IR (thin film): \({\bar{\nu }}\) = 3361 (NH), 3289 (indole NH), 1741, 1698 (C=O), 1657 (C=N) cm−1.

2-[3-Benzyl-5-(5-methoxy-3-phenyl-1H-indol-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl]-2,3-dihydrophthalazine-1,4-dione (11c, C34H24N8O3)

Pale yellow crystals; 0.36 g (73%); m.p.: 282 °C; R f = 0.77 (benzene/ethyl acetate 8:2 v/v); 1H NMR (400 MHz, DMSO-d 6 ): δ = 11.52 (s, 1H, indole NH), 10.92 (NH), 7.26–7.50 (m, 17H, Ar–H), 4.91 (s, 2H, CH2), 3.63 (s, 3H, OCH3) ppm; 13C NMR (100 MHz, CDCl3): δ = 171.85, 167.91, 164.64, 160.32, 154.55, 154.25, 140.23, 135.32, 134.32, 133.78, 132.91, 131.66, 130.99, 130.21, 129.75, 128.89, 128.71, 128.25, 127.44, 126.74, 124.51, 123.85, 123.51, 123.41, 113.91, 51.23, 48.15 ppm; IR (thin film): \({\bar{\nu }}\) = 3366 (NH), 3294 (indole NH), 1743, 1705 (C=O), 1662 (C=N) cm−1.

In vitro antimicrobial activities

The in vitro biological screening of the synthesized compounds 3–11 was carried out against bacteria species namely, S. aureus, E. coli, and P. aeruginosa and fungal species namely, A. niger, A. flavus, and A. oryzae by Cup-plate method [25]. An aliquot of 0.1 cm3 of each bacterial strain was spread on nutrient agar, while 0.1 cm3 of the fungal spore suspension was spread on potatoes dextrose agar. An agar well diffusion test was performed in each case. In these tests 6 mm wells were produced using a sterile cork borer and each well was then inoculated with 1000, 500, 250, and 125 µg cm−3 of each test compound in DMF. The plates were incubated at 37 °C for 24 and 72 h in case of antibacterial and antifungal activity, respectively. The zone of inhibition around the well was determined (in mm). Streptomycin and fluconazole were used as standards for antibacterial and antifungal activities, respectively.

Antioxidant activity

1,1-Diphenyl-2-picryl hydrazil (DPPH) Radical Scavenging Activity (RSA)

The RSA of the test compounds in methanolic solution at concentrations 25, 50, 75, and 100 µg cm−3 containing freshly prepared DPPH solution (0.004% w/v) was carried out and compared with those of standards BHA, TBHQ, and AA using reported method [26]. All the test analyses were performed on three replicates were averaged. The results in percentage are expressed as the ratio of absorption decrease in the presence of test compounds and absorption of DPPH solution in the absence of test compounds at 517 nm on ELICO SL 171 minispect spectrometer. The percentage scavenging activity of the DPPH free radical was determined using the following equation:

$$\% {\text{DPPH RSA}} = \frac{{{\text{Absorbance of control}} - {\text{Absorbance of sample}}}}{\text{Absorbance of control}} \times 100.$$

Ferric ion (Fe3+) reducing antioxidant power (FRAP)

The reducing power of the synthesized compounds was determined according to the literature method [27]. Different concentrations of samples (25, 50, 75, and 100 µg cm−3) in 1 cm3 DMSO were mixed with 2.5 cm3 of phosphate buffer (0.2 M, pH 6.6) and 2.5 cm3 of potassium ferricyanide (1%). The mixture was incubated at 50 °C for 20 min after which 2.5 cm3 of trichloroacetic acid (10%) was added to the mixture and centrifuged for 10 min at 1000 rpm. The upper layer solution (2.5 cm3) was mixed with 2.5 cm3 of distilled water and 0.5 cm3 of ferric chloride (0.1%). Then the absorbance at 700 nm was measured in spectrophotometer. Higher absorbance of the reaction mixture indicated greater reducing power.

Ferrous (Fe2+) ion metal chelating activity

The chelating activity of the ferrous ions by the synthesized compounds and standards was estimated by following reported method [28]. The test samples (25, 50, 75, and 100 µg cm−3) in 0.4 cm3 of ethanolic solution were added to a solution of ferrous chloride (0.05 cm3, 2 mM). The reaction was initiated by the addition of 0.2 cm3 ferrozine (5 mM) and the total volume was adjusted to 4 cm3 with ethanol. Ferrozine reacted with the divalent iron to form stable magenta complex species that were very soluble in water. The mixture was shaken vigorously and kept at room temperature for 10 min. Then the absorbance of the solution was measured spectrophotometrically at 562 nm. All test analyses were run in triplicate and averaged. The percentage of the inhibition of the ferrozine Fe2+ complex formations was calculated using the formula:

$$\% {\text{Ferrous ion chelation}} = \frac{{{\text{Absorbance of control}} - {\text{Absorbance of sample}}}}{\text{Absorbance of control}} \times 100.$$