Introduction

The spirooxindole framework is an important structural motif found in many natural products and bioactive compounds [1–5]. These spirooxindole-based molecules have shown to possess varieties of important biological activities, such as antimicrobial [6–8], anti-inflammatory [9], antimalarial [10], antimycobacterial [11], antitubercular [12], antitumor and anticancer [13, 14], and MDM2 inhibitor activity [15–17]. In addition, they are widely used as building blocks for the synthesis of bioactive natural products [18–20].

Due to their prominent biological and pharmacological activities, we became interested in the synthesis of a variety of spirooxindole derivatives using multicomponent reactions. Recently, we have developed simple and facile synthetic methods for the preparation of spirooxindole derivatives bearing hexahydroquinolines [21], dihydroquinazolinones [22], and 4-chromenes [23]. As a part of ongoing study on other spirooxindole derivatives, herein we examined four-component reactions of \(\beta \)-ketoesters, isatins, phenylhydrazines, and malononitrile to afford spirooxindole derivatives bearing pyrano[2,3-c]pyrazoles. It should be noted that a number of synthetic approaches using the three-component reaction of \(\beta \)-ketoesters with 3-methyl-2-pyrazolin-5-ones and malononitrile has been reported using \(\mathrm{InCl}_{3}\) [24], ZnS [25], \([\hbox {Ch}\hbox {-}\hbox {OSO}_{3}\hbox {H}]_{3}\hbox {W}_{12}\mathrm{PO}_{40}\) [26], CAN/sonication [13], NaCl/sonication [27], \(\hbox {I}_{2}\) [28], L-proline [29–35], 4-DMAP [36], and electrolysis [37] (Scheme 1). Also, several synthetic approaches to spirooxindole derivatives bearing pyrano[2,3-c]pyrazoles based on four-component reactions of \(\beta \)-ketoesters, isatins, phenylhydrazines, and malononitrile have been reported using \(\beta \)-cyclodextrin [38], chitosan/ionic liquid [39], piperidine [40, 41], L-proline [42], and \(\mathrm{ZrO}_{2}\) [43] conditions. Still, there is a demand for more efficient and environmentally benign synthetic approaches to spirooxindole derivatives bearing pyrano[2,3-c]pyrazoles.

Scheme 1
scheme 1

Reported three-component synthesis of spirooxindole derivative

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Scheme 2
scheme 2

\(\hbox {CeO}_2\) nanoparticle-catalyzed four-component reactions for the synthesis of 5

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Green and sustainable chemical processes with reduction or even elimination of the use and production of hazardous materials are in high demand. Consequently, the use of non-toxic catalysts and pollution abatement solvents has become a prime choice for the researchers in both academia and industry. Recently, cerium oxide nanoparticles have emerged as environmentally benign and economical heterogeneous catalysts [44–49]. They have exhibited various advantages, such as sustainability in water, low corrosiveness and toxicity, high catalytic reactivity, recoverability and reusability, and ease of handling [50, 51]. Because of these advantages, \(\mathrm{CeO}_{2}\)-NPs, including core-metal/shell \(\mathrm{CeO}_{2}\) nanoparticles, have been extensively used as efficient and useful catalysts in various organic transformations [52–57]. Moreover, commercially available \(\mathrm{CeO}_{2}\)-NPs are also being used in fluorescent applications [58], fuel cells [59], sunscreens [60], as antioxidants in cell model culture [61] and as gas sensors [62]. To the best of our knowledge, \(\mathrm{CeO}_{2}\) nanoparticle-catalyzed reactions of \(\beta \)-ketoesters with phenylhydrazines, malononitrile, and isatins for the construction of spirooxindoles have not been reported so far.

Herein, we describe a one-pot synthesis of biologically interesting spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole] derivatives using \(\mathrm{CeO}_{2}\) nanoparticle-catalyzed four-component reaction of \(\beta \)-ketoesters, phenylhydrazines, malononitrile, and isatins in water (Scheme 2). In addition, we report on the antibacterial and antioxidant activities of the synthesized spirooxindole derivatives.

Results and discussion

The four-component reaction of methyl acetoacetate (1a), phenylhydrazine (2a), malononitrile (3), and isatin (4a) was first examined in the presence of several catalysts and solvents (Table 1). Initially, reaction in the absence of catalyst in water at \(90\,^{\circ }\mathrm{C}\) afforded the product 5a in only 19 % yield (entry 1). Adding 30 mol% of ceric ammonium nitrate (CAN) and \(\mathrm{CeCl}_{3}\) at \(90\,^{\circ }\mathrm{C}\) allowed to increase the yield of 5a to 47 and 35 %, respectively (entries 2 and 3). Further reactions were attempted with \(\mathrm{CeO}_{2}\)-NPs in several solvents. The best yield (93 %) was obtained in the presence of 30 mol% of \(\mathrm{CeO}_{2}\)-NPs in water at \(90\,^{\circ }\mathrm{C}\) (entry 5). Moreover, in polar solvents, such as ethanol and acetonitrile, 5a was produced in a 65 and 81 % yield, respectively (entries 6 and 7) and in a non-polar solvent toluene, 5a was obtained only in trace amounts (entry 8). The decrease or increase in loading of the catalyst (\(\mathrm{CeO}_{2}\)-NPs) did not improve the yield of 5a (entries 9, 10, and 11). Using 20 mol % of Lewis acids such as \(\hbox {FeCl}_{3}\), \(\hbox {In}(\hbox {OTf})_{3}\) and \(\hbox {Cu}(\hbox {OTf})_{2}\) also gave the desired product in diminished yields (entries 12, 13, and 14). The identity of 5a was confirmed by analysis of its spectroscopic data in comparison to reported values [25]. The \({}^{1}\mathrm{H}\) NMR of 5a shows a methyl peak (\(\delta = 1.54\hbox { ppm}\), singlet) and an amide proton (\(\delta = 10.74\hbox { ppm}\), singlet). The \({}^{13}\hbox {C}\) NMR exhibits a characteristic quaternary carbon peak at 47.7 ppm and an amide carbon peak at 177.4 ppm.

Table 1 Optimization of reaction conditions for the synthesis of \({\mathbf{5a}}^{\mathrm{a}}\)
figure a
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Under the optimized reaction condition, the generality of this multicomponent reaction was further explored by employing various \(\beta \)-ketoesters 1a–1d, phenylhydrazines 2a–2e, and isatins 4a–4k (Table 2). Reactions of methyl 3-oxobutanoate (1a) with 2a, 3, and isatin 4b or 4c bearing electron-donating groups provided products 5b and 5c in an 84 and 86 % yield, respectively, whereas those with isatin 4d, 4e, or 4f bearing electron-withdrawing groups afforded the desired products 5d–5f, in a 91, 90, and an 87 % yield, respectively. Reactions of methyl and acetyl substituted isatins 4g and 4h provided products 5g–5h in an 80 and 89 % yield, respectively. With other \(\beta \)-ketoesters of methyl 3-oxohexanoate (1b) or methyl 4-methyl-3-oxopentanoate (1c), the desired products 5i–5q were produced in 86–94 % yields. Next, treatment of methyl 3-oxo-3-phenylpropanoate (1d) with phenylhydrazine (2a), malononitrile (3), and isatins 4a, 4g, or 4h afforded the desired products 5r–5t in 75–84 % yields.

Table 2 Additional reactions for the synthesis of a variety of spirooxindole derivatives bearing pyrano[2,3 c]pyrazoles
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In addition, further reactions of substituted phenylhydrazines 2b–2e bearing electron-donating or -withdrawing substituents on various positions of benzene ring were successful. For example, treatment of 1a with 4-methyl phenylhydrazine (2b), 3, and 4a provided the desired product 5u in an 84 % yield, whereas that of 1c with 2-ethyl phenylhydrazine (2c), 3, and 4h afforded the product 5v in a 79 % yield. Moreover, treatment of 1c or 1a with 4-chloro phenylhydrazine (2d) or 2-chloro phenylhydrazine (2e), 3, and 4a gave products 5w and 5x in 73 and 74 % yields. However, when malononitrile was replaced by cyanoesters like methyl cyanoacetate or ethyl cyanoacetate, no desired products were obtained, instead intractable mixtures were produced. Our procedure provides a rapid synthetic route to a variety of highly functionalized spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole] derivatives in good yield. Moreover, most of the synthesized compounds of 5f, 5i, 5k–5q, and 5t–5w are novel and reported for the first time.

The formation of 5a can be explained by the mechanism as shown in Scheme 3 [40, 41]. In the presence of \(\mathrm{CeO}_{2}\)-NPs, the intermediate 7 is first formed by condensation of methyl acetoacetate (1a) with phenylhydrazine (2a). The Knoevenagel condensation of 3a, derived from 3 and 4a, provides the intermediate 8, which is reacted with 7a to furnish 9 through Michael reaction. Tautomerism of 9 followed by intramolecular cyclization gives intermediate 10, which undergoes further isomerization to furnish the final product 5a.

Scheme 3
scheme 3

Proposed mechanism for the formation of 5a

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In vitro antioxidant activity

Furthermore, the synthesized spirooxindoles were screened for their antioxidant activity by using ferric reducing/antioxidant tests [63]. The FRAP assay measures the ability of a compound to reduce the ferric 2,4,6-tripyridyl-s-triazine complex to the colored ferrous complex with development of intense blue color at the maximum wavelength of 593 nm. FRAP values were obtained by comparing the absorbance change in test reaction mixtures with those containing ferrous ions of known concentration. The results of the antioxidant test are expressed as Trolox equivalent antioxidant capacity (TEAC) values as shown in Fig. 1. A higher value of TEAC suggests a higher antioxidant capacity. The majority of the tested compounds in the series revealed moderate interactions with the FRAP reagent. Compounds 5f, 5h, 5k, and 5t exhibited superior activity as compared to other synthesized compounds.

The antioxidant activity of organic compounds seems to be related to the presence of hydroxyl groups, double bond conjugation, and resonance effects [64]. The synthesized compounds containing both electron-withdrawing and -donating groups showed moderate activity. Compounds containing electron-withdrawing or -donating group on ortho or para positions to the spirooxindolic NH or acetyl group on spirooxindolic nitrogen atom showed higher activity than other compounds. In FRAP tests, the antioxidant activities of the compounds are well correlated with their \(\mathrm{EC}_{50}\) (half maximal effective concentration) values, as shown in Fig. 2.

Fig. 1
figure 1

Antioxidant activity of the synthesized spirooxindoles expressed in TEAC values

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Fig. 2
figure 2

\(\hbox {EC}_{50}\) (\(\upmu \hbox {g/mL}\)) of the synthesized spirooxindoles. Abbreviation: T Trolox, EC effective concentration

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Table 3 Antimicrobial activity of synthesized spirooxindoles against several standard strains
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Antibacterial activity

The antibacterial activity of the synthesized spirooxindoles was tested against two gram-negative Escherichia coli (KCTC-1924) and Pseudomonas aeruginosa (KCTC-2004) and two gram-positive Staphylococcus aureus (KCTC-1916) and Bacillus cereus (KCTC-1012) bacteria, respectively, by using a modified Kirby-Bauer disk diffusion method [65]. The inhibition zone against the growth of the verified bacteria for the compounds is reported in Table 3. Aliquots of bacterial suspension (\(100\,\upmu \hbox {L}\)) were spread on \(\hbox {Difco}^{\mathrm{TM}}\) nutrient broth containing the test microorganism with an optical density of 0.7 at 595 nm. From the results, synthesized compounds showed antibacterial activity toward the investigated bacterial strains. In particular, compounds 5m and 5t exhibited excellent activity toward the gram-negative bacteria compared to standard ciprofloxacin. Compounds 5m, 5t, and 5v displayed moderate levels of antimicrobial activity toward the gram- positive bacteria. Compounds containing electron-withdrawing group on spirooxindolic NH or its para position showed higher activity than other compounds.

Conclusions

A green and efficient protocol for the construction of spirooxindole derivatives bearing pyrano[2,3-c]pyrazole] by a multicomponent coupling of \(\beta \)-ketoesters, phenylhydrazines, malononitrile, and isatins was developed. This method offers several advantages such as mild reaction conditions, ease of handling, high yields, and the use of an effective green catalyst. The synthesized spirooxindole derivatives show potent antioxidant and antibacterial activities.

Experimental

All experiments were conducted under a nitrogen atmosphere. Merck precoated silica gel plates (Art. 5554) with a fluorescent indicator were used for analytical TLC. Flash column chromatography was performed using silica gel 9385 (Merck). The melting points were determined using micro-cover glasses on a Fisher-Johns apparatus and were uncorrected. The \({}^{1}\hbox {H}\) NMR spectra were recorded on a Varian-VNS (300 MHz), DPX (300 MHz), and VNS (600 MHz) spectrometer in DMSO-\(d_{6}\) setting the solvent chemical shift at 2.50 ppm. The \({}^{13}\hbox {C}\) NMR spectra were recorded on a Varian-VNS (75 MHz), DPX (75 MHz), and VNS (150 MHz) spectrometer in DMSO-\(d_{6}\) setting the solvent chemical shift at 39.5 ppm. Chemical shifts (\(\delta \)) are expressed in units of ppm and J values are given in Hz. Multiplicities are abbreviated as follows: \(\hbox {s} = \hbox {singlet}\), \(\hbox {d} = \hbox {doublet}\), \(\hbox {t} = \hbox {triplet}\), \(\hbox {q} = \hbox {quartet}\), \(\hbox {br s} = \hbox {broad}\) singlet, \(\hbox {dd} = \hbox {doublet}\) of doublets, \(\hbox {tt} = \hbox {triplet}\) of triplet, and \(\hbox {m} = \hbox {multiplet}\). The IR spectra were recorded on PerkinElmer FT-IR spectrometer Spectrum \(\hbox {Two}^{\mathrm{TM}}\). High-resolution mass spectrometry (HRMS) was obtained with a JEOL JMS-700 spectrometer (EI) at the Korea Basic Science Institute.

General procedure for the synthesis of spirooxindole derivatives (5a–5x)

A mixture of \(\beta \)-ketoester 1 (1 mmol), phenylhydrazine 2 (1 mmol), malononitrile 3 (1 mmol), isatin 4 (1 mmol), and \(\mathrm{CeO}_{2}\)-NPs (30 mol%) in water (5 mL) was stirred at \(90\,^{\circ }\mathrm{C}\) for the time mentioned, until the completion of reaction as indicated by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature. The precipitated product was then filtered and dissolved in EtOAc. The solution was then dried over \(\mathrm{MgSO}_{4}\) and filtered. After evaporating solvent, the residue was recrystallized from EtOAc to provide pure product.

6\(^\prime \)-Amino-3\(^\prime \)-methyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5a)

Compound 5a (342 mg, 93 %) was obtained as a white solid: mp 237–\(239\,^{\circ }\mathrm{C}\); \({}^{1}\hbox {H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.74 (1H, s), 7.78 (2H, d, \(J= 7.8\hbox { Hz}\)), 7.56–7.49 (4H, m), 7.37–7.26 (2H, m), 7.17 (1H, d, \(J = 7.2\hbox { Hz}\)), 7.02 (1H, t, \(J = 7.5\hbox { Hz}\)), 6.95 (1H, d, \(J = 7.5\hbox { Hz}\)), 1.54 (3H, s); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.4, 161.0, 144.9, 143.8, 141.6, 137.2, 132.1, 129.4, 129.2, 126.5, 124.8, 122.7, 120.1, 117.8, 109.8, 96.3, 56.2, 47.7, 11.6; IR (ATR) 3451, 3254, 3080, 2247, 1891, 1739, 1562, 1428, 1348, 1236, 1099, 988, \(931\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{21}\hbox {H}_{15}\hbox {N}_{5}\hbox {O}_{2}\): 369.1226. Found: 369.1229.

6\(^\prime \)-Amino-3\(^\prime \),5-dimethyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5b)

Compound 5b (320 mg, 84 %) was obtained as a white solid: mp 288–\(289\,^{\circ }\mathrm{C}\); \({}^{1}\hbox {H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.63 (1H, s), 7.79 (2H, d, \(J = 8.1\hbox { Hz}\)), 7.52 (4H, t, \(J= 8.1\hbox { Hz}\)), 7.35 (1H, t, \(J= 7.5\hbox { Hz}\)), 7.08 (1H, d, \(J= 7.5\hbox { Hz}\)), 7.0 (1H, s), 6.83 (1H, d, \(J= 7.8\hbox { Hz}\)), 2.23 (3H, s), 1.56 (3H, s); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.4, 161.0, 144.9, 144.0, 139.1, 137.3, 132.3, 131.6, 129.5, 129.4, 126.5, 125.3, 120.1, 118.0, 109.6, 96.5, 56.3, 47.8, 20.6, 11.7; IR (ATR) 3420, 3254, 3089, 2246, 1875, 1732, 1673, 1554, 1427, 1347, 1237, 1158, \(989\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{22}\hbox {H}_{17}\hbox {N}_{5}\hbox {O}_{2}\): 383.1382. Found: 383.1384.

6\(^\prime \)-Amino-5-methoxy-3\(^\prime \)-methyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5c)

Compound 5c (342 mg, 86 %) was obtained as a white solid: mp 213–\(215\,^{\circ }\mathrm{C}\); \({}^{1}\hbox {H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.54 (1H, s), 7.79 (2H, d, \(J= 8.1\hbox { Hz}\)), 7.54–7.49 (4H, m), 7.34 (1H, t, \(J = 7.8\hbox { Hz}\)), 6.8 (3H, d, \(J= 8.1\hbox { Hz}\)), 3.68 (3H, s), 1.56 (3H, s); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.5, 161.0, 155.7, 145.0, 144.0, 137.3, 134.8, 133.4, 129.5, 126.5, 120.1, 118.0, 114.4, 111.3, 110.4, 96.4, 56.3, 55.5, 48.3, 11.7; IR (ATR) 3318, 3184, 3062, 2203, 1696, 1486, 1392, 1293, 1199, 1029, \(962\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{22}\hbox {H}_{17}\hbox {N}_{5}\hbox {O}_{3}\): 399.1331. Found: 399.1328.

6\(^\prime \)-Amino-4-bromo-3\(^\prime \)-methyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5d)

Compound 5d (405 mg, 91 %) was obtained as a white solid: mp 268–\(270\,^{\circ }\mathrm{C}\); \({}^{1}\hbox {H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 11.02 (1H, s), 7.78 (2H, d, \(J = 9.0\hbox { Hz}\)), 7.65 (2H, s), 7.51 (2H, t, \(J= 7.8\hbox { Hz}\)), 7.35 (1H, t, \(J= 7.2\hbox { Hz}\)), 7.25 (1H, t, \(J= 7.8\hbox { Hz}\)), 7.18 (1H, d, \(J= 7.8\hbox { Hz}\)), 6.98 (1H, d, \(J= 7.8\hbox { Hz}\)), 1.60 (3H, s); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 176.7, 161.7, 145.5, 143.7, 143.6, 137.2, 131.3, 129.5, 128.6, 126.7, 126.1, 120.1, 119.5, 117.7, 109.5, 94.1, 53.9, 49.4, \(11.7\hbox { cm}^{-1}\); IR (ATR) 3320, 3193, 2198, 1723, 1655, 1583, 1527, 1447, 1394, 1221, 1127, \(1037\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{21}\mathrm{H}_{14}\hbox {BrN}_{5}\hbox {O}_{2}\): 447.0331. Found: 447.0328.

6\(^\prime \)-Amino-5-chloro-3\(^\prime \)-methyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5e)

Compound 5e (362 mg, 90 %) was obtained as a white solid: mp 229–\(231\,^{\circ }\mathrm{C}\); \({}^{1}\hbox {H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.89 (1H, s), 7.79 (2H, d, \(J= 7.2\hbox { Hz}\)), 7.63 (2H, s), 7.53–7.49 (2H, m), 7.35–7.32 (3H, m), 6.97 (1H, d, \(J= 7.5\hbox { Hz}\)), 1.59 (3H, s); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.3, 161.1, 145.0, 143.8, 140.4, 137.2, 134.3, 129.4, 129.2, 126.7, 126.6, 125.2, 120.3, 117.9, 111.3, 95.7, 55.6, 48.0, 11.7; IR (ATR) 3305, 3181, 3015, 2194, 1842, 1725, 1646, 1451, 1382, 1216, 1123, \(1068\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{21}\mathrm{H}_{14}\mathrm{ClN}_{5}\mathrm{O}_{2}\): 403.0836. Found: 403.0835.

6\(^\prime \)-Amino-7-chloro-3\(^\prime \)-methyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5f)

Compound 5f (350 mg, 87 %) was obtained as a white solid: mp 235–\(237\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 11.19 (1H, s), 7.78 (2H, d, \(J = 7.5\hbox { Hz}\)), 7.63 (2H, s), 7.52 (2H, t, \(J = 7.5\hbox { Hz}\)), 7.35 (2H, t, \(J = 7.5\hbox { Hz}\)), 7.18 (1H, d, \(J= 7.2\hbox { Hz}\)), 7.06 (1H, t, \(J= 7.8\hbox { Hz}\)), 1.57 (3H, s); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.5, 161.0, 144.9, 143.7, 139.3, 137.1, 133.9, 129.4, 129.2, 126.6, 123.9, 123.6, 120.2, 117.7, 114.1, 95.8, 55.7, 48.6, 11.7; IR (ATR) 3439, 3253, 3093, 2248, 1969, 1737, 1600, 1430, 1352, 1236, 1160, \(992\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{21}\mathrm{H}_{14}\mathrm{ClN}_{5}\mathrm{O}_{2}\): 403.0836. Found: 403.0837.

6\(^\prime \)-Amino-1,3\(^\prime \)-dimethyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5g)

Compound 5g (306 mg, 80 %) was obtained as a white solid: mp 212–\(214\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 7.78 (2H, d, \(J= 8.4\hbox { Hz}\)), 7.60 (2H, s), 7.51 (2H, t, \(J= 8.4\hbox { Hz}\)), 7.39 (1H, t, \(J = 7.2\hbox { Hz}\)), 7.35 (1H, t, \(J= 7.2\hbox { Hz}\)), 7.23 (1H, d, \(J = 7.8\hbox { Hz}\)), 7.15 (1H, d, \(J= 7.8\hbox { Hz}\)), 7.11 (1H, t, \(J= 7.2\hbox { Hz}\)), 3.24 (3H, s), 1.45 (3H, s); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 175.8, 161.2, 144.9, 143.8, 143.0, 137.2, 131.4, 129.5, 126.6, 124.6, 123.4, 120.2, 117.8, 108.9, 96.2, 55.8, 47.4, 26.5, 11.7; IR (ATR) 3445, 3236, 3095, 2229, 1780, 1541, 1419, 1331, 1214, \(952\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{22}\mathrm{H}_{17}\hbox {N}_{5}\mathrm{O}_{2}\): 383.1382. Found: 383.1381.

1-Acetyl-6\(^\prime \)-amino-3\(^\prime \)-methyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5h)

Compound 5h (364 mg, 89 %) was obtained as a white solid: mp 224–\(226\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 8.20 (1H, d, \(J = 8.1\hbox { Hz}\)), 7.8 (4H, d, \(J= 8.1\hbox { Hz}\)), 7.55–7.43 (3H, m), 7.39–7.29 (3H, m), 2.64 (3H, s), 1.51 (3H, s); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.2, 170.6, 161.2, 144.9, 144.0, 139.3, 137.1, 130.4, 129.7, 129.5, 126.8, 126.2, 125.1, 120.4, 117.6, 115.8, 95.9, 56.0, 48.5, 26.3, 11.9; IR (ATR) 3379, 3319, 3192, 2924, 2202, 1723, 1651, 1517, 1393, 1257, 1162, 1031, \(908\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{23}\mathrm{H}_{17}\hbox {N}_{5}\mathrm{O}_{3}\): 411.1331. Found: 411.1335.

1-Acetyl-6\(^\prime \)-amino-2-oxo-1\(^\prime \)-phenyl-3\(^\prime \)-propyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5i)

Compound 5i (394 mg, 90 %) was obtained as a white solid: mp 224–\(226\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 8.20 (1H, d, \(J= 8.1\hbox { Hz}\)), 7.83–7.81 (4H, m), 7.56–7.44 (3H, m), 7.41–7.29 (3H, m), 2.64 (3H, s), 1.80 (2H, t, \(J= 7.5\hbox { Hz}\)), 1.21–1.02 (2H, m), 0.58 (3H, t, \(J= 7.5\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.6, 170.4, 161.0, 147.8, 144.8, 139.1, 137.1, 130.8, 129.7, 129.4, 126.8, 126.2, 125.2, 120.4, 117.6, 115.8, 95.5, 56.2, 48.6, 28.5, 26.2, 20.8, 13.6; IR (ATR) 3313, 3198, 2947, 2204, 1757, 1648, 1458, 1394, 1262, 1153, 1071, \(907\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{25}\mathrm{H}_{21}\hbox {N}_{5}\mathrm{O}_{3}\): 439.1644. Found: 439.1641.

6\(^\prime \)-Amino-3\(^\prime \)-isopropyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5j)

Compound 5j (372 mg, 94 %) was obtained as a white solid: mp 217–\(219\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.75 (1H, s), 7.80 (2H, d, \(J= 7.8\hbox { Hz}\)), 7.53–7.51 (4H, m), 7.35 (1H, t, \(J= 6.6\hbox { Hz}\)), 7.27 (1H, t, \(J= 7.2\hbox { Hz}\)), 7.19 (1H, d, \(J= 7.2\hbox { Hz}\)), 7.02 (1H, t, \(J = 7.2\hbox { Hz}\)), 6.94 (1H, d, \(J= 7.8\hbox { Hz}\)), 2.10-2.05 (1H, m), 1.01 (3H, d, \(J = 6.6\hbox { Hz}\)), 0.69 (3H, d, \(J= 6.6\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 178.0, 160.7, 153.1, 144.7, 141.5, 137.3, 132.8, 129.4, 129.3, 126.5, 125.0, 122.6, 120.2, 117.9, 109.9, 95.0, 56.7, 47.8, 26.4, 21.4, 21.0; IR (ATR) 3412, 3254, 3091, 2248, 1891, 1675, 1619, 1425, 1345, 1437, 1160, 1096, \(989\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{23}\mathrm{H}_{19}\mathrm{N}_{5}\mathrm{O}_{2}\): 397.1539. Found: 397.1540.

6\(^\prime \)-Amino-3\(^\prime \)-isopropyl-5-methoxy-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5k)

Compound 5k (388 mg, 91 %) was obtained as a white solid: mp 221–\(223\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.57 (1H, s), 7.81 (2H, d, \(J= 8.1\hbox { Hz}\)), 7.54–7.52 (4H, m), 7.34 (1H, t, \(J= 7.2\hbox { Hz}\)), 6.85 (3H, s), 3.67 (3H, s), 2.16–2.07 (1H, m), 1.01 (3H, d, \(J= 6.9\)), 0.74 (3H, d, \(J= 6.6\)); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.9, 160.7, 155.7, 153.2, 144.7, 137.4, 134.7, 134.1, 129.4, 126.5, 120.2, 118.0, 114.5, 111.4, 110.4, 95.1, 56.8, 55.6, 48.4, 26.4, 21.5, 21.1; IR (ATR) 3313, 3188, 2964, 2193, 1839, 1704, 1645, 1475, 1390, 1199, 1030, \(760\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{24}\mathrm{H}_{21}\mathrm{N}_{5}\mathrm{O}_{3}\): 427.1644. Found: 427.1642.

6\(^\prime \)-Amino-5-bromo-3\(^\prime \)-isopropyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5l)

Compound 5l (436 mg, 92 %) was obtained as a white solid: mp 200–\(202\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.90 (1H, s), 7.80 (2H, d, \(J= 7.5\hbox { Hz}\)), 7.57–7.45 (6H, m), 7.36 (1H, t, \(J= 7.5\hbox { Hz}\)), 6.90 (1H, d, \(J= 8.1\hbox { Hz}\)), 2.13–2.06 (1H, m), 1.02 (3H, d, \(J= 6.9\hbox { Hz}\)), 0.74 (3H, d, \(J= 6.9\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.6, 160.8, 152.9, 144.8, 140.7, 137.3, 135.4, 132.1, 129.4, 128.0, 126.6, 120.4, 117.8, 114.3, 111.9, 94.4, 56.0, 48.0, 26.5, 21.4, 21.1; IR (ATR) 3313, 3289, 2972, 2193, 1707, 1640, 1452, 1393, 1215, \(1085\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{23}\mathrm{H}_{18}\hbox {BrN}_{5}\mathrm{O}_{2}\): 475.0644. Found: 475.0641.

6\(^\prime \)-Amino-5-chloro-3\(^\prime \)-isopropyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5m)

Compound 5m (396 mg, 92 %) was obtained as a white solid: mp 203–\(205\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.90 (1H, s), 7.80 (2H, d, \(J= 8.1\hbox { Hz}\)), 7.58–7.50 (4H, m), 7.38–7.32 (3H, m), 6.96 (1H, d, \(J= 8.1\hbox { Hz}\)), 2.14–2.05 (1H, m), 1.02 (3H, d, \(J= 6.9\hbox { Hz}\)), 0.74 (3H, d, \(J= 6.6\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.8, 160.8, 152.9, 144.8, 140.3, 137.3, 135.1, 129.4, 129.3, 126.7, 126.6, 125.3, 120.5, 117.9, 111.4, 94.4, 55.9, 48.1, 26.5, 21.5, 21.1; IR (ATR) 3345, 3225, 3016, 2248, 1751, 1680, 1561, 1423, 1351, 1220, 1095, \(920\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{23}\mathrm{H}_{18}\mathrm{ClN}_{5}\mathrm{O}_{2}\): 431.1149. Found: 431.1153.

6\(^\prime \)-Amino-5-fluoro-3\(^\prime \)-isopropyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5n)

Compound 5n (385 mg, 93 %) was obtained as a white solid: mp 226–\(228\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.79 (1H, s), 7.80 (2H, d, \(J= 7.8\hbox { Hz}\)), 7.57–7.49 (4H, m), 7.35 (1H, t, \(J= 7.2\hbox { Hz}\)), 7.22–7.19 (1H, m), 7.14–7.07 (1H, m), 6.96–6.91 (1H, m), 2.14–2.07 (1H, m), 1.02 (3H, d, \(J= 6.9\hbox { Hz}\)), 0.74 (3H, d, \(J= 6.9\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 178.0, 160.8, 158.7 (d, \(J= 237.9\hbox { Hz}\)), 153.0, 144.7, 137.5 (d, \(J= 46.1\hbox { Hz}\)), 134.7 (d, \(J= 15.5\hbox { Hz}\)), 129.4, 126.6, 120.3, 117.8, 115.7 (d, \(J= 46.1\hbox { Hz}\)), 112.8 (d, \(J= 48.5\hbox { Hz}\)), 110.9, 94.5, 56.2, 48.4, 26.4, 21.5, 21.0; IR (ATR) 3318, 3191, 2972, 2192, 1762, 1645, 1471, 1391, 1179, 1082, \(757\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{23}\mathrm{H}_{18}\hbox {FN}_{5}\mathrm{O}_{2}\): 415.1445. Found: 415.1443.

6\(^\prime \)-Amino-3\(^\prime \)-isopropyl-1-methyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5o)

Compound 5o (353 mg, 86 %) was obtained as a white solid: mp 221–\(223\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 7.80 (2H, d, \(J= 7.8\hbox { Hz}\)), 7.56 (2H, s), 7.52 (2H, t, \(J= 7.8\hbox { Hz}\)), 7.40–7.34 (2H, m), 7.26 (1H, d, \(J= 7.8\hbox { Hz}\)), 7.16 (1H, d, \(J= 8.4\hbox { Hz}\)), 7.11 (1H, t, \(J= 7.8\hbox { Hz}\)), 3.33 (3H, s), 1.92–1.87 (1H, m), 0.91 (3H, d, \(J= 7.2\hbox { Hz}\)), 0.69 (3H, d, \(J= 6.6\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 176.2, 160.8, 152.9, 144.7, 142.9, 137.3, 132.0, 129.4, 124.7, 123.3, 120.4, 120.2, 117.7, 108.9, 108.8, 94.8, 56.2, 47.5, 26.4, 26.4, 21.4, 20.7; IR (ATR) 3306, 3181, 2965, 2201, 1703, 1654, 1457, 1397, 1083, \(937\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{24}\mathrm{H}_{21}\mathrm{N}_{5}\mathrm{O}_{2}\): 411.1695. Found: 411.1697.

1-Acetyl-6\(^\prime \)-amino-3\(^\prime \)-isopropyl-2-oxo-1\(^\prime \)-phenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5p)

Compound 5p (394 mg, 90 %) was obtained as a white solid: mp 221–\(223\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 7.80 (2H, d, \(J= 7.8\hbox { Hz}\)), 7.56 (2H, s), 7.52 (2H, t, \(J= 7.8\hbox { Hz}\)), 7.40–7.34 (2H, m), 7.26 (1H, d, \(J= 7.8\hbox { Hz}\)), 7.16 (1H, d, \(J= 8.4\hbox { Hz}\)), 7.11 (1H, t, \(J= 7.8\hbox { Hz}\)), 3.33 (3H, s), 1.92–1.87 (1H, m), 0.91 (3H, d, \(J= 7.2\hbox { Hz}\)), 0.69 (3H, d, \(J= 6.6\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.7, 170.3, 160.7, 152.9, 144.7, 139.0, 137.2, 131.1, 129.7, 129.4, 126.7, 126.1, 125.2, 120.5, 117.4, 115.8, 94.5, 56.5, 48.6, 26.5, 26.1, 21.5, 20.7; IR (ATR) 3422, 3254, 3093, 2247, 1875, 1675, 1610, 1425, 1343, 1243, 1160, 1099, \(989\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{25}\mathrm{H}_{21}\mathrm{N}_{5}\mathrm{O}_{3}\): 439.1644. Found: 439.1641.

6\(^\prime \)-Amino-3\(^\prime \)-isopropyl-2-oxo-1,1\(^\prime \)-diphenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5q)

Compound 5q (435 mg, 92 %) was obtained as a white solid: mp 238–\(240\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 7.82 (2H, d, \(J= 7.5\hbox { Hz}\)), 7.66–7.62 (4H, m), 7.55–7.53 (3H, m), 7.40–7.31 (5H, m), 7.18–7.14 (1H, m), 6.8 (1H, d, \(J = 7.8\hbox { Hz}\)), 2.19-2.12 (1H, m), 1.02 (3H, d, \(J= 6.6\hbox { Hz}\)), 0.78 (3H, d, \(J= 6.6\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 175.8, 160.6, 153.0, 144.8, 142.5, 137.3, 134.0, 131.8, 130.0, 129.5, 129.3, 128.5, 126.6, 126.3, 125.2, 124.0, 120.3, 117.6, 109.2, 94.5, 56.6, 47.7, 26.6, 21.6, 20.7; IR (ATR) 3456, 3278, 3160, 2962, 2191, 1704, 1651, 1595, 1489, 1384, 1211, 1084, \(933\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{29}\mathrm{H}_{23}\mathrm{N}_{5}\mathrm{O}_{2}\): 473.1852. Found: 473.1850.

6\(^\prime \)-Amino-2-oxo-1\(^\prime \),3\(^\prime \)-diphenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5r)

Compound 5r (357 mg, 83 %) was obtained as a white solid: mp 208–\(210\,^{\circ }\mathrm{C}{}^{ 1}; \mathrm{H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.60 (1H, s), 7.90 (2H, d, \(J = 7.8\hbox { Hz}\)), 7.59–7.54 (4H, m), 7.42 (1H, t, \(J= 7.2\hbox { Hz}\)), 7.23–7.19 (3H, m), 7.13 (2H, t, \(J= 7.2\hbox { Hz}\)), 6.97–6.92 (3H, m), 6.79 (1H, d, \(J= 7.8\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.6, 160.3, 147.4, 145.7, 141.7, 137.1, 133.5, 132.0, 129.5, 129.2, 128.3, 127.9, 127.3, 124.8, 122.5, 121.0, 120.8, 117.6, 109.8, 95.6, 57.5, 48.1; IR (ATR) 3408, 3254, 3095, 2246, 1891, 1816, 1730, 1671, 1549, 1422, 1345, 1158, 1099, \(989\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{26}\mathrm{H}_{17}\mathrm{N}_{5}\mathrm{O}_{2}\): 431.1382 . Found: 431.1379.

6\(^\prime \)-Amino-1-methyl-2-oxo-1\(^\prime \),3\(^\prime \)-diphenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5s)

Compound 5s (333 mg, 75 %) was obtained as a white solid: mp 198–\(200\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 7.89 (2H, d, \(J= 7.8\hbox { Hz}\)), 7.61 (2H, s), 7.57 (2H, t, \(J = 7.8\hbox { Hz}\)), 7.43–7.41 (1H, m), 7.28 (1H, t, \(J= 7.8\hbox { Hz}\)), 7.23 (2H, t, \(J= 7.8\hbox { Hz}\)), 7.14 (2H, t, \(J= 7.8\hbox { Hz}\)), 7.02 (1H, t, \(J= 7.8\hbox { Hz}\)), 6.93 (1H, d, \(J= 7.8\hbox { Hz}\)), 6.80 (2H, d, \(J = 7.2\hbox { Hz}\)), 2.98 (3H, s); \({}^{13}\)C NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 175.9, 160.5, 147.4, 145.4, 142.8, 137.1, 132.6, 131.8, 129.5, 129.3, 128.3, 127.9, 127.3, 127.2, 124.4, 123.2, 120.9, 117.6, 108.7, 95.8, 56.7, 47.7, 26.2; IR (ATR) 3364, 3185, 3068, 2199, 1863, 1660, 1515, 1382, 1249, \(1079\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{27}\mathrm{H}_{19}\mathrm{N}_{5}\mathrm{O}_{2}\): 445.1539. Found: 445.1542.

1-Acetyl-6\(^\prime \)-amino-2-oxo-1\(^\prime \),3\(^\prime \)-diphenyl-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5t)

Compound 5t (397 mg, 84 %) was obtained as a white solid: mp 204–\(206\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 7.97–7.90 (3H, m), 7.81 (2H, s), 7.58 (2H, t, \(J= 7.8\hbox { Hz}\)), 7.46–7.34 (3H, m), 7.27-7.21 (2H, m), 7.11 (2H, t, \(J= 7.8\hbox { Hz}\)), 6.79 (2H, d, \(J= 7.5\hbox { Hz}\)), 2.39 (3H, s); \({}^{13}\hbox {C}\) NMR (75 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.4, 169.9, 160.6, 147.5, 145.3, 139.0, 137.0, 131.8, 131.5, 129.5, 128.6, 128.0, 127.4, 127.3, 126.0, 125.0, 121.0, 117.4, 115.6, 96.0, 56.7, 48.8, 25.7; IR (ATR) 3317, 3197, 2925, 2200, 1717, 1645, 1454, 1385, 1682, 1153, 1027, \(913\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{28}\mathrm{H}_{19}\mathrm{N}_{5}\mathrm{O}_{3}\): 473.1488. Found: 473.1487.

6\(^\prime \)-Amino-3\(^\prime \)-methyl-2-oxo-1\(^\prime \)-(p-tolyl)-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5u)

Compound 5u (321 mg, 84 %) was obtained as a white solid: mp 242–\(244\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.72 (1H, s), 7.65 (2H, d, \(J = 8.4\hbox { Hz}\)), 7.54 (2H, s), 7.31–7.26 (3H, m), 7.16 (1H, d, \(J= 7.2\hbox { Hz}\)), 7.02 (1H, t, \(J= 7.2\hbox { Hz}\)), 6.94 (1H, d, \(J= 7.8\hbox { Hz}\)), 2.35 (3H, s), 1.53 (3H, s); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.5, 161.0, 144.7, 143.6, 141.6, 136.0, 134.9, 132.2, 129.9, 129.7, 129.2, 124.8, 122.6, 120.2, 120.1, 118.0, 109.8, 96.1, 56.2, 47.8, 20.5, 11.6; IR (ATR) 3443, 3326, 3155, 2189, 1707, 1471, 1394, 1328, 1224, 1068, 1044, 933, 751, \(615\hbox { cm}^{-1}\); HRMS m / z (\(M^{+}\)) calcd for \(\mathrm{C}_{22}\mathrm{H}_{17}\mathrm{N}_{5}\mathrm{O}_{2}\): 383.1382. Found: 383.1385.

1-Acetyl-6\(^\prime \)-amino-1\(^\prime \)-(2-ethylphenyl)-3\(^\prime \)-isopropyl-2-oxo-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5v)

Compound 5v (368 mg, 79 %) was obtained as a white solid: mp 211–\(213\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 8.18 (1H, d, \(J= 8.4\hbox { Hz}\)), 7.59 (2H, s), 7.50–7.44 (4H, m), 7.41–7.38 (1H, m), 7.36–7.34 (2H, m), 2.63 (3H, s), 2.45–2.41 (2H, m), 1.97–1.92 (1H, m), 1.04 (3H, t, \(J= 7.2\hbox { Hz}\)), 0.91 (3H, d, \(J= 6.6\hbox { Hz}\)), 0.65 (3H, d, \(J = 6.6\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.8, 170.4, 160.8, 152.4, 145.5, 141.3, 139.0, 134.8, 131.3, 129.8, 129.6, 127.6, 126.8, 126.2, 124.9, 117.5, 115.8, 92.7, 56.6, 48.8, 26.5, 26.1, 23.9, 21.6, 20.8, 14.4; IR (ATR) 3395, 3322, 2965, 2204, 1766, 1648, 1526, 1393, 1331, 1268, 1155, 1090, \(1040\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{27}\mathrm{H}_{25}\mathrm{N}_{5}\mathrm{O}_{3}\): 467.1957. Found: 467.1954.

1-Acetyl-6\(^\prime \)-amino-1\(^\prime \)-(4-chlorophenyl)-3\(^\prime \)-isopropyl-2-oxo-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5w)

Compound 5w (357 mg, 83 %) was obtained as a white solid: mp 250–\(252\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (300 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.75 (1H, s), 7.84 (2H, d, \(J= 8.7\hbox { Hz}\)), 7.58–7.54 (4H, m), 7.27 (1H, t, \(J= 7.5\hbox { Hz}\)), 7.19 (1H, d, \(J = 7.2\hbox { Hz}\)), 7.01 (1H, t, \(J= 7.5\hbox { Hz}\)), 6.90 (1H, d, \(J= 7.8\hbox { Hz}\)), 2.11–2.02 (1H, s), 0.99 (3H, d, \(J= 6.9\hbox { Hz}\)), 0.68 (3H, d, \(J = 6.9\hbox { Hz}\)); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.8, 160.6, 153.5, 144.7, 141.4, 136.2, 132.6, 130.6, 129.3, 125.0, 122.6, 121.6, 117.7, 109.9, 95.3, 56.7, 47.8, 26.3, 21.3, 20.9; IR (ATR) 3460, 3334, 3186, 2190, 1852, 1709, 1644, 1508, 1391, 1329, 1221, 1092, 1044, \(931\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\mathrm{C}_{23}\mathrm{H}_{18}\mathrm{ClN}_{5}\mathrm{O}_{2}\): 431.1149. Found: 431.1146.

6\(^\prime \)-Amino-1\(^\prime \)-(2-chlorophenyl)-3\(^\prime \)-methyl-2-oxo-1\(^\prime \)H-spiro[indoline-3,4\(^\prime \)-pyrano[2,3-c]pyrazole]-5\(^\prime \)-carbonitrile (5x)

Compound 5x (298 mg, 74 %) was obtained as a white solid: mp 250–\(252\,^{\circ }\mathrm{C}\); \({}^{1}\mathrm{H}\) NMR (600 MHz, DMSO-\(d_{6}\)) \(\delta \) 10.72 (1H, s), 7.72 (1H, d, \(J= 7.8\hbox { Hz}\)), 7.64 (1H, d, \(J= 6.6\hbox { Hz}\)), 7.59–7.53 (2H, m), 7.40 (2H, s), 7.29 (1H, t, \(J= 7.8\hbox { Hz}\)), 7.11 (1H, d, \(J= 7.2\hbox { Hz}\)), 7.04 (1H, t, \(J= 7.2\hbox { Hz}\)), 6.94 (1H, d, \(J = 7.2\hbox { Hz}\)), 1.53 (3H, s); \({}^{13}\hbox {C}\) NMR (150 MHz, DMSO-\(d_{6}\)) \(\delta \) 177.4, 161.0, 146.1, 144.2, 141.6, 133.9, 132.2, 131.3, 130.8, 130.4, 129.9, 129.3, 128.3, 124.7, 122.6, 118.0, 109.8, 95.0, 56.2, 48.0, 11.7; IR (ATR) 3315, 3160, 2922, 2198, 1711, 1531, 1473, 1381, 1128, 1042, 932, 750, \(623\hbox { cm}^{-1}\); HRMS m / z (\(\hbox {M}^{+}\)) calcd for \(\hbox {C}_{21}\mathrm{H}_{14}\hbox {ClN}_{5}\mathrm{O}_{2}\): 403.0836. Found: 403.0833.

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was carried out according to the procedure described by Benzie and Strain [63]. FRAP reagent was prepared freshly by adding 10 vol. of 300 mM acetate buffer, pH 3.6 (3.1 g sodium acetate and 16 mL glacial acetic acid) and 1 vol. of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) prepared in 40 mM HCl and 1 vol. of 20 mM \(\hbox {FeCl}_{3}\). For each assay, 3 mL of FRAP reagent was mixed with 0.1 mL diluted synthesized compound. The mixture was shaken and incubated at \(37\,^{\circ }\mathrm{C}\) for 30 mins. Absorbance of the reaction mixture was measured at 593 nm. The standard calibration curve was carried out using Trolox and values expressed in terms of TEAC (\(\upmu \hbox {M}\)). For calculating \(\mathrm{EC}_{50}\) values, aliquots of the different concentrations of the synthesized compounds were prepared and incubated as described above.

Antibacterial activity

The antibacterial activities of synthesized compounds were determined using a modified Kirby-Bauer disk diffusion method [65]. Briefly, the test bacteria were grown in 10 mL of fresh \(\hbox {Difco}^{\mathrm{TM}}\) nutrient broth for 24 h. Optical density of test bacteria was measured using an Optizer 3220 (Double beam) UV–Vis spectrophotometer and found to be 0.7 at 595 nm. Aliquots of above bacterial suspension (\(100\,\upmu \hbox {L}\)) were then spread on \(\hbox {Difco}^{\mathrm{TM}}\) nutrient broth agar, which corresponded to the broth in which they were maintained. Two gram-negative bacteria Escherichia coli (KCTC-1924) and Pseudomonas aeruginosa (KCTC-2004) and two gram-positive bacteria Staphylococcus aureus (KCTC-1916) and Bacillus cereus (KCTC-1012) were obtained from the Korean Collection for Type Cultures (KCTC). The bacteria were incubated at \(37\,^{\circ }\mathrm{C}\) for 20–36 h, and then the diameters of the inhibition zones were measured in millimeters. Two mg of each test compound was dissolved in DMSO and further diluted to \(100\,\upmu \hbox {g/mL}\). Standard disks of ciprofloxacin served as positive controls and filter disks impregnated with DMSO as negative controls. Further, the depth of the agar in the plate is a factor to be considered in the disk diffusion method. Blank paper disks of diameter of 8.0 mm were impregnated with \(10\,\upmu \hbox {L}\) of above diluted sample solutions. When a filter disk impregnated with a tested chemical is placed on agar, the chemical diffuses from the disk into the agar. The solubility of the chemical and its molecular size determine the size of the area of infiltration. When an organism is placed on the agar, it will not grow in the area around the disk, if the chemical is active. This area of no growth around the disk is referred to as the zone of inhibition.