The structural motif of 3-substituted-3-hydroxyisatin is present in several bioactive natural products as well as clinical drugs such as paratunamide A, CPC-1 and sporidesmin (Fig. 1) [1-3].The medicinal properties of these compounds are derived from the C3 substituent and the absolute configuration of the chiral center [4].The development of efficient and practical methods to prepare such compounds is of paramount importance and it is an active area of research in asymmetric catalysis [5-7].One of the simplest preparative procedures for 3-substituted-3-hydroxyisatins is the catalytic addition of nucleophiles to readily available isatins, which grants access to appealing molecular scaffolds possessing quaternary carbon centers [8, 9].Furthermore, organic reactions employing water as a medium hold great promise from a green chemistry perspective [10].For example, Dash et al. [11, 12] have previously reported the water-catalyzed diastereoselective aldol reaction of thiazolidinediones with isatin and other aldehydes.As part of our ongoing interest in developing new methodologies for the synthesis of heterocycles [13-24], coupled with the reality that creatinine is present in numerous natural products [25], we envisaged the replacement of thiazolidinediones with structurally relevant creatinine in the aldolization of isatins. In 2010, Crooks et al. [26-29] reported the diastereoselective aldol addition of isatins with creatinine. However, this methodology suffers from the need to use NaOOCH₃ (NaOAc) and CH₃COOH (AcOH) in stoichiometric quantities, which prompted us to revisit this transformation with particular emphasis on performing the reaction under gold catalysis in water without compromising the diastereoselectivity. Herein, we report our study on the gold (Ⅲ)-catalyzed diastereoselective aldol addition of isatins with creatinine under aqueous condition leading to 3-hydroxyisatin derivatives. In view of the extensive biological properties of 3-hydroxyisatins, all the compounds were screened for their free radical scavenging activity. The gold-catalyzed protocol was also extended to a three-component reaction between isatins, malononitrile and creatinine through a tandem condensation / conjugate addition.

Solvents and reagents were purchased from SRL chemicals, India Pvt. Ltd, India and were used without further purification. Melting points (m.p.) were determined in open capillary tubes and are uncorrected. Infrared (IR) spectra were recorded on a Jasco FT-IR spectrophotometer as KBr pellets. ¹H and ¹³C NMR spectra were obtained in CDCl₃ or DMSO-d₆solutions on a Bruker spectrometer at 400 and 100 MHz, respectively. The proton chemical shifts (δ) are relative to tetramethylsilane (TMS, δ = 0.00) as internal standard and are expressed in parts per million (ppm). The spin multiplicities are given as s (singlet), d (doublet), t (triplet) and m (multiplet). The coupling constants (J) are given in hertz. Mass spectra were recorded on a PE-SCIEX API 3000 mass spectrometer. Elemental analyses were recorded using a ThermoFinnigan FLASH EA 1112CHN analyzer. All compounds gave C, H and N analysis within ±0.5% of the theoretical values. Analytical TLC was performed on precoated plastic sheets of silica gel G/UV-254 of 0.2 mm thickness (Macherey-Nagel, Germany) using analytical grade solvents and visualized with iodine spray (10% (w/w) I₂ in silica gel) or UV light (λ = 254 and 365 nm). The absorbance was measured at 517 nm using a Systronics 118 model spectrophotometer.

Water (15 mL) was added to a mixture of 1.0 mmol of isatin derivative, 1.2 mmol of creatinine (for 2p, 2.3 mmol of creatinine) and 1 mol% of HAuCl₄ and the resulting suspension was heated to reflux for 30 min. The clear reaction mixture was cooled to 15-20 ℃. The precipitated aldol product was filtered and washed with copious amount of water and then with methanol and ethyl acetate (EtOAc). The obtained product was thoroughly dried under vacuum to afford the pure product 2a-2p.

3-Hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl) i-ndolin-2-one (2a): Yellow solid; m.p. = 225-227 ℃; IR (KBr): 3557, 3384, 3242, 2795, 1718, 1689, 1672, 1242, 755 cm^-1. ¹H NMR (DMSO-d₆): δ 3.14 (s, 3H, -CH₃), 4.04 (s, 1H, CH), 6.31 (brs, 1H, OH), 6.70-6.74 (d, J = 7.6 Hz, 1H, -C₇H), 6.81-6.88 (t, J = 7.6 Hz, 1H, -C₆H), 7.06-7.07 (d, J = 7.6 Hz, 1H, -C₄H), 7.17-7.20 (t, J = 7.8 Hz, 1H, -C₅H), 7.51 (brs, 2H, NH₂), 10.23 (brs, 1H, NH); ¹³C NMR (DMSO-d₆): δ 32.6, 69.4, 76.3, 109.5, 121.1, 123.9, 127.9, 129.3, 142.6, 171.8, 175.7, 182.3. MS (ESI): m/z = 261[M+H]⁺; Anal. Calcd. for C₁₂H₁₂N₄O₃: C, 55.38%; H, 4.65%; N, 21.53%. Found C, 55.55%; H, 4.61%; N, 21.45%.

5-Fluoro-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidi-n-4-yl) indolin-2-one (2b): Pale yellow solid; m.p. = 250-252 ℃; IR (KBr): 3358, 3174, 3047, 2697, 1731, 1701, 1645, 1435, 806 cm^-1. ¹H NMR (DMSO-d₆): δ 3.14 (s, 3H, CH₃), 4.07 (s, 1H, CH), 6.52 (bs, 1H, OH), 6.70-6.75 (m, 1H, -C₇H), 6.80-6.84 (dd, J = 8.1 Hz, J = 2.37 Hz 1H, -C₄H), 6.99-7.06 (m, 1H, -C₆H), 7.51 (brs, 2H, NH₂), 10.28 (brs, 1H, NH); ¹³C NMR (DMSO-d₆): δ 32.2, 69.8, 76.5, 111.0, 124.2, 126.0, 129.9, 130.4, 141.9, 172.7, 175.8, 182.1. MS (ESI): m/z = 279[M+H]⁺; Anal. Calcd. for C₁₂H₁₁FN₄O₃: C, 51.80%; H, 3.98%; N, 20.14%. Found C, 52.09%; H, 3.92%; N, 20.05%.

5-Chloro-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazoli-din-4-yl) indolin-2-one (2c):Pale brown solid; m.p. = 267-269 ℃; IR (KBr): 3384, 3177, 2782, 2672, 1731, 1707, 1618, 1586, 1083, 818 cm^-1; ¹H NMR (DMSO-d₆): δ 3.16 (s, 3H, CH₃), 4.07 (s, 1H, CH), 6.52 (brs, 1H, OH), 6.74-6.77 (d, J = 8.1 Hz, 1H, -C₇H), 6.99-7.00 (d, J = 2.4 Hz, 1H, -C₄H), 7.23-7.24 (dd, J = 8.1 Hz, J = 2.1 Hz, 1H, -C₆H), 7.55 (brs, 2H, NH₂), 10.39 (brs, 1H, NH); ¹³C NMR (DMSO-d₆): δ 32.8, 69.6, 76.3, 110.9, 123.9, 125.0, 129.1, 129.9, 141.6, 172.0, 175.3, 182.1. MS (ESI): m/z = 295[M+H]⁺, 297 [M+H]²⁺; Anal. Calcd. for C₁₂H₁₁ClN₄O₃: C, 48.91%; H, 3.76%; N, 19.01%. Found C, 49.05%; H, 3.72%; N, 18.94%.

5-Bromo-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazoli-din-4-yl) indolin-2-one (2d): Brown solid; m.p. = 246-248 ℃; IR (KBr): 3384, 3176, 2980, 2672, 1731, 1707, 1566, 1186, 818 cm^-1; ¹H NMR (DMSO-d₆): δ 3.16 (s, 3H, CH₃), 4.07 (s, 1H, CH), 6.53 (brs, 1H, OH), 6.70-6.73 (d, J = 8.1 Hz, 1H, -C₆H), 7.11 (s, 1H, -C₄H), 7.36-7.38 (dd, J = 8.1 Hz, J = 1.5 Hz, 1H, -C₇H), 7.76 (brs, 2H, NH₂), 10.42 (brs, 1H, NH); ¹³C NMR (DMSO-d₆): δ 32.9, 69.6, 76.4, 111.5, 112.7, 126.6, 130.4, 132.0, 142.1, 172.1, 175.2, 182.1. MS (ESI): m/z = 339[M+H]⁺, 241[M+H]²⁺; Anal. Calcd. for C₁₂H₁₁Br₄O₃: C, 42.50%; H, 3.27%; N, 16.52%. Found C, 42.35%; H, 3.33%; N, 16.60%.

5-Iodo-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl) indolin-2-one (2e):Dark brown solid; m.p. = 202-204 ℃; IR (KBr): 3394, 3176, 2973, 2768, 1730, 1707, 1583, 1308, 1184, 817 cm^-1; ¹H NMR (DMSO-d₆): δ 3.17 (s, 3H, CH₃), 4.06 (s, 1H, CH). 6.44 (brs, 1H, OH), 6.66-6.71 (d, J = 8.4 Hz, 1H, -C₆H), 7.09 (s, 1H, -C₄H), 7.37-7.40 (dd, J = 8.2 Hz; J = 1.4 Hz, 1H, -C₇H), 7.13 (s, 1H, -C₄H), 7.44-7.49 (dd, J = 8.4 Hz, J = 1.6 Hz, 1H, -C₇H); ¹³C NMR (DMSO-d₆): δ 33.0, 69.8, 76.5, 111.5, 112.8, 126.9, 130.4, 132.2, 142.3, 172.5, 175.5, 182.6. MS (ESI): m/z = 387[M+H]⁺; Anal. Calcd. for C₁₂H₁₁IN₄O₃: C, 37.32%; H, 2.87%; N, 14.51%. Found C, 37.51%; H, 2.81%; N, 14.44%.

5-Nitro-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl) indolin-2-one (2f):Dark yellow solid; m.p. > 300 ℃; IR (KBr): 3421, 3345, 3190, 2922, 1734, 1707, 1583, 1313, 1207, 846 cm^-1; ¹H NMR (DMSO-d₆): δ 3.23 (s, 3H, CH₃), 4.15 (s, 1H, CH), 6.74 (s, 1H, OH), 6.95-6.98 (d, J =8.7 Hz, 1H, -C₇H), 7.67 (bs, 2H, NH₂), 7.83-7.84 (d, J =2.7 Hz, 1H, -C₄H), 7.81-7.82 (dd, J = 8.7 Hz, J = 2.4 Hz, 1H, -C₇H), 11.04 (s, 1H, NH); ¹³C NMR (DMSO-d₆): δ 33.1, 69.9, 75.8, 109.8, 119.2, 126.9, 128.9, 141.5, 149.4, 172.3, 176.0, 181.9. MS (ESI): m/z = 305[M+H]⁺; Anal. Calcd. for C₁₂H₁₂N₅O₅: C, 47.22%; H, 3.63%; N, 22.94%. Found C, 46.99%; H, 3.68%; N, 23.04%.

3-Hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)-1-methylindolin-2-one (2g): Pale yellow solid; m.p. = 230-232 ℃; IR (KBr): 3603, 3384, 3174, 2884, 1722, 1711, 1648, 1102, 754 cm^-1; ¹H NMR (DMSO-d₆): δ 3.05 (s, 3H, CH₃), 3.18 (s, 3H, CH₃), 4.10 (s, 1H, CH), 6.45 (brs, 1H, OH), 6.91-6.97 (m, 2H, -C₅H, -C₆H), 7.07-7.09 (d, J = 7.5 Hz, 1H, C₇H), 7.25-7.31 (t, J = 8.1 Hz, 1H, -C₄H), 7.55 (brs, 2H, NH₂); ¹³C NMR (DMSO-d₆): δ 25.9, 32.6, 69.9, 76.3, 109.2, 111.2, 115.9, 129.0, 140.4, 156.4, 171.9, 174.0, 181.9. MS (ESI): m/z = 275[M+H]⁺; Anal. Calcd. for C₁₃H₁₄N₄O₃: C, 56.93%; H, 5.14%; N, 20.43%. Found C, 57.08%; H, 5.09%, N, 19.99%.

3-Hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)-1-ethylindolin-2-one (2h): Pale yellow solid; m.p. = 196-198 ℃; IR (KBr): 3603, 3381, 3176, 2985, 1726, 1710, 1698, 1338, 1102, 754 cm^-1; ¹H NMR (DMSO-d₆): δ 1.32 (t, J = 7.6 Hz, 3H, CH₂), 3.12 (s, 3H, CH₃), 3.22 (s, 3H, CH₃), 4.15 (s, 1H, CH), 6.52 (brs, 1H, OH), 6.88-6.94 (m, 2H, -C₅H, -C₆H), 7.07-7.09 (d, J = 7.6 Hz, 1H, C₇H), 7.27-7.33 (t, J = 8.1 Hz, 1H, -C₄H), 7.64 (brs, 2H, NH₂); ¹³C NMR (DMSO-d₆): δ 19.1, 25.9, 32.9, 70.5, 76.3, 109.7, 111.9, 115.4, 129.0, 140.8, 156.8, 172.3, 174.5, 182.5. MS (ESI): m/z = 289[M+H]⁺; Anal. Calcd. for C₁₄H₁₆N₄O₃: C, 58.32%; H, 5.59%; N, 19.43%. Found C, 57.99%; H, 5.65%; N, 19.60%.

3-Hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)-1-hexylindolin-2-one (2i): Pale yellow solid; m.p. = 183-185 ℃; IR (KBr): 3366, 3314, 3173, 3063, 2956, 1696, 1652, 1498, 763 cm^-1; ¹H NMR (DMSO-d₆): δ 0.87 (t, J = 7.6 Hz, 3H, -CH₃), 1.22-1.35 (m, 6H, -(CH₂)₃-Me), 1.40-1.44 (m, 2H, -CH₂-); 3.25 (s, 3H, NMe), 3.44 (t, J = 7.6 Hz, 2H); 4.22 (s, 1H, CH), 5.99 (brs, 1H, OH), 6.95 (d, J = 8.4 Hz, 1H, ArH); 7.19-7.24 (m, 2H, ArH), 7.36-7.41 (m, 1H, ArH) 7.77 (brs, 2H, -NH₂). ¹³C NMR (DMSO-d₆): δ 13.9, 21.9, 26.5, 27.2, 28.1, 31.4, 44.4, 77.7, 88.4, 115.4, 125.2, 125.7, 127.7, 128.9, 144.5, 159.2, 174.1, 179.9. MS (ESI): m/z = 345[M+H]⁺; Anal. Calcd. for C₁₈H₂₄N₄O₃: C, 62.77%; H, 7.02%; N, 16.27%. Found C, 63.01%; H, 6.96%; N, 16.20%.

3-Hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)-1-phenylindolin-2-one (2j): Pale yellow solid; m.p. = 229-231 ℃; IR (KBr): 3402, 3199, 2948, 1722, 1700, 1132, 843 cm^-1; ¹H NMR (DMSO-d₆): δ 3.25 (s, 3H, CH₃), 4.22 (s, 1H, CH), 6.62 (brs, 1H, OH), 6.64-6.65 (d, J = 0.6 Hz, 1H, C₄H), 6.98-7.03 (t, J = 7.6 Hz, 1H, Ar-H), 7.17-7.23 (m, 2H, C₅H, C₆H), 7.44-7.60 (m, 5H, Ar-H, C₇H), 7.70 (brs, 2H, NH₂); ¹³C NMR (DMSO-d₆): δ 33.0, 70.6, 76.3, 108.9, 122.7, 124.4, 126.9, 127.1, 127.2, 128.1, 129.6, 129.7, 129.9, 134.4, 143.9, 171.9, 174.1, 182.5. MS (ESI): m/z = 337[M+H]⁺; Anal. Calcd. for C₁₈H₁₆N₄O: C, 64.28%; H, 4.79%; N, 16.66%. Found C, 63.99%; H, 4.71%; N, 16.57%.

1-Benzyl-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazoli-din-4-yl) indolin-2-one (2k): Pale brown solid; m.p. = 207-209 ℃; IR (KBr): 3377, 3312, 3199, 3065, 3031, 2824, 1705, 1644, 1575, 1215, 798 cm^-1; ¹H NMR (DMSO-d₆): δ 3.20 (s, 3H, NMe), 4.04 (s, 1H, CH), 5.22 (s, 2H, -CH₂Ph), 6.21 (brs, 1H, OH), 6.93 (d, J = 7.6 Hz, 1H, ArH), 7.19-7.27 (m, 5 H, ArH), 7.34-7.39 (m, 3H), 7.69 (brs, 2H, NH₂). ¹³C NMR (DMSO-d₆): δ 35.1, 55.5, 83.3, 94.1, 118.1, 124.7, 126.0, 127.0, 127.9, 128.4, 128.5, 147.5, 171.2, 175.1, 180.9. MS (ESI): m/z = 351[M+H]⁺; Anal. Calcd. for C₁₉H₁₈N₄O₃: C, 65.13%; H, 5.18%; N, 15.99%. Found C. 64.95%; H, 5.25%; N, 16.11%.

1-Acetyl-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazoli-di n-4-yl) indolin-2-one (2l): Colorless solid; m.p. = 175-177 ℃; IR (KBr): 3603, 3381, 3175, 2985, 2939, 1727, 1710, 1698, 1649, 1375, 754 cm^-1; ¹H NMR (DMSO-d₆): δ 2.58 (s, 3H, -COCH₃), 3.23 (s, 3H, CH₃), 4.24 (s, 1H, CH), 6.77 (brs, 1H, OH), 7.17-7.38 (m, 3H, C₄H, C₅H, C₆H), 7.46 (brs, 1H, NH), 7.56 (brs, 1H, NH), 8.04-8.06 (d, J = 8.1 Hz, 1H, C₇H); ¹³C NMR (DMSO-d₆): δ 26.1, 32.9, 71.0, 76.3, 117.3, 123.9, 128.7, 129.2, 129.7, 139.1, 169.9, 172.3, 175.4, 181.9. MS (ESI): m/z = 303[M+H]⁺; Anal. Calcd. for C₁₄H₁₄N₄O₄: C, 55.63%; H, 4.67%; N, 18.53%. Found C, 55.75%; H, 4.65%; N, 18.45%.

1-Benzoyl-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazo-lidin-4-yl) indolin-2-one (2m): Colorless solid; m.p. = 224-226 ℃; IR (KBr): 3600, 3380, 3166, 2989, 2925, 1730, 1709, 1700, 1645, 1379, 755 cm^-1; ¹H NMR (DMSO-d₆): δ 3.17 (s, 3H, CH₃), 4.21 (s, 1H, CH), 4.74-4.91 (ABq, J = 16.2 Hz), 6.57 (brs, 1H, OH), 6.64-6.67 (d, J = 8.1 HZ, 1H, -C₄H), 6.91-6.96 (t, J = 7.6 Hz, 1H, -C₅H), 7.11-7.34 (m, 5H, -C₆H, -C₇H and Ar-H), 7.45-7.47 (d, J = 7.2 Hz, 2H, Ar-H), 7.56 (brs, 2H, NH₂); ¹³C NMR (DMSO-d₆): δ 32.7, 42.9, 69.5, 76.0, 109.0, 121.9, 123.7, 127.0, 127.2, 127.3, 127.4, 128.2, 128.3, 129.4, 136.0, 143.2, 171.9, 174.4, 182.3. MS (ESI): m/z = 365[M+H]⁺; Anal. Calcd. for C₁₉H₁₈N₄O₃: C, 62.63%; H, 4.43%; N, 15.38%. Found C, 62.75%; H, 4.38%; N, 15.30%.

3-Hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)-1-(phenylsulfonyl) indolin-2-one (2n): Colorless solid; m.p. = 230-232 ℃; IR (KBr): 3444, 3389, 3267, 2968, 1768, 1725, 1701, 1682, 1544, 1108, 858 cm^-1; ¹H NMR (DMSO-d₆): δ 3.13 (s, 3H, CH₃), 4.17 (s, 1H, CH), 6.93 (brs, 1H, OH), 7.15-7.17 (m, 2H, C₅H, C₆H), 7.36-7.42 (m, 2H, Ar-H), 7.62-7.84 (m, 5H, Ar-H, NH₂), 8.05-8.08 (m, 2H, C₄H, C₇H); ¹³C NMR (DMSO-d₆): δ 33.32, 70.7, 78.3, 122.4, 124.3, 124.5, 126.9, 127.0, 127.1, 128.3, 128.4, 129.5, 135.4, 142.1, 171.9 (C=N), 173.9, 182.6. MS (ESI): m/z = 401[M+H]⁺; Anal. Calcd. for C₁₈H₁₆N₄O₅S: C, 53.99%; H, 4.03%; N, 13.99%; S, 8.01%. Found C, 60.11%; H, 3.99%; N, 14.07%; S 7.95%.

5, 6-Dibromo-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidaz-olidin-4-yl) indolin-2-one (2o): Brown solid; m.p. = 196-198 ℃; IR (KBr): 3391, 3350, 3246, 3176, 2782, 2607, 1731, 1706, 1585, 1477, 1082, 818 cm^-1; ¹H NMR (DMSO-d₆): δ 3.17 (s, 3H, CH₃), 4.10 (s, 1H, CH), 6.69 (brs, 1H, OH), 7.10 (s, 1H, -C₆H), 7.52 (brs, 1H, NH), 7.67 (d, 1H, J = 1.8 Hz, -C₄H), 7.85 (bs, 1H, NH), 10.80 (brs, 1H, NH); ¹³C NMR (DMSO-d₆): δ 32.9, 69.8, 77.0, 102.8, 113.2, 125.7, 131.6, 133.9, 141.7, 172.2, 175.1, 181.9. MS (ESI): m/z = 417[M+H]⁺, 419[M+H]²⁺, 421[M+H]⁴⁺; Anal. Calcd. for C₁₂H₁₀Br₂N₄O₃: C, 34.48%, H, 2.41%, N, 13.40%. Found C, 34.59%; H, 2.36%; N, 13.33%.

1, 1'-(Propane-1, 3-diyl) bis (3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl) indolin-2-one (2p):Pale brown solid; m.p. > 300 ℃; IR (KBr): 3361, 3189, 1708, 1644, 1467, 1200, 755 cm^-1; mp 287-289 ℃; ¹H NMR (DMSO-d₆): δ 1.55-1.61 (m, 2H), 3.01 (s, 3H, NMe), 4.02 (t, J = 7.6 Hz, 4H), 4.27 (s, 2H, CH), 6.05 (brs, 2H, OH), 7.00 (d, 2H, J = 8.4 Hz, Ar-H), 7.21 (d, 2H, J = 8.4 Hz, Ar-H), 7.29-7.42 (m, 4H, Ar-H), 7.83 (brs, 2H, NH), 9.89 (brs, 2H, NH). ¹³C NMR (DMSO-d₆): δ 22.9, 35.7, 39.9, 82.1, 90.7, 115, 1, 124.2, 124.8, 126.7, 127.1, 142.6, 153.8, 174.7, 179.9. MS (ESI): m/z = 561[M+H]⁺; Anal. Calcd. for C₂₇H₂₈N₈O₆: C, 57.85%; H, 5.03%; N, 19.99%. Found C, 58.04%; H, 4.99%; N, 19.82%.

A mixture of 1.0 mmol of isatin, 1.0 mmol of malononitrile and 1 mol% of chloroauric acid in 15 mL of water was stirred at room temperature for 15 min. To this mixture was added 1.2 mmol of creatinine and the resulting suspension was heated to reflux for 30 min and then cooled to room temperature. The precipitated product was filtered and washed with copious amount of water and then with methanol and ethyl acetate. The obtained product was thoroughly dried under vacuum to afford the crude product. The crude product was purified by column chromatography using hexane/EtOAc eluent system to afford the inseparable diastereoisomeric mixture of 3a-3e.

2-(3-(2-Imino-3-methyl-5-oxoimidazolidin-4-yl)-2-oxoind-olin-3-yl) malononitrile (3a):Pale yellow solid; m.p. = 201-203 ℃; IR (KBr): 3324, 2912, 2255, 1725, 1702, 1305, 877 cm^-1; ¹H NMR (DMSO-d₆): δ 2.33 (s, 3H, NMe), 4.34 (CH-CO), 5.93 (s, 1H, CH (CN)₂), 6.96 (d, J = 6.0 Hz, 1H), 7.15 (s, 1H, ArH), 7.40 (s, 1H, ArH), 7.49 (s, 1H, ArH), 7.50 (brs, 1H, -NH), 8.03 (brs, 1H, -NH), 11.07 (brs, 1H, NH-CO). ¹³C NMR (DMSO-d₆): δ 33.2, 43.9, 53.3, 66.1, 111.0, 111.6, 112.3, 123.1, 124.5, 125.0, 131.4, 142.8, 172.7, 173.4, 183.1. MS (ESI): m/z = 309[M+H]⁺; Anal. Calcd. for C₁₅H₁₂N₆O₂: C, 58.44%; H, 3.92%; N, 27.26%. Found C, 58.89%; H, 3.85%; N, 27.10%.

2-(5-Fluoro-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)-2-oxoindolin-3-yl) malononitrile (3b):Colorless solid; m.p. = 165-167 ℃; IR (KBr): 3329, 2922, 2233, 1717, 1632, 1314, 799 cm^-1; ¹H NMR (DMSO-d₆): δ 2.50 (s, 3H, NCH₃), 4.38 (s, 1H, CH-CO), 5.95 (s, 1H, CH (CN)₂), 6.96-6.99 (m, 1H, ArH), 7.25-7.32 (m, 2H, ArH), 7.59 (brs, 1H, NH), 8.07 (brs, 1H, NH), 11.11 (brs, 1H, NH-CO). ¹³C NMR (DMSO-d₆): δ 33.3, 53.6, 66.1, 111.2, 111.8, 112.3, 112.4 (J_C-F = 32.0 Hz), 112.6, 118.0, 118.2, 126.0, 139.1, 157.2, 159.6 (J_C-F = 152.0 Hz), 172.6, 173.0, 182.7. MS (ESI): m/z = 327[M+H]⁺; Anal. Calcd. for C₁₅H₁₁FN₆O₂: C, 55.22%; H, 3.40%; N, 25.76%. Found C, 54.90%; H, 3.45%; N, 25.91%.

2-(5-Chloro-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)-2-oxoindolin-3-yl) malononitrile (3c):Colorless solid; m.p. = 189-191 ℃; IR (KBr): 3325, 2978, 2248, 1728, 1699, 1344, 813 cm^-1; ¹H NMR (DMSO-d₆): δ 2.62 (s, 3H, NCH₃), 4.39 (s, 1H, CH-CO), 5.98 (1H, s, CH (CN)₂), 6.98 (d, J = 6.9 Hz, 1H, ArH), 7.46 (d, J = 8.3 Hz, 2H, ArH), 7.62 (brs, 1H, NH), 8.09 (brs, 1H, NH), 11.22 (brs, 1H, NH-CO). ¹³C NMR (DMSO-d₆): δ 27.0, 53.0, 66.0, 111.0, 111.6, 112.1, 124.3, 126.1, 126.5, 130.9, 141.5, 172.1, 173.2, 182.3. MS (ESI): m/z = 343[M+H]⁺, 345[M+H]²⁺; Anal. Calcd. for C₁₅H₁₁ClN₆O₂: C, 52.56%; H, 3.23%; N, 24.52%. Found C, 52.90%; H, 3.17%; N, 24.41%.

2-(5-Nitro-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)-2-oxoindolin-3-yl) malononitrile (3d):Dark yellow solid; m.p. = 297-299 ℃; IR (KBr): 3308, 2951, 2251, 1701, 1689, 1378, 798 cm^-1; ¹H NMR (DMSO-d₆): δ 2.68 (s, 3H, -NCH₃), 4.48 (s, 1H, CH-CO), 6.15 (s, 1H, CH (CN)₂), 7.19 (d, J = 8.4 Hz, 1H, ArH), 8.16-8.55 (m, 2H, ArH), 9.31 (brs, 1H, NH), 10.17 (brs, 1H, NH), 11.87 (brs, 1H, NH-CO). ¹³C NMR (DMSO-d₆): δ 34.0, 52.7, 66.9, 110.9, 111.5, 120.3, 124.9, 128.1, 142.4, 149.2, 173.0, 174.0, 182.1. MS (ESI): m/z = 354[M+H]⁺; Anal. Calcd. for C₁₅H₁₁N₇O₄: C, 50.99%; H, 3.14%; N, 27.75%. Found C, 51.22%; H, 3.08%; N, 27.60%.

2-(3-(2-Imino-3-methyl-5-oxoimidazolin-4-yl)-5-methy-l-2-oxoindolin-3-yl) malononitrile (3e): Light yellow solid; m.p. = 213-215 ℃; IR (KBr): 3301, 2934, 2250, 1711, 1678, 1311, 843 cm^-1; ¹H NMR (DMSO-d₆): δ 2.31 (s, 3H, -CH₃), 2.39 (s, 3H, -NCH₃), 4.29 (s, 1H, CH-CO), 5.90 (s, 1H, CH (CN)₂), 6.86 (d, J = 7.1 Hz, 1H, ArH), 7.21 (d, J = 6.92 Hz, 1H, ArH), 7.33 (s, 1H, ArH), 7.49 (brs, 1H, NH), 8.00 (brs, 1H, NH), 10.95 (brs, 1H, NH-CO). ¹³C NMR (DMSO-d₆): δ 21.1, 22.3, 44.0, 53.3, 66.0, 110.8, 111.6, 112.3, 125.0, 131.6, 132.2, 140.3, 172.6, 173.2, 183.2. MS (ESI): m/z = 323[M+H]⁺; Anal. Calcd. for C₁₆H₁₄N₆O₂: C, 56.92%; H, 4.38%; N, 26.07%. Found C, 57.05%; H, 4.32%; N, 25.99%.

To a 3 mL ethanolic solution of 1, 1-diphenyl-2-picryl-hydra-zil (DPPH, 200 μmol/L), 0.05 mL of different concentration (50, 500, 1000 μg/mL) of test samples and 20 μg of ascorbic acid were added. The solutions were incubated at 37 ℃ for 30 min. The absorbance was measured at 517 nm using a Systronics 118 model spectrophotometer. The percentage inhibition of the DPPH radical was calculated by comparing the results of the test with those of the control using the formula: inhibition (%) = (A_C-A_T)/A_C × 100, where A_C is the absorbance of the control sample and A_T is the absorbance of test sample.

Hydrogen peroxide radical scavenging activity was performed by dissolving 10 μg of each 3-hydroxyisatin (2a-2o) in 3.4 mL of 0.1 mol/L phosphate buffer (pH = 7.4) and mixing with 600 μL of 43 mmol/L solution of H₂O₂. butylated hydroxy toluene (BHT, 20 μg) was used as a standard and the stock solution was prepared in the same buffer. The absorbance value (230 nm) of the reaction mixture was recorded at 10 min intervals between 0 and 40 min. For each concentration, a blank sample was used for background subtraction. The percentage of inhibition was calculated by comparing the results of the test with those of the control using the formula: inhibition (%) = (A_C-A_T)/A_C× 100, where A_C is the absorbance of the control sample and A_T is the absorbance of test sample.

Before commencing our studies, we took into consideration that commonly used gold precatalysts are either unstable (AuCl₃) or sparingly soluble (AuCl and AuBr₃) in water. Therefore, our attention was focused on the use of chloroauric acid (HAuCl₄), which happens to be the precursor for most of the commercially available gold salts. A mixture of isatin 1a, creatinine and chloroauric acid (1 mol%) in water was heated to reflux (Scheme 1). Even though, the starting materials were not completely soluble in water, the pure product 2a was obtained after 30 min, as confirmed by NMR analysis (Table 1, entry 1). Most importantly, the product was obtained stereoselectively as a single diastereoisomer, which was in sharp agreement to Crook's protocol [26].As chloroauric acid undergoes hydrolysis in water to form HCl and subsequently catalyze the whole process, a control experiment with substrate 1a was performed by using a 10% HCl solution. After 30 min at reflux temperature, only 10% of the product 2a was formed, as indicated by NMR analysis of the crude product. Screening with other potentially oxophilic catalysts such as Zn (OTf)₂, Cu (OTf)₂ and In (OTf)₃ under similar conditions did not lead to the formation of the product. Pleased with these initial results, we applied chloroauric acid as a catalyst for the reaction of a range of isatin derivatives. Isatin derivatives possessing substituents with a different electronic nature on the nitrogen as well as on the periphery were tolerated and gave excellent yields of the aldol product 2a-2o (Table 1). All the reactions were performed in completely demineralized water (pH = 7) as slight acidity or basicity of the reaction medium could also influence the stereochemical outcome. A blank reaction without chloroauric acid did not proceed at all. One of the beneficial advantages of our protocol is that products with a bromo or an iodo substituent (2d, 2e and 2o) could also be realized. These could potentially serve as synthetic precursors for organometallic cross-coupling reactions [30].The structural characterization of all the products was established from their spectral data (FTIR, ¹H NMR, ¹³C NMR and MS) and elemental analyses. As an illustrative example, the IR spectrum of compound 3a exhibited sharp peaks at 1718 and 1689 cm^-1 corresponding to the carbonyl stretching of oxindole and creatinine cores, respectively. Broad stretching bands between 3384 and 3314 cm^-1 suggested the presence of various amide functionalities. A broad parabolic peak between 3557 and 3444 cm^-1 revealed the presence of an intermolecularly bonded hydroxyl group. The ¹H NMR spectrum recorded in DMSO-d₆ showed a singlet at δ = 4.04 ppm, which corresponded to the 4'-methyne hydrogen and a broad singlet at 6.31 ppm, which indicated the presence of a hydroxyl group, which was D₂O exchangeable. In the ¹³C NMR spectrum, peaks at 175.7 and 182.3 ppm indicated the presence of the carbonyl carbons of isatin and creatinine, respectively. Additionally, the peaks at 69.4 and 76.3 ppm were assigned to the tertiary aliphatic carbon and quaternary carbon (the carbons of the bond between the isatin and creatinine moieties), respectively. Finally, the mass spectrum exhibited a molecular ion peak at m/z = 261[M+H]⁺, which strongly supported the formation of product 2a. The exclusive diastereoselectivity (an equimolar mixture of SS and RR isomers) of 2a can be explained by the Zimmerman-Traxler model, which favors anti-selective products over the syn-isomers [31].

Up to this point, we have only discussed the aldolization chemistry of monosubstituted systems. We also used our methodology for the synthesis of more complex systems. As depicted in Scheme 2, we applied our gold (Ⅲ)-catalyzed procedure for the synthesis of a complex bis-isatinyl system 1p. Thus, treatment of 1, 1'-(propane-1, 3-diyl) bis-isatin (1.0 mmol) with creatinine (2.3 mmol) under our optimized conditions produced the corresponding bis-aldol product 2 in 60% yield after reflux for 1 h.

In an attempt to display the synthetic embellishment of our chemistry, we probed the reaction of several isatins with malononitrile and creatinine to realize a sequential condensation/Michael addition (Scheme 3). The reaction resulted in the formation of multicomponent adducts 3a-3e in satisfactory yields (Table 2).

All products were thoroughly characterized by performing spectroscopic analysis. For example, the ¹H NMR spectrum of compound 3c exhibited singlets at δ = 5.98 and 4.39 ppm, which correspond to the CH-proton of the CH (CN)₂ group and creatinine moieties, respectively. In the ¹³C NMR spectrum, the peaks at δ = 43.9 and 66.1 ppm corresponded to the carbon flanked by the geminal cyano groups and the tertiary carbon of the creatinine core, respectively. In the mass spectrum, two Cl isotope peaks at m/z = 343[M+H]⁺and 345[M+H]²⁺confirmed the formation of product 3c. To establish the stereochemistry of the products, a single crystal of 3e was obtained and used for X-ray diffraction. Analysis of the crystal structure revealed that compound 3e was formed as the anti-isomer (R, R and S, S enantiomeric pairs) and no syn-stereochemistry was observed (Fig. 2). The exclusive formation of the anti-isomer over syn-isomer was in contrast to our aldolization study.

Because of our continuing interest in chemical biology [32-48] coupled with the information about radical scavenging properties of isatin derivatives [49], we investigated the antioxidant potency of the isatin derivatives 2a-2o by DPPH radical scavenging and hydrogen peroxide methods. The radical scavenging activity[50] of 3-hydroxy isatins was determined spectrophotometrically[51] byusing Blois's protocol [52]. All compounds were tested for their interaction with stable free radical DPPH, which specifies their radical scavenging activity. The percentage of inhibition was compared with that of standard L-ascorbic acid (Table 3). The results as a percentage (%) are expressed as the ratio of absorbance decrease at 517 nm and the absorbance of DPPH solution in the absence of compounds. A lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The capability to scavenge the DPPH radical was calculated using the equation: DPPH scavenging effect (%) = (A_c-A_t/A_c) × 100, where A_c is the absorbance of the control reaction and A_t is the absorbance in the presence of samples or standards. Analysis of the screening results revealed that the radical scavenging activity of the compounds on DPPH radicals increases with respect to the concentration (Table 3). Compounds possessing 5-Cl (2c), 5-Br (2d) and 5-Ⅰ (2e) moieties showed maximum activity at a concentration of 1000 μg/mL. This could be attributed to the better homolysis tendency of the carbon-halide bond leading to the respective free radicals. The radical scavenging activity of compounds possessing N-Et (2h) and N-Bn (2k) groups was less potent compared with the standard. Hydrogen peroxide radical scavenging activity[50] was performed using a solution of hydroxyisatins 2a-2o in a mixture of phosphate buffer and a solution of H₂O₂ by a spectrophotometric method and compared with the standard BHT. The absorbance value (230 nm) of the reaction mixture was recorded at 10 min intervals between 0 and 40 min. For each concentration, a blank sample was used for background subtraction and the corresponding absorbance value for 2a-2o is given in Table 4. The compound containing a 5-iodo group (2e) exhibited maximum antioxidant potential after 40 min. Compounds 2d and 2c emerged as the second most active compounds among those tested, with an absorbance value of 71% and 70%, respectively. This observation was in agreement with the results obtained using the DPPH protocol (compare Table 3). However, the antioxidant properties of the compounds possessing N-Et (2h) and N-Bn (2k) and no substitution (2a) were poor, as evidenced by their low absorbance values. The other compounds screened in this study exhibited inhibition that was comparable with the standard, BHT.

In summary, we prepared of 3-hydroxy-3-(2-imino-3-meth-yl-5-oxoimidazolidin-4-yl) indolin-2-one derivatives in excellent yields as single diastereoisomers (syn-selectivity) through the chloroauric acid catalyzed aldolization between isatins and creatinine under aqueous conditions. The reaction was facile, high-yielding, and did not require solvent extraction and column chromatography. The radical scavenging activity of the prepared 3-hydroxyisatin derivatives revealed that compounds possessing halo groups at the C5-carbon (2c, 2d and 2c) exhibited comparable antioxidant potency to the standard. Furthermore, we applied our reaction conditions to a tandem reaction between isatin, creatinine and malononitrile. In this case, the products were obtained in excellent yield with complete anti-selectivity. Further studies aimed towards the preparation of more complex systems as well as kinetic studies to investigate the reaction mechanism are currently underway in our laboratory and will be reported in due course.

Acknowledgments

K. P thanks the Central Council for Research in Siddha (CCRS), India and T. P thanks the management of Inogent Laboratories Ltd., India. C. P. is grateful to the Science and Engineering Research Board (SERB) for the financial support (SERB/F/5666/2015-16).