Chemistry
All the chemicals were purchased from Aldrich-Sigma Chemical Company (St. Louis, MO, USA) and Alfa-Aesar Chemical Company (Lancashire, Heysham, England) and used without further purification. All reactions were routinely monitored by TLC on Merck F254 silica gel plates. Silica gel (70–230 mesh, Silicacycle) was used for column chromatography. The 1H- and 13C-NMR spectra were determined on an Agilent Varian-400 NMR (Agilent Technologies, Santa Clara, CA, USA) instrument in CDCl3, acetone-d
6, methanol-d
4, or acetic acid-d
6 unless otherwise noted. Chemical shifts (δ) were expressed as parts per million (ppm) downfield from tetramethylsilane (TMS) as the internal standard (σ 0.00), and coupling constants (J) were given in hertz (Hz). High-resolution mass spectra (HRMS) using a Bruker Impact HD (ESI) were performed in the Instrument Center of the Ministry of Science and Technology at the National Chiao-Tung University, Taiwan. Dry tetrahydrofuran (THF) was freshly distilled from lithium aluminum hydride (LAH) before use. All the other solvents were obtained from commercial sources and purified before use if necessary. Images were acquired with a Leica DM1000 LED microscope (Leica Microsystems, Wetzlar, Hessen, Germany). UV–VIS spectra were recorded on a Thermo Multiskan Go Microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). IR spectra were registered on a Thermo Nicolet iS5 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with attenuated total reflection (ATR) method. The purities of the final compounds were all greater than 95% as determined by analytical reverse-phase HPLC.
General procedure for the preparation of compounds 4–15
The key intermediate 3 for preparation of target compounds was synthesized according to the method reported previously [19]. To each stirred solution containing 3 (263 mg, 1.00 mmol) in EtOH (15 ml) was added dropwise a solution of 5-substituted oxindole (1.00 mmol) in EtOH (2 ml) and then piperidine (0.1 ml) was added. After stirring at room temperature for 6 h, the precipitate formed was filtrated, washed with EtOH, and purified by column chromatography (silica gel, 90:10:1 EtOAc–MeOH–TEA).
(Z)-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-2-oxoindoline-5-sulfonamide (4)
The requisite oxindole-5-sulfonamide, for condensation with 3 to form target compound 4, was prepared in 60% yield by a modified method of amidation of 2-oxoindoline-5-sulfonyl chloride with ammonium hydroxide solution [32].
Yield of 4: 58%, orange solids. Mp: 252–255 °C, UV λmax (MeOH), nm (logɛ): 418 (4.21). IR (ATR), cm−1: 3239, 2976, 1600, 1562. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 8.26 (d, 1H, J = 1.2 Hz ArH), 7.74–7.77 (m, 2H, –C=CH–, ArH), 7.12 (d, 1H, J = 8.4 Hz, ArH), 4.56 (s, 2H, Ar–CH
2
), 4.00 (t, 2H, J = 5.6 Hz, –NCH
2
CH2–), 3.50 (t, 2H, J = 5.6 Hz, –NCH2CH
2
–), 3.37 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.51 (s, 3H, Ar–CH
3
), 1.31 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.5, 169.5, 150.2, 142.0, 137.7, 133.8, 128.1, 127.4, 126.9, 126.5, 121.3, 117.9, 116.5, 111.2, 51.8, 48.1, 45.7, 39.5, 9.9, 8.7. HRMS m/z (ESI): calcd., 458.1869.1744 [M + H]+; found, 458.1857 [M + H]+.
(Z)-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-N,N-dimethyl-2-oxoindoline-5-sulfonamide (5)
The requisite N,N-dimethyl-2-oxoindoline-5-sulfonamide, for condensation with 3 to form target compound 5, was prepared in 74% yield by a modified method of amidation of 2-oxoindoline-5-sulfonyl chloride with dimethylamine in methanol [32].
Yield of 5: 56%, orange solids. Mp: 244–247 °C, UV λmax (MeOH), nm (logɛ): 428 (4.53). IR (ATR), cm−1: 2971, 1657, 1586. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 8.19 (d, 1H, J = 1.6 Hz, ArH), 7.86 (s, 1H, –C=CH–), 7.63 (dd, 1H, J = 8.0, 1.6 Hz, ArH), 7.18 (d, 1H, J = 1.6 Hz, ArH), 4.57 (s, 2H, Ar–CH
2
), 3.96 (t, 2H, J = 6.0 Hz, –NCH
2
CH2–), 3.50 (t, 2H, J = 6.0 Hz, –NCH2CH
2
–), 3.39 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.68 (s, 6H, –N(CH
3
)2), 2.50 (s, 3H, Ar–CH
3
), 1.33 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.5, 169.5, 150.2, 142.4, 133.8, 129.7, 128.2, 128.1, 127.7, 127.1, 121.3, 119.5, 116.4, 111.3, 51.7, 48.5, 48.0, 39.4, 38.3, 10.0, 8.7. HRMS m/z (ESI): calcd., 486.2172 [M + H]+; found, 486.2170 [M + H]+.
(Z)-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-N,N-diethyl-2-oxoindoline-5-sulfonamide (6)
2-Oxoindoline-5-sulfonyl chloride (2.32 g, 10.0 mmol), prepared by a modified method [32], was suspended in dichloromethane (20 ml). The resulting mixture was added dropwise a solution of triethylamine (1.21 g, 12.0 mmol) in dichloromethane (5 ml) and then a solution of diethylamine (0.90 g, 12.0 mmol) in dichloromethane (5 ml) was added. After stirring at room temperature for 8 h, the precipitate formed was filtrated, washed with dichloromethane, and purified by column chromatography (silica gel, 1:4 EtOAc–Hexane) to yield 1.90 g (71%) of N,N-diethyl-2-oxoindoline5-sulfonamide (16) as pale yellow crystals. Mp: 155–156 °C, UV λmax (MeOH), nm (logɛ): 289 (4.64). IR (ATR), cm−1: 3159, 1707, 1615. 1H NMR (400 MHz, methanol-d
4) δ, ppm: 7.70 (d, 1H, J = 6.8 Hz, ArH), 7.69 (s, 1H, ArH), 7.02 (d, 1H, J = 6.8 Hz, ArH) 3.62 (s, 2H, ArCH
2
), 3.21 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 1.13 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, methanol-d
4) δ, ppm: 179.6, 148.9, 134.7, 129.1, 128.2, 124.6, 110.6, 47.8, 43.4, 14.7. HRMS m/z (EI): calcd., 268.0882 [M]+; found, 268.0872 [M]+.
N,N-Diethyl-2-oxoindoline5-sulfonamide (16) obtained from above was used to condense with 3 to afford target compound 6 as described in general synthesis. Yield of 6: 57%, orange solids. Mp: 245–247 °C, UV λmax (MeOH), nm (logɛ): 429 (4.54). IR (ATR), cm−1: 2968, 1659, 1589. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 8.20 (d, 1H, J = 2.0 Hz, ArH), 7.79 (s, 1H, –C=CH–), 7.63 (dd, 1H, J = 8.0, 2.0 Hz, ArH), 7.11 (d, 1H, J = 8.0 Hz, ArH), 4.52 (s, 2H, Ar–CH
2
), 3.92 (t, 2H, J = 7.2 Hz, –NCH
2
CH2–), 3.47 (t, 2H, J = 7.2 Hz, –NCH2CH
2
–), 3.36 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 3.21 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.47 (s, 3H, Ar–CH
3
), 1.31 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2), 1.10 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.4, 169.4, 150.1, 142.1, 134.7, 133.8, 128.1, 127.5, 127.2, 127.0, 121.3, 118.8, 116.5, 111.4, 51.7, 48.5, 48.1, 43.2, 39.4, 14.7, 10.0, 8.7. HRMS m/z (ESI): calcd., 514.2497 [M + H]+; found, 514.2483 [M + H]+.
(Z)-N,N-Bis(2-chloroethyl)-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-2-oxoindoline-5-sulfonamide (7)
2-Oxoindoline-5-sulfonyl chloride (2.32 g, 10.0 mmol), prepared by a modified method [32], and bis(2-choroethyl)amine hydrochloride (2.14 g, 12.0 mmol) were suspended in dichloromethane (20 ml) and then was added dropwise a solution of triethylamine (2.23 g, 22.0 mmol) in dichloromethane (10 ml). After stirring at room temperature for 8 h, the precipitate formed was filtrated, washed with dichloromethane, and purified by column chromatography (silica gel, 1:4 EtOAc–Hexane). 2.13 g (63%) of N,N-bis(2-chloroethyl)-2-oxoindoline-5-sulfonamide (17) as pale yellow solids. Mp: 193–195 °C, UV λmax (MeOH), nm (logɛ): 289 (4.60). IR (ATR), cm−1: 3157, 1704, 1615. 1H NMR (400 MHz, CDCl3) δ, ppm: 7.76 (d, 1H, J = 8.4, ArH), 7.71 (s, 1H, ArH), 6.98 (d, 1H, J = 8.4, ArH), 3.70 (t, 4H, J = 7.0 Hz, –N(CH2
CH
2
Cl)2), 3.62 (s, 2H, ArCH
2
) 3.49 (t, 4H, J = 7.0 Hz, –N(CH
2
CH2Cl)2). 13C-NMR (100 MHz, dmso-d
6) δ, ppm: 176.4, 148.2, 130.3, 128.0, 123.3, 117.5, 113.5, 109.2, 50.3, 42.3, 39.5.
N,N-Bis(2-chloroethyl)-2-oxoindoline-5-sulfonamide (17) obtained from above was used to condense with 3 to afford target compound 7 as described in general synthesis. Purification of 7 was performed by recrystallization from THF. Yield of 7: 54%, orange solids. Mp: 236–237 °C, UV λmax (MeOH), nm (logɛ): 428 (4.58). IR (ATR), cm−1: 2917, 1651, 1574. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 8.29 (s, 1H, –C=CH–), 7.87 (s, 1H, ArH), 7.73 (d, 1H, J = 8.4 Hz, ArH,), 7.18 (d, 1H, J = 8.4, ArH), 4.60 (s, 2H, Ar–CH
2
), 3.98 (2H, J = 7.2 Hz, –NCH
2
CH2–) 3.73 (t, 4H, J = 7.2 Hz, –N(CH2CH
2
Cl)2), 3.53 (t, 6H, J = 7.2 Hz, –NCH2CH
2
–, –N(CH
2
CH2Cl)2), 3.39 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.50 (s, 3H, Ar–CH
3
), 1.31 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.5, 169.5, 150.4, 142.6, 133.8, 133.4, 128.4, 127.9, 127.6, 127.3, 121.4, 119.0, 116.3, 111.5, 52.2, 51.8, 48.5, 48.1, 43.1, 39.4, 10.0, 8.7. HRMS m/z (ESI): calcd., 582.1718 [M + H]+; found, 582.1703 [M + H]+.
(Z)-3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-2-oxo-N-phenylindoline-5-sulfonamide (8)
The requisite 2-oxo-N-phenylindoline-5-sulfonamide, for condensation with 3 to form target compound 8, was prepared in 83% yield by a modified method of amidation of 2-oxoindoline-5-sulfonyl chloride with aniline [32].
Yield of 8: 46%, orange solids. Mp: 225–228 °C, UV λmax (MeOH), nm (logɛ): 429 (4.47). IR (ATR), cm−1: 3213, 2963, 1667, 1557. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 8.12 (d, 1H, J = 1.6, ArH), 7.71 (s, 1H, –C=CH–), 7.59 (dd, 1H, J = 8.4, 1.6 Hz, ArH), 7.22–7.14, (m, 5H, ArH), 7.02 (d, 1H, J = 8.4 Hz, ArH), 4.56 (s, 2H, Ar–CH
2
), 3.95 (t, 2H, J = 6.0 Hz, –NCH
2
CH2–), 3.48 (t, 2H, J = 6.0 Hz, –NCH2CH
2
–), 3.37 (q, 4H, J = 7.0 Hz, –N(CH
2
CH3)2), 2.48 (s, 3H, Ar–CH
3
), 1.31 (t, 6H, J = 7.0 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.3, 169.4, 150.2, 142.3, 138.4, 133.9, 133.7, 130.2, 128.1, 127.5, 127.3, 126.8, 125.9, 122.2, 121.3, 118.8, 116.2, 111.1, 51.7, 48.5, 48.1, 39.4, 10.0, 8.7. HRMS m/z (ESI): calcd., 534.2182 [M + H]+; found, 534.2181 [M + H]+.
(Z)-3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-2-oxo-N-(4-(trifluoromethyl)phenyl)indoline-5-sulfonamide (9)
The requisite 2-oxo-N-(4-(trifluoromethyl)phenyl)indoline-5-sulfonamide, for condensation with 3 to form target compound 9, was prepared in 77% yield by a modified method of amidation of 2-oxoindoline-5-sulfonyl chloride with 4-(trifluoromethyl)aniline [32].
Yield of 9: 46%, orange solids. Mp: 231–234 °C, UV λmax (MeOH), nm (logɛ): 429 (4.32). IR (ATR), cm−1: 3162, 1634, 1582. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 8.21 (s, 1H, ArH), 7.75 (s, 1H, –C=CH–), 7.68 (dd, 1H, J = 8.0, 1.6 Hz, ArH), 7.52 (d, 2H, J = 8.4 Hz, ArH), 7.34 (d, 2H, J = 8.4 Hz, ArH), 7.05 (d, 1H, J = 8.0 Hz, ArH), 4.58 (s, 2H, Ar-CH
2
), 3.97 (t, 2H, J = 7.2 Hz, –NCH
2
CH2–), 3.50 (t, 2H, J = 7.2 Hz, –NCH2CH
2
–), 3.38 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.50 (s, 3H, Ar–CH
3
), 1.32 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2).
13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.4, 169.5, 150.4, 142.7, 142.3, 133.7, 128.3, 127.6, 127.5, 127.4, 127.4, 127.1, 126.9, 126.6, 126.6, 124.0, 121.4, 120.6, 118.8, 111.3, 51.8, 48.2, 48.1, 39.4, 39.5, 10.0, 8.7. HRMS m/z (ESI): calcd., 602.2060 [M + H]+; found, 602.2043 [M + H]+.
(Z)-N-(3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-2-oxoindolin-5-yl)methanesulfonamide (10)
The requisite N-(2-oxoindolin-5-yl)methanesulfonamide, for condensation with 3 to form target compound 10, was prepared in 86% yield by a modified method of mesylation of 5-aminooxindole with methanesulfonyl chloride [33].
Yield of 10: 54%, orange solids. Mp: 245–247 °C, UV λmax (MeOH), nm (logɛ): 395 (4.43). IR (ATR), cm−1: 3539, 3260, 1681, 1586. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 7.65 (s, 1H, –C=CH–), 7.61 (d, 1H, J = 2.0 Hz, ArH), 7.16 (dd, 1H, J = 8.4, 2.0 Hz, ArH), 6.99 (d, 1H, J = 8.4 Hz, ArH), 4.58 (s, 2H, Ar–CH
2
), 3.98 (t, 2H, J = 6.0 Hz, –NCH
2
CH2-), 3.51 (t, 2H, J = 6.0 Hz, –NCH2CH
2
–), 3.89 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 3.00 (s, 3H, –SO2CH
3
), 2.49 (s, 3H, Ar-CH
3
), 1.33 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.5, 169.8, 149.7, 137.2, 133.6, 133.1, 127.3, 126.9, 126.3, 123.4, 121.0, 117.9, 115.1, 111.9, 52.0, 48.6, 48.2, 39.5, 10.0, 8.8. HRMS m/z (ESI): calcd., 472.2028 [M + H]+; found, 472.2013 [M + H]+.
(Z)-3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-5-(trifluoromethyl)indolin-2-one (11)
Commercially available 5-trifluoromethyl-2-oxindoe was condensed with 7 to afford target compound 11 in a manner described above. Yield of 11: 60%, orange solids. Mp: 212–215 °C, UV λmax (MeOH), nm (logɛ): 415 (4.47). IR (ATR), cm−1: 3180, 1667, 1583. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 7.71 (d, 1H, J = 2.0 Hz, ArH), 7.57 (s, 1H, –C=CH–), 7.11 (dd, 1H, J = 8.4, 2.0 Hz, ArH), 7.04 (d, 1H, J = 8.4 Hz, ArH), 4.60 (s, 2H, Ar–CH
2
), 3.97 (t, 2H, J = 6.4 Hz, –NCH
2
CH2–), 3.49 (t, 2H, J = 6.4 Hz, –NCH2CH
2
–), 3.37 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.48 (s, 3H, Ar–CH
3
), 1.31 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.6, 169.7, 150.1, 145.8, 137.9, 133.6, 127.8, 127.7, 127.0, 123.1, 121.3, 117.3, 113.3, 112.0, 51.8, 48.6, 48.1, 39.5, 9.9, 8.7. HRMS m/z (ESI): calcd., 464.2241 [M + NH4]+; found, 464.1999 [M + NH4]+.
(Z)-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-5-nitroindolin-2-one (12)
The requisite 5-nitrooxindole, for condensation with 3 to form target compound 12, was prepared in 96% yield by a modified method of nitration of oxindole with HNO3/H2SO4 [33].
Yield of 12: 62%, light yellow solids. Mp: 229–230 °C, UV λmax (MeOH), nm (logɛ): 249 (4.62). IR (ATR), cm−1: 2971, 1671, 1553, 1551. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 8.52 (d, 1H, J = 2.0 Hz, ArH), 8.11 (dd, 1H, J = 8.4, 2.0 Hz, ArH), 7.82 (s, 1H, –C=CH–), 7.11 (d, 1H, J = 8.4 Hz, ArH), 4.60 (s, 2H, Ar–CH
2
), 3.99 (t, 2H, J = 6.0 Hz, –NCH
2
CH2–), 3.52 (t, 2H, J = 6.0 Hz, –NCH2CH
2
–), 3.40 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.51 (s, 3H, Ar–CH
3
), 1.34 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.6, 169.4, 150.5, 144.6, 143.8, 133.7, 128.7, 127.9, 127.1, 124.1, 121.5, 115.9, 115.2, 111.0, 51.8, 48.6, 48.1, 39.4, 10.0, 8.7. HRMS m/z (ESI): calcd., 424.1979 [M + H]+; found, 424.1992 [M + H]+.
(Z)-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-5-methoxyindolin-2-one (13)
The requisite 5-methoxyoxindole, for condensation with 3 to form target compound 13, was prepared in 78% yield by a modified method of Wolff-Kishner reduction of 5-methoxyisatin in the presence of N2H4 under basic conditions [34].
Yield of 13: 59%, orange solids. Mp: 214–216 °C, UV λmax (MeOH), nm (logɛ): 396 (4.69). IR (ATR), cm−1: 3028, 1672, 1577. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 7.62 (s, 1H, –C=CH–), 7.25 (d, 1H, J = 2.0 Hz, ArH), 6.91 (d, 1H, J = 8.4 Hz, ArH), 6.79 (dd, 1H, J = 8.4, 2.0 Hz, ArH), 4.59 (s, 2H, Ar–CH
2
), 3.98 (t, 2H, J = 6.0 Hz, –NCH
2
CH2–), 3.82 (s, 3H, –OCH
3
), 3.51 (t, 2H, J = 6.0 Hz, –NCH2CH
2
–), 3.39 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.47 (s, 3H, Ar–CH
3
), 1.33 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.5, 169.9, 157.1, 149.4, 133.5, 133.2, 127.3, 126.2, 125.5, 120.9, 118.9, 114.3, 112.0, 105.8, 55.6, 51.9, 49.2, 48.3, 39.5, 9.9, 8.8. HRMS m/z (ESI): calcd., 409.2234 [M + H]+; found, 409.2241 [M + H]+.
(Z)-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-5-hydroxyindolin-2-one (14)
The requisite 5-hydroxyoxindole, for condensation with 3 to form target compound 14, was prepared in 56% yield by a modified method of demethylation of 5-methoxyoxindole with a solution of hydrobromic acid in acetic acid [30].
Yield of 14: 59%, orange solids. Mp: 240–242 °C, UV λmax (MeOH), nm (logɛ): 359 (4.70). IR (ATR), cm−1: 3368, 3171, 1657, 1581. 1H-NMR (400 MHz, acetic acid -d
4) δ, ppm: 7.50 (s, 1H, –C=CH–), 7.11 (d, 1H, J = 2.0 Hz, ArH), 6.82 (d, 1H, J = 8.4 Hz, ArH), 6.72 (dd, 1H, J = 8.4, 2.0 Hz, ArH), 4.53 (s, 2H, Ar–CH
2
), 3.96 (t, 2H, J = 6.0 Hz, –NCH
2
CH2–), 3.50 (t, 2H, J = 6.0 Hz, –NCH2CH
2
–), 3.38 (q, 4H, J = 7.2 Hz, –N(CH
2
CH3)2), 2.45 (s, 3H, Ar–CH
3
), 1.33 (t, 6H, J = 7.2 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.4, 169.9, 153.4, 149.3, 133.5, 132.6, 127.4, 125.9, 125.2, 120.7, 118.9, 115.4, 111.9, 106.9, 51.9, 48.6, 48.5, 48.2, 39.5, 9.9, 8.8. HRMS m/z (ESI): calcd., 395.2067 [M + H]+; found, 395.2078 [M + H]+.
(Z)-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)methylene)-5-mercaptoindolin-2-one (15)
The requisite 5-mercaptooxindole, for condensation with 3 to form target compound 15, was prepared in 90% yield by treatment of 2-oxoindoline-5-sulfonyl chloride with triphenylphosphine [29].
Yield: 62%, orange solids. Mp: 257–260 °C, UV λmax (MeOH), nm (logɛ): 394 (4.43). IR (ATR), cm−1: 3368, 3171, 1657, 1581. 1H-NMR (400 MHz, acetic acid-d
4) δ, ppm: 7.66 (s, 1H, –C=CH–), 7.51–7.48 (m, 2H, ArH), 7.06 (d, 1H, J = 8.4 Hz, ArH), 4.67 (s, 2H, Ar–CH2), 4.00 (t, 2H, J = 5.6 Hz, –NCH
2
CH2–), 3.49 (t, 2H, J = 5.6 Hz, –NCH2CH
2
–), 3.39 (q, 4H, J = 7.0 Hz, –N(CH
2
CH3)2), 2.31 (s, 3H, Ar–CH
3
), 1.33 (t, 6H, J = 7.0 Hz, –N(CH2CH
3
)2). 13C-NMR (100 MHz, acetic acid-d
4) δ, ppm: 171.3, 169.4, 149.8, 140.0, 133.5, 132.9, 131.8, 127.2, 127.0, 125.9, 123.9, 120.9, 117.2, 112.2, 51.8, 48.6, 48.1, 39.5, 9.9, 8.7. HRMS m/z (ESI): calcd., 410.1779 [M]+; found, 410.1771 [M]+.
Biology
Cell culture
The HCT116 (human colon cancer cells, BCRC 60349) and Detroit 551 (human normal fibroblast cells, BCRC 60118) were maintained in DMEM (Gibco, Grand Island, NY, USA) containing 10% FBS (HyClone, Logan, UT, USA). NCI-H460 (BCRC 60373) and 786-O (BCRC 60243) cells were maintained in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10% FBS (HyClone, Logan, UT, USA). HUVEC (BCRC, H-UV001) was maintained in medium 199 (Sigma, St. Louis MO, USA) with 25 U/ml heparin (Sigma, St. Louis MO, USA), 30 μg/ml endothelial cell growth supplement (ECGS, Sigma, St. Louis MO, USA) containing 10% FBS (HyClone, Logan, UT, USA), and incubated at 37 °C in a 5% CO2 atmosphere.
Cell proliferation assay
The cells incubated as above were plated at a density of 2000 cells/well (cancer cells) [41, 44, 45] or 10,000 cells/well (Detroit 551) [46] on a 96-well plate for 24 h. Serial dilutions of indicated compounds were added and incubated for additional 72 h. At the end of the incubation, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MTT formazan crystals formed were dissolved in DMSO, and the absorbance at 570 nm was recorded using a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [46].
Acute cytotoxicity
The acute cytotoxicity effect of compounds 7 and 13–15, and sunitinib was determined by Cell-Counting-Kit-8 (Dojindo, Rockville, MD, USA) assay on HCT116, NCI-460, 786-O, and Detroit 551 cells according to the manufacturer’s protocol. Cells were seeded at 5000 cells/well on a 96-well plate for 24 h. The indicated compounds in different concentrations (100 μl) were added to cells. After 6 h, old medium was aspirated, and the cells were washed three times with PBS. WST-8 (Dojindo, Rockville, MD, USA) (10 µl) was added to each well, and the absorbance of the plate was recorded at 450 nm on a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Image cytometry
Cell cycle profiles of HCT116 cells were determined with an NC-3000 image cytometer (ChemoMetec, Allerod, Denmark) in accordance with manufacturer’s protocol. Briefly, cells were seeded at 200,000 cells/well on a 6-well plate for 24 h. Two ml of indicated compounds (5.0 μM for sunitinib and 7, 3.0 μM for 13–15) were added to cells. After incubation for 24 h, 100,000 cells were harvested and centrifuged at 400 g at room temperature for 5 min, washed once with PBS (50 μM), and resuspended in lysis buffer (50 μl) (ChemoMetec, Allerod, Denmark) containing 10 μg/ml of 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI, ChemoMetec, Allerod, Denmark). The cells were incubated at 37 °C for 5 min and then stabilization buffer (50 μl) (ChemoMetec, Allerod, Denmark) was added to the mixture. The cellular fluorescence was measured with an NC-3000 image cytometer using NC-SlideA8 (ChemoMetec, Allerod, Denmark). The NC-3000 software (ChemoMetec, Allerod, Denmark) was used for image acquisition, image analysis and quantification, and data visualization.
In-vitro tube formation assay
The in vitro tube formation assay was assessed using ibidi μ-Slides (15-well, ibidi GmbH, Martinsried, Germany) in accordance with manufacturer’s protocol. Briefly, growth factor reduced Matrigel (10 μl) (Sigma, St. Louis MO) was added to the inner well of ibidi μ-Slides, and incubated at 37 °C for 1 h. HUVEC cells were harvested by centrifugation, and the cell suspension was adjusted to 200,000 cells/ml by 10 ng/ml VEGF contained growth medium (M199) with or without indicated compounds 7, 13, 14, 15, or sunitinib in different concentrations (1.0, 0.50 and 0.10 μM). 10,000 HUVEC cells in 50 μl of above growth medium was added to Matrigel (Sigma, St. Louis MO, USA) coated ibidi μ-Slides. After 6 h of incubation at 37 °C, the supernatant was discarded, and 50 μl of serum-free medium with diluted calcein AM (6.25 μg/ml) was added to above ibidi μ-Slides. After incubation in the dark at room temperature for 30 min, the μ-Slides were washed with PBS (50 μl) and fluorescence pictures were taken at 485 nm with a Leica DM1000 LED microscope (Leica Microsystems, Wetzlar, Hessen, Germany).
In-vitro kinase assay
The Reaction Biology Corporation (http://www.reactionbiology.com) HotSpot assay platform was used to determine the inhibitory activity of 7, 13, 14, 15, and sunitinib against VEGFR-2, PDGFRβ, and Aurora A, measured by quantifying the amount of 33P incorporated into the substrate in the presence of the test compound [47]. Briefly, specific kinase and substrate and required cofactors were prepared in reaction buffer. Test compounds were added to the reaction and after 20 min a mixture of ATP (Sigma, St. Louis MO, USA) and 33P ATP (Perkin Elmer, Waltham MA, USA) was added to make a final concentration of 10.0 μM. Reactions were stood at room temperature for 120 min, and then the reactions were spotted onto a P81 ion exchange filter paper (Whatman Inc., Piscataway, NJ, USA). Unbound phosphate was removed by extensive washing of filters in 0.1% phosphoric acid. Kinase activity data was reported as the percent remaining kinase activity in test compounds compared to the solvent control dimethyl sulfoxide (DMSO).
Molecular modeling
Ligands-receptor docking calculation was carried out in accordance with the LibDock protocol. Briefly, receptor active site and ligands were characterized into polar and apolar hotspots. The ligand poses were placed into the receptor site in accordance with hotspots map. In this study CHARMm force field was used for energy minimization of the ligand molecules and ligand–receptor binding. The binding sphere was defined based on the protein data bank (PDB) definition. Conformations of ligands were generated by the BEST method.
Statistical analysis
Statistical calculations were carried out with GraphPad Prism vision 5. Results are reported as the mean ± SD. Statistical significance was determined by the unpaired student’s t test.