Synthesis and biological evaluation of novel indole-2-one and 7-aza-2-oxindole derivatives as anti-inflammatory agents

Abstract

Sepsis, a typically acute inflammatory disease, is the biggest cause of death in ICU (intensive care unit). Novel anti-inflammatory alternatives are still in urgent need. In this study, we designed and synthesized 30 indole-2-one and 7-aza-2-oxindole derivatives based on the skeleton of tenidap, and their anti-inflammatory activity was determined by evaluating the inhibitory potency against lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF)-α and interleukin (IL)-6 release in RAW264.7 macrophages. Quantitative SAR (structure–activity relationship) analysis revealed that a high molecular polarizability and low lipid/water partition coefficient (ALogP) in indole-2-one are beneficial for anti-inflammatory activity. Moreover, compounds 7i and 8e inhibited the expression of TNF-α, IL-6, COX-2, PGES, and iNOS in LPS-stimulated macrophages, and 7i exhibited a significant protection from LPS-induced septic death in mouse models. These data present a series of new indole-2-one compounds with potential therapeutic effects in acute inflammatory diseases.

Keywords:

• anti-inflammation
• macrophages
• sepsis

Supplementary materials

The spectral data of new or unreported compounds

(Z)-3-[4-(allyloxy)-2-hydroxybenzylidene]-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6a): Yellow powder, 32.2% yield, mp: 212.5°C–213.3°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 4.67 (2H, d, J=6.6 Hz, O−CH₂), 5.29 (1H, dd, J₁=1.8 Hz, J₂=10.2 Hz, −C=CH₂), 5.44 (1H, dd, J₁=1.8 Hz, J₂=17.4 Hz, −C=CH₂), 6.04–6.07 (1H, m, −CH=C), 6.72 (1H, d, J=8.4 Hz, Ar-H⁵), 6.74 (1H, s, Ar-H³), 6.89–6.91 (1H, m, 7-aza-2-oxindole-H⁵), 7.67 (1H, d, J=8.4 Hz, 7-aza-2-oxindole-H⁴), 7.77 (1H, d, J=8.4 Hz, 7-aza-2-oxindole-H⁶), 7.86 (1H, d, J=8.4 Hz, Ar-H⁶), 7.98 (1H, s, Ar-CH). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 168.76, 162.08, 158.67, 156.72, 156.65, 147.62, 133.49, 131.19, 129.12, 123.79, 118.16, 117.44, 115.55, 106.83, 104.91, 100.45, 69.04. ESI-MS m/z: 293.5 (M − 1)⁻.

(Z)-3-[4-(allyloxy)benzylidene]-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6b): Yellow powder, 30.4% yield, mp: 135.6°C–136.9°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 4.67 (2H, d, J=4.8 Hz, O−CH₂), 5.30 (1H, dd, J₁=1.8 Hz, J₂=10.2 Hz, −C=CH₂), 5.44 (1H, dd, J₁=1.8 Hz, J₂=17.4 Hz, −C=CH₂), 6.04–6.10 (1H, m, −CH=C), 6.91–6.93 (1H, m, 7-aza-2-oxindole-H⁵), 7.12 (2H, d, J=9.0 Hz, Ar-H^3,5), 7.72 (2H, d, J=9.0 Hz, Ar-H^2,6), 7.83 (1H, s, Ar-CH), 7.93 (1H, d, J=7.8 Hz, 7-aza-2-oxindole-H⁴), 8.46 (1H, d, J=8.4 Hz, 7-aza-2-oxindole-H⁶). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 168.74, 160.09, 156.94, 154.74, 147.84, 138.16, 134.87, 133.52, 132.10, 129.20, 126.54, 124.04, 122.37, 118.07, 117.47, 115.30, 68.63. ESI-MS m/z: 279.0 (M + 1)⁺.

(Z)-3-(2,4,6-trimethoxybenzylidene)-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6c): Yellow powder, 77.2% yield, mp: 176.7°C–178.1°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 3.79 (9H, s, O−CH₃ ×3), 6.19 (2H, s, Ar-H^3,5), 6.81–6.83 (1H, m, 7-aza-2-oxindole-H⁵), 7.15 (1H, d, J=6.0 Hz, 7-aza-2-oxindole-H⁴), 7.88 (1H, s, Ar-CH), 8.04 (1H, d, J=6.0 Hz, 7-aza-2-oxindole-H⁶). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 175.92, 166.24, 165.93, 163.64 ×2, 159.23, 143.65, 130.65, 127.75, 116.04, 108.12, 105.04, 91.03 ×2, 55.86 ×3. ESI-MS m/z: 313.2 (M + 1)⁺.

(Z)-3-(2-chlorobenzylidene)-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6d): Yellow powder, 55.2% yield, mp: 198.5°C–199.5°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 6.79–6.81 (1H, m, 7-aza-2-oxindole-H⁵), 7.35–7.43 (3H, m, Ar-H^4–6), 7.53 (1H, d, J=7.8 Hz, Ar-H³), 7.55 (1H, d, J=6.0 Hz, 7-aza-2-oxindole-H⁴), 7.66 (1H, d, J=6.0 Hz, 7-aza-2-oxindole-H⁶), 7.99 (1H, s, Ar-CH). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 167.92, 157.45, 148.91, 133.57, 133.30, 132.59, 131.90, 130.59, 130.17, 129.92, 128.29, 127.81, 117.74, 115.16. ESI-MS m/z: 256.9 (M + 1)⁺.

(Z)-3-[(1-methyl-1H-indol-3-yl)methylene]-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6e): Yellow powder, 50.1% yield, mp: 296.4°C–297.5°C. ESI-MS m/z: 276.0 (M + 1)⁺ (see Wood et al¹).

(Z)-3-[4-(piperazin-1-yl)benzylidene]-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6f): Orange powder, 74.3% yield, mp: 72.3°C–74.1°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 2.82 (4H, t, J=5.4 Hz, N−CH₂ ×2), 3.26 (4H, t, J=5.4 Hz, −N−CH₂ ×2), 6.91–6.93 (1H, m, 7-aza-2-oxindole-H⁵), 7.04 (2H, d, J=9.0 Hz, Ar-H^3,5), 7.64 (2H, d, J=9.0 Hz, Ar-H^2,6), 7.72 (1H, s, Ar-CH), 7.95 (1H, d, J=4.8 Hz, 7-aza-2-oxindole-H⁴), 8.05 (1H, d, J=4.8 Hz, 7-aza-2-oxindole-H⁶). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.11, 156.48, 152.76, 147.07, 138.95, 135.06, 132.33 ×2, 128.75, 121.34, 117.35, 113.94 ×2, 113.22, 63.01 ×2, 47.94 ×2. ESI-MS m/z: 306.8 (M + 1)⁺.

(Z)-3-[4-(4-methylpiperazin-1-yl)benzylidene]-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6g): Orange oil, 53.7% yield. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 2.24 (3H, s, N−CH₃), 2.46 (4H, t, J=5.4 Hz, C−N−CH₂ ×2), 3.38 (4H, t, J=5.4 Hz, −N−CH₂ ×2), 6.91–6.93 (1H, m, 7-aza-2-oxindole-H⁵), 7.06 (2H, d, J=9.0 Hz, Ar-H^3,5), 7.65 (2H, d, J=9.0 Hz, Ar-H^2,6), 7.72 (1H, s, Ar-CH), 7.95 (1H, d, J=7.2 Hz, 7-aza-2-oxindole-H⁴), 8.01 (1H, d, J=7.2 Hz, 7-aza-2-oxindole-H⁶). ¹³C-NMR (600 MHz, DMSO-d₆): δ (ppm): 169.09, 156.48, 152.27, 147.13, 138.90, 135.02, 132.28 ×2, 123.05, 121.55, 117.36, 116.19, 114.13 ×2, 54.49 ×2, 46.77 ×2, 45.85. ESI-MS m/z: 321.01 (M + 1)⁺.

(Z)-3-(4-morpholinobenzylidene)-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6h): Orange powder, 77.1% yield, mp: 57.7°C–58.5°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 3.32 (4H, t, J=5.4 Hz, O−CH₂ ×2), 3.71 (4H, t, J=5.4 Hz, −N−CH₂ ×2), 6.91–6.94 (1H, m, 7-aza-2-oxindole-H⁵), 7.03 (2H, d, J=9.0 Hz, Ar-H^3,5), 7.64 (1H, s, Ar-CH), 7.72 (2H, d, J=9.0 Hz, Ar-H^2,6), 8.00 (1H, d, J=6.0 Hz, 7-aza-2-oxindole-H⁴), 8.04 (1H, d, J=6.0 Hz, 7-aza-2-oxindole-H⁶). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.02, 156.59, 155.15, 147.24, 138.80, 134.90, 131.62 ×2, 128.81, 126.93, 121.91, 117.35, 113.42 ×2, 66.03 ×2, 46.82 ×2. ESI-MS m/z: 307.91 (M + 1)⁺.

(Z)-4-{4-[(2-oxo-1H-pyrrolo[2,3-b]pyridin-3(2H)-ylidene)methyl]phenyl}piperazine-1-carbaldehyde (6i): Yellow powder, 32.3% yield, mp: 105.3°C–106.6°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 3.52 (8H, t, J=5.4 Hz, −N−CH₂ ×4), 6.91–6.94 (1H, m, 7-aza-2-oxindole-H⁵), 7.11 (2H, d, J=9.0 Hz, Ar-H^3,5), 7.67 (2H, d, J=9.0 Hz, Ar-H^2,6), 7.74 (1H, s, Ar-CH), 7.95 (1H, d, J=7.2 Hz, 7-aza-2-oxindole-H⁴), 8.00 (1H, d, J=7.2 Hz, 7-aza-2-oxindole-H⁶), 8.10 (1H, s, −N−CHO). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.01, 161.44 ×3, 147.27, 138.72, 134.96, 132.24 ×2, 128.84, 123.64, 117.36, 114.77 ×2, 113.97, 45.64 ×2, 44.53 ×2. ESI-MS m/z: 334.98 (M + 1)⁺.

(Z)-3-[4-(4-acryloylpiperazin-1-yl)benzylidene]-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6j): Orange oil, 31.9% yield. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 3.75 (8H, t, J=6.6 Hz, −N−CH₂ ×4), 5.72 (1H, dd, J₁=1.8 Hz, J₂=10.8 Hz, CO−C=CH), 6.15 (1H, dd, J₁=1.8 Hz, J₂=14.4 Hz, CO−C=CH), 6.81–6.88 (1H, m, CO−CH=C), 6.92–6.94 (1H, m, 7-aza-2-oxindole-H⁵), 7.04 (2H, d, J=7.8 Hz, Ar-H^3,5), 7.08 (1H, d, J=9.0 Hz, 7-aza-2-oxindole-H⁴), 7.66 (2H, d, J=7.8 Hz, Ar-H^2,6), 7.73 (1H, s, Ar-CH), 8.06 (1H, d, J=7.8 Hz, 7-aza-2-oxindole-H⁶). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 171.77, 167.20, 164.58, 151.88, 147.22, 138.80, 134.98, 132.28 ×2, 131.74, 128.87, 128.35, 127.80, 126.32, 117.36, 114.32 ×2, 45.88 ×2, 37.39 ×2. ESI-MS m/z: 360.9 (M + 1)⁺.

(Z)-3-{4-[4-(3-chloropropanoyl)piperazin-1-yl]benzylidene}-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (6k): Orange powder, 34.4% yield, mp: 45.4°C–46.9°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 3.05 (2H, t, J=7.2 Hz, OC−CH₂), 3.39 (4H, t, J=6.0 Hz, OC−N−CH₂ ×2), 3.62 (4H, t, J=6.0 Hz, −N−CH₂ ×2), 3.82 (2H, t, J=7.2 Hz, −CH₂−Cl), 6.91–6.93 (1H, m, 7-aza-2-oxindole-H⁵), 7.07 (2H, d, J=9.0 Hz, Ar-H^3,5), 7.66 (2H, d, J=9.0 Hz, Ar-H^2,6), 7.73 (1H, s, Ar-CH), 7.95 (1H, d, J=7.8 Hz, 7-aza-2-oxindole-H⁴), 8.00 (1H, d, J=7.8 Hz, 7-aza-2-oxindole-H⁶). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.04, 167.28, 156.56, 154.24, 139.74, 138.80, 134.99, 132.28 ×2, 128.35, 127.79, 117.36, 114.31 ×2, 113.52, 45.66 ×4, 40.78, 35.45. ESI-MS m/z: 397.33 (M + 1)⁺.

(Z)-3-[4-(allyloxy)-2-hydroxybenzylidene]indolin-2-one (7a): Yellow oil, 37.2% yield. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 4.59 (2H, d, J=4.8 Hz, O−CH₂), 5.29 (1H, dd, J₁ =1.8 Hz, J₂ =10.2 Hz, −C=CH₂), 5.42 (1H, dd, J₁=1.8 Hz, J₂=16.2 Hz, −C=CH₂), 6.02–6.08 (1H, m, −CH=C), 6.53 (1H, s, Ar-H³), 6.57 (1H, d, J=8.4 Hz, Ar-H⁵), 6.85 (1H, d, J=7.8 Hz, indolin-2-one-H⁷), 7.14–7.20 (2H, m, indolin-2-one-H^5,6), 7.62 (1H, d, J=8.4 Hz, Ar-H⁶), 7.66 (1H, s, Ar-CH), 8.88 (1H, d, J=8.4 Hz, indolin-2-one-H⁴). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.31, 161.47, 158.54, 142.52, 139.12, 133.62, 132.54, 130.86, 129.35, 124.55, 122.19, 121.11, 117.94, 114.55, 110.04, 106.10, 102.15, 68.50. ESI-MS m/z: 292.7 (M − 1)⁻.

(Z)-3-[4-(allyloxy)benzylidene]indolin-2-one (7b): Yellow powder, 60.1% yield, mp: 165.7°C–166.3°C. ESI-MS m/z: 278.3 (M + 1)⁺ (see Chen et al²).

(Z)-3-(2,4,6-trimethoxybenzylidene)indolin-2-one (7c): Yellow powder, 83.1% yield, mp: 178.1°–179.0°C. ESI-MS m/z: 311.9 (M + 1)⁺ (see Yang et al³).

(Z)-3-[2-(trifluoromethyl)benzylidene]indolin-2-one (7d): Yellow powder, 73% yield, mp: 149.5°C–151.0°C. ESI-MS m/z: 290.2 (M + 1)⁺ (see Zhang et al⁴).

(Z)-3-[4-(piperazin-1-yl)benzylidene]indolin-2-one (7e): Orange powder, 80.4% yield, mp: 225.4°C–226.9°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 2.82 (4H, t, J=5.4 Hz, N−CH₂ ×2), 3.25 (4H, t, J=5.4 Hz, −N−CH₂ ×2), 6.80 (2H, d, J=7.8 Hz, Ar-H^3,5), 7.02 (1H, d, J=8.4 Hz, indole-2-one-H⁷), 7.11–7.14 (1H, m, indole-2-one-H⁵), 7.17–7.20 (1H, m, indole-2-one-H⁶), 7.52 (1H, s, Ar-CH), 7.76 (2H, d, J=7.8 Hz, Ar-H^2,6), 8.42 (1H, d, J=8.4 Hz, indole-2-one-H⁴). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.47, 142.49, 139.94, 134.68, 131.84 ×2, 129.24, 127.56, 126.10, 123.99, 123.40, 121.11, 113.97 ×2, 113.30, 48.17 ×2, 45.65 ×2. ESI-MS m/z: 306.1 (M + 1)⁺.

(Z)-3-[4-(4-methylpiperazin-1-yl)benzylidene]indolin-2-one (7f): Orange powder, 82.3% yield, mp: 191.6°C–193.0°C. ESI-MS m/z: 320.19 (M + 1)⁺ (see Furuta et al⁵).

(Z)-3-(4-morpholinobenzylidene)indolin-2-one (7g): Orange powder, 85.3% yield, mp: 239.3°C–240.1°C. ESI-MS m/z: 307.09 (M + 1)⁺ (see Vieth et al⁶).

(Z)-4-{4-[(2-oxoindolin-3-ylidene)methyl]phenyl)}piperazine-1-carbaldehyde (7h): Yellow powder, 45.7% yield, mp: 204.7°C–205.6°C. ESI-MS m/z: 334.09 (M + 1)⁺ (see Ravindranathan et al⁷).

(Z)-3-[4-(4-acryloylpiperazin-1-yl)benzylidene]indolin-2-one (7i): Orange powder, 27.5% yield, mp: 135.6°C–136.9°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 3.69 (8H, t, J=5.4 Hz, −N−CH₂ ×4), 5.71 (1H, dd, J₁=1.8 Hz, J₂ =10.8 Hz, CO−C=CH), 6.15 (1H, dd, J₁=1.8 Hz, J₂=16.8 Hz, CO−C=CH), 6.80–6.84 (1H, m, CO-CH=C), 6.88 (2H, d, J=7.8 Hz, Ar-H^3,5), 7.04 (1H, d, J=9.0 Hz, indole-2-one-H⁷), 7.12–7.15 (1H, m, indole-2-one-H⁵), 7.18–7.21 (1H, m, indole-2-one-H⁶), 7.53 (1H, s, Ar-CH), 7.74 (2H, d, J=7.8 Hz, Ar-H^2,6), 8.44 (1H, d, J=9.0 Hz, indole-2-one-H⁴). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.44, 164.57, 151.54, 142.56, 136.84, 134.63, 131.79 ×2, 129.38, 128.29, 127.79, 123.99, 122.11, 121.74, 121.15, 114.37 ×2, 113.66, 47.43 ×2, 44.75 ×2. ESI-MS m/z: 360.41 (M + 1)⁺. HRMS (ESI) calculated for C₂₂H₂₁N₃O₂(MH+), 360.1707; found, 360.1711.

(Z)-3-{4-[4-(3-chloropropanoyl)piperazin-1-yl]benzylidene} indolin-2-one (7j): Orange powder, 31.8% yield, mp: 77.3°C–78.5°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 2.88 (2H, t, J=6.6 Hz, CO−CH₂), 3.38 (4H, t, J=5.4 Hz, CO−N−CH₂ ×2), 3.68 (4H, t, J=5.4 Hz, −N−CH₂ ×2), 3.88 (2H, t, J=6.6 Hz, −CH₂−Cl), 6.87 (2H, d, J=7.8 Hz, Ar-H^3,5), 6.96 (1H, d, J=9.0 Hz, indole-2-one-H⁷), 7.01–7.04 (1H, m, indole-2-one-H⁵), 7.18–7.21 (1H, m, indole-2-one-H⁶), 7.75 (1H, s, Ar-CH), 7.81 (2H, d, J=7.8 Hz, Ar-H^2,6), 8.34 (1H, d, J=9.0 Hz, indole-2-one-H⁴). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.40, 168.14, 142.57, 136.81, 134.63, 131.79 ×2, 129.37, 126.00, 124.00, 121.74, 121.13, 120.92, 114.38 ×2, 110.12, 47.20, 46.90, 44.48, 44.42, 40.79, 35.46. ESI-MS m/z: 395.95 (M + 1)⁺.

(Z)-3-[4-(4-benzoylpiperazin-1-yl)benzylidene]indolin-2-one (7k): Orange powder, 40.3% yield, mp: 204.2°C–205.3°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 3.47 (4H, t, J=5.4 Hz, −N−CH₂ ×2), 3.77 (4H, t, J=5.4 Hz, CO−N−CH₂ ×2), 6.79 (2H, d, J=7.8 Hz, Ar-H^3,5), 7.06 (1H, d, J=9.0 Hz, indole-2-one-H⁷), 7.12–7.14 (1H, m, indole-2-one-H⁵), 7.18–7.20 (1H, m, indole-2-one-H⁶), 7.45–7.47 (3H, m, CO-Ar-H^3–5), 7.53 (1H, s, Ar-CH), 7.74 (2H, d, J=7.8 Hz, Ar-H^2,6), 7.62–7.66 (2H, m, CO-Ar-H^2,6), 8.44 (1H, d, J=9.0 Hz, indole-2-one-H⁴). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.32, 151.59, 142.58, 136.79, 135.99, 131.78, 129.86, 129.40, 128.69 ×2, 127.24 ×3, 125.99, 124.11, 124.07, 122.09, 121.73, 121.13, 114.52 ×2, 113.80, 47.63 ×2, 44.55 ×2. ESI-MS m/z: 409.9 (M + 1)⁺.

(Z)-5-chloro-3-[(2-mercapto-1H-benzo[d]imidazol-6-yl) imino]indolin-2-one (8a): Orange powder, 67.3% yield, mp: >300°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 6.87 (1H, d, J=10.2 Hz, benzoimidazol-H⁵), 6.98 (1H, d, J=10.2 Hz, indolin-2-one-H⁶), 7.04 (1H, s, benzoimidazol-H⁷), 7.09 (1H, d, J=10.2 Hz, benzoimidazol-H⁴), 7.46 (1H, d, J=10.2 Hz, indolin-2-one-H⁷), 7.55 (1H, s, indolin-2-one-H⁴). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.03, 163.53, 154.28, 145.96, 145.04, 133.85, 133.35, 130.58, 125.40, 124.58, 122.24, 117.12, 113.09, 110.47, 101.84. ESI-MS m/z: 329.78 (M + 1)⁺.

(Z)-1-{2-[2-(5-chloro-2-oxoindolin-3-ylidene)hydrazinyl]-2-oxoethyl}pyridin-1-iumchloride (8b): Yellow powder, 78.9% yield, mp: 235.5°C–236.7°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 6.22 (2H, s, −N−CO−CH₂−), 7.05 (1H, d, J=8.4 Hz, indolin-2-one-H⁶), 7.48 (1H, d, J=8.4 Hz, indolin-2-one-H⁷), 7.59 (1H, s, indolin-2-one-H⁴), 8.27 (2H, m, pyridin-H^3,5), 8.73 (1H, m, pyridin-H⁴), 9.11 (2H, d, J=5.4 Hz, pyridin-H^2,6). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 169.04, 163.53, 153.28, 146.91, 146.72, 134.85, 133.35, 125.41, 125.30, 123.24, 118.22, 115.21, 113.09, 110.50, 40.35. ESI-MS m/z: 315.19 (M − Cl)⁺.

(Z)-5-chloro-3-[(2-methyl-1H-indol-5-yl)imino]indolin-2-one (8c): Yellow powder, 79.0% yield, mp: 167.6°C–168.5°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 2.36 (3H, s, methylindole-CH₃), 6.50 (1H, s, methylindole-H³), 6.74 (1H, d, J=8.4 Hz, methylindole-H⁶), 6.84 (1H, d, J=7.8 Hz, indolin-2-one-H⁶), 6.90 (1H, d, J=8.4 Hz, methylindole-H⁷), 7.13 (1H, s, indolin-2-one-H⁴), 7.21 (1H, s, methylindole-H⁴), 7.33 (1H, d, J=7.8 Hz, indolin-2-one-H⁷). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 178.34, 163.98, 152.30, 145.49, 141.21, 135.96, 133.70, 133.46, 129.09, 126.05, 125.62, 124.85, 124.17, 117.19 ×2, 111.39, 13.44. ESI-MS m/z: 310.45 (M + 1)⁺.

(Z)-5-chloro-3-(quinolin-6-ylimino)indolin-2-one (8d): Yellow powder, 79.8% yield, mp: 197.3°C–198.2°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 7.49–7.51 (2H, m, quinolin-H^7,8), 7.56–7.58 (2H, m, indolin-2-one-H⁶, quinolin-H³), 7.65 (1H, s, indolin-2-one-H⁴), 7.94 (1H, d, J=8.4 Hz, indolin-2-one-H⁷), 8.15 (1H, s, quinolin-H⁵), 8.35 (1H, d, J=7.8 Hz, quinolin-H⁴), 8.90 (1H, d, J=7.8 Hz, quinolin-H²). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 163.35, 154.67, 150.26, 147.88, 146.20, 145.99, 135.99, 134.24, 130.95, 128.28, 125.51, 124.81, 123.93, 122.47, 117.11 ×2, 113.48. ESI-MS m/z: 308.37 (M + 1)⁺.

(Z)-5-chloro-3-[(2,3-dihydro-1H-inden-5-yl)imino]indolin-2-one (8e): Orange powder, 83.7% yield, mp: 269.5°C–270.9°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 2.03–2.09 (2H, m, inden-H⁴), 2.90 (4H, t, J=7.2 Hz, inden-H^3.5), 6.46 (1H, s, inden-H²), 6.76 (1H, d, J=8.4 Hz, inden-H⁷), 6.91 (1H, d, J=8.4 Hz, inden-H⁶), 7.31 (1H, d, J=8.4 Hz, indolin-2-one-H⁶), 7.40 (1H, d, J=8.4 Hz, indolin-2-one-H⁷), 7.54 (1H, s, indolin-2-one-H⁴). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 163.51, 153.83, 148.40, 145.82, 145.60, 141.24, 133.78, 125.43, 125.29, 124.83, 117.09, 115.51, 114.00, 113.28, 32.66, 32.10, 25.46. ESI-MS m/z: 297.23 (M + 1)⁺. HRMS (ESI) calculated for C₁₇H₁₃ClN₂O (MH+), 297.0789; found, 297.0785.

(Z)-5-chloro-3-[(9-ethyl-9H-carbazol-3-yl)imino]indolin-2-one (8f): Yellow powder, 81.2% yield, mp: 266.6°C–267.5°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 1.35 (3H, t, J=7.2 Hz, −NH−C−CH₃), 4.50 (2H, q, J=7.2 Hz, −NH−CH₂−C), 6.93 (1H, d, J=8.4 Hz, indolin-2-one-H⁶), 7.19–7.24 (2H, m, carbazol-H^7,8), 7.38 (1H, d, J=8.4 Hz, carbazol-H⁹), 7.50 (1H, d, J=8.4 Hz, carbazol-H²), 7.65 (1H, d, J=8.4 Hz, carbazol-H³), 7.74 (1H, d, J=8.4 Hz, indolin-2-one-H⁷), 7.92 (1H, s, indolin-2-one-H⁴), 8.16 (1H, d, J=8.4 Hz, carbazol-H⁶), 8.19 (1H, s, carbazol-H⁵). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 153.17, 145.74, 141.63, 140.38, 138.15, 133.58, 126.50, 125.35, 124.35, 124.09, 122.96, 122.71, 122.40, 120.97, 119.19, 117.62, 115.09, 113.24, 110.09, 109.71, 37.43, 13.90. ESI-MS m/z: 374.90 (M + 1)⁺.

(Z)-3-[(1-acetylindolin-5-yl)imino]-5-chloroindolin-2-one (8g): Crimson powder, 75.4% yield, mp: 270.7°C–270.9°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 2.18 (3H, s, N−CO−CH₃), 3.18 (2H, t, J=8.4 Hz, acetyindolin-H³), 4.15 (2H, t, J=8.4 Hz, acetyindolin-H²), 6.92 (1H, d, J=8.4 Hz, acetyindolin-H⁷), 6.97 (1H, s, acetyindolin-H⁴), 7.41 (1H, d, J=8.4 Hz, acetyindolin-H⁶), 7.45 (1H, d, J=8.4 Hz, indolin-2-one-H⁶), 7.52 (1H, s, indolin-2-one-H⁴), 8.10 (1H, d, J=8.4 Hz, indolin-2-one-H⁷). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 168.78, 163.55, 153.47, 145.83, 141.25, 133.80, 126.56, 125.46, 124.53, 122.07, 120.76, 117.12, 116.40, 115.72, 113.30, 48.65, 27.62, 24.17. ESI-MS m/z: 340.45 (M + 1)⁺.

(Z)-3-[(2-amino-6-morpholinopyridin-3-yl)imino]-5-chloroindolin-2-one (8h): Purple powder, 75.7% yield, mp: 213.8°C–215.1°C. ¹H-NMR (600 MHz, DMSO-d₆) δ (ppm): 3.64–3.66 (8H, m, morpholino-CH₂ ×4), 6.16 (1H, d, J=9.6 Hz, pyrdin-H⁵), 6.78 (1H, d, J=9.6 Hz, pyrdin-H⁴), 7.19 (1H, d, J=8.4 Hz, indolin-2-one-H⁶), 7.61 (1H, d, J=8.4 Hz, indolin-2-one-H⁷), 7.93 (1H, s, indolin-2-one-H⁴), 8.93(2H, s, −NH2). ¹³C-NMR (600 MHz, DMSO-d₆) δ (ppm): 159.34, 153.47, 150.51, 143.48, 139.96, 133.71, 130.07, 126.02, 120.86, 117.64, 111.97, 95.97, 66.21 ×2, 44.80 ×2. ESI-MS m/z: 358.59 (M + 1)⁺.

Figure S1 Original spectrums of ¹H-NMRand ¹³C-NMR data of the active compounds.

Notes: (A) ¹H-NMR of compound 7i. (B) ¹³C-NMR of compound 7i. (C) ¹H-NMR of compound 8e. (D) ¹³C-NMR of compound 8e.
Abbreviations: ¹H-NMR, ¹H-nuclear magnetic resonance; ¹³C-NMR, ¹³C-nuclear magnetic resonance; ppm, parts per million; t1, relaxation time.

Figure S2 LC-MS of the active compounds.

Notes: (A) LC-MS of compound 7i. (B) LC-MS of compound 8e.
Abbreviations: LC-MS, liquid chromatograph mass spectrometer; MS, mass spectrometer; m/z, mass charge ratio; UV, ultraviolet rays.

Figure S3 HPLC determination for purity of the representative compounds.

Note: (A) RP-HPLC spectrum of compound 7b: purity 98.73%. (B) RP-HPLC spectrum of compound 7c: purity 96.75%. (C) RP-HPLC spectrum of compound 7i: purity 96.32%. (D) RP-HPLC spectrum of compound 7k: purity 96.25%. (E) RP-HPLC spectrum of compound 8d: purity 99.18%. (F) RP-HPLC spectrum of compound 8e: purity 99.93%. (G) RP-HPLC spectrum of compound 8f: purity 99.80%. (H) RP-HPLC spectrum of compound 8g: purity 98.60%. The values 1, 2, and 5 beside the peak in each graph denotes the compound is the first, second, or fifth peak shown in the graph. HPLC conditions: column, XDB-C18 (4.6 mm ID ×150 mm L); column temperature, 25°C; mobile phase, H₂O/CH₃OH =50:50–10:90; flow rate, 1.0 mL/min; wavelength, UV 400 nm. BB, BV, MM, and VV refer to the type of peak.
Abbreviations: AU, absorbance unit; D, data; HJ, method name; HPLC, high performance liquid chromatography; ID, inside diameter; L, length; RP-HPLC, reversed-phase high performance liquid chromatography; mins, minutes; min, minimum; UV, ultraviolet; VWD1A, variable wavelength UV detector.

Figure S4 The cytotoxicity of active compounds 7i and 8e in RAW264.7 macrophages, MPMs, and NRK-52E cells.

Abbreviations: DMSO, dimethyl sulfoxide; MPM, mouse peritoneal macrophage.

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Acknowledgments

Financial support was provided by the National Natural Science Funding of China (21272179 and 21202124), High-level Innovative Talent Funding of Zhejiang Department of Health (GL), Project of Wenzhou Sci&Tech Bureau (Y20120061), Zhejiang Natural Science Funding (Q12H300009), Qianjiang Talent Project of Zhejiang Province (2013R10020), the project sponsored by SRF for ROCS and SEM, and Zhejiang Key Group Project in Scientific Innovation (2010R50042 to TD).

Disclosure

The authors report no conflicts of interest in this work.