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Ⅰ-1		H	CHO		Ⅲ-1	H	H
Ⅰ-2		H	CHO	Ⅲ-2	H	H
Ⅰ-3		H	CHO	Ⅲ-3	H	H
Ⅰ-4		H	CHO	Ⅳ-1	H
Ⅱ-1	H		CHO	Ⅳ-2	H
Ⅱ-2	H		CHO	Ⅳ-3	H
Ⅱ-3	H		CHO	Ⅳ-4	H
Ⅱ-4	H		CHO	Ⅳ-5	H
Ⅱ-5	H		CHO	Ⅳ-6	H
Ⅱ-6	H		CHO


Ⅰ-1		H	CHO		Ⅲ-1	H	H
Ⅰ-2		H	CHO	Ⅲ-2	H	H
Ⅰ-3		H	CHO	Ⅲ-3	H	H
Ⅰ-4		H	CHO	Ⅳ-1	H
Ⅱ-1	H		CHO	Ⅳ-2	H
Ⅱ-2	H		CHO	Ⅳ-3	H
Ⅱ-3	H		CHO	Ⅳ-4	H
Ⅱ-4	H		CHO	Ⅳ-5	H
Ⅱ-5	H		CHO	Ⅳ-6	H
Ⅱ-6	H		CHO

Compd.	Mp, ¹H NMR, ¹³C NMR, HRMS-ESI
Ⅰ-1	mp: 239-242 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 12.57 (s, 1H), 10.03 (s, 1H), 9.45 (s, 1H), 8.04 (s, 1H), 7.72 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 3.98 (s, 3H), 2.05 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.00, 168.90, 146.46, 142.69, 139.76, 132.68, 129.16, 127.64, 127.50, 125.26, 124.02, 123.94, 119.87, 119.00, 117.92, 103.76, 56.01, 24.53; HRMS-ESI: m/z Calcd. for C₁₈H₁₆N₂O₃ [M+H]⁺ 309.123 4, found 309.123 5
Ⅰ-2	mp: 190-193 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.58 (s, 1H), 10.42 (s, 1H), 9.54 (s, 1H), 8.13 (s, 1H), 7.75-7.62 (m, 2H), 7.28 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 3.99 (d, J = 3.2 Hz, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 189.65, 184.17, 160.96, 146.45, 137.32, 135.38, 134.69, 128.37, 127.96, 126.46, 124.57, 124.48, 123.99, 119.34, 112.82, 104.18, 56.53, 56.04; HRMS-ESI: m/z Calcd. for C₁₈H₁₅NO₄ [M+H]⁺ 310.107 4, found 310.107 0
Ⅰ-3	mp: 222-225 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.61 (s, 1H), 9.53 (s, 1H), 8.14 (s, 1H), 7.99 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 4.00 (s, 3H), 3.89 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 184.13, 166.73, 147.25, 146.88, 135.48, 129.75, 129.75, 129.38, 129.38, 128.24, 128.00, 126.80, 124.69, 124.24, 119.27, 104.20, 56.09, 52.57; HRMS-ESI: m/z Calcd. for C₁₈H₁₅NO₄ [M+H]⁺ 310.107 4, found 310.107 4
Ⅰ-4	mp: 173-175 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.61 (s, 1H), 9.46 (s, 1H), 8.53 (d, J = 5.3 Hz, 1H), 8.49 (s, 1H), 8.19 (d, J = 3.1 Hz, 1H), 7.57 (d, J = 5.3 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 4.01 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 183.72, 151.45, 149.51, 147.07, 143.21, 138.02, 137.48, 127.77, 125.45, 124.70, 124.19, 121.43, 119.90, 104.23, 56.10; HRMS-ESI: m/z Calcd. for C₁₅H₁₁ClN₂O₂ [M+H]⁺ 287.058 2, found 287.058 0
Ⅱ-1	mp: 155-158 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.95 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.20-7.14 (m, 3H), 6.67 (d, J = 7.9 Hz, 1H), 5.68 (s, 2H), 3.71 (s, 3H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.70, 147.46, 147.12, 145.15, 138.85, 127.78, 127.15, 127.15, 126.65, 124.25, 124.10, 124.10, 119.02, 114.65, 105.45, 55.42, 53.08; HRMS-ESI: m/z Calcd. for C₁₇H₁₄N₂O₄ [M+H]⁺ 311.102 6, found 311.102 3
Ⅱ-2	mp: 103-105 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.90 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.14 (t, J = 7.9 Hz, 1H), 6.93 (d, J = 8.3 Hz, 2H), 6.67 (d, J = 7.9 Hz, 1H), 5.53 (s, 2H), 3.76 (s, 3H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.70, 147.31, 138.90, 136.68, 131.96, 131.96, 128.58, 128.58, 127.74, 126.84, 123.97, 121.80, 118.69, 114.54, 105.34, 55.45, 53.02; HRMS-ESI: m/z Calcd. for C₁₇H₁₄BrNO₂ [M+H]⁺ 344.028 1, found 344.027 6
Ⅱ-3	mp: 203-206 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.94 (s, 1H), 8.28-8.06 (m, 3H), 7.73 (d, J = 7.5 Hz, 1H), 7.48 (t, J = 8.9 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.05 (s, 2H), 3.58 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 192.73, 185.32, 167.15, 147.47, 142.73, 131.72, 131.69, 131.57, 131.47, 127.23, 124.02, 117.96, 116.78, 116.56, 114.15, 105.77, 56.43, 56.03; HRMS-ESI: m/z Calcd. for C₁₈H₁₄FNO₃ [M+H]⁺ 312.103 0, found 312.102 9
Ⅱ-4	mp: 151-154 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.94 (s, 1H), 8.40 (s, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.3 Hz, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 5.75 (s, 2H), 3.82 (s, 3H), 3.75 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.31, 166.41, 147.58, 144.36, 142.19, 129.95, 129.95, 129.13, 127.55, 127.33, 127.33, 126.62, 124.25, 118.14, 114.07, 106.01, 55.95, 52.94, 52.58; HRMS-ESI: m/z Calcd. for C₁₉H₁₇NO₄ [M+H]⁺ 324.123 0, found 324.123 0
Ⅱ-5	mp: 118-121 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.90 (s, 1H), 8.34 (s, 1H), 7.84 (d, J = 7.1 Hz, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 4.85 (t, J = 4.9 Hz, 2H), 4.66 (t, J = 4.9 Hz, 2H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.14, 165.80, 147.52, 142.42, 133.91, 129.74, 129.57, 129.57, 129.15, 129.15, 127.48, 126.60, 124.03, 118.03, 114.05, 105.71, 64.82, 56.04, 49.04; HRMS-ESI: m/z Calcd. for C₁₉H₁₇NO₄ [M+H]⁺ 324.123 0, found 324.122 6
Ⅱ-6	mp: 159-160 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.98 (s, 1H), 7.88 (d, J = 5.7 Hz, 2H), 7.20 (t, J = 7.9 Hz, 1H), 6.75 (d, J = 7.9 Hz, 1H), 5.30 (s, 2H), 3.94 (s, 3H), 2.56 (t, J = 2.6 Hz, 1H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.77, 147.34, 138.01, 127.67, 126.45, 123.97, 118.57, 114.59, 105.25, 77.40, 75.25, 55.64, 39.47; HRMS-ESI: m/z Calcd. for C₁₃H₁₁NO₂ [M+H]⁺ 214.086 3, found 214.086 2
Ⅲ-1	mp: 230-233 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 8.77 (s, 1H), 8.02 (d, J = 15.5 Hz, 1H), 7.66 (dd, J = 8.3, 1.8 Hz, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.52 (d, J = 5.3 Hz, 1H), 7.50 (d, J = 2.6 Hz, 1H), 7.15 (t, J = 7.9 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 3.91 (d, J = 3.6 Hz, 9H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 189.07, 152.78, 149.13, 146.38, 138.24, 132.09, 129.26, 127.73, 126.66, 122.60, 122.25, 117.56, 115.07, 113.21, 110.83, 109.98, 109.93, 103.36, 56.09, 55.46; HRMS-ESI: m/z Calcd. for C₂₀H₁₉NO₄ [M+H]⁺ 338.138 7, found 338.139 1
Ⅲ-2	mp: 181-184 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 11.99 (s, 1H), 8.08-7.95 (m, 2H), 7.80 (d, J = 8.1 Hz, 1H), 7.68-7.53 (m, 3H), 7.14 (t, J = 7.9 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 6.16 (s, 2H), 3.95 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 187.14, 151.34, 148.35, 146.99, 138.98, 133.66, 133.04, 128.22, 127.38, 124.67, 122.20, 115.37, 113.79, 113.33, 108.52, 108.17, 103.77, 102.35, 55.76; HRMS-ESI: m/z Calcd. for C₁₉H₁₅NO₄ [M+H]⁺ 322.107 4, found 322.107 4
Ⅲ-3	mp: 140-143 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 12.45 (s, 1H), 8.41 (d, J = 13.4 Hz, 1H), 8.17 (s, 1H), 7.98 (d, J = 13.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 7.9 Hz, 1H), 3.94 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 146.99, 136.04, 135.39, 131.58, 127.96, 126.63, 123.31, 113.40, 109.21, 104.59, 55.83; HRMS-ESI: m/z Calcd. for C₁₁H₁₀N₂O₃ [M+H]⁺ 219.076 4, found 219.076 2
Ⅳ-1	mp: 200-203 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.45-8.30 (m, 2H), 8.20 (d, J = 8.7 Hz, 2H), 8.01 (d, J = 13.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 5.79 (s, 2H), 3.76 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 147.81, 147.22, 146.55, 139.55, 134.17, 132.47, 128.21, 128.17, 128.17, 126.93, 124.28, 124.28, 124.04, 113.73, 108.64, 106.01, 56.02, 52.72; HRMS-ESI: m/z Calcd. for C₁₈H₁₅N₃O₅ [M+H]⁺ 354.108 4, found 354.108 0
Ⅳ-2	mp: 193-195 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.35 (d, J = 13.4 Hz, 1H), 8.31 (s, 1H), 7.99 (d, J = 13.4 Hz, 1H), 7.53 (t, J = 8.0 Hz, 3H), 7.18 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 6.85 (d, J = 7.9 Hz, 1H), 5.61 (s, 2H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 147.86, 139.48, 138.21, 134.28, 132.22, 131.95, 131.95, 129.52, 129.52, 128.18, 126.89, 123.94, 121.09, 113.66, 108.49, 105.92, 56.02, 52.54; HRMS-ESI: m/z Calcd. for C₁₈H₁₅BrN₂O₃ [M+H]⁺ 387.033 9, found 387.033 7
Ⅳ-3	mp: 209-212 ℃; ¹H NMR (400 MHz, methanol-d₄) δ_H: 9.20 (d, J = 13.4 Hz, 1H), 8.98 (dd, J = 8.8, 5.5 Hz, 2H), 8.90 (s, 1H), 8.80 (d, J = 13.4 Hz, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.28 (t, J = 8.8 Hz, 2H), 8.00 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 6.84 (s, 2H), 4.39 (s, 3H); ¹³C NMR (100 MHz, methanol-d₄) δ_C: 193.49, 148.57, 141.28, 135.37, 132.86, 132.55, 132.52, 132.39, 132.29, 128.63, 128.49, 124.61, 117.55, 117.34, 114.68, 109.07, 106.63, 57.27, 56.90; HRMS-ESI: m/z Calcd. for C₁₉H₁₅FN₂O₄ [M+H]⁺ 355.108 9, found 355.108 7
Ⅳ-4	mp: 220-222 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.44-8.27 (m, 2H), 8.00 (d, J = 13.4 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 5.73 (s, 2H), 3.79 (d, J = 22.2 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 166.39, 147.88, 144.26, 139.61, 134.24, 132.31, 129.97, 129.97, 129.15, 128.20, 127.32, 127.32, 127.02, 123.95, 113.69, 108.51, 105.98, 56.01, 52.97, 52.58; HRMS-ESI: m/z Calcd. for C₂₀H₁₈N₂O₅ [M+H]⁺ 367.128 8, found 367.128 6
Ⅳ-5	mp: 134-136 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.36-8.24 (m, 2H), 7.95 (d, J = 13.4 Hz, 1H), 7.83 (d, J = 7.1 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.48 (dt, J = 15.5, 7.8 Hz, 3H), 7.17 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 4.81 (t, J = 4.7 Hz, 2H), 4.62 (t, J = 4.8 Hz, 2H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 165.82, 147.83, 139.88, 134.31, 133.92, 132.00, 129.69, 129.60, 129.60, 129.14, 129.14, 128.14, 126.99, 123.77, 113.64, 108.28, 105.69, 64.81, 56.09, 49.12; HRMS-ESI: m/z Calcd. for C₂₀H₁₈N₂O₅ [M+H]⁺ 367.128 8, found 367.129 0
Ⅳ-6	mp: 163-166 ℃; ¹H NMR (600 MHz, chloroform-d) δ_H: 8.15 (d, J = 13.5 Hz, 1H), 7.65 (d, J = 11.7 Hz, 2H), 7.25 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 5.23 (d, J = 2.5 Hz, 2H), 3.89 (s, 3H), 2.47 (t, J = 2.5 Hz, 1H); ¹³C NMR (150 MHz, chloroform-d) δ_C: 147.95, 135.08, 133.21, 132.55, 128.09, 126.98, 123.58, 113.18, 108.82, 105.21, 77.45, 75.11, 55.74, 39.42; HRMS-ESI: m/z Calcd. for C₁₄H₁₂N₂O₃ [M+H]⁺ 257.092 1, found 257.091 8

Compd.	Mp, ¹H NMR, ¹³C NMR, HRMS-ESI
Ⅰ-1	mp: 239-242 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 12.57 (s, 1H), 10.03 (s, 1H), 9.45 (s, 1H), 8.04 (s, 1H), 7.72 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 3.98 (s, 3H), 2.05 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.00, 168.90, 146.46, 142.69, 139.76, 132.68, 129.16, 127.64, 127.50, 125.26, 124.02, 123.94, 119.87, 119.00, 117.92, 103.76, 56.01, 24.53; HRMS-ESI: m/z Calcd. for C₁₈H₁₆N₂O₃ [M+H]⁺ 309.123 4, found 309.123 5
Ⅰ-2	mp: 190-193 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.58 (s, 1H), 10.42 (s, 1H), 9.54 (s, 1H), 8.13 (s, 1H), 7.75-7.62 (m, 2H), 7.28 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 3.99 (d, J = 3.2 Hz, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 189.65, 184.17, 160.96, 146.45, 137.32, 135.38, 134.69, 128.37, 127.96, 126.46, 124.57, 124.48, 123.99, 119.34, 112.82, 104.18, 56.53, 56.04; HRMS-ESI: m/z Calcd. for C₁₈H₁₅NO₄ [M+H]⁺ 310.107 4, found 310.107 0
Ⅰ-3	mp: 222-225 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.61 (s, 1H), 9.53 (s, 1H), 8.14 (s, 1H), 7.99 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 4.00 (s, 3H), 3.89 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 184.13, 166.73, 147.25, 146.88, 135.48, 129.75, 129.75, 129.38, 129.38, 128.24, 128.00, 126.80, 124.69, 124.24, 119.27, 104.20, 56.09, 52.57; HRMS-ESI: m/z Calcd. for C₁₈H₁₅NO₄ [M+H]⁺ 310.107 4, found 310.107 4
Ⅰ-4	mp: 173-175 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.61 (s, 1H), 9.46 (s, 1H), 8.53 (d, J = 5.3 Hz, 1H), 8.49 (s, 1H), 8.19 (d, J = 3.1 Hz, 1H), 7.57 (d, J = 5.3 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 4.01 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 183.72, 151.45, 149.51, 147.07, 143.21, 138.02, 137.48, 127.77, 125.45, 124.70, 124.19, 121.43, 119.90, 104.23, 56.10; HRMS-ESI: m/z Calcd. for C₁₅H₁₁ClN₂O₂ [M+H]⁺ 287.058 2, found 287.058 0
Ⅱ-1	mp: 155-158 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.95 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.20-7.14 (m, 3H), 6.67 (d, J = 7.9 Hz, 1H), 5.68 (s, 2H), 3.71 (s, 3H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.70, 147.46, 147.12, 145.15, 138.85, 127.78, 127.15, 127.15, 126.65, 124.25, 124.10, 124.10, 119.02, 114.65, 105.45, 55.42, 53.08; HRMS-ESI: m/z Calcd. for C₁₇H₁₄N₂O₄ [M+H]⁺ 311.102 6, found 311.102 3
Ⅱ-2	mp: 103-105 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.90 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.14 (t, J = 7.9 Hz, 1H), 6.93 (d, J = 8.3 Hz, 2H), 6.67 (d, J = 7.9 Hz, 1H), 5.53 (s, 2H), 3.76 (s, 3H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.70, 147.31, 138.90, 136.68, 131.96, 131.96, 128.58, 128.58, 127.74, 126.84, 123.97, 121.80, 118.69, 114.54, 105.34, 55.45, 53.02; HRMS-ESI: m/z Calcd. for C₁₇H₁₄BrNO₂ [M+H]⁺ 344.028 1, found 344.027 6
Ⅱ-3	mp: 203-206 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.94 (s, 1H), 8.28-8.06 (m, 3H), 7.73 (d, J = 7.5 Hz, 1H), 7.48 (t, J = 8.9 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.05 (s, 2H), 3.58 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 192.73, 185.32, 167.15, 147.47, 142.73, 131.72, 131.69, 131.57, 131.47, 127.23, 124.02, 117.96, 116.78, 116.56, 114.15, 105.77, 56.43, 56.03; HRMS-ESI: m/z Calcd. for C₁₈H₁₄FNO₃ [M+H]⁺ 312.103 0, found 312.102 9
Ⅱ-4	mp: 151-154 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.94 (s, 1H), 8.40 (s, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.3 Hz, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 5.75 (s, 2H), 3.82 (s, 3H), 3.75 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.31, 166.41, 147.58, 144.36, 142.19, 129.95, 129.95, 129.13, 127.55, 127.33, 127.33, 126.62, 124.25, 118.14, 114.07, 106.01, 55.95, 52.94, 52.58; HRMS-ESI: m/z Calcd. for C₁₉H₁₇NO₄ [M+H]⁺ 324.123 0, found 324.123 0
Ⅱ-5	mp: 118-121 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.90 (s, 1H), 8.34 (s, 1H), 7.84 (d, J = 7.1 Hz, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 4.85 (t, J = 4.9 Hz, 2H), 4.66 (t, J = 4.9 Hz, 2H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.14, 165.80, 147.52, 142.42, 133.91, 129.74, 129.57, 129.57, 129.15, 129.15, 127.48, 126.60, 124.03, 118.03, 114.05, 105.71, 64.82, 56.04, 49.04; HRMS-ESI: m/z Calcd. for C₁₉H₁₇NO₄ [M+H]⁺ 324.123 0, found 324.122 6
Ⅱ-6	mp: 159-160 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.98 (s, 1H), 7.88 (d, J = 5.7 Hz, 2H), 7.20 (t, J = 7.9 Hz, 1H), 6.75 (d, J = 7.9 Hz, 1H), 5.30 (s, 2H), 3.94 (s, 3H), 2.56 (t, J = 2.6 Hz, 1H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.77, 147.34, 138.01, 127.67, 126.45, 123.97, 118.57, 114.59, 105.25, 77.40, 75.25, 55.64, 39.47; HRMS-ESI: m/z Calcd. for C₁₃H₁₁NO₂ [M+H]⁺ 214.086 3, found 214.086 2
Ⅲ-1	mp: 230-233 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 8.77 (s, 1H), 8.02 (d, J = 15.5 Hz, 1H), 7.66 (dd, J = 8.3, 1.8 Hz, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.52 (d, J = 5.3 Hz, 1H), 7.50 (d, J = 2.6 Hz, 1H), 7.15 (t, J = 7.9 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 3.91 (d, J = 3.6 Hz, 9H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 189.07, 152.78, 149.13, 146.38, 138.24, 132.09, 129.26, 127.73, 126.66, 122.60, 122.25, 117.56, 115.07, 113.21, 110.83, 109.98, 109.93, 103.36, 56.09, 55.46; HRMS-ESI: m/z Calcd. for C₂₀H₁₉NO₄ [M+H]⁺ 338.138 7, found 338.139 1
Ⅲ-2	mp: 181-184 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 11.99 (s, 1H), 8.08-7.95 (m, 2H), 7.80 (d, J = 8.1 Hz, 1H), 7.68-7.53 (m, 3H), 7.14 (t, J = 7.9 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 6.16 (s, 2H), 3.95 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 187.14, 151.34, 148.35, 146.99, 138.98, 133.66, 133.04, 128.22, 127.38, 124.67, 122.20, 115.37, 113.79, 113.33, 108.52, 108.17, 103.77, 102.35, 55.76; HRMS-ESI: m/z Calcd. for C₁₉H₁₅NO₄ [M+H]⁺ 322.107 4, found 322.107 4
Ⅲ-3	mp: 140-143 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 12.45 (s, 1H), 8.41 (d, J = 13.4 Hz, 1H), 8.17 (s, 1H), 7.98 (d, J = 13.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 7.9 Hz, 1H), 3.94 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 146.99, 136.04, 135.39, 131.58, 127.96, 126.63, 123.31, 113.40, 109.21, 104.59, 55.83; HRMS-ESI: m/z Calcd. for C₁₁H₁₀N₂O₃ [M+H]⁺ 219.076 4, found 219.076 2
Ⅳ-1	mp: 200-203 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.45-8.30 (m, 2H), 8.20 (d, J = 8.7 Hz, 2H), 8.01 (d, J = 13.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 5.79 (s, 2H), 3.76 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 147.81, 147.22, 146.55, 139.55, 134.17, 132.47, 128.21, 128.17, 128.17, 126.93, 124.28, 124.28, 124.04, 113.73, 108.64, 106.01, 56.02, 52.72; HRMS-ESI: m/z Calcd. for C₁₈H₁₅N₃O₅ [M+H]⁺ 354.108 4, found 354.108 0
Ⅳ-2	mp: 193-195 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.35 (d, J = 13.4 Hz, 1H), 8.31 (s, 1H), 7.99 (d, J = 13.4 Hz, 1H), 7.53 (t, J = 8.0 Hz, 3H), 7.18 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 6.85 (d, J = 7.9 Hz, 1H), 5.61 (s, 2H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 147.86, 139.48, 138.21, 134.28, 132.22, 131.95, 131.95, 129.52, 129.52, 128.18, 126.89, 123.94, 121.09, 113.66, 108.49, 105.92, 56.02, 52.54; HRMS-ESI: m/z Calcd. for C₁₈H₁₅BrN₂O₃ [M+H]⁺ 387.033 9, found 387.033 7
Ⅳ-3	mp: 209-212 ℃; ¹H NMR (400 MHz, methanol-d₄) δ_H: 9.20 (d, J = 13.4 Hz, 1H), 8.98 (dd, J = 8.8, 5.5 Hz, 2H), 8.90 (s, 1H), 8.80 (d, J = 13.4 Hz, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.28 (t, J = 8.8 Hz, 2H), 8.00 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 6.84 (s, 2H), 4.39 (s, 3H); ¹³C NMR (100 MHz, methanol-d₄) δ_C: 193.49, 148.57, 141.28, 135.37, 132.86, 132.55, 132.52, 132.39, 132.29, 128.63, 128.49, 124.61, 117.55, 117.34, 114.68, 109.07, 106.63, 57.27, 56.90; HRMS-ESI: m/z Calcd. for C₁₉H₁₅FN₂O₄ [M+H]⁺ 355.108 9, found 355.108 7
Ⅳ-4	mp: 220-222 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.44-8.27 (m, 2H), 8.00 (d, J = 13.4 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 5.73 (s, 2H), 3.79 (d, J = 22.2 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 166.39, 147.88, 144.26, 139.61, 134.24, 132.31, 129.97, 129.97, 129.15, 128.20, 127.32, 127.32, 127.02, 123.95, 113.69, 108.51, 105.98, 56.01, 52.97, 52.58; HRMS-ESI: m/z Calcd. for C₂₀H₁₈N₂O₅ [M+H]⁺ 367.128 8, found 367.128 6
Ⅳ-5	mp: 134-136 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.36-8.24 (m, 2H), 7.95 (d, J = 13.4 Hz, 1H), 7.83 (d, J = 7.1 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.48 (dt, J = 15.5, 7.8 Hz, 3H), 7.17 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 4.81 (t, J = 4.7 Hz, 2H), 4.62 (t, J = 4.8 Hz, 2H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 165.82, 147.83, 139.88, 134.31, 133.92, 132.00, 129.69, 129.60, 129.60, 129.14, 129.14, 128.14, 126.99, 123.77, 113.64, 108.28, 105.69, 64.81, 56.09, 49.12; HRMS-ESI: m/z Calcd. for C₂₀H₁₈N₂O₅ [M+H]⁺ 367.128 8, found 367.129 0
Ⅳ-6	mp: 163-166 ℃; ¹H NMR (600 MHz, chloroform-d) δ_H: 8.15 (d, J = 13.5 Hz, 1H), 7.65 (d, J = 11.7 Hz, 2H), 7.25 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 5.23 (d, J = 2.5 Hz, 2H), 3.89 (s, 3H), 2.47 (t, J = 2.5 Hz, 1H); ¹³C NMR (150 MHz, chloroform-d) δ_C: 147.95, 135.08, 133.21, 132.55, 128.09, 126.98, 123.58, 113.18, 108.82, 105.21, 77.45, 75.11, 55.74, 39.42; HRMS-ESI: m/z Calcd. for C₁₄H₁₂N₂O₃ [M+H]⁺ 257.092 1, found 257.091 8

Compd.	IC₅₀/μmol·L^-1		Compd.	IC₅₀/μmol·L^-1
Ⅰ-1	36.0		Ⅲ-1	33.3
Ⅰ-2	27.4	Ⅲ-2	47.3
Ⅰ-3	> 50.	Ⅲ-3	2.0
Ⅰ-4	34.1	Ⅳ-1	3.2
Ⅱ-1	27.6	Ⅳ-2	10.0
Ⅱ-2	8.1	Ⅳ-3	> 50.
Ⅱ-3	> 50.	Ⅳ-4	> 50.
Ⅱ-4	> 50.	Ⅳ-5	2.5
Ⅱ-5	> 50.	Ⅳ-6	2.4
Ⅱ-6	20.5

Compd.	IC₅₀/μmol·L^-1		Compd.	IC₅₀/μmol·L^-1
Ⅰ-1	36.0		Ⅲ-1	33.3
Ⅰ-2	27.4	Ⅲ-2	47.3
Ⅰ-3	> 50.	Ⅲ-3	2.0
Ⅰ-4	34.1	Ⅳ-1	3.2
Ⅱ-1	27.6	Ⅳ-2	10.0
Ⅱ-2	8.1	Ⅳ-3	> 50.
Ⅱ-3	> 50.	Ⅳ-4	> 50.
Ⅱ-4	> 50.	Ⅳ-5	2.5
Ⅱ-5	> 50.	Ⅳ-6	2.4
Ⅱ-6	20.5

7-甲氧基吲哚类化合物的设计、合成及抗炎活性评价

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徐梓菡 ¹ , 李慧琼 ¹ , 沈天择 ¹^,² , 丁玲 ¹ , 肖伟烈 ¹^,² , 张兴杰 ¹^,^* , 张芮菡 ¹^,^*

药学学报 | 研究论文 2025,60(10): 3111-3119

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药学学报 |研究论文 2025 , 60 (10) : 3111 -3119

7-甲氧基吲哚类化合物的设计、合成及抗炎活性评价

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徐梓菡¹, 李慧琼¹, 沈天择¹^,², 丁玲¹, 肖伟烈¹^,², 张兴杰¹^,^*, 张芮菡¹^,^*

作者信息

¹云南大学药学院, 化学科学与工程学院, 教育部自然资源药物化学重点实验室, 云南昆明 650500

²西南联合研究生院, 云南昆明 650092

通讯作者:

张兴杰, Tel: 18087353165, E-mail: zhangxj@ynu.edu.cn

张芮菡, Tel: 13808730113, E-mail: zhangruihan@ynu.edu.cn

作者简介:

^#共同第一作者.

作者贡献:

徐梓菡负责化合物的设计合成、数据整理及论文撰写; 李慧琼负责生物活性测试及数据分析; 沈天择负责实验技术指导; 丁玲负责部分生物活性测试; 张兴杰负责生物实验指导; 张芮菡和肖伟烈负责课题指导及论文指导。

Design, synthesis and anti-inflammatory activity evaluation of 7-methoxyindoles

Zi-han XU¹, Hui-qiong LI¹, Tian-ze SHEN¹^,², Ling DING¹, Wei-lie XIAO¹^,², Xing-jie ZHANG¹^,^*, Rui-han ZHANG¹^,^*

Affiliations

¹Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Pharmacy and School of Chemical Science and Technology, Yunnan University, Kunming 650500, China

²Southwest United Graduate School, Kunming 650092, China

出版时间: 2025-10-12 doi: 10.16438/j.0513-4870.2025-0030

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摘要

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核苷酸结合寡聚化结构域样受体蛋白3 (NOD-like receptor protein 3, NLRP3) 炎症小体的异常活化与多种炎症和自身免疫疾病的发生相关。本研究发现吲哚类化合物A-3能够抑制NLRP3炎症小体通路的激活。对该化合物进行结构修饰, 共合成19个衍生物Ⅰ-1~Ⅰ-4、Ⅱ-1~Ⅱ-6、Ⅲ-1~Ⅲ-3、Ⅳ-1~Ⅳ-6, 有4个化合物Ⅲ-3、Ⅳ-1、Ⅳ-5、Ⅳ-6在细胞水平能够较好地抑制炎症小体活化。初步机制研究表明, 化合物Ⅳ-5能够抑制J774A.1细胞的焦亡。此外, 基于3D药效团模型对该系列化合物的构效关系进行了分析。本研究为新型NLRP3炎症小体通路抑制剂的开发提供了苗头骨架。

关键词

吲哚类化合物 / NLRP3炎症小体通路 / 结构修饰 / 抗炎活性 / 3D药效团

Abstract

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The abnormal activation of NOD-like receptor protein 3 (NLRP3) inflammasome is related to the development of a variety of inflammatory and autoimmune diseases. In this study, the indole compound A-3 was found to inhibit the activation of the NLRP3 inflammasome pathway. A total of 19 derivatives Ⅰ-1-Ⅰ-4, Ⅱ-1-Ⅱ-6, Ⅲ-1-Ⅲ-3, Ⅳ-1-Ⅳ-6 were synthesized by structural modification of A-3, and 4 compounds Ⅲ-3, Ⅳ-1, Ⅳ-5, Ⅳ-6 could inhibit inflammasome activation at the cellular level. Preliminary mechanistic studies have shown that the compound Ⅳ-5 can inhibit pyroptosis in J774A.1 cell. In addition, we analyzed the structure-activity relationship of the series of compounds based on 3D pharmacophore model. This study provides lead compounds for the development of novel NLRP3 inflammasome pathway inhibitors.

Key words

indole / NLRP3 inflammasome pathway / structural modification / anti-inflammatory activity / 3D pharmacophore

引用本文

徐梓菡, 李慧琼, 沈天择, 丁玲, 肖伟烈, 张兴杰, 张芮菡. 7-甲氧基吲哚类化合物的设计、合成及抗炎活性评价. 药学学报, 2025 , 60 (10) : 3111 -3119 . DOI: 10.16438/j.0513-4870.2025-0030

Zi-han XU, Hui-qiong LI, Tian-ze SHEN, Ling DING, Wei-lie XIAO, Xing-jie ZHANG, Rui-han ZHANG. Design, synthesis and anti-inflammatory activity evaluation of 7-methoxyindoles[J]. Acta Pharmaceutica Sinica, 2025 , 60 (10) : 3111 -3119 . DOI: 10.16438/j.0513-4870.2025-0030

正文

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炎症小体是先天性免疫系统的受体和传感器, 可调节半胱氨酸天冬氨酸蛋白水解酶1 (cysteinyl aspartate specific proteinase 1, caspase-1) 的活化, 并在感染性微生物和宿主蛋白质分子的作用下诱发炎症^[1]。在众多的炎症小体中, NLRP3炎症小体是目前研究最为深入的一种, 它作为一种多蛋白复合物, 介导白细胞介素-1β (interleukin, IL-1β) 和白细胞介素-18 (IL-18) 的成熟和释放^[2]。然而, 炎症小体的异常激活可能会导致炎症的放大, 从而引发一系列疾病, 包括炎症性肠病^[3]、心血管疾病^{[4, 5]}、类风湿性关节炎^[6]、阿尔茨海默症^[7]、癌症^[8]、痛风^[9]等。

研究表明^{[10, 11]}, MCC950作为一种有效的NLRP3特异性抑制剂, 在NACTH结构域的Walker B motif处可逆地结合NLRP3, 阻断NLRP3炎症小体的激活。但该分子在治疗类风湿性关节炎的Ⅱ期临床试验中发现可能引起肝毒性, 目前研究已中止^[12]。此外, 还有许多直接或间接抑制NLRP3炎症小体激活的小分子抑制剂例如穿心莲内酯(andrographolide)^[13]、曲尼司特(tranilast)^[14]、达潘三腈(dapansutrile)^[15]等已经开发并进入临床试验(图1), 但目前仍然未出现任何靶向上市药物^[16], 并且大多数抑制剂存在骨架单一、同质化严重等问题。因此, 广泛开发抑制NLRP3炎症小体活化的新型小分子药物具有十分重要的意义。

吲哚类化合物结构复杂多样, 表现出显著的生物活性和成药性, 如抗糖尿病、抗癌、抗菌、抗炎、抗病毒等^{[17, 18]}, 已成为开发新型药物的重要来源。许多吲哚类生物碱具有显著的抗炎效果, 如长春胺(vincamine) 通过抑制核因子-κB通路发挥抗炎作用^[19], 吲哚美辛(indometacin) 通过抑制环氧合酶活性发挥抗炎作用^[20]等。目前7-甲氧基吲哚类化合物在抗炎作用方面未见任何报道, 具有潜在的研究价值。该研究前期从课题组内吲哚生物碱化合物库筛选发现, 化合物A-3显示出良好的抗NLRP3炎症小体活性, 其化学名为4-溴-7-甲氧基-1H-吲哚-3-甲醛(图2)。以A-3为苗头分子出发, 进行多个位点的结构修饰, 以此获得更好活性的抗炎化合物。

结果

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1　苗头化合物A-3活性的测试

利用小鼠巨噬细胞J774A.1评估了化合物A-3对NLRP3炎症小体活化的抑制能力, 结果显示其具有良好的抗炎活性, IC₅₀为8.3 μmol·L^-1。通过对该化合物的C-1、C-3、C-4位点进行修饰改造, 旨在发现具有更强抗炎活性的目标分子。

2　目标化合物的设计

为了提高该类型化合物的生物活性, 分别对A、B、C 3个位点进行设计与改造(图2), A部分保留C-3位的醛基, 在C-4位通过Suzuki反应^[21]引入不同基团进行探讨; B部分在氨基位点通过卤素取代反应引入不同基团进行探讨; C部分在C-3位点通过羟醛缩合反应^[22]引入不同基团进行探讨。

3　目标化合物的合成

以7-甲氧基吲哚为原料出发, 冰浴条件下与POCl₃和DMF作用得到中间体A-2。A-2和NBS反应后的中间体A-3与带有不同取代基的硼酸反应得到Ⅰ类化合物; A-2上的氨基与带有不同取代基的苄溴在碱性作用下得到Ⅱ类化合物; A-2上的醛基与带有不同取代基的酮反应得到Ⅲ-1和Ⅲ-2, 与硝基甲烷和乙酸铵在120 ℃下得到Ⅲ-3; 根据Ⅲ-3的反应条件, Ⅱ类化合物可通过类似方法得到Ⅳ类化合物。所有路线(合成路线1) 共合成19个目标分子, 其中Ⅰ类化合物4个, Ⅱ类化合物6个, Ⅲ类化合物3个, Ⅳ类化合物6个, 目标结构上各个位点的取代基团见表1。其结构经¹H NMR、¹³C NMR、HR-MS确定, 理化性质及谱图数据见表2。

4　体外抗炎活性评价

4.1　目标化合物对J774A.1细胞释放乳酸脱氢酶的影响

实验中使用小鼠巨噬细胞J774A.1评价了目标化合物对NLRP3炎症小体的抑制作用, 以穿心莲内酯为阳性对照, 实验结果如图3所示。化合物Ⅱ-2、Ⅲ-3、Ⅳ-1、Ⅳ-5、Ⅳ-6在浓度为10 μmol·L^-1时对乳酸脱氢酶(lactate dehydrogenase, LDH) 的释放表现出良好的抑制作用, 其中化合物Ⅲ-3、Ⅳ-1、Ⅳ-5、Ⅳ-6以剂量依赖性的方式降低LDH水平, IC₅₀值分别为2.00 ± 0.49、3.22 ± 0.57、2.49 ± 0.43、2.36 ± 0.66 μmol·L^-1, 均优于阳性对照穿心莲内酯(IC₅₀值为10.47 ± 0.78 μmol·L^-1)。

4.2　目标化合物抑制NLRP3炎症小体的激活

NLRP3炎症小体激活后, 活化的caspase-1将白细胞介素-1β前体(pro-IL-1β) 转化为IL-1β, 释放到胞外。为了进一步研究化合物的抗炎活性, 本研究利用蛋白免疫印迹(Western blot, WB) 检测了化合物Ⅲ-3、Ⅳ-1、Ⅳ-5、Ⅳ-6对炎性蛋白Caspase-1、IL-1β表达的影响, 实验结果如图4所示。当使用LPS + Nigericin激活NLRP3炎症小体后, caspase-1和IL-1β蛋白大量表达, 而目标化合物能明显抑制caspase-1和IL-1β的成熟和释放。

4.3　目标化合物Ⅳ-5抑制J774A.1细胞焦亡

NLRP3炎症小体活化后激活caspase-1, 活化的caspase-1还会剪切Gasdermin D (GSDMD) 蛋白, 使其N端活化, 活化后的GSDMD-NT会在细胞膜上打孔使细胞焦亡。碘化丙啶(propidium iodide, PI) 染料是一种细胞核染色试剂, 当细胞焦亡时其膜通透性发生改变, PI染料可以透过细胞膜, 特异性的与细胞内的DNA结合, 使焦亡细胞释放红色荧光。用化合物Ⅳ-5处理LPS + Nigericin刺激的J774A.1细胞, PI/Hoechst 33342染料进行染色, 使用倒置荧光显微镜观察。结果表明(图5A、B), 化合物Ⅳ-5能剂量依赖性的减少PI阳性细胞的数量。WB的结果显示(图5C、D), 化合物Ⅲ-3、Ⅳ-1、Ⅳ-5、Ⅳ-6能剂量依赖的抑制GSDMD-NT的产生, 表明目标化合物能抑制nigericin诱导的J774A.1巨噬细胞焦亡。

5　构效关系研究

I和Ⅱ类化合物的抗炎活性并不理想(表3), 只有化合物Ⅱ-2的活性达到10 μmol·L^-1以下。而在Ⅲ类化合物中, Ⅲ-3的抗炎活性得到明显提升, 可能是因为化合物上的硝基对活性的影响较大。为了进一步验证硝基对化合物活性的提升作用, 作者在Ⅱ类化合物的C-3位点引入硝基后得到Ⅳ类化合物, 并将Ⅳ类化合物与Ⅱ类化合物进行分析比较: Ⅳ-1、Ⅳ-5、Ⅳ-6与Ⅱ-1、Ⅱ-5、Ⅱ-6相比, 在C-3位点引入硝基后, 化合物的活性得到明显提升, 进一步论证硝基对化合物活性的影响较大; 与Ⅳ-2相比, Ⅳ-3和Ⅳ-4没有活性可能是由于体积过大的R₂基团严重影响这类化合物的活性, 因而在引入硝基后化合物活性并未发生明显变化; 与Ⅳ-3和Ⅳ-4相比, Ⅳ-5的活性有明显提升可能是由于R₂基团的链长增加, 导致化合物的柔性增大, 更有利于活性的提升。

由于该类化合物的靶标尚未明确, 作者利用3D药效团模型(3D pharmacophore) 方法^[23]对该类化合物的构效关系进行了定性研究。理想的药效团模型应该与活性好(IC₅₀较小) 的化合物有较高的匹配值(FitValue较大), 而对活性差的化合物有较低的匹配值。采用包含12个化合物的训练集生成了9个候选药效团, 然后用由5个化合物组成的测试集对候选药效团的性能进行评价, pharmacophore 3和9能够较好地区分活性与非活性化合物(图6A)。

由于药效团3与9的特征基本一致, 均包含两个氢键受体和一个疏水中心。以药效团9为例, 与活性较好的化合物Ⅳ-6 (2.4 μmol·L^-1) 进行叠合(图6B、C), Ⅳ-6上的硝基和甲氧基分别作为氢键受体, 而吲哚上的苯环作为疏水中心。

6　小结

通过筛选组内化合物库的方法鉴定出一种有效抑制NLRP3炎症小体活化的化合物A-3, 设计并合成了19个7-甲氧基吲哚类衍生物的目标分子, 其中有18个化合物为新化合物(化合物Ⅲ-3除外)。体外活性实验表明, 化合物Ⅲ-3、Ⅳ-1、Ⅳ-5和Ⅳ-6对NLRP3炎症小体显示出良好的抑制活性。进一步的WB和PI染色实验显示化合物Ⅳ-5能够抑制J774A.1细胞的焦亡。此外, 利用3D药效团方法进行初步研究, 为进一步基于该苗头骨架开发炎症小体通路抑制剂奠定基础。

实验部分

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1　主要仪器与试剂

所涉及化学试剂均为分析纯规格, 其中有机溶剂均通过3A分子筛进行脱水处理。针对需隔绝空气的反应体系均采取氮气保护措施。反应进程采用薄层色谱法(TLC) 进行监测, 使用青岛海洋化工厂生产的GF25429硅胶板完成。分离纯化采用200~300目硅胶进行柱层析。化合物的结构表征依托Bruker AM-400 MHz或600 MHz核磁共振波谱仪完成并选用DMSO-d₆或CDCl₃作为氘代溶剂, 化学位移值以δ (ppm) 相对于内参比四甲基硅烷作为单位报告。化合物的相对分子质量通过Agilent LC/MSD TOF飞行时间质谱仪完成。化合物的熔点测定均使用SGW^®X-4显微熔点仪。小鼠单核巨噬细胞J774A.1购自昆明动物研究所。细胞检测使用荧光显微镜(Axio Observer 3, 德国Zeiss公司) 观察。

2　化学合成

2.1　I类化合物的合成步骤

以N-(3-(3-醛基-7-甲氧基-1H-吲哚-4-基)苯基)乙酰胺(Ⅰ-1) 为例, 向市售的7-甲氧基吲哚(1 mmol, 131 μL) 加入2 mL DMF溶液稀释, 冰浴下倒入盛有350 μL POCl₃的DMF (12 mol·L^-1) 溶液中, 常温搅拌3 h, TLC监测反应完全, 用2 mol·L^-1的NaOH溶液调节反应液的pH值, 乙酸乙酯萃取, 饱和NaCl溶液洗涤3次, 无水硫酸钠干燥, 旋干, 得到乳白色固体粉末, 为中间体A-2, 产率94.2%。

将中间体A-2 (1 mmol, 175 mg) 溶解在5 mL DMF溶液中, 冰浴下缓慢加入NBS (1.2 mmol, 214 mg), 常温搅拌1 h, TLC监测反应完全, 倒入30 mL水, 乙酸乙酯萃取, 饱和NaCl溶液洗涤3次, 无水硫酸钠干燥, 旋干, 得到黄色油状物, 为中间体A-3, 产率88.5%。

将中间体A-3 (1 mmol, 254 mg)、3-乙酰氨基苯硼酸(1.2 mmol, 215 mg) 溶解在8 mL 1, 4-二氧六环中, 再加入3 mol·L^-1 K₂CO₃溶液2 mL, 氮气保护下加入(dppf)₂PdCl₂催化剂(0.15 mmol, 110 mg), 90 ℃下回流7~8 h, TLC监测反应完全, 乙酸乙酯萃取, 饱和NaCl溶液洗涤3次, 无水硫酸钠干燥, 旋干, 使用PE∶EtOAc = 1∶1硅胶柱层析, 得到灰色固体粉末, 为目标化合物Ⅰ-1, 产率78.7%。

类似方法制备Ⅰ-2~Ⅰ-4。

2.2　Ⅱ类化合物的合成步骤

以7-甲氧基-1-(4-硝基苄基)-1H-吲哚-3-甲醛(Ⅱ-1) 为例, 将中间体A-2 (1 mmol, 175 mg) 溶解在5 mL DMF中, 冰浴下缓慢加入NaH (1.5 mmol, 36 mg), 常温搅拌0.5 h后, 加入4-硝基苄溴(2 mmol, 432 mg), 50 ℃搅拌6 h, TLC监测反应完全, 倒入30 mL水, 乙酸乙酯萃取, 饱和NaCl溶液洗涤3次, 无水硫酸钠干燥, 旋干, 使用PE∶EtOAc = 3∶1硅胶柱层析, 得到淡黄色固体粉末, 为目标化合物Ⅱ-1, 产率85.7%。

类似方法制备Ⅱ-2~Ⅱ-6。

2.3　Ⅲ类化合物的合成步骤

以(E)-1-(3, 4-二甲氧基苯基)-3-(7-甲氧基-1H-吲哚-3-基)丙-2-烯-1-酮(Ⅲ-1) 为例, 将中间体A-2 (1 mmol, 175 mg)、3, 4-二甲氧基苯乙酮(1.2 mmol, 216 mg) 和甲醇钠(3 mmol, 540 μL) 溶解在8 mL甲醇溶液中, 50 ℃下回流7~8 h, TLC监测反应结束, 旋干, 使用PE∶EtOAc = 2∶1硅胶柱层析, 得到橙红色固体粉末, 产率54.8%。

类似方法制备Ⅲ-2。

将化合物A-2 (1 mmol, 175 mg)、乙酸铵(3 mmol, 230 mg) 溶解在硝基甲烷(100 mmol, 5.4 mL) 中, 120 ℃下回流2~3 h, TLC监测反应完全, 倒入30 mL水, 抽滤, 得到红色固体, 为目标化合物Ⅲ-3, 产率91.4%。

2.4　Ⅳ类化合物的合成步骤

以(E)-7-甲氧基-1-(4-硝基苄基)-3-(2-硝基乙烯基)-1H-吲哚(Ⅳ-1) 为例, 将化合物Ⅱ-1 (0.5 mmol, 107 mg)、乙酸铵(1.5 mmol, 115 mg) 溶解在硝基甲烷(50 mmol, 2.7 mL) 中, 120 ℃下回流2~3 h, TLC监测反应完全, 倒入10 mL水, 抽滤, 得到橙红色固体粉末, 为目标化合物Ⅳ-1, 产率86.9%。

类似方法制备Ⅳ-2~Ⅳ-6。

3　生物活性实验

3.1　NLRP3炎症小体的激活

将培养瓶中生长密度为80%~90%的细胞以每孔2×10⁵个细胞接种于24孔板中, 放置于37 ℃, 含5% CO₂的培养箱中孵育过夜, 第二天弃去上清, 加入Ultra-Fectin培养基配置的脂多糖(LPS, 200 ng·mL^-1) 培养3~3.5 h, 再加入穿心莲内酯(30 μmol·L^-1) 或化合物(10 μmol·L^-1) 孵育30 min, 最后使用浓度为10 μmol·L^-1的Nigericin刺激1 h, 激活NLRP3炎症小体。

3.2　LDH测定

NLRP3炎症小体激活后, 收集细胞上清液, 用LDH检测试剂盒测定J774A.1细胞上清液中LDH的释放率。实验设置空白组、模型组、样品组。

3.3　细胞焦亡的检测

激活NLRP3炎症小体后, 弃去上清液, 37 ℃下, 每孔加入3 μg·mL^-1 PI和0.5 μg·mL^-1 Hoechst 33342混合液200 μL对细胞染色15 min。立即用倒置荧光显微镜观察, 随机选择5个不同的视野来计算焦亡细胞的比例^[24]。

3.4　WB检测

激活NLRP3炎症小体后, 收集细胞上清液和细胞RIPA裂解物。细胞裂解物中加入6倍蛋白上样缓冲液, 并在沸水中加热10 min。上清液蛋白沉淀加入2倍蛋白上样缓冲液, 沸水浴10 min, -20 ℃保存。在10%或12% SDS-PAGE凝胶上分离蛋白质样品, 并使用湿式转印系统将蛋白质转印到硝酸纤维素膜上。在室温下将膜在5%脱脂牛奶中封闭1 h。然后, 将其与在5%脱脂牛奶中稀释的一抗在4 ℃下孵育过夜, TBST中洗涤3次, 将膜加入由5%脱脂牛奶制备的HRP偶联的二抗中, 并在室温下摇床孵育1 h, 再用TBST洗涤3次。使用增强化学发光试剂盒检测蛋白质条带, 并使用Tanon-5200Multi自动化学发光图像分析系统成像^[25]。

3.5　统计学方法

采用GraphPad Prism 10软件对各实验结果进行统计分析, 并作图。所有结果均以3个独立实验的平均值±标准差(SD) 值表示。对于多组间的数据比较, 采用单因素方差分析方法进行差异分析。当P < 0.05时, 认为具有统计学意义。

4　3D药效团的生成与分析

采用软件Discovery Studio 2019 (DS) 进行药效团模型的生成与构效关系分析。选择12个化合物Ⅰ-2、Ⅰ-3、Ⅱ-1、Ⅱ-4、Ⅱ-5、Ⅱ-6、Ⅲ-1、Ⅲ-3、Ⅳ-2、Ⅳ-3、Ⅳ-4、Ⅳ-6作为训练集, 5个化合物Ⅰ-1、Ⅰ-4、Ⅱ-3、Ⅳ-1、Ⅳ-5作为测试集。采用DS中Pharmacophore-3D QSAR Pharmacophore Generation模块进行药效团的建立。针对抑制活性较低(IC₅₀值大于50 μmol·L^-1) 的化合物, 设置活性阈值为100, 作为非抑制活性判断标准。基于高活性化合物的结构特点, 在Feature Mapping模块中选择3种化学特征: 分别为氢键受体、氢键供体、疏水作用。在构象分析阶段采用BEST方法使化合物生成不超过200的能量优化构象。经训练集数据建模共生成9个候选药效团模型, 并通过Ligand Profiler模块将测试集数据绘入药效团模型进行验证评估, 并以实验测定的化合物活性数据与预测FitValue的相关性为评价指标, 比较不同的药效团模型的预测性能, 最终筛选出最优药效团模型。

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doi: 10.16438/j.0513-4870.2025-0030

接收时间：2025-01-10
首发时间：2026-09-20
出版时间：2025-10-12

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收稿日期：2025-01-10
修回日期：2025-03-25

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作者信息

¹云南大学药学院, 化学科学与工程学院, 教育部自然资源药物化学重点实验室, 云南昆明 650500

²西南联合研究生院, 云南昆明 650092

通讯作者:

张兴杰, Tel: 18087353165, E-mail: zhangxj@ynu.edu.cn

张芮菡, Tel: 13808730113, E-mail: zhangruihan@ynu.edu.cn

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https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2025-0030

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Ⅰ-1		H	CHO		Ⅲ-1	H	H
Ⅰ-2		H	CHO	Ⅲ-2	H	H
Ⅰ-3		H	CHO	Ⅲ-3	H	H
Ⅰ-4		H	CHO	Ⅳ-1	H
Ⅱ-1	H		CHO	Ⅳ-2	H
Ⅱ-2	H		CHO	Ⅳ-3	H
Ⅱ-3	H		CHO	Ⅳ-4	H
Ⅱ-4	H		CHO	Ⅳ-5	H
Ⅱ-5	H		CHO	Ⅳ-6	H
Ⅱ-6	H		CHO

Compd.

R₁

R₂

R₃

Compd.

R₁

R₂

R₃

Ⅰ-1

CHO

Ⅲ-1

Ⅰ-2

CHO

Ⅲ-2

Ⅰ-3

CHO

Ⅲ-3

Ⅰ-4

CHO

Ⅳ-1

Ⅱ-1

CHO

Ⅳ-2

Ⅱ-2

CHO

Ⅳ-3

Ⅱ-3

CHO

Ⅳ-4

Ⅱ-4

CHO

Ⅳ-5

Ⅱ-5

CHO

Ⅳ-6

Ⅱ-6

CHO

Compd.	Mp, ¹H NMR, ¹³C NMR, HRMS-ESI
Ⅰ-1	mp: 239-242 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 12.57 (s, 1H), 10.03 (s, 1H), 9.45 (s, 1H), 8.04 (s, 1H), 7.72 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 3.98 (s, 3H), 2.05 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.00, 168.90, 146.46, 142.69, 139.76, 132.68, 129.16, 127.64, 127.50, 125.26, 124.02, 123.94, 119.87, 119.00, 117.92, 103.76, 56.01, 24.53; HRMS-ESI: m/z Calcd. for C₁₈H₁₆N₂O₃ [M+H]⁺ 309.123 4, found 309.123 5
Ⅰ-2	mp: 190-193 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.58 (s, 1H), 10.42 (s, 1H), 9.54 (s, 1H), 8.13 (s, 1H), 7.75-7.62 (m, 2H), 7.28 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 3.99 (d, J = 3.2 Hz, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 189.65, 184.17, 160.96, 146.45, 137.32, 135.38, 134.69, 128.37, 127.96, 126.46, 124.57, 124.48, 123.99, 119.34, 112.82, 104.18, 56.53, 56.04; HRMS-ESI: m/z Calcd. for C₁₈H₁₅NO₄ [M+H]⁺ 310.107 4, found 310.107 0
Ⅰ-3	mp: 222-225 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.61 (s, 1H), 9.53 (s, 1H), 8.14 (s, 1H), 7.99 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 4.00 (s, 3H), 3.89 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 184.13, 166.73, 147.25, 146.88, 135.48, 129.75, 129.75, 129.38, 129.38, 128.24, 128.00, 126.80, 124.69, 124.24, 119.27, 104.20, 56.09, 52.57; HRMS-ESI: m/z Calcd. for C₁₈H₁₅NO₄ [M+H]⁺ 310.107 4, found 310.107 4
Ⅰ-4	mp: 173-175 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.61 (s, 1H), 9.46 (s, 1H), 8.53 (d, J = 5.3 Hz, 1H), 8.49 (s, 1H), 8.19 (d, J = 3.1 Hz, 1H), 7.57 (d, J = 5.3 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 4.01 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 183.72, 151.45, 149.51, 147.07, 143.21, 138.02, 137.48, 127.77, 125.45, 124.70, 124.19, 121.43, 119.90, 104.23, 56.10; HRMS-ESI: m/z Calcd. for C₁₅H₁₁ClN₂O₂ [M+H]⁺ 287.058 2, found 287.058 0
Ⅱ-1	mp: 155-158 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.95 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.20-7.14 (m, 3H), 6.67 (d, J = 7.9 Hz, 1H), 5.68 (s, 2H), 3.71 (s, 3H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.70, 147.46, 147.12, 145.15, 138.85, 127.78, 127.15, 127.15, 126.65, 124.25, 124.10, 124.10, 119.02, 114.65, 105.45, 55.42, 53.08; HRMS-ESI: m/z Calcd. for C₁₇H₁₄N₂O₄ [M+H]⁺ 311.102 6, found 311.102 3
Ⅱ-2	mp: 103-105 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.90 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.14 (t, J = 7.9 Hz, 1H), 6.93 (d, J = 8.3 Hz, 2H), 6.67 (d, J = 7.9 Hz, 1H), 5.53 (s, 2H), 3.76 (s, 3H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.70, 147.31, 138.90, 136.68, 131.96, 131.96, 128.58, 128.58, 127.74, 126.84, 123.97, 121.80, 118.69, 114.54, 105.34, 55.45, 53.02; HRMS-ESI: m/z Calcd. for C₁₇H₁₄BrNO₂ [M+H]⁺ 344.028 1, found 344.027 6
Ⅱ-3	mp: 203-206 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.94 (s, 1H), 8.28-8.06 (m, 3H), 7.73 (d, J = 7.5 Hz, 1H), 7.48 (t, J = 8.9 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.05 (s, 2H), 3.58 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 192.73, 185.32, 167.15, 147.47, 142.73, 131.72, 131.69, 131.57, 131.47, 127.23, 124.02, 117.96, 116.78, 116.56, 114.15, 105.77, 56.43, 56.03; HRMS-ESI: m/z Calcd. for C₁₈H₁₄FNO₃ [M+H]⁺ 312.103 0, found 312.102 9
Ⅱ-4	mp: 151-154 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.94 (s, 1H), 8.40 (s, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.3 Hz, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 5.75 (s, 2H), 3.82 (s, 3H), 3.75 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.31, 166.41, 147.58, 144.36, 142.19, 129.95, 129.95, 129.13, 127.55, 127.33, 127.33, 126.62, 124.25, 118.14, 114.07, 106.01, 55.95, 52.94, 52.58; HRMS-ESI: m/z Calcd. for C₁₉H₁₇NO₄ [M+H]⁺ 324.123 0, found 324.123 0
Ⅱ-5	mp: 118-121 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.90 (s, 1H), 8.34 (s, 1H), 7.84 (d, J = 7.1 Hz, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 4.85 (t, J = 4.9 Hz, 2H), 4.66 (t, J = 4.9 Hz, 2H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.14, 165.80, 147.52, 142.42, 133.91, 129.74, 129.57, 129.57, 129.15, 129.15, 127.48, 126.60, 124.03, 118.03, 114.05, 105.71, 64.82, 56.04, 49.04; HRMS-ESI: m/z Calcd. for C₁₉H₁₇NO₄ [M+H]⁺ 324.123 0, found 324.122 6
Ⅱ-6	mp: 159-160 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.98 (s, 1H), 7.88 (d, J = 5.7 Hz, 2H), 7.20 (t, J = 7.9 Hz, 1H), 6.75 (d, J = 7.9 Hz, 1H), 5.30 (s, 2H), 3.94 (s, 3H), 2.56 (t, J = 2.6 Hz, 1H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.77, 147.34, 138.01, 127.67, 126.45, 123.97, 118.57, 114.59, 105.25, 77.40, 75.25, 55.64, 39.47; HRMS-ESI: m/z Calcd. for C₁₃H₁₁NO₂ [M+H]⁺ 214.086 3, found 214.086 2
Ⅲ-1	mp: 230-233 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 8.77 (s, 1H), 8.02 (d, J = 15.5 Hz, 1H), 7.66 (dd, J = 8.3, 1.8 Hz, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.52 (d, J = 5.3 Hz, 1H), 7.50 (d, J = 2.6 Hz, 1H), 7.15 (t, J = 7.9 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 3.91 (d, J = 3.6 Hz, 9H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 189.07, 152.78, 149.13, 146.38, 138.24, 132.09, 129.26, 127.73, 126.66, 122.60, 122.25, 117.56, 115.07, 113.21, 110.83, 109.98, 109.93, 103.36, 56.09, 55.46; HRMS-ESI: m/z Calcd. for C₂₀H₁₉NO₄ [M+H]⁺ 338.138 7, found 338.139 1
Ⅲ-2	mp: 181-184 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 11.99 (s, 1H), 8.08-7.95 (m, 2H), 7.80 (d, J = 8.1 Hz, 1H), 7.68-7.53 (m, 3H), 7.14 (t, J = 7.9 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 6.16 (s, 2H), 3.95 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 187.14, 151.34, 148.35, 146.99, 138.98, 133.66, 133.04, 128.22, 127.38, 124.67, 122.20, 115.37, 113.79, 113.33, 108.52, 108.17, 103.77, 102.35, 55.76; HRMS-ESI: m/z Calcd. for C₁₉H₁₅NO₄ [M+H]⁺ 322.107 4, found 322.107 4
Ⅲ-3	mp: 140-143 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 12.45 (s, 1H), 8.41 (d, J = 13.4 Hz, 1H), 8.17 (s, 1H), 7.98 (d, J = 13.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 7.9 Hz, 1H), 3.94 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 146.99, 136.04, 135.39, 131.58, 127.96, 126.63, 123.31, 113.40, 109.21, 104.59, 55.83; HRMS-ESI: m/z Calcd. for C₁₁H₁₀N₂O₃ [M+H]⁺ 219.076 4, found 219.076 2
Ⅳ-1	mp: 200-203 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.45-8.30 (m, 2H), 8.20 (d, J = 8.7 Hz, 2H), 8.01 (d, J = 13.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 5.79 (s, 2H), 3.76 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 147.81, 147.22, 146.55, 139.55, 134.17, 132.47, 128.21, 128.17, 128.17, 126.93, 124.28, 124.28, 124.04, 113.73, 108.64, 106.01, 56.02, 52.72; HRMS-ESI: m/z Calcd. for C₁₈H₁₅N₃O₅ [M+H]⁺ 354.108 4, found 354.108 0
Ⅳ-2	mp: 193-195 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.35 (d, J = 13.4 Hz, 1H), 8.31 (s, 1H), 7.99 (d, J = 13.4 Hz, 1H), 7.53 (t, J = 8.0 Hz, 3H), 7.18 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 6.85 (d, J = 7.9 Hz, 1H), 5.61 (s, 2H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 147.86, 139.48, 138.21, 134.28, 132.22, 131.95, 131.95, 129.52, 129.52, 128.18, 126.89, 123.94, 121.09, 113.66, 108.49, 105.92, 56.02, 52.54; HRMS-ESI: m/z Calcd. for C₁₈H₁₅BrN₂O₃ [M+H]⁺ 387.033 9, found 387.033 7
Ⅳ-3	mp: 209-212 ℃; ¹H NMR (400 MHz, methanol-d₄) δ_H: 9.20 (d, J = 13.4 Hz, 1H), 8.98 (dd, J = 8.8, 5.5 Hz, 2H), 8.90 (s, 1H), 8.80 (d, J = 13.4 Hz, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.28 (t, J = 8.8 Hz, 2H), 8.00 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 6.84 (s, 2H), 4.39 (s, 3H); ¹³C NMR (100 MHz, methanol-d₄) δ_C: 193.49, 148.57, 141.28, 135.37, 132.86, 132.55, 132.52, 132.39, 132.29, 128.63, 128.49, 124.61, 117.55, 117.34, 114.68, 109.07, 106.63, 57.27, 56.90; HRMS-ESI: m/z Calcd. for C₁₉H₁₅FN₂O₄ [M+H]⁺ 355.108 9, found 355.108 7
Ⅳ-4	mp: 220-222 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.44-8.27 (m, 2H), 8.00 (d, J = 13.4 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 5.73 (s, 2H), 3.79 (d, J = 22.2 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 166.39, 147.88, 144.26, 139.61, 134.24, 132.31, 129.97, 129.97, 129.15, 128.20, 127.32, 127.32, 127.02, 123.95, 113.69, 108.51, 105.98, 56.01, 52.97, 52.58; HRMS-ESI: m/z Calcd. for C₂₀H₁₈N₂O₅ [M+H]⁺ 367.128 8, found 367.128 6
Ⅳ-5	mp: 134-136 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.36-8.24 (m, 2H), 7.95 (d, J = 13.4 Hz, 1H), 7.83 (d, J = 7.1 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.48 (dt, J = 15.5, 7.8 Hz, 3H), 7.17 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 4.81 (t, J = 4.7 Hz, 2H), 4.62 (t, J = 4.8 Hz, 2H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 165.82, 147.83, 139.88, 134.31, 133.92, 132.00, 129.69, 129.60, 129.60, 129.14, 129.14, 128.14, 126.99, 123.77, 113.64, 108.28, 105.69, 64.81, 56.09, 49.12; HRMS-ESI: m/z Calcd. for C₂₀H₁₈N₂O₅ [M+H]⁺ 367.128 8, found 367.129 0
Ⅳ-6	mp: 163-166 ℃; ¹H NMR (600 MHz, chloroform-d) δ_H: 8.15 (d, J = 13.5 Hz, 1H), 7.65 (d, J = 11.7 Hz, 2H), 7.25 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 5.23 (d, J = 2.5 Hz, 2H), 3.89 (s, 3H), 2.47 (t, J = 2.5 Hz, 1H); ¹³C NMR (150 MHz, chloroform-d) δ_C: 147.95, 135.08, 133.21, 132.55, 128.09, 126.98, 123.58, 113.18, 108.82, 105.21, 77.45, 75.11, 55.74, 39.42; HRMS-ESI: m/z Calcd. for C₁₄H₁₂N₂O₃ [M+H]⁺ 257.092 1, found 257.091 8

Compd.

Mp, ¹H NMR, ¹³C NMR, HRMS-ESI

Ⅰ-1

mp: 239-242 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 12.57 (s, 1H), 10.03 (s, 1H), 9.45 (s, 1H), 8.04 (s, 1H), 7.72 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 3.98 (s, 3H), 2.05 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.00, 168.90, 146.46, 142.69, 139.76, 132.68, 129.16, 127.64, 127.50, 125.26, 124.02, 123.94, 119.87, 119.00, 117.92, 103.76, 56.01, 24.53; HRMS-ESI: m/z Calcd. for C₁₈H₁₆N₂O₃ [M+H]⁺ 309.123 4, found 309.123 5

Ⅰ-2

mp: 190-193 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.58 (s, 1H), 10.42 (s, 1H), 9.54 (s, 1H), 8.13 (s, 1H), 7.75-7.62 (m, 2H), 7.28 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 3.99 (d, J = 3.2 Hz, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 189.65, 184.17, 160.96, 146.45, 137.32, 135.38, 134.69, 128.37, 127.96, 126.46, 124.57, 124.48, 123.99, 119.34, 112.82, 104.18, 56.53, 56.04; HRMS-ESI: m/z Calcd. for C₁₈H₁₅NO₄ [M+H]⁺ 310.107 4, found 310.107 0

Ⅰ-3

mp: 222-225 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.61 (s, 1H), 9.53 (s, 1H), 8.14 (s, 1H), 7.99 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 4.00 (s, 3H), 3.89 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 184.13, 166.73, 147.25, 146.88, 135.48, 129.75, 129.75, 129.38, 129.38, 128.24, 128.00, 126.80, 124.69, 124.24, 119.27, 104.20, 56.09, 52.57; HRMS-ESI: m/z Calcd. for C₁₈H₁₅NO₄ [M+H]⁺ 310.107 4, found 310.107 4

Ⅰ-4

mp: 173-175 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 12.61 (s, 1H), 9.46 (s, 1H), 8.53 (d, J = 5.3 Hz, 1H), 8.49 (s, 1H), 8.19 (d, J = 3.1 Hz, 1H), 7.57 (d, J = 5.3 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 4.01 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 183.72, 151.45, 149.51, 147.07, 143.21, 138.02, 137.48, 127.77, 125.45, 124.70, 124.19, 121.43, 119.90, 104.23, 56.10; HRMS-ESI: m/z Calcd. for C₁₅H₁₁ClN₂O₂ [M+H]⁺ 287.058 2, found 287.058 0

Ⅱ-1

mp: 155-158 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.95 (s, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.20-7.14 (m, 3H), 6.67 (d, J = 7.9 Hz, 1H), 5.68 (s, 2H), 3.71 (s, 3H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.70, 147.46, 147.12, 145.15, 138.85, 127.78, 127.15, 127.15, 126.65, 124.25, 124.10, 124.10, 119.02, 114.65, 105.45, 55.42, 53.08; HRMS-ESI: m/z Calcd. for C₁₇H₁₄N₂O₄ [M+H]⁺ 311.102 6, found 311.102 3

Ⅱ-2

mp: 103-105 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.90 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.14 (t, J = 7.9 Hz, 1H), 6.93 (d, J = 8.3 Hz, 2H), 6.67 (d, J = 7.9 Hz, 1H), 5.53 (s, 2H), 3.76 (s, 3H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.70, 147.31, 138.90, 136.68, 131.96, 131.96, 128.58, 128.58, 127.74, 126.84, 123.97, 121.80, 118.69, 114.54, 105.34, 55.45, 53.02; HRMS-ESI: m/z Calcd. for C₁₇H₁₄BrNO₂ [M+H]⁺ 344.028 1, found 344.027 6

Ⅱ-3

mp: 203-206 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.94 (s, 1H), 8.28-8.06 (m, 3H), 7.73 (d, J = 7.5 Hz, 1H), 7.48 (t, J = 8.9 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.05 (s, 2H), 3.58 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 192.73, 185.32, 167.15, 147.47, 142.73, 131.72, 131.69, 131.57, 131.47, 127.23, 124.02, 117.96, 116.78, 116.56, 114.15, 105.77, 56.43, 56.03; HRMS-ESI: m/z Calcd. for C₁₈H₁₄FNO₃ [M+H]⁺ 312.103 0, found 312.102 9

Ⅱ-4

mp: 151-154 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.94 (s, 1H), 8.40 (s, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.3 Hz, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 5.75 (s, 2H), 3.82 (s, 3H), 3.75 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.31, 166.41, 147.58, 144.36, 142.19, 129.95, 129.95, 129.13, 127.55, 127.33, 127.33, 126.62, 124.25, 118.14, 114.07, 106.01, 55.95, 52.94, 52.58; HRMS-ESI: m/z Calcd. for C₁₉H₁₇NO₄ [M+H]⁺ 324.123 0, found 324.123 0

Ⅱ-5

mp: 118-121 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 9.90 (s, 1H), 8.34 (s, 1H), 7.84 (d, J = 7.1 Hz, 2H), 7.72 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.17 (t, J = 7.9 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 4.85 (t, J = 4.9 Hz, 2H), 4.66 (t, J = 4.9 Hz, 2H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 185.14, 165.80, 147.52, 142.42, 133.91, 129.74, 129.57, 129.57, 129.15, 129.15, 127.48, 126.60, 124.03, 118.03, 114.05, 105.71, 64.82, 56.04, 49.04; HRMS-ESI: m/z Calcd. for C₁₉H₁₇NO₄ [M+H]⁺ 324.123 0, found 324.122 6

Ⅱ-6

mp: 159-160 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 9.98 (s, 1H), 7.88 (d, J = 5.7 Hz, 2H), 7.20 (t, J = 7.9 Hz, 1H), 6.75 (d, J = 7.9 Hz, 1H), 5.30 (s, 2H), 3.94 (s, 3H), 2.56 (t, J = 2.6 Hz, 1H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 184.77, 147.34, 138.01, 127.67, 126.45, 123.97, 118.57, 114.59, 105.25, 77.40, 75.25, 55.64, 39.47; HRMS-ESI: m/z Calcd. for C₁₃H₁₁NO₂ [M+H]⁺ 214.086 3, found 214.086 2

Ⅲ-1

mp: 230-233 ℃; ¹H NMR (400 MHz, chloroform-d) δ_H: 8.77 (s, 1H), 8.02 (d, J = 15.5 Hz, 1H), 7.66 (dd, J = 8.3, 1.8 Hz, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.52 (d, J = 5.3 Hz, 1H), 7.50 (d, J = 2.6 Hz, 1H), 7.15 (t, J = 7.9 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 3.91 (d, J = 3.6 Hz, 9H); ¹³C NMR (100 MHz, chloroform-d) δ_C: 189.07, 152.78, 149.13, 146.38, 138.24, 132.09, 129.26, 127.73, 126.66, 122.60, 122.25, 117.56, 115.07, 113.21, 110.83, 109.98, 109.93, 103.36, 56.09, 55.46; HRMS-ESI: m/z Calcd. for C₂₀H₁₉NO₄ [M+H]⁺ 338.138 7, found 338.139 1

Ⅲ-2

mp: 181-184 ℃; ¹H NMR (600 MHz, DMSO-d₆) δ_H: 11.99 (s, 1H), 8.08-7.95 (m, 2H), 7.80 (d, J = 8.1 Hz, 1H), 7.68-7.53 (m, 3H), 7.14 (t, J = 7.9 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 6.16 (s, 2H), 3.95 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 187.14, 151.34, 148.35, 146.99, 138.98, 133.66, 133.04, 128.22, 127.38, 124.67, 122.20, 115.37, 113.79, 113.33, 108.52, 108.17, 103.77, 102.35, 55.76; HRMS-ESI: m/z Calcd. for C₁₉H₁₅NO₄ [M+H]⁺ 322.107 4, found 322.107 4

Ⅲ-3

mp: 140-143 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 12.45 (s, 1H), 8.41 (d, J = 13.4 Hz, 1H), 8.17 (s, 1H), 7.98 (d, J = 13.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 7.9 Hz, 1H), 3.94 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 146.99, 136.04, 135.39, 131.58, 127.96, 126.63, 123.31, 113.40, 109.21, 104.59, 55.83; HRMS-ESI: m/z Calcd. for C₁₁H₁₀N₂O₃ [M+H]⁺ 219.076 4, found 219.076 2

Ⅳ-1

mp: 200-203 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.45-8.30 (m, 2H), 8.20 (d, J = 8.7 Hz, 2H), 8.01 (d, J = 13.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 5.79 (s, 2H), 3.76 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 147.81, 147.22, 146.55, 139.55, 134.17, 132.47, 128.21, 128.17, 128.17, 126.93, 124.28, 124.28, 124.04, 113.73, 108.64, 106.01, 56.02, 52.72; HRMS-ESI: m/z Calcd. for C₁₈H₁₅N₃O₅ [M+H]⁺ 354.108 4, found 354.108 0

Ⅳ-2

mp: 193-195 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.35 (d, J = 13.4 Hz, 1H), 8.31 (s, 1H), 7.99 (d, J = 13.4 Hz, 1H), 7.53 (t, J = 8.0 Hz, 3H), 7.18 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 6.85 (d, J = 7.9 Hz, 1H), 5.61 (s, 2H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 147.86, 139.48, 138.21, 134.28, 132.22, 131.95, 131.95, 129.52, 129.52, 128.18, 126.89, 123.94, 121.09, 113.66, 108.49, 105.92, 56.02, 52.54; HRMS-ESI: m/z Calcd. for C₁₈H₁₅BrN₂O₃ [M+H]⁺ 387.033 9, found 387.033 7

Ⅳ-3

mp: 209-212 ℃; ¹H NMR (400 MHz, methanol-d₄) δ_H: 9.20 (d, J = 13.4 Hz, 1H), 8.98 (dd, J = 8.8, 5.5 Hz, 2H), 8.90 (s, 1H), 8.80 (d, J = 13.4 Hz, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.28 (t, J = 8.8 Hz, 2H), 8.00 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 6.84 (s, 2H), 4.39 (s, 3H); ¹³C NMR (100 MHz, methanol-d₄) δ_C: 193.49, 148.57, 141.28, 135.37, 132.86, 132.55, 132.52, 132.39, 132.29, 128.63, 128.49, 124.61, 117.55, 117.34, 114.68, 109.07, 106.63, 57.27, 56.90; HRMS-ESI: m/z Calcd. for C₁₉H₁₅FN₂O₄ [M+H]⁺ 355.108 9, found 355.108 7

Ⅳ-4

mp: 220-222 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.44-8.27 (m, 2H), 8.00 (d, J = 13.4 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 5.73 (s, 2H), 3.79 (d, J = 22.2 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 166.39, 147.88, 144.26, 139.61, 134.24, 132.31, 129.97, 129.97, 129.15, 128.20, 127.32, 127.32, 127.02, 123.95, 113.69, 108.51, 105.98, 56.01, 52.97, 52.58; HRMS-ESI: m/z Calcd. for C₂₀H₁₈N₂O₅ [M+H]⁺ 367.128 8, found 367.128 6

Ⅳ-5

mp: 134-136 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 8.36-8.24 (m, 2H), 7.95 (d, J = 13.4 Hz, 1H), 7.83 (d, J = 7.1 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.48 (dt, J = 15.5, 7.8 Hz, 3H), 7.17 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 4.81 (t, J = 4.7 Hz, 2H), 4.62 (t, J = 4.8 Hz, 2H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 165.82, 147.83, 139.88, 134.31, 133.92, 132.00, 129.69, 129.60, 129.60, 129.14, 129.14, 128.14, 126.99, 123.77, 113.64, 108.28, 105.69, 64.81, 56.09, 49.12; HRMS-ESI: m/z Calcd. for C₂₀H₁₈N₂O₅ [M+H]⁺ 367.128 8, found 367.129 0

Ⅳ-6

mp: 163-166 ℃; ¹H NMR (600 MHz, chloroform-d) δ_H: 8.15 (d, J = 13.5 Hz, 1H), 7.65 (d, J = 11.7 Hz, 2H), 7.25 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 5.23 (d, J = 2.5 Hz, 2H), 3.89 (s, 3H), 2.47 (t, J = 2.5 Hz, 1H); ¹³C NMR (150 MHz, chloroform-d) δ_C: 147.95, 135.08, 133.21, 132.55, 128.09, 126.98, 123.58, 113.18, 108.82, 105.21, 77.45, 75.11, 55.74, 39.42; HRMS-ESI: m/z Calcd. for C₁₄H₁₂N₂O₃ [M+H]⁺ 257.092 1, found 257.091 8

Compd.	IC₅₀/μmol·L^-1		Compd.	IC₅₀/μmol·L^-1
Ⅰ-1	36.0		Ⅲ-1	33.3
Ⅰ-2	27.4	Ⅲ-2	47.3
Ⅰ-3	> 50.	Ⅲ-3	2.0
Ⅰ-4	34.1	Ⅳ-1	3.2
Ⅱ-1	27.6	Ⅳ-2	10.0
Ⅱ-2	8.1	Ⅳ-3	> 50.
Ⅱ-3	> 50.	Ⅳ-4	> 50.
Ⅱ-4	> 50.	Ⅳ-5	2.5
Ⅱ-5	> 50.	Ⅳ-6	2.4
Ⅱ-6	20.5

Compd.

IC₅₀/μmol·L^-1

Compd.

IC₅₀/μmol·L^-1

Ⅰ-1

36.0

Ⅲ-1

33.3

Ⅰ-2

27.4

Ⅲ-2

47.3

Ⅰ-3

> 50.

Ⅲ-3

2.0

Ⅰ-4

34.1

Ⅳ-1

3.2

Ⅱ-1

27.6

Ⅳ-2

10.0

Ⅱ-2

8.1

Ⅳ-3

> 50.

Ⅱ-3

> 50.

Ⅳ-4

> 50.

Ⅱ-4

> 50.

Ⅳ-5

2.5

Ⅱ-5

> 50.

Ⅳ-6

2.4

Ⅱ-6

20.5