药学学报

Prodrug	Appearance	Yield/%	mp/℃	¹H NMR, ¹³C NMR, HR-MS
A1	Yellow solid	57	172.2—172.9	¹H NMR δ 13.30 (br, 1H), 12.05 (br, 1H), 8.84 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.91 (t, J = 8.0 Hz, 2H), 7.66 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.38 — 7.34 (m, 2H).¹³C NMR δ 167.92, 167.01, 147.26, 141.03, 134.55, 133.23, 131.94, 131.82, 131.06, 130.70, 130.34, 128.80, 128.74, 128.60, 127.22, 125.96, 109.26, 61.23, 14.10. HR-MS (ESI) Calcd. for C₁₉H₁₇N₃O₂SCl [M+H]⁺ 386.073 0, Found 386.073 8.
A2	Yellow solid	58	170.1—171.2	¹H NMR δ 12.40 (s, 1H), 8.71 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.88 (dd, J = 7.6, 2.4 Hz, 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.52 (q, J = 7.9 Hz, 2H), 7.46—7.32 (m, 3H), 4.26 (t, J = 6.5 Hz, 2H), 1.76 (h, J = 7.1 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H). ¹³C NMR δ 167.68, 167.03, 147.63, 140.16, 134.95, 133.6, 132.71, 131.53, 131.22, 130.85, 130.59, 129.52, 129.4, 129.38, 127.73, 126.74, 109.35, 67.06, 21.96, 10.87. HR-MS (ESI) Calcd. for C₂₀H₁₈N₃O₂SCl [M+H]⁺ /span>400.080 8, Found 400.088 8.
A3	Yellow solid	60	159.5—160.3	¹H NMR δ 12.40 (s, 1H), 8.69 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.90 —7.81 (m, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.55—7.46 (m, 2H), 7.44—7.32 (m, 3H), 5.16 (hept, J = 6.3 Hz, 1H), 1.35 (d, J = 6.1 Hz, 6H). ¹³C NMR δ 205.55, 167.21, 166.04, 147.14, 139.59, 134.28, 133.20, 132.07, 131.02, 130.72, 130.35, 130.07, 129.35, 129.00, 128.84, 127.21, 126.11, 108.82, 68.85, 21.53. HR-MS (ESI) Calcd. for C₂₀H₁sClN₃O₂S [M+H]⁺ 400.080 8, Found 400.086 8.
A4	Yellow solid	55	144.3—145.4	¹H NMR δ 12.40 (s, 1H), 8.71 (s, 1H), 8.00 (dd, J = 8.0, 1.2 Hz, 1H), 7.89—7.84 (m, 2H), 7.67 (td, J = 7.6, 1.3 Hz, 1H), 7.56—7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.39 —7.33 (m, 2H), 4.30 (t, J = 6.6 Hz, 2H), 1.72 (p, 2H), 1.42 (h, 2H), 0.94 (t, J = 7.4 Hz, 3H). ¹³C NMR δ 167.68, 167.02, 147.63, 140.16, 134.95, 133.70, 132.70, 131.53, 131.22, 130.85, 130.58, 129.52, 129.40, 129.37, 127.73, 126.77, 109.35, 65.32, 30.57, 19.23, 14.09. HR-MS (ESI) Calcd. for C₂₁H₂₀N₃O₂SCl [M+H]⁺ 414.096 5, Found 414.104 4.
A5	Yellow solid	52	137.4—138.2	¹H NMR δ 12.41 (s, 1H), 8.71 (s, 1H), 8.00 (dd, J = 7.9, 1.2 Hz, 1H), 7.90 —7.84 (m, 2H), 7.69—7.64 (m, 1H), 7.56—7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.39—7.33 (m, 2H), 5.03 (h, J = 6.3 Hz, 1H), 1.77—1.63 (m, 2H), 1.33 (d, J = 6.3 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H). ¹³C NMR δ 167.71, 166.60, 147.64, 140.09, 134.89, 133.70, 132.64, 131.53, 131.22, 130.86, 130.52, 129.74, 129.52, 129.39, 127.73, 126.67, 109.35, 73.65, 28.66, 19.65, 10.07. HR-MS (ESI) Calcd. for C₂₁H₂₀N₃O₂SCl [M+H]⁺ 414.096 5, Found 414.098 8.
A6	Yellow solid	52	98.1—99.0	¹H NMR δ 12.39 (s, 1H), 8.71 (s, 1H), 7.99 (dd, J = 8.0, 1.2 Hz, 1H), 7.86 (ddd, J = 7.8, 6.1, 1.6 Hz, 2H), 7.66 (td, J = 7.7, 1.4 Hz, 1H), 7.55 —7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.38 —7.33 (m, 2H), 4.29 (t, J = 6.7 Hz, 2H), 1.74 (p, J = 6.8 Hz, 2H), 1.42—1.30 (m, 4H), 0.89 (t, J = 7.0 Hz, 3H). ¹³C NMR δ 167.68, 167.02, 147.63, 140.19, 134.95, 133.70, 132.69, 131.52, 131.22, 130.85, 130.57, 129.52, 129.40, 129.38, 127.73, 126.80, 109.33, 65.60, 28.21, 28.14, 22.29, 14.34. HR-MS (ESI) Calcd. for C₂₂H₂₂N₃O₂SCl [M+H]⁺ 428.112 1, Found 428.120 1.
A7	Yellow solid	59	92.3—93.1	¹H NMR δ 12.45 (s, 1H), 8.76 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.96 —7.87 (m, 2H), 7.72 (t, J = 7.7 Hz, 1H), 7.62—7.53 (m, 2H), 7.51—7.37 (m, 3H), 4.34 (t, J = 6.6 Hz, 2H), 1.79 (t, J = 7.4 Hz, 2H), 1.49—1.28 (m, 6H), 0.92 (t, J = 6.7 Hz, 3H). ¹³C NMR δ 167.56, 166.92, 147.53, 140.10, 134.85, 133.60, 132.56, 131.41, 131.12, 130.74, 130.44, 129.39, 129.30, 129.27, 127.60, 126.71, 109.20, 65.51, 31.27, 28.36, 25.52, 22.36, 14.22. HR-MS (ESI) Calcd. for C₂₃H₂₄N₃O₂SCl [M+H]⁺ 442.127 8, Found 442.135 5.
A8	Yellow solid	50	153.4—154.6	¹H NMR δ 12.42 (s, 1H), 8.70 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.90 —7.83 (m, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.56—7.47 (m, 2H), 7.42 (td, J = 7.5, 1.2 Hz, 1H), 7.39 —7.33 (m, 2H), 5.00—4.91 (m, 1H), 1.98—1.89 (m, 2H), 1.78—1.68 (m, 2H), 1.64—1.49 (m, 3H), 1.47—1.25 (m, 3H). ¹³C NMR δ 167.72, 166.37, 140.10, 134.84, 132.62, 131.52, 131.22, 130.86, 130.60, 129.80, 129.52, 129.39, 127.73, 126.67, 109.34, 73.85, 31.44, 25.34, 23.69. HR-MS (ESI) Calcd. for C₂₃H₂₂N₃O₂SCl [M+H]⁺ 440.112 1, Found 440.110 1.
A9	Yellow solid	46	168.3—169.2	¹H NMR δ 12.41 (s, 1H), 8.78 (s, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.92 —7.86 (m, 2H), 7.70—7.65 (m, 1H), 7.56—7.49 (m, 4H), 7.45—7.40 (m, 3H), 7.39 —7.34 (m, 3H), 5.38 (s, 2H). ¹³C NMR δ 166.20, 147.14, 139.61, 135.86, 134.72, 133.20, 132.41, 131.04, 130.73, 130.35, 130.21, 129.01, 128.92, 128.51, 128.37, 128.16, 128.08, 127.22, 126.35, 108.87, 66.59. HR-MS (ESI) Calcd. for C₂₄H₁₈N₃O₂SCl [M+H]⁺ 448.080 8, Found 448.088 8.
A10	Yellow solid	51	116.1—117.0	¹H NMR δ 12.46 (s, 1H), 8.79 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.46—7.34 (m, 6H), 7.32—7.25 (m, 1H), 4.57 (t, J = 6.8 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H). ¹³C NMR δ 167.58, 166.74, 147.55, 140.03, 138.30, 134.93, 133.61, 132.66, 131.43, 131.13, 130.75, 130.45, 129.41, 129.24, 129.03, 128.78, 127.62, 126.80, 126.67, 109.25, 65.98, 34.61. HR-MS (ESI) Calcd. for C₂₅H₂₀N₃O₂SCl [M+H]⁺ 462.096 5, Found 462.104 1.
A11	Yellow solid	49	114.4—115.6	¹H NMR δ 12.43 (s, 1H), 8.73 (s, 1H), 8.03 (dd, J = 8.0, 1.2 Hz, 1H), 7.89—7.83 (m, 2H), 7.74—7.68 (m, 1H), 7.56—7.51 (m, 2H), 7.42 (td, J = 7.5, 1.4 Hz, 1H), 7.39 —7.33 (m, 2H), 5.98 (s, 2H), 1.18 (s, 9H). ¹³C NMR δ 167.13, 164.91, 139.23, 135.19, 133.04, 131.04, 130.73, 130.35, 130.31, 129.04, 129.02, 127.24, 127.06, 126.51, 108.96, 80.13, 38.26, 26.47. HR-MS (ESI) Calcd. for C₂₃H₂₂N₃O₄SCl [M+H]⁺ 472.102 0, Found 472.109 6.
A12	Yellow solid	53	110.3—111.0	¹H NMR δ 12.44 (s, 1H), 8.76 (s, 1H), 8.04 (dd, J = 8.0, 1.2 Hz, 1H), 7.93 —7.85 (m, 2H), 7.73 (td, J = 7.6, 1.3 Hz, 1H), 7.57—7.51 (m, 2H), 7.43 (td, J = 7.5, 1.5 Hz, 1H), 7.40 —7.33 (m, 2H), 5.96 (s, 2H), 4.85 (hept, J = 6.2 Hz, 1H), 1.26 (d, J = 6.2 Hz, 6H). ¹³C NMR δ 167.62, 165.32, 153.25, 147.67, 139.76, 135.83, 133.66, 131.54, 131.23, 131.01, 130.85, 129.54, 129.49, 127.74, 127.30, 126.99, 109.47, 83.02, 73.24, 21.80. HR-MS (ESI) Calcd. for C₂₂H₂₀N₃O₅SCl [M+H]⁺ 474.081 2, Found 474.088 9.

Prodrug

Appearance

Yield/%

mp/℃

¹H NMR, ¹³C NMR, HR-MS

Yellow solid

172.2—172.9

¹H NMR δ 13.30 (br, 1H), 12.05 (br, 1H), 8.84 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.91 (t, J = 8.0 Hz, 2H), 7.66 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.38 — 7.34 (m, 2H).¹³C NMR δ 167.92, 167.01, 147.26, 141.03, 134.55, 133.23, 131.94, 131.82, 131.06, 130.70, 130.34, 128.80, 128.74, 128.60, 127.22, 125.96, 109.26, 61.23, 14.10. HR-MS (ESI) Calcd. for C₁₉H₁₇N₃O₂SCl [M+H]⁺ 386.073 0, Found 386.073 8.

Yellow solid

170.1—171.2

¹H NMR δ 12.40 (s, 1H), 8.71 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.88 (dd, J = 7.6, 2.4 Hz, 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.52 (q, J = 7.9 Hz, 2H), 7.46—7.32 (m, 3H), 4.26 (t, J = 6.5 Hz, 2H), 1.76 (h, J = 7.1 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H). ¹³C NMR δ 167.68, 167.03, 147.63, 140.16, 134.95, 133.6, 132.71, 131.53, 131.22, 130.85, 130.59, 129.52, 129.4, 129.38, 127.73, 126.74, 109.35, 67.06, 21.96, 10.87. HR-MS (ESI) Calcd. for C₂₀H₁₈N₃O₂SCl [M+H]⁺ /span>400.080 8, Found 400.088 8.

Yellow solid

159.5—160.3

¹H NMR δ 12.40 (s, 1H), 8.69 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.90 —7.81 (m, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.55—7.46 (m, 2H), 7.44—7.32 (m, 3H), 5.16 (hept, J = 6.3 Hz, 1H), 1.35 (d, J = 6.1 Hz, 6H). ¹³C NMR δ 205.55, 167.21, 166.04, 147.14, 139.59, 134.28, 133.20, 132.07, 131.02, 130.72, 130.35, 130.07, 129.35, 129.00, 128.84, 127.21, 126.11, 108.82, 68.85, 21.53. HR-MS (ESI) Calcd. for C₂₀H₁sClN₃O₂S [M+H]⁺ 400.080 8, Found 400.086 8.

Yellow solid

144.3—145.4

¹H NMR δ 12.40 (s, 1H), 8.71 (s, 1H), 8.00 (dd, J = 8.0, 1.2 Hz, 1H), 7.89—7.84 (m, 2H), 7.67 (td, J = 7.6, 1.3 Hz, 1H), 7.56—7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.39 —7.33 (m, 2H), 4.30 (t, J = 6.6 Hz, 2H), 1.72 (p, 2H), 1.42 (h, 2H), 0.94 (t, J = 7.4 Hz, 3H). ¹³C NMR δ 167.68, 167.02, 147.63, 140.16, 134.95, 133.70, 132.70, 131.53, 131.22, 130.85, 130.58, 129.52, 129.40, 129.37, 127.73, 126.77, 109.35, 65.32, 30.57, 19.23, 14.09. HR-MS (ESI) Calcd. for C₂₁H₂₀N₃O₂SCl [M+H]⁺ 414.096 5, Found 414.104 4.

Yellow solid

137.4—138.2

¹H NMR δ 12.41 (s, 1H), 8.71 (s, 1H), 8.00 (dd, J = 7.9, 1.2 Hz, 1H), 7.90 —7.84 (m, 2H), 7.69—7.64 (m, 1H), 7.56—7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.39—7.33 (m, 2H), 5.03 (h, J = 6.3 Hz, 1H), 1.77—1.63 (m, 2H), 1.33 (d, J = 6.3 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H). ¹³C NMR δ 167.71, 166.60, 147.64, 140.09, 134.89, 133.70, 132.64, 131.53, 131.22, 130.86, 130.52, 129.74, 129.52, 129.39, 127.73, 126.67, 109.35, 73.65, 28.66, 19.65, 10.07. HR-MS (ESI) Calcd. for C₂₁H₂₀N₃O₂SCl [M+H]⁺ 414.096 5, Found 414.098 8.

Yellow solid

98.1—99.0

¹H NMR δ 12.39 (s, 1H), 8.71 (s, 1H), 7.99 (dd, J = 8.0, 1.2 Hz, 1H), 7.86 (ddd, J = 7.8, 6.1, 1.6 Hz, 2H), 7.66 (td, J = 7.7, 1.4 Hz, 1H), 7.55 —7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.38 —7.33 (m, 2H), 4.29 (t, J = 6.7 Hz, 2H), 1.74 (p, J = 6.8 Hz, 2H), 1.42—1.30 (m, 4H), 0.89 (t, J = 7.0 Hz, 3H). ¹³C NMR δ 167.68, 167.02, 147.63, 140.19, 134.95, 133.70, 132.69, 131.52, 131.22, 130.85, 130.57, 129.52, 129.40, 129.38, 127.73, 126.80, 109.33, 65.60, 28.21, 28.14, 22.29, 14.34. HR-MS (ESI) Calcd. for C₂₂H₂₂N₃O₂SCl [M+H]⁺ 428.112 1, Found 428.120 1.

Yellow solid

92.3—93.1

¹H NMR δ 12.45 (s, 1H), 8.76 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.96 —7.87 (m, 2H), 7.72 (t, J = 7.7 Hz, 1H), 7.62—7.53 (m, 2H), 7.51—7.37 (m, 3H), 4.34 (t, J = 6.6 Hz, 2H), 1.79 (t, J = 7.4 Hz, 2H), 1.49—1.28 (m, 6H), 0.92 (t, J = 6.7 Hz, 3H). ¹³C NMR δ 167.56, 166.92, 147.53, 140.10, 134.85, 133.60, 132.56, 131.41, 131.12, 130.74, 130.44, 129.39, 129.30, 129.27, 127.60, 126.71, 109.20, 65.51, 31.27, 28.36, 25.52, 22.36, 14.22. HR-MS (ESI) Calcd. for C₂₃H₂₄N₃O₂SCl [M+H]⁺ 442.127 8, Found 442.135 5.

Yellow solid

153.4—154.6

¹H NMR δ 12.42 (s, 1H), 8.70 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.90 —7.83 (m, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.56—7.47 (m, 2H), 7.42 (td, J = 7.5, 1.2 Hz, 1H), 7.39 —7.33 (m, 2H), 5.00—4.91 (m, 1H), 1.98—1.89 (m, 2H), 1.78—1.68 (m, 2H), 1.64—1.49 (m, 3H), 1.47—1.25 (m, 3H). ¹³C NMR δ 167.72, 166.37, 140.10, 134.84, 132.62, 131.52, 131.22, 130.86, 130.60, 129.80, 129.52, 129.39, 127.73, 126.67, 109.34, 73.85, 31.44, 25.34, 23.69. HR-MS (ESI) Calcd. for C₂₃H₂₂N₃O₂SCl [M+H]⁺ 440.112 1, Found 440.110 1.

Yellow solid

168.3—169.2

¹H NMR δ 12.41 (s, 1H), 8.78 (s, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.92 —7.86 (m, 2H), 7.70—7.65 (m, 1H), 7.56—7.49 (m, 4H), 7.45—7.40 (m, 3H), 7.39 —7.34 (m, 3H), 5.38 (s, 2H). ¹³C NMR δ 166.20, 147.14, 139.61, 135.86, 134.72, 133.20, 132.41, 131.04, 130.73, 130.35, 130.21, 129.01, 128.92, 128.51, 128.37, 128.16, 128.08, 127.22, 126.35, 108.87, 66.59. HR-MS (ESI) Calcd. for C₂₄H₁₈N₃O₂SCl [M+H]⁺ 448.080 8, Found 448.088 8.

A10

Yellow solid

116.1—117.0

¹H NMR δ 12.46 (s, 1H), 8.79 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.46—7.34 (m, 6H), 7.32—7.25 (m, 1H), 4.57 (t, J = 6.8 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H). ¹³C NMR δ 167.58, 166.74, 147.55, 140.03, 138.30, 134.93, 133.61, 132.66, 131.43, 131.13, 130.75, 130.45, 129.41, 129.24, 129.03, 128.78, 127.62, 126.80, 126.67, 109.25, 65.98, 34.61. HR-MS (ESI) Calcd. for C₂₅H₂₀N₃O₂SCl [M+H]⁺ 462.096 5, Found 462.104 1.

A11

Yellow solid

114.4—115.6

¹H NMR δ 12.43 (s, 1H), 8.73 (s, 1H), 8.03 (dd, J = 8.0, 1.2 Hz, 1H), 7.89—7.83 (m, 2H), 7.74—7.68 (m, 1H), 7.56—7.51 (m, 2H), 7.42 (td, J = 7.5, 1.4 Hz, 1H), 7.39 —7.33 (m, 2H), 5.98 (s, 2H), 1.18 (s, 9H). ¹³C NMR δ 167.13, 164.91, 139.23, 135.19, 133.04, 131.04, 130.73, 130.35, 130.31, 129.04, 129.02, 127.24, 127.06, 126.51, 108.96, 80.13, 38.26, 26.47. HR-MS (ESI) Calcd. for C₂₃H₂₂N₃O₄SCl [M+H]⁺ 472.102 0, Found 472.109 6.

A12

Yellow solid

110.3—111.0

¹H NMR δ 12.44 (s, 1H), 8.76 (s, 1H), 8.04 (dd, J = 8.0, 1.2 Hz, 1H), 7.93 —7.85 (m, 2H), 7.73 (td, J = 7.6, 1.3 Hz, 1H), 7.57—7.51 (m, 2H), 7.43 (td, J = 7.5, 1.5 Hz, 1H), 7.40 —7.33 (m, 2H), 5.96 (s, 2H), 4.85 (hept, J = 6.2 Hz, 1H), 1.26 (d, J = 6.2 Hz, 6H). ¹³C NMR δ 167.62, 165.32, 153.25, 147.67, 139.76, 135.83, 133.66, 131.54, 131.23, 131.01, 130.85, 129.54, 129.49, 127.74, 127.30, 126.99, 109.47, 83.02, 73.24, 21.80. HR-MS (ESI) Calcd. for C₂₂H₂₀N₃O₅SCl [M+H]⁺ 474.081 2, Found 474.088 9.

Prodrug	Appearance	Yield/%	mp/℃	¹H NMR, ¹³C NMR, HR-MS
A1	Yellow solid	57	172.2—172.9	¹H NMR δ 13.30 (br, 1H), 12.05 (br, 1H), 8.84 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.91 (t, J = 8.0 Hz, 2H), 7.66 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.38 — 7.34 (m, 2H).¹³C NMR δ 167.92, 167.01, 147.26, 141.03, 134.55, 133.23, 131.94, 131.82, 131.06, 130.70, 130.34, 128.80, 128.74, 128.60, 127.22, 125.96, 109.26, 61.23, 14.10. HR-MS (ESI) Calcd. for C₁₉H₁₇N₃O₂SCl [M+H]⁺ 386.073 0, Found 386.073 8.
A2	Yellow solid	58	170.1—171.2	¹H NMR δ 12.40 (s, 1H), 8.71 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.88 (dd, J = 7.6, 2.4 Hz, 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.52 (q, J = 7.9 Hz, 2H), 7.46—7.32 (m, 3H), 4.26 (t, J = 6.5 Hz, 2H), 1.76 (h, J = 7.1 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H). ¹³C NMR δ 167.68, 167.03, 147.63, 140.16, 134.95, 133.6, 132.71, 131.53, 131.22, 130.85, 130.59, 129.52, 129.4, 129.38, 127.73, 126.74, 109.35, 67.06, 21.96, 10.87. HR-MS (ESI) Calcd. for C₂₀H₁₈N₃O₂SCl [M+H]⁺ /span>400.080 8, Found 400.088 8.
A3	Yellow solid	60	159.5—160.3	¹H NMR δ 12.40 (s, 1H), 8.69 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.90 —7.81 (m, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.55—7.46 (m, 2H), 7.44—7.32 (m, 3H), 5.16 (hept, J = 6.3 Hz, 1H), 1.35 (d, J = 6.1 Hz, 6H). ¹³C NMR δ 205.55, 167.21, 166.04, 147.14, 139.59, 134.28, 133.20, 132.07, 131.02, 130.72, 130.35, 130.07, 129.35, 129.00, 128.84, 127.21, 126.11, 108.82, 68.85, 21.53. HR-MS (ESI) Calcd. for C₂₀H₁sClN₃O₂S [M+H]⁺ 400.080 8, Found 400.086 8.
A4	Yellow solid	55	144.3—145.4	¹H NMR δ 12.40 (s, 1H), 8.71 (s, 1H), 8.00 (dd, J = 8.0, 1.2 Hz, 1H), 7.89—7.84 (m, 2H), 7.67 (td, J = 7.6, 1.3 Hz, 1H), 7.56—7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.39 —7.33 (m, 2H), 4.30 (t, J = 6.6 Hz, 2H), 1.72 (p, 2H), 1.42 (h, 2H), 0.94 (t, J = 7.4 Hz, 3H). ¹³C NMR δ 167.68, 167.02, 147.63, 140.16, 134.95, 133.70, 132.70, 131.53, 131.22, 130.85, 130.58, 129.52, 129.40, 129.37, 127.73, 126.77, 109.35, 65.32, 30.57, 19.23, 14.09. HR-MS (ESI) Calcd. for C₂₁H₂₀N₃O₂SCl [M+H]⁺ 414.096 5, Found 414.104 4.
A5	Yellow solid	52	137.4—138.2	¹H NMR δ 12.41 (s, 1H), 8.71 (s, 1H), 8.00 (dd, J = 7.9, 1.2 Hz, 1H), 7.90 —7.84 (m, 2H), 7.69—7.64 (m, 1H), 7.56—7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.39—7.33 (m, 2H), 5.03 (h, J = 6.3 Hz, 1H), 1.77—1.63 (m, 2H), 1.33 (d, J = 6.3 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H). ¹³C NMR δ 167.71, 166.60, 147.64, 140.09, 134.89, 133.70, 132.64, 131.53, 131.22, 130.86, 130.52, 129.74, 129.52, 129.39, 127.73, 126.67, 109.35, 73.65, 28.66, 19.65, 10.07. HR-MS (ESI) Calcd. for C₂₁H₂₀N₃O₂SCl [M+H]⁺ 414.096 5, Found 414.098 8.
A6	Yellow solid	52	98.1—99.0	¹H NMR δ 12.39 (s, 1H), 8.71 (s, 1H), 7.99 (dd, J = 8.0, 1.2 Hz, 1H), 7.86 (ddd, J = 7.8, 6.1, 1.6 Hz, 2H), 7.66 (td, J = 7.7, 1.4 Hz, 1H), 7.55 —7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.38 —7.33 (m, 2H), 4.29 (t, J = 6.7 Hz, 2H), 1.74 (p, J = 6.8 Hz, 2H), 1.42—1.30 (m, 4H), 0.89 (t, J = 7.0 Hz, 3H). ¹³C NMR δ 167.68, 167.02, 147.63, 140.19, 134.95, 133.70, 132.69, 131.52, 131.22, 130.85, 130.57, 129.52, 129.40, 129.38, 127.73, 126.80, 109.33, 65.60, 28.21, 28.14, 22.29, 14.34. HR-MS (ESI) Calcd. for C₂₂H₂₂N₃O₂SCl [M+H]⁺ 428.112 1, Found 428.120 1.
A7	Yellow solid	59	92.3—93.1	¹H NMR δ 12.45 (s, 1H), 8.76 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.96 —7.87 (m, 2H), 7.72 (t, J = 7.7 Hz, 1H), 7.62—7.53 (m, 2H), 7.51—7.37 (m, 3H), 4.34 (t, J = 6.6 Hz, 2H), 1.79 (t, J = 7.4 Hz, 2H), 1.49—1.28 (m, 6H), 0.92 (t, J = 6.7 Hz, 3H). ¹³C NMR δ 167.56, 166.92, 147.53, 140.10, 134.85, 133.60, 132.56, 131.41, 131.12, 130.74, 130.44, 129.39, 129.30, 129.27, 127.60, 126.71, 109.20, 65.51, 31.27, 28.36, 25.52, 22.36, 14.22. HR-MS (ESI) Calcd. for C₂₃H₂₄N₃O₂SCl [M+H]⁺ 442.127 8, Found 442.135 5.
A8	Yellow solid	50	153.4—154.6	¹H NMR δ 12.42 (s, 1H), 8.70 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.90 —7.83 (m, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.56—7.47 (m, 2H), 7.42 (td, J = 7.5, 1.2 Hz, 1H), 7.39 —7.33 (m, 2H), 5.00—4.91 (m, 1H), 1.98—1.89 (m, 2H), 1.78—1.68 (m, 2H), 1.64—1.49 (m, 3H), 1.47—1.25 (m, 3H). ¹³C NMR δ 167.72, 166.37, 140.10, 134.84, 132.62, 131.52, 131.22, 130.86, 130.60, 129.80, 129.52, 129.39, 127.73, 126.67, 109.34, 73.85, 31.44, 25.34, 23.69. HR-MS (ESI) Calcd. for C₂₃H₂₂N₃O₂SCl [M+H]⁺ 440.112 1, Found 440.110 1.
A9	Yellow solid	46	168.3—169.2	¹H NMR δ 12.41 (s, 1H), 8.78 (s, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.92 —7.86 (m, 2H), 7.70—7.65 (m, 1H), 7.56—7.49 (m, 4H), 7.45—7.40 (m, 3H), 7.39 —7.34 (m, 3H), 5.38 (s, 2H). ¹³C NMR δ 166.20, 147.14, 139.61, 135.86, 134.72, 133.20, 132.41, 131.04, 130.73, 130.35, 130.21, 129.01, 128.92, 128.51, 128.37, 128.16, 128.08, 127.22, 126.35, 108.87, 66.59. HR-MS (ESI) Calcd. for C₂₄H₁₈N₃O₂SCl [M+H]⁺ 448.080 8, Found 448.088 8.
A10	Yellow solid	51	116.1—117.0	¹H NMR δ 12.46 (s, 1H), 8.79 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.46—7.34 (m, 6H), 7.32—7.25 (m, 1H), 4.57 (t, J = 6.8 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H). ¹³C NMR δ 167.58, 166.74, 147.55, 140.03, 138.30, 134.93, 133.61, 132.66, 131.43, 131.13, 130.75, 130.45, 129.41, 129.24, 129.03, 128.78, 127.62, 126.80, 126.67, 109.25, 65.98, 34.61. HR-MS (ESI) Calcd. for C₂₅H₂₀N₃O₂SCl [M+H]⁺ 462.096 5, Found 462.104 1.
A11	Yellow solid	49	114.4—115.6	¹H NMR δ 12.43 (s, 1H), 8.73 (s, 1H), 8.03 (dd, J = 8.0, 1.2 Hz, 1H), 7.89—7.83 (m, 2H), 7.74—7.68 (m, 1H), 7.56—7.51 (m, 2H), 7.42 (td, J = 7.5, 1.4 Hz, 1H), 7.39 —7.33 (m, 2H), 5.98 (s, 2H), 1.18 (s, 9H). ¹³C NMR δ 167.13, 164.91, 139.23, 135.19, 133.04, 131.04, 130.73, 130.35, 130.31, 129.04, 129.02, 127.24, 127.06, 126.51, 108.96, 80.13, 38.26, 26.47. HR-MS (ESI) Calcd. for C₂₃H₂₂N₃O₄SCl [M+H]⁺ 472.102 0, Found 472.109 6.
A12	Yellow solid	53	110.3—111.0	¹H NMR δ 12.44 (s, 1H), 8.76 (s, 1H), 8.04 (dd, J = 8.0, 1.2 Hz, 1H), 7.93 —7.85 (m, 2H), 7.73 (td, J = 7.6, 1.3 Hz, 1H), 7.57—7.51 (m, 2H), 7.43 (td, J = 7.5, 1.5 Hz, 1H), 7.40 —7.33 (m, 2H), 5.96 (s, 2H), 4.85 (hept, J = 6.2 Hz, 1H), 1.26 (d, J = 6.2 Hz, 6H). ¹³C NMR δ 167.62, 165.32, 153.25, 147.67, 139.76, 135.83, 133.66, 131.54, 131.23, 131.01, 130.85, 129.54, 129.49, 127.74, 127.30, 126.99, 109.47, 83.02, 73.24, 21.80. HR-MS (ESI) Calcd. for C₂₂H₂₀N₃O₅SCl [M+H]⁺ 474.081 2, Found 474.088 9.

Prodrug

Appearance

Yield/%

mp/℃

¹H NMR, ¹³C NMR, HR-MS

Yellow solid

172.2—172.9

Yellow solid

170.1—171.2

Yellow solid

159.5—160.3

Yellow solid

144.3—145.4

Yellow solid

137.4—138.2

Yellow solid

98.1—99.0

Yellow solid

92.3—93.1

Yellow solid

153.4—154.6

Yellow solid

168.3—169.2

A10

Yellow solid

116.1—117.0

A11

Yellow solid

114.4—115.6

A12

Yellow solid

110.3—111.0

Compd.	Inhibition rate/%	^aIC₅₀/μmol·L¹
A	94.60	0.02 ± 0.00
A1	73.08	2.34 ± 0.05
A2	91.04	1.41 ± 0.07
A3	75.96	1.86 ± 0.02
A4	61.82	3.53 ± 0.15
A5	61.32	4.65 ± 0.03
A6	29.53	> 50.00
A7	28.62	> 50.00
A8	36.82	> 50.00
A9	30.25	> 50.00
A10	34.80	> 50.00
A11	20.22	> 50.00
A12	21.52	> 50.00
Brequinar	92.91	0.01 ± 0.00

Compd.

Inhibition rate/%

^aIC₅₀/μmol·L¹

94.60

0.02 ± 0.00

73.08

2.34 ± 0.05

91.04

1.41 ± 0.07

75.96

1.86 ± 0.02

61.82

3.53 ± 0.15

61.32

4.65 ± 0.03

29.53

> 50.00

28.62

> 50.00

36.82

> 50.00

30.25

> 50.00

A10

34.80

> 50.00

A11

20.22

> 50.00

A12

21.52

> 50.00

Brequinar

92.91

0.01 ± 0.00

Compd.	Inhibition rate/%	^aIC₅₀/μmol·L¹
A	94.60	0.02 ± 0.00
A1	73.08	2.34 ± 0.05
A2	91.04	1.41 ± 0.07
A3	75.96	1.86 ± 0.02
A4	61.82	3.53 ± 0.15
A5	61.32	4.65 ± 0.03
A6	29.53	> 50.00
A7	28.62	> 50.00
A8	36.82	> 50.00
A9	30.25	> 50.00
A10	34.80	> 50.00
A11	20.22	> 50.00
A12	21.52	> 50.00
Brequinar	92.91	0.01 ± 0.00

Compd.

Inhibition rate/%

^aIC₅₀/μmol·L¹

94.60

0.02 ± 0.00

73.08

2.34 ± 0.05

91.04

1.41 ± 0.07

75.96

1.86 ± 0.02

61.82

3.53 ± 0.15

61.32

4.65 ± 0.03

29.53

> 50.00

28.62

> 50.00

36.82

> 50.00

30.25

> 50.00

A10

34.80

> 50.00

A11

20.22

> 50.00

A12

21.52

> 50.00

Brequinar

92.91

0.01 ± 0.00

Compd.	Percent of unchanged compounds/%
A	98.64	97.85	86.13
A1	91.64	82.91	81.26
A2	92.83	85.94	83.27
A3	92.94	84.97	82.55
A4	93.64	86.93	84.62
A5	95.66	87.71	84.26
A6	89.03	75.62	72.75
A7	90.12	88.26	82.73
A8	89.13	85.62	81.45
A9	90.03	88.62	82.75
A10	88.03	74.93	70.18
A11	94.11	87.54	85.91
A12	100.00	100.00	100.00

Compd.

Percent of unchanged compounds/%

24 h

48 h

72 h

98.64

97.85

86.13

91.64

82.91

81.26

92.83

85.94

83.27

92.94

84.97

82.55

93.64

86.93

84.62

95.66

87.71

84.26

89.03

75.62

72.75

90.12

88.26

82.73

89.13

85.62

81.45

90.03

88.62

82.75

A10

88.03

74.93

70.18

A11

94.11

87.54

85.91

A12

100.00

Compd.	Percent of unchanged compounds/%
A	98.64	97.85	86.13
A1	91.64	82.91	81.26
A2	92.83	85.94	83.27
A3	92.94	84.97	82.55
A4	93.64	86.93	84.62
A5	95.66	87.71	84.26
A6	89.03	75.62	72.75
A7	90.12	88.26	82.73
A8	89.13	85.62	81.45
A9	90.03	88.62	82.75
A10	88.03	74.93	70.18
A11	94.11	87.54	85.91
A12	100.00	100.00	100.00

Compd.

Percent of unchanged compounds/%

24 h

48 h

72 h

98.64

97.85

86.13

91.64

82.91

81.26

92.83

85.94

83.27

92.94

84.97

82.55

93.64

86.93

84.62

95.66

87.71

84.26

89.03

75.62

72.75

90.12

88.26

82.73

89.13

85.62

81.45

90.03

88.62

82.75

A10

88.03

74.93

70.18

A11

94.11

87.54

85.91

A12

100.00

Compd.	Percent of unchanged compounds/%
A1	0.00	0.00	0.00	0.00
A2	22.24	11.90	0.00	0.00
A3	31.53	19.36	9.43	0.00
A4	29.42	17.92	9.10	0.00
A5	32.24	16.50	6.22	0.00
A6	36.62	19.56	10.30	0.00
A7	41.25	22.78	12.36	1.26
A8	40.12	18.91	11.51	0.00
A9	88.24	77.90	56.36	45.26
A10	95.32	91.80	89.62	58.22
A11	99.38	98.30	96.19	87.26
A12	99.76	0.00	0.00	0.00

Compd.

Percent of unchanged compounds/%

3 h

6 h

12 h

24 h

0.00

22.24

11.90

0.00

31.53

19.36

9.43

0.00

29.42

17.92

9.10

0.00

32.24

16.50

6.22

0.00

36.62

19.56

10.30

0.00

41.25

22.78

12.36

1.26

40.12

18.91

11.51

0.00

88.24

77.90

56.36

45.26

A10

95.32

91.80

89.62

58.22

A11

99.38

98.30

96.19

87.26

A12

99.76

0.00

Compd.	Percent of unchanged compounds/%
A1	0.00	0.00	0.00	0.00
A2	22.24	11.90	0.00	0.00
A3	31.53	19.36	9.43	0.00
A4	29.42	17.92	9.10	0.00
A5	32.24	16.50	6.22	0.00
A6	36.62	19.56	10.30	0.00
A7	41.25	22.78	12.36	1.26
A8	40.12	18.91	11.51	0.00
A9	88.24	77.90	56.36	45.26
A10	95.32	91.80	89.62	58.22
A11	99.38	98.30	96.19	87.26
A12	99.76	0.00	0.00	0.00

Compd.

Percent of unchanged compounds/%

3 h

6 h

12 h

24 h

0.00

22.24

11.90

0.00

31.53

19.36

9.43

0.00

29.42

17.92

9.10

0.00

32.24

16.50

6.22

0.00

36.62

19.56

10.30

0.00

41.25

22.78

12.36

1.26

40.12

18.91

11.51

0.00

88.24

77.90

56.36

45.26

A10

95.32

91.80

89.62

58.22

A11

99.38

98.30

96.19

87.26

A12

99.76

0.00

Compd.	Percent of unchanged compounds/%
A1	76.48	58.48	39.36	17.69
A2	80.96	66.09	47.08	25.11
A3	88.25	76.08	60.17	36.32
A4	82.92	69.28	57.18	35.71
A5	85.13	70.29	55.04	33.18
A6	81.42	67.19	49.68	26.13
A7	78.37	63.14	45.03	23.52
A8	83.92	71.05	52.08	26.12
A9	65.96	47.09	37.68	19.11
A10	55.18	33.05	14.65	3.12
A11	0.00	0.00	0.00	0.00
A12	0.00	0.00	0.00	0.00

Compd.

Percent of unchanged compounds/%

3 h

6 h

12 h

24 h

76.48

58.48

39.36

17.69

80.96

66.09

47.08

25.11

88.25

76.08

60.17

36.32

82.92

69.28

57.18

35.71

85.13

70.29

55.04

33.18

81.42

67.19

49.68

26.13

78.37

63.14

45.03

23.52

83.92

71.05

52.08

26.12

65.96

47.09

37.68

19.11

A10

55.18

33.05

14.65

3.12

A11

0.00

A12

0.00

Compd.	Percent of unchanged compounds/%
A1	76.48	58.48	39.36	17.69
A2	80.96	66.09	47.08	25.11
A3	88.25	76.08	60.17	36.32
A4	82.92	69.28	57.18	35.71
A5	85.13	70.29	55.04	33.18
A6	81.42	67.19	49.68	26.13
A7	78.37	63.14	45.03	23.52
A8	83.92	71.05	52.08	26.12
A9	65.96	47.09	37.68	19.11
A10	55.18	33.05	14.65	3.12
A11	0.00	0.00	0.00	0.00
A12	0.00	0.00	0.00	0.00

Compd.

Percent of unchanged compounds/%

3 h

6 h

12 h

24 h

76.48

58.48

39.36

17.69

80.96

66.09

47.08

25.11

88.25

76.08

60.17

36.32

82.92

69.28

57.18

35.71

85.13

70.29

55.04

33.18

81.42

67.19

49.68

26.13

78.37

63.14

45.03

23.52

83.92

71.05

52.08

26.12

65.96

47.09

37.68

19.11

A10

55.18

33.05

14.65

3.12

A11

0.00

A12

0.00

Compd.	IC₅₀/μmol·L^-1
Brequinar	0.50 ± 0.01
A	1.52 ± 0.01
A1	> 50.00
A2	> 50.00
A3	> 50.00
A4	> 50.00
A5	> 50.00
A6	> 50.00
A7	> 50.00
A8	> 50.00
A9	> 50.00
A10	> 50.00
A11	14.23 ± 0.63
A12	0.63 ± 0.01

Compd.

IC₅₀/μmol·L^-1

Brequinar

0.50 ± 0.01

1.52 ± 0.01

> 50.00

A10

> 50.00

A11

14.23 ± 0.63

A12

0.63 ± 0.01

Compd.	IC₅₀/μmol·L^-1
Brequinar	0.50 ± 0.01
A	1.52 ± 0.01
A1	> 50.00
A2	> 50.00
A3	> 50.00
A4	> 50.00
A5	> 50.00
A6	> 50.00
A7	> 50.00
A8	> 50.00
A9	> 50.00
A10	> 50.00
A11	14.23 ± 0.63
A12	0.63 ± 0.01

Compd.

IC₅₀/μmol·L^-1

Brequinar

0.50 ± 0.01

1.52 ± 0.01

> 50.00

A10

> 50.00

A11

14.23 ± 0.63

A12

0.63 ± 0.01

二氢乳清酸脱氢酶抑制剂前药的合成和稳定性及活性评价

PDF下载

单继伟 , 齐甜甜 , 李洪林 , 朱丽丽 , 赵振江 ^*

药学学报 | 研究论文·药物化学 2018,53(3): 410-415

收起

药学学报 | 研究论文·药物化学 2018, 53(3): 410-415

二氢乳清酸脱氢酶抑制剂前药的合成和稳定性及活性评价

全屏

单继伟, 齐甜甜, 李洪林, 朱丽丽, 赵振江^*

作者信息

华东理工大学药学院, 上海市新药设计重点实验室, 上海 200237

通讯作者:

* 赵振江, Tel:86-21-64253962, E-mail:zhjzhao@ecust.edu.cn

Synthesis, stability and activity of the prodrugs of dihydroorotate dehydrogenase inhibitors

Ji-wei SHAN, Tian-tian QI, Hong-lin LI, Li-li ZHU, Zhen-jiang ZHAO^*

Affiliations

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China

出版时间: 2018-03-12 doi: 10.16438/j.0513-4870.2017-0992

文章导航

摘要

收起

本文以课题组前期发现的高活性DHODH抑制剂A为基础，依据前药合成策略，对其羧基进行酯化修饰得到12个前药分子，旨在优化化合物A的稳定性。分子水平抑制活性显示12个前药分子中只有A1~A5有微弱活性，与其作用机制和前药设计相符；随后评价了A1~A12在有机溶剂甲醇和pH为2.0、9.0的缓冲液中的稳定性，结果显示A12在甲醇中能较好的抑制先导分子内成环，A1~A8在酸性条件下易于水解，A9~A12在碱性条件易于水解；最后评价了A1~A12的细胞增殖抑制活性，其中A12表现出很好活性，其IC₅₀值为0.63 μmol·L^-1，与布奎那相当。这些结果为开展进一步体内活性研究奠定了基础。

关键词

二氢乳清酸脱氢酶抑制剂 / 前药设计 / 药物稳定性 / 活性评价

Abstract

收起

This study was conducted to improve structural instability of a highly active DHODH inhibitor A found in our group. Twelve prodrugs were synthesized by modifying the carboxyl group. The enzyme activity test of 12 prodrugs A1-A12 demonstrated that A1-A5 displayed weak inhibitory activity, and A6-A12 displayed no activity, which met the action mechanism of designed prodrug. The structural stability of A1-A12 in methanol and pH 2.0, 9.0 buffers were tested, and the results showed that A12 could avoid intramolecular ring-formation in CH₃OH, A1-A8 were easily hydrolyzed under acidic conditions, and A9-A12 were inclined to hydrolyze under alkaline conditions. The cell proliferation inhibitory activity of 12 prodrugs were evaluated, in which compound A12 displayed excellent activity (IC₅₀=0.63 μmol·L^-1) similar to brequinar. These results laid a good foundation for conducting further vivo studies.

Key words

dihydroorotate dehydrogenase inhibitor / prodrug design / drug stability / inhibitory activity evaluation

引用本文

单继伟, 齐甜甜, 李洪林, 朱丽丽, 赵振江. 二氢乳清酸脱氢酶抑制剂前药的合成和稳定性及活性评价. 药学学报, 2018 , 53 (3) : 410 -415 . DOI: 10.16438/j.0513-4870.2017-0992

Ji-wei SHAN, Tian-tian QI, Hong-lin LI, Li-li ZHU, Zhen-jiang ZHAO. Synthesis, stability and activity of the prodrugs of dihydroorotate dehydrogenase inhibitors[J]. Acta Pharmaceutica Sinica, 2018 , 53 (3) : 410 -415 . DOI: 10.16438/j.0513-4870.2017-0992

正文

收起

二氢乳清酸脱氢酶(dihydroorotate dehydrogenase, DHODH)是嘧啶从头合成的关键酶^[1]。它是一种含铁的黄素依赖性线粒体酶, 对细胞内嘧啶合成起决定作用。细胞内的嘧啶来源主要有两个途径:从头合成途径和补救合成途径。从头合成途径所需的氮源来自谷氨酰胺和天冬氨酸, 碳源来自二氧化碳^[2]。补救合成途径是细胞内已有的嘧啶或自身代谢的嘧啶核苷、碱基的重复利用从而保证细胞的正常运转。对于恶性增殖的细胞或癌细胞来说, 补救合成途径生成的嘧啶核苷酸的量无法维持细胞生存, 细胞只能依赖嘧啶的从头合成途径^[3]。因此, 抑制了DHODH的活性即可阻止DNA和RNA的合成, 进一步抑制活化细胞的增殖和代谢, 从而起到治疗疾病的作用。DHODH已被确证是多种疾病的治疗靶点, 如癌症、病毒、自身免疫性疾病(类风湿关节炎和多发性硬化)等^[3]。

1998年, 来氟米特(leflunomide, 图 1)被美国食品药品监督管理局(FDA)批准上市^[4]。来氟米特是一个含有异恶唑环的前体药物, 自身没有生物活性, 它的体内代谢产物A771726 (特立氟胺, 图 1)是其药物活性的来源^[5]。来氟米特的作用机制是通过抑制DHODH的活性来抑制细胞呼吸链的电子传递, 致使嘧啶合成受阻, 阻断了DNA复制, 从而导致细胞凋亡^[6]。布奎那(brequinar, 图 1)是以喹啉为母核的化合物, 其作用机制和来氟米特相同, 均是作用于CoQ结合通道^[7]。起初布奎那是用来治疗多种人类实体瘤, 但是人们在研究过程中发现布奎那有很强的不良反应, 因此其在肿瘤方面的研究已被FDA叫停。人们进一步研究发现布奎那可以用来治疗自身性免疫疾病和病毒性感染^[8]。

本课题组前期发现并合成了高活性的DHODH抑制剂: (E)-4-(2-氯苯基)-2-(2-羧基苄叉肼基)噻唑(A), 化合物A的IC₅₀值为29 nmol·L^-1, 并表现出较好的开发前景, 随后课题组得到了A-DHODH复合物的晶体结构并阐明其作用机制^[9]。化合物A的羧基可以与酶口袋内部质子化的Arg136的胍基形成很好的氢键相互作用, 这是化合物A具有高活性的决定因素。

在进一步的研究中发现化合物A在特定环境下羧基与肼基的NH之间有分子内成环形成哒嗪环B的可能性^[9]。为此, 本文以羧基为切入点, 依据前药策略设计合成了12个前药分子, 并探究了12个前药分子的酶抑制活性、化学环境稳定性和细胞增殖抑制活性, 为进一步的前药体内药效研究奠定基础。前药分子的合成路线如合成路线1所示。

结果与讨论

收起

1 前药化合物的设计合成

根据前药设计方法对母体分子A的羧基进行修饰得到一系列酯类化合物, 期望改善药物的稳定性等理化性质。

本文先合成了8个不同取代基的脂肪酯A1~A8。为了探究引入苯环对化合物活性的影响, 合成了化合物A的苯甲酯A9和苯乙酯A10; 参考双酯前药的合成策略, 合成了化合物A的特戊酰氧基甲酯A11和异丙氧基羰氧基甲酯A12。

2 前药分子的表征

前药分子的表征见表 1。

3 前药分子的活性评价

3.1 前药分子的酶活

根据表 2中酶活数据可以看出, 除了A1~A5有微弱抑制活性, 其余7个前药均未表现出对DHODH的抑制活性。DHODH的CoQ结合口袋可分为两个区域—内部的亲水区和外部的疏水区, 化合物A的羧基可以与口袋内部亲水区的Arg136的质子化胍基形成氢键, 这是化合物A生物活性的来源。较小体积的烷基酯A1~A5的活性较化合物A降低明显, 原因在于酯基虽然能与Arg136形成氢键, 却不能形成作用力更强的盐桥作用。随着取代基团的增大, 前药不能占据DHODH的亲水结合口袋, 因此A6~A12均没表现出对DHODH的抑制活性。

3.2 前药分子的稳定性

本文随后测量了12个前药分子和化合物A在甲醇中的稳定性, 主要是为了探究前药对化合物A的分子内成环的抑制, 结果见表 3。化合物A在甲醇中72 h内会有13.87%的环化产物B生成, A1~A11在72 h内也有不同程度的环化, 但A12未发生任何变化。这表明化合物A成不同的酯后在醇体系中的稳定性区别很大。

为了探究前药分子的水解趋势, 本文选取pH为2.0、9.0的两个缓冲体系来探究其在24 h内的水解情况(表 4、5)。综合比较两个表中的数据: A1~A8为化合物A的脂肪酯。在酸性条件下, 8个分子在24 h时有7个完全水解为化合物A, A7也仅有1.26%未水解, 对比A1~A8在pH = 9.0的缓冲体系中分别有17.69%、25.11%、36.32%、35.71%、33.18%、26.13%、23.52%、26.12%的剩余, 表明A1~A8在酸性条件下易于水解为化合物A; A9~A12分别为A的苯甲酯、苯乙酯、特戊酸甲酯、异丙氧基羰基氧基甲酯, 对比两个表中的数据, 发现这4个化合物在碱性条件下更易于水解为化合物A。

3.3 前药分子的细胞增殖抑制活性

本文对12个前药分子进行了细胞增殖抑制活性的评价。根据表 6所示:在BXPC-3细胞测试中A12表现出较好抑制活性, 其IC₅₀值为0.63 μmol·L^-1, 与阳性对照brequinar的IC₅₀值0.50 μmol·L^-1相当, 同时与先导物A比较活性也有改善, 这表明化合物A12可以有效的改善化合物A的理化性能以便更好发挥细胞抑制增殖作用。化合物A12为化合物A的异丙氧基羰基氧基甲酯, 为双酯前药, 其水解可分为两个过程, 先是末端酯键位阻较小易于水解, 随后得到羟甲酯可以自行水解到A。这表明前药A12在进入肿瘤细胞的过程中, 酯基可以在较好的时机释放出原药。

小结

收起

本文基于课题组前期发现的高活性DHODH抑制剂A, 依据前药合成策略, 针对母体分子A的结构特征, 对其活性基团羧基进行修饰得到了12个酯类前药。首先对12个前药分子进行了酶活测试, 结果显示12个前药分子中除了A1~A5有些许活性, 其余7个前药分子均没有酶抑制活性, 结果与其作用机制和前药设计符合; 进一步对12个前药分子的稳定性做了测试, 发现羧基成不同的酯后可以调控化合物水解及分子内成环; 对12个前药分子开展了细胞增殖抑制活性评价, 结果显示化合物A12的细胞增殖抑制活性较好, 与布奎那活性相当。为进一步的体内活性研究奠定了基础。

实验部分

收起

实验中用到的化学试剂均为市售分析纯, 纯度符合实验要求, 无需纯化, 可直接使用; 柱色谱硅胶(300~400目)为泰坦科技售出; 薄层硅胶制备板由青岛海洋化工厂生产; 化合物的核磁氢谱(¹H NMR)数据和核磁碳谱(¹³C NMR)数据均由Brucker AM-400 (400 MHz)测定, TMS为内标; 化合物质谱数据由Micromass GCT CA 055质谱仪测定。

1 合成部分[前药的合成(以A1为例)]

(E)-4-(2-氯苯基)-2-(2-乙氧基甲酰基苄叉肼基)噻唑(A1):称取化合物A (0.2 g, 0.5 mmol)于50 mL两口瓶中, 加入碳酸铯(0.4 g, 1.1 mmol)和N, N-二甲基甲酰胺(DMF) 10 mL, 电磁搅拌下加入碘乙烷(0.2 g, 1.1 mmol), 加热升温至70 ℃, 反应过程中用TLC检测, 反应5 h后原料反应完全, 停止加热, 向反应瓶中加入20 mL水, 用二氯甲烷(DCM)萃取反应液, 静置分层取有机相, 水相用DCM萃取3次, 合并有机相, 干燥, 旋干溶剂, 过柱纯化, 得到黄色粉末固体123.0 mg。

2 前药分子的稳定性及体外活性测试

2.1 前药分子的DHODH抑制活性

二氢乳清酸脱氢酶(DHODH)抑制剂前药的酶活测试方法为DCIP比色法, 其作用机制是:将纯化酶、CoQ和DCIP三者混合后点进96孔板内, 25 ℃下孵育10 min, 随后加入原料二氢乳清酸开始反应。正常态的DCIP在600 nm处有最大吸收, 而还原态的DCIP在600 nm处无吸收^[10]。根据这个特性, 可以通过检测反应液在600 nm处吸光度的减弱速率大小来衡量二氢乳清酸的氧化程度, 进一步推测化合物的活性强弱。速率越大说明反应进行越快, 酶的受抑制程度越小, 相对应的抑制剂活性就低。实验以brequinar为阳性对照, 将12个前药分子和母体分子A共13个样品稀释为8个浓度梯度, 每组均设3个平行组。

2.2 前药分子的稳定性

稳定性测试采用的是HPLC测试的方法。稳定性测试的化学环境是甲醇、pH = 2.0的盐酸溶液、pH = 9.0的缓冲液(氢氧化钠和磷酸氢二钾)。甲醇溶液的配置:前药分子A1~A12和化合物A各取0.5 mg溶于1 mL甲醇中得到甲醇溶液。甲醇溶液分别在0、24、48和72 h的时间段HPLC进样10 µL, 根据溶液中化合物的峰面积变化, 进而推测其结构稳定性; 酸性溶液和碱性溶液的配置:称取化合物1 mg配制成1 mg·mL^-1的母液。母液各取1 mL加入0.5 mL的缓冲溶液配制样品, 分别在0、3、6、12和24 h的时间段取样, 最后比较其在缓冲溶液中的峰面积变化, 进而推测其结构稳定性。测试条件:色谱柱为Zorbax XDB-C18 (4.6 mm × 250 mm); 流动相为纯化水(0.1%TFA)与99%甲醇; 洗脱时间10 min; 流速为0.85 mL·min^-1; 柱温为35℃; 检测波长254 nm。

2.3 前药分子的BXPC-3细胞抑制活性

实验选用的细胞为原位胰腺腺癌细胞(BXPC-3), 采用了MTT比色法^[10], 用于检测细胞存活和生长。MTT[3-(4, 5-二甲基噻唑-2)-2, 5-二苯基四氮唑溴盐]是一种黄颜色的染料, 其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲瓒并沉积在细胞中, 而死细胞无此功能。二甲基亚砜(DMSO)能溶解细胞中的甲瓒, 用酶联免疫检测仪在490 nm波长处测定其光吸收值, 可间接反映活细胞数量。在一定细胞数范围内, MTT结晶形成的量与细胞数成正比。检测化合物对BXPC-3细胞的增殖抑制能力, 即对BXPC-3细胞的半数致死率。具体操作步骤为使用含有10% FBS的1640培养基培养细胞, 并以每孔5 000个细胞接种至96孔板, 置于37℃、5% CO₂培养箱中培养, 孵育24 h, 分别加入不同浓度梯度的药物, 继续孵育48 h; 之后每孔加入终浓度为5 mg·mL^-1的MTT溶液混匀, 最后在490 nm波长下测定各孔光吸收值。使用Origin软件处理实验数据并计算化合物的IC₅₀值。实验过程采用brequinar为阳性对照, 每次实验3次平行。

基金

收起

国家自然科学基金资助项目(21372078)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

Singh A, Maqbool M, Mobashir M, et al. Dihydroorotate dehydrogenase:an inevitable drug target for the development of antimalarials[J]. Eur J Med Chem, 2016, 125:640-651.

[2]

Jones ME. Pyrimidine nucleotide biosynthesis in animals:genes, enzymes, and regulation of UMP biosynthesis[J]. Annu Rev Biochem, 1980, 49:253-279.

[3]

Liu S, Neidhardt EA, Grossman TH, et al. Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents[J]. Structure, 2000, 8:25-33.

[4]

Breedveld FC, Dayer JM. Leflunomide:mode of action in the treatment of rheumatoid arthritis[J]. Ann Rheum Dis, 2000, 59:841-849.

[5]

Baumann P, Mandlweber S, V lkl A, et al. Dihydroorotate dehydrogenase inhibitor A771726(leflunomide) induces apoptosis and diminishes proliferation of multiple myeloma cells[J]. Mol Cancer Ther, 2009, 8:366-375.

[6]

Hansen M, Le Nours J, Johansson E, et al. Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain[J]. Protein Sci, 2004, 13:1031-1042.

[7]

Thomson AW, Lemster B, Fung JJ, et al. Potentiation of the antiproliferative activity of brequinar sodium for murine lymphocytes by exogenous cytidine[J]. Transplant Proc, 1993, 25:704-705.

[8]

Makowka L, Sher LS, Cramer DV. The development of brequinar as an immunosuppressive drug for transplantation[J]. Immunol Rev, 1993, 136:51-70.

[9]

Song W, Li SL, et al. Structure based design of potent human dihydroorotate dehydrogenase inhibitors as anticancer agents[J]. Med Chem Commun, 2016, 7:1441-1448.

[10]

Diao Y, Lu W, Jin H, et al. Discovery of diverse human dihydroorotate dehydrogenase inhibitors as immunosuppressive agents by structure based virtual screening[J] J Med Chem, 2012, 55:8341-8349.

2018年第53卷第3期

PDF下载

118

引用本文

BibTeX

文章信息

doi: 10.16438/j.0513-4870.2017-0992

接收时间：2017-10-12
首发时间：2026-01-15
出版时间：2018-03-12

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2017-10-12
修回日期：2018-01-12

基金

国家自然科学基金资助项目(21372078)

作者信息

华东理工大学药学院, 上海市新药设计重点实验室, 上海 200237

通讯作者:

* 赵振江, Tel:86-21-64253962, E-mail:zhjzhao@ecust.edu.cn

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2017-0992

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

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

关闭全屏

BibTeX
EndNote
RefWorks
TxT

Prodrug	Appearance	Yield/%	mp/℃	¹H NMR, ¹³C NMR, HR-MS
A1	Yellow solid	57	172.2—172.9	¹H NMR δ 13.30 (br, 1H), 12.05 (br, 1H), 8.84 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.91 (t, J = 8.0 Hz, 2H), 7.66 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.38 — 7.34 (m, 2H).¹³C NMR δ 167.92, 167.01, 147.26, 141.03, 134.55, 133.23, 131.94, 131.82, 131.06, 130.70, 130.34, 128.80, 128.74, 128.60, 127.22, 125.96, 109.26, 61.23, 14.10. HR-MS (ESI) Calcd. for C₁₉H₁₇N₃O₂SCl [M+H]⁺ 386.073 0, Found 386.073 8.
A2	Yellow solid	58	170.1—171.2	¹H NMR δ 12.40 (s, 1H), 8.71 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.88 (dd, J = 7.6, 2.4 Hz, 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.52 (q, J = 7.9 Hz, 2H), 7.46—7.32 (m, 3H), 4.26 (t, J = 6.5 Hz, 2H), 1.76 (h, J = 7.1 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H). ¹³C NMR δ 167.68, 167.03, 147.63, 140.16, 134.95, 133.6, 132.71, 131.53, 131.22, 130.85, 130.59, 129.52, 129.4, 129.38, 127.73, 126.74, 109.35, 67.06, 21.96, 10.87. HR-MS (ESI) Calcd. for C₂₀H₁₈N₃O₂SCl [M+H]⁺ /span>400.080 8, Found 400.088 8.
A3	Yellow solid	60	159.5—160.3	¹H NMR δ 12.40 (s, 1H), 8.69 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.90 —7.81 (m, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.55—7.46 (m, 2H), 7.44—7.32 (m, 3H), 5.16 (hept, J = 6.3 Hz, 1H), 1.35 (d, J = 6.1 Hz, 6H). ¹³C NMR δ 205.55, 167.21, 166.04, 147.14, 139.59, 134.28, 133.20, 132.07, 131.02, 130.72, 130.35, 130.07, 129.35, 129.00, 128.84, 127.21, 126.11, 108.82, 68.85, 21.53. HR-MS (ESI) Calcd. for C₂₀H₁sClN₃O₂S [M+H]⁺ 400.080 8, Found 400.086 8.
A4	Yellow solid	55	144.3—145.4	¹H NMR δ 12.40 (s, 1H), 8.71 (s, 1H), 8.00 (dd, J = 8.0, 1.2 Hz, 1H), 7.89—7.84 (m, 2H), 7.67 (td, J = 7.6, 1.3 Hz, 1H), 7.56—7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.39 —7.33 (m, 2H), 4.30 (t, J = 6.6 Hz, 2H), 1.72 (p, 2H), 1.42 (h, 2H), 0.94 (t, J = 7.4 Hz, 3H). ¹³C NMR δ 167.68, 167.02, 147.63, 140.16, 134.95, 133.70, 132.70, 131.53, 131.22, 130.85, 130.58, 129.52, 129.40, 129.37, 127.73, 126.77, 109.35, 65.32, 30.57, 19.23, 14.09. HR-MS (ESI) Calcd. for C₂₁H₂₀N₃O₂SCl [M+H]⁺ 414.096 5, Found 414.104 4.
A5	Yellow solid	52	137.4—138.2	¹H NMR δ 12.41 (s, 1H), 8.71 (s, 1H), 8.00 (dd, J = 7.9, 1.2 Hz, 1H), 7.90 —7.84 (m, 2H), 7.69—7.64 (m, 1H), 7.56—7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.39—7.33 (m, 2H), 5.03 (h, J = 6.3 Hz, 1H), 1.77—1.63 (m, 2H), 1.33 (d, J = 6.3 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H). ¹³C NMR δ 167.71, 166.60, 147.64, 140.09, 134.89, 133.70, 132.64, 131.53, 131.22, 130.86, 130.52, 129.74, 129.52, 129.39, 127.73, 126.67, 109.35, 73.65, 28.66, 19.65, 10.07. HR-MS (ESI) Calcd. for C₂₁H₂₀N₃O₂SCl [M+H]⁺ 414.096 5, Found 414.098 8.
A6	Yellow solid	52	98.1—99.0	¹H NMR δ 12.39 (s, 1H), 8.71 (s, 1H), 7.99 (dd, J = 8.0, 1.2 Hz, 1H), 7.86 (ddd, J = 7.8, 6.1, 1.6 Hz, 2H), 7.66 (td, J = 7.7, 1.4 Hz, 1H), 7.55 —7.48 (m, 2H), 7.42 (td, J = 7.5, 1.5 Hz, 1H), 7.38 —7.33 (m, 2H), 4.29 (t, J = 6.7 Hz, 2H), 1.74 (p, J = 6.8 Hz, 2H), 1.42—1.30 (m, 4H), 0.89 (t, J = 7.0 Hz, 3H). ¹³C NMR δ 167.68, 167.02, 147.63, 140.19, 134.95, 133.70, 132.69, 131.52, 131.22, 130.85, 130.57, 129.52, 129.40, 129.38, 127.73, 126.80, 109.33, 65.60, 28.21, 28.14, 22.29, 14.34. HR-MS (ESI) Calcd. for C₂₂H₂₂N₃O₂SCl [M+H]⁺ 428.112 1, Found 428.120 1.
A7	Yellow solid	59	92.3—93.1	¹H NMR δ 12.45 (s, 1H), 8.76 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.96 —7.87 (m, 2H), 7.72 (t, J = 7.7 Hz, 1H), 7.62—7.53 (m, 2H), 7.51—7.37 (m, 3H), 4.34 (t, J = 6.6 Hz, 2H), 1.79 (t, J = 7.4 Hz, 2H), 1.49—1.28 (m, 6H), 0.92 (t, J = 6.7 Hz, 3H). ¹³C NMR δ 167.56, 166.92, 147.53, 140.10, 134.85, 133.60, 132.56, 131.41, 131.12, 130.74, 130.44, 129.39, 129.30, 129.27, 127.60, 126.71, 109.20, 65.51, 31.27, 28.36, 25.52, 22.36, 14.22. HR-MS (ESI) Calcd. for C₂₃H₂₄N₃O₂SCl [M+H]⁺ 442.127 8, Found 442.135 5.
A8	Yellow solid	50	153.4—154.6	¹H NMR δ 12.42 (s, 1H), 8.70 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.90 —7.83 (m, 2H), 7.66 (t, J = 7.4 Hz, 1H), 7.56—7.47 (m, 2H), 7.42 (td, J = 7.5, 1.2 Hz, 1H), 7.39 —7.33 (m, 2H), 5.00—4.91 (m, 1H), 1.98—1.89 (m, 2H), 1.78—1.68 (m, 2H), 1.64—1.49 (m, 3H), 1.47—1.25 (m, 3H). ¹³C NMR δ 167.72, 166.37, 140.10, 134.84, 132.62, 131.52, 131.22, 130.86, 130.60, 129.80, 129.52, 129.39, 127.73, 126.67, 109.34, 73.85, 31.44, 25.34, 23.69. HR-MS (ESI) Calcd. for C₂₃H₂₂N₃O₂SCl [M+H]⁺ 440.112 1, Found 440.110 1.
A9	Yellow solid	46	168.3—169.2	¹H NMR δ 12.41 (s, 1H), 8.78 (s, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.92 —7.86 (m, 2H), 7.70—7.65 (m, 1H), 7.56—7.49 (m, 4H), 7.45—7.40 (m, 3H), 7.39 —7.34 (m, 3H), 5.38 (s, 2H). ¹³C NMR δ 166.20, 147.14, 139.61, 135.86, 134.72, 133.20, 132.41, 131.04, 130.73, 130.35, 130.21, 129.01, 128.92, 128.51, 128.37, 128.16, 128.08, 127.22, 126.35, 108.87, 66.59. HR-MS (ESI) Calcd. for C₂₄H₁₈N₃O₂SCl [M+H]⁺ 448.080 8, Found 448.088 8.
A10	Yellow solid	51	116.1—117.0	¹H NMR δ 12.46 (s, 1H), 8.79 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.46—7.34 (m, 6H), 7.32—7.25 (m, 1H), 4.57 (t, J = 6.8 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H). ¹³C NMR δ 167.58, 166.74, 147.55, 140.03, 138.30, 134.93, 133.61, 132.66, 131.43, 131.13, 130.75, 130.45, 129.41, 129.24, 129.03, 128.78, 127.62, 126.80, 126.67, 109.25, 65.98, 34.61. HR-MS (ESI) Calcd. for C₂₅H₂₀N₃O₂SCl [M+H]⁺ 462.096 5, Found 462.104 1.
A11	Yellow solid	49	114.4—115.6	¹H NMR δ 12.43 (s, 1H), 8.73 (s, 1H), 8.03 (dd, J = 8.0, 1.2 Hz, 1H), 7.89—7.83 (m, 2H), 7.74—7.68 (m, 1H), 7.56—7.51 (m, 2H), 7.42 (td, J = 7.5, 1.4 Hz, 1H), 7.39 —7.33 (m, 2H), 5.98 (s, 2H), 1.18 (s, 9H). ¹³C NMR δ 167.13, 164.91, 139.23, 135.19, 133.04, 131.04, 130.73, 130.35, 130.31, 129.04, 129.02, 127.24, 127.06, 126.51, 108.96, 80.13, 38.26, 26.47. HR-MS (ESI) Calcd. for C₂₃H₂₂N₃O₄SCl [M+H]⁺ 472.102 0, Found 472.109 6.
A12	Yellow solid	53	110.3—111.0	¹H NMR δ 12.44 (s, 1H), 8.76 (s, 1H), 8.04 (dd, J = 8.0, 1.2 Hz, 1H), 7.93 —7.85 (m, 2H), 7.73 (td, J = 7.6, 1.3 Hz, 1H), 7.57—7.51 (m, 2H), 7.43 (td, J = 7.5, 1.5 Hz, 1H), 7.40 —7.33 (m, 2H), 5.96 (s, 2H), 4.85 (hept, J = 6.2 Hz, 1H), 1.26 (d, J = 6.2 Hz, 6H). ¹³C NMR δ 167.62, 165.32, 153.25, 147.67, 139.76, 135.83, 133.66, 131.54, 131.23, 131.01, 130.85, 129.54, 129.49, 127.74, 127.30, 126.99, 109.47, 83.02, 73.24, 21.80. HR-MS (ESI) Calcd. for C₂₂H₂₀N₃O₅SCl [M+H]⁺ 474.081 2, Found 474.088 9.

Prodrug

Appearance

Yield/%

mp/℃

¹H NMR, ¹³C NMR, HR-MS

Yellow solid

172.2—172.9

Yellow solid

170.1—171.2

Yellow solid

159.5—160.3

Yellow solid

144.3—145.4

Yellow solid

137.4—138.2

Yellow solid

98.1—99.0

Yellow solid

92.3—93.1

Yellow solid

153.4—154.6

Yellow solid

168.3—169.2

A10

Yellow solid

116.1—117.0

A11

Yellow solid

114.4—115.6

A12

Yellow solid

110.3—111.0

Compd.	Inhibition rate/%	^aIC₅₀/μmol·L¹
A	94.60	0.02 ± 0.00
A1	73.08	2.34 ± 0.05
A2	91.04	1.41 ± 0.07
A3	75.96	1.86 ± 0.02
A4	61.82	3.53 ± 0.15
A5	61.32	4.65 ± 0.03
A6	29.53	> 50.00
A7	28.62	> 50.00
A8	36.82	> 50.00
A9	30.25	> 50.00
A10	34.80	> 50.00
A11	20.22	> 50.00
A12	21.52	> 50.00
Brequinar	92.91	0.01 ± 0.00

Compd.

Inhibition rate/%

^aIC₅₀/μmol·L¹

94.60

0.02 ± 0.00

73.08

2.34 ± 0.05

91.04

1.41 ± 0.07

75.96

1.86 ± 0.02

61.82

3.53 ± 0.15

61.32

4.65 ± 0.03

29.53

> 50.00

28.62

> 50.00

36.82

> 50.00

30.25

> 50.00

A10

34.80

> 50.00

A11

20.22

> 50.00

A12

21.52

> 50.00

Brequinar

92.91

0.01 ± 0.00

Compd.	Percent of unchanged compounds/%
A	98.64	97.85	86.13
A1	91.64	82.91	81.26
A2	92.83	85.94	83.27
A3	92.94	84.97	82.55
A4	93.64	86.93	84.62
A5	95.66	87.71	84.26
A6	89.03	75.62	72.75
A7	90.12	88.26	82.73
A8	89.13	85.62	81.45
A9	90.03	88.62	82.75
A10	88.03	74.93	70.18
A11	94.11	87.54	85.91
A12	100.00	100.00	100.00

Compd.

Percent of unchanged compounds/%

24 h

48 h

72 h

98.64

97.85

86.13

91.64

82.91

81.26

92.83

85.94

83.27

92.94

84.97

82.55

93.64

86.93

84.62

95.66

87.71

84.26

89.03

75.62

72.75

90.12

88.26

82.73

89.13

85.62

81.45

90.03

88.62

82.75

A10

88.03

74.93

70.18

A11

94.11

87.54

85.91

A12

100.00

Compd.	Percent of unchanged compounds/%
A1	0.00	0.00	0.00	0.00
A2	22.24	11.90	0.00	0.00
A3	31.53	19.36	9.43	0.00
A4	29.42	17.92	9.10	0.00
A5	32.24	16.50	6.22	0.00
A6	36.62	19.56	10.30	0.00
A7	41.25	22.78	12.36	1.26
A8	40.12	18.91	11.51	0.00
A9	88.24	77.90	56.36	45.26
A10	95.32	91.80	89.62	58.22
A11	99.38	98.30	96.19	87.26
A12	99.76	0.00	0.00	0.00

Compd.

Percent of unchanged compounds/%

3 h

6 h

12 h

24 h

0.00

22.24

11.90

0.00

31.53

19.36

9.43

0.00

29.42

17.92

9.10

0.00

32.24

16.50

6.22

0.00

36.62

19.56

10.30

0.00

41.25

22.78

12.36

1.26

40.12

18.91

11.51

0.00

88.24

77.90

56.36

45.26

A10

95.32

91.80

89.62

58.22

A11

99.38

98.30

96.19

87.26

A12

99.76

0.00

Compd.	Percent of unchanged compounds/%
A1	76.48	58.48	39.36	17.69
A2	80.96	66.09	47.08	25.11
A3	88.25	76.08	60.17	36.32
A4	82.92	69.28	57.18	35.71
A5	85.13	70.29	55.04	33.18
A6	81.42	67.19	49.68	26.13
A7	78.37	63.14	45.03	23.52
A8	83.92	71.05	52.08	26.12
A9	65.96	47.09	37.68	19.11
A10	55.18	33.05	14.65	3.12
A11	0.00	0.00	0.00	0.00
A12	0.00	0.00	0.00	0.00

Compd.

Percent of unchanged compounds/%

3 h

6 h

12 h

24 h

76.48

58.48

39.36

17.69

80.96

66.09

47.08

25.11

88.25

76.08

60.17

36.32

82.92

69.28

57.18

35.71

85.13

70.29

55.04

33.18

81.42

67.19

49.68

26.13

78.37

63.14

45.03

23.52

83.92

71.05

52.08

26.12

65.96

47.09

37.68

19.11

A10

55.18

33.05

14.65

3.12

A11

0.00

A12

0.00

Compd.	IC₅₀/μmol·L^-1
Brequinar	0.50 ± 0.01
A	1.52 ± 0.01
A1	> 50.00
A2	> 50.00
A3	> 50.00
A4	> 50.00
A5	> 50.00
A6	> 50.00
A7	> 50.00
A8	> 50.00
A9	> 50.00
A10	> 50.00
A11	14.23 ± 0.63
A12	0.63 ± 0.01

Compd.

IC₅₀/μmol·L^-1

Brequinar

0.50 ± 0.01

1.52 ± 0.01

> 50.00

A10

> 50.00

A11

14.23 ± 0.63

A12

0.63 ± 0.01