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Compd.	Yield /%	mp /℃	¹H NMR (600 MHz, CD₃OD) δ	¹³C NMR (150 MHz, CD₃OD) δ	HR-ESI-MS m/s Formula Calcd. Found
7a	38	78.8-80.8	8.14-8.13 (m, 1H), 7.81 (t, J = 8.1 Hz, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.48-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.34-7.30 (m, 4H), 7.25-7.22 (m, 1H), 4.70-4.64 (m, 1H), 3.79 (t, J = 6.1 Hz, 1H), 3.00 (dd, J = 13.7, 7.0 Hz, 1H), 2.92 (dd, J = 13.7, 7.1 Hz, 1H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.88-1.73 (m, 2H), 1.69-1.60 (m, 2H).	170.3, 168.1, 137.7, 135.7, 133.8, 130.8, 129.1 (2), 128.3, 128.2 (2), 127.2, 127.1, 126.4, 126.2, 124.8, 120.1, 116.8, 52.4, 48.9, 40.4, 40.2, 38.5, 28.4, 22.3.	[M+H]⁺ : C₂₅H₃₁N₄O₂ 419.244 2 419.242 2
7b	12	112.2-114.0	8.18 (m, 1H), 7.80-7.75 (m, 3H), 7.55 (dd, J = 8.8, 2.1 Hz, 1H), 7.46-7.43 (m, 1H), 7.40-7.37 (m, 1H), 7.35-7.33 (m, 2H), 7.23-7.22 (m, 2H), 4.66-4.61 (m, 1H), 2.95-2.87 (m, 2H), 2.74-2.65 (m, 3H), 2.61 (dd, J = 14.5, 8.8 Hz, 1H), 1.69-1.48 (m, 5H), 1.28 (s, 9H).	161.8, 161.6, 135.8, 134.8, 133.8, 128.9 (2), 128.1, 127.2, 127.1, 126.1, 125.0 (2), 124.7, 120.0, 117.8, 116.5, 115.9, 53.8, 48.5, 48.2, 40.8, 38.9, 33.8, 31.3, 30.4 (3), 23.6.	[M+H]⁺ : C₂₉H₃₉N₄O₂ 475.306 8 475.305 2
7c	24	67.2-69.1	8.12 (m, 1H), 7.80 (t, J = 8.7 Hz, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.41-7.39 (m, 3H), 7.37-7.34 (m, 2H), 7.31-7.28 (m, 1H), 7.23-7.20 (m, 2H), 6.95-6.94 (m, 2H), 5.02 (s, 2H), 4.64-4.61 (m, 1H), 3.88-3.86 (m, 1H), 2.94 (dd, J = 13.8, 6.7 Hz, 1H), 2.85 (dd, J = 13.8, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.58-2.54 (m, 1H), 1.91-1.78 (m, 2H), 1.70-1.64 (m, 2H).	170.3, 167.5, 157.8, 137.4, 135.7, 133.8, 130.8, 130.1 (2), 129.8, 128.3, 128.1 (2), 127.5, 127.3, 127.1 (3), 126.2, 124.8, 120.1, 116.8, 114.7 (2), 69.6, 52.3, 49.1, 40.5, 39.5, 38.5, 28.1, 22.2.	[M+H]⁺ : C₃₂H₃₇N₄O₃ 525.286 0 525.286 1
7d	21	85.9-87.9	8.14-8.13 (m, 1H), 7.81 (t, J = 8.8 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.69-7.68 (m, 2H), 7.53-7.49 (m, 3H), 7.48-7.45 (m, 1H), 7.43-7.37 (m, 1H), 4.75-4, 69 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 3.05 (dd, J = 7.0, 3.1 Hz, 2H), 2.77 (dd, J = 14.7, 4.4 Hz, 1H), 2.74-2.69 (m, 2H), 2.59 (dd, J = 14.7, 9.4 Hz, 1H), 1.91-1.76 (m, 2H), 1.69-1.62 (m, 2H).	169.9, 167.6, 143.7, 135.6, 133.8, 132.0 (2), 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 120.0, 118.3, 116.8, 110.3, 52.2, 40.6, 40.2, 38.5, 28.1, 27.6, 22.2.	[M+H]⁺ : C₂₆H₃₀N₅O₂ 444.239 4 444.237 6
7e	34	78.4-80.4	8.12 (m, 1H), 7.81 (t, J = 7.9 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.51 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.19-7.12 (m, 4H), 4.67-4.61 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 2.96 (dd, J = 13.7, 6.8 Hz, 1H), 2.87 (dd, J = 13.7, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.55 (dd, J = 14.7, 9.5 Hz, 1H), 2.29 (s, 3H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	170.3, 167.5, 136.1, 135.6, 134.5, 133.8, 130.8, 129.0 (2), 128.8 (2), 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 52.3, 49.0, 40.4, 39.9, 38.5, 28.1, 22.2, 19.7.	[M+H]⁺ : C₂₆H₃₃N₄O₂ 433.259 8 433.258 1
7f	21	46.7-48.7	8.13 (m, 1H), 7.82-7.79 (m, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.32-7.30 (m, 2H), 7.06-7.02 (m, 2H), 4.66-4.63 (m, 1H), 3.86 (t, J = 6.1 Hz, 1H), 2.95 (qd, J = 13.9, 7.0 Hz, 2H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.89-1.79 (m, 2H), 1.69-1.64 (m, 2H).	170.2, 167.6, 161.9, 135.6, 133.8, 133.6, 130.8, 130.8, 130.7, 128.3, 127.3, 127.1, 126.2, 124.8, 120.1, 116.8, 114.9, 114.8, 52.3, 48.9, 40.5, 39.3, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₅H₃₀FN₄O₂ 437.234 7 437.233 3
7g	8	72.8-74.8	8.21-8.18 (m, 2H), 8.13 (m, 1H), 7.81 (t, J = 9.3 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.56-7.51 (m, 3H), 7.48-7.45 (m, 1H), 7.42-7.39 (m, 1H), 4.78-4.73 (m, 1H), 3.87-3.85 (m, 1H), 3.14-3.06 (m, 2H), 2.82-2.72 (m, 3H), 2.64-2.60 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	169.9, 167.6, 147.0, 145.7, 135.6, 133.8, 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 123.2 (2), 120.0, 116.8, 52.2, 48.5, 40.7, 39.9, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₅H₃₀N₅O₄ 464.229 2 464.227 8
7h	11	67.3-68.6	8.13 (s, 1H), 7.82-7.79 (m, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.22-7.20 (m, 2H), 6.88-6.86 (m, 2H), 4.64-4.60 (m, 1H), 3.87-3.86 (m, 1H), 3.75 (m, 3H), 2.93 (dd, J = 13.9, 6.9 Hz, 1H), 2.87-2.83 (m, 1H), 2.75-2.70 (m, 3H), 2.58-2.53 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	170.4, 167.5, 158.7, 135.7, 133.8, 130.8, 130.0 (2), 129.5, 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 113.6 (2), 54.2, 52.3, 49.1, 40.4, 39.4, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₆H₃₃N₄O₃ 449.254 7 449.253 3
7i	4	56.0-58.0	8.97-8.96 (m, 1H), 8.63-8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.89 (m, 1H), 7.72-7.69 (m, 1H), 7.63-7.60 (m, 1H), 7.32-7.29 (m, 4H), 7.23-7.20 (m, 1H), 4.68-4.66 (m, 1H), 3.89-3.87 (m, 1H), 3.03 (dd, J = 13.8, 7.1 Hz, 1H), 2.94 (dd, J = 13.7, 7.1 Hz, 1H), 2.86-2.83 (m, 2H), 2.78 (dd, J = 14.9, 4.6 Hz, 1H), 2.68-2.64 (m, 1H), 1.95-1.90 (m, 1H), 1.86-1.80 (m, 1H), 1.76-1.71 (m, 2H).	170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₃₀N₅O₂ 420.239 4 420.238 3
7j	33	98.5-100.5	8.97-8.96 (m, 1H), 8.63-8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.89 (m, 1H), 7.72-7.69 (m, 1H), 7.63-7.60 (m, 1H), 7.32-7.29 (m, 4H), 7.23-7.20 (m, 1H), 4.68-4.66 (m, 1H), 3.89-3.87 (m, 1H), 3.03 (dd, J = 13.8, 7.1 Hz, 1H), 2.94 (dd, J = 13.7, 7.1 Hz, 1H), 2.86-2.83 (m, 2H), 2.78 (dd, J = 14.9, 4.6 Hz, 1H), 2.68-2.64 (m, 1H), 1.95-1.90 (m, 1H), 1.86-1.80 (m, 1H), 1.76-1.71 (m, 2H).	170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₂₉N₆O₄ 465.224 5 465.222 7
7k	33	84.8-85.7	8.99 (s, 1H), 8.64 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.91-7.90 (m, 1H), 7.73-7.70 (m, 1H), 7.64-7.61 (m, 1H), 7.30 (m, 4H), 4.68-4.63 (m, 1H), 3.88 (s, 1H), 3.00-2.93 (m, 2H), 2.85- 2.77 (m, 3H), 2.65 (dd, J = 14.3, 8.6 Hz, 1H), 1.92 (s, 1H), 1.84-1.73 (m, 3H).	170.5, 167.8, 144.2, 144.1, 136.4, 132.5, 132.3, 130.7 (2), 128.5, 128.3 (3), 127.5 (2), 127.3, 124.5, 52.3, 48.7, 40.5, 39.3, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₂₉ClN₅O₂ 454.200 4 454.199 3
7l	7	68.3-69.9	8.97-8.96 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.69 (m, 1H), 7.62-7.60 (m, 1H), 7.37-7.28 (m, 5H), 7.23-7.19 (m, 2H), 6.92 (d, J = 7.4 Hz, 2H), 4.98 (s, 2H), 4.65-4.60 (m, 1H), 3.90-3.88 (m, 1H), 2.96 (dd, J = 13.8, 6.8 Hz, 1H), 2.87-2.81 (m, 3H), 2.76 (dd, J = 14.5, 5.2 Hz, 1H), 2.62 (dd, J = 14.8, 8.7 Hz, 1H), 1.95-1.80 (m, 2H), 1.76-1.71 (m, 2H).	170.7, 167.7, 157.8, 144.2, 144.1, 137.3, 132.5, 130.1 (2), 129.8, 128.5, 128.3, 128.1 (2), 127.5(3), 127.3, 127.1 (2), 124.5, 114.6 (2), 69.6, 52.3, 49.0, 40.4, 39.4, 38.6, 28.2, 22.4.	[M+H]⁺ : C₃₁H₃₆N₅O₃ 526.281 3 526.280 3
7m	33	91.5-92.8	8.97 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.70 (m, 1H), 7.63-7.60 (m, 1H), 7.34 (d, J = 7.6 Hz, 2H), 7.24-7.22 (m, 2H), 4.68-4.63 (m, 1H), 3.91-3.89 (m, 1H), 3.00 (dd, J = 13.7, 6.5Hz, 1H), 2.90-2.84 (m, 3H), 2.77 (dd, J =14.9, 4.8 Hz, 1H), 2.62 (dd, J = 14.8, 8.8 Hz, 1H), 1.96-1.84 (m, 2H), 1.77-1.73 (m, 2H), 1.26 (s, 9H).	170.7, 167.7, 149.4, 144.2, 144.1, 134.4, 132.5, 128.8 (2), 128.5, 128.3, 127.5, 127.4, 127.3, 125.1 (2), 124.4, 52.3, 48.8, 40.3, 39.6, 38.6, 33.8, 30.4 (3), 28.2, 22.4.	[M+H]⁺ : C₂₈H₃₈N₅O₂ 476.302 0 476.301 7
12a	11	162.3-164.2	8.11 (m, 1H), 7.85-7.79 (m, 5H), 7.77-7.75 (m, 2H), 7.51-7.43 (m, 5H), 7.42-7.39 (m, 1H), 4.89-4.76 (m, 1H), 3.84 (t, J = 6.1 Hz, 1H), 3.18 (dd, J = 13.7, 7.2 Hz, 1H), 3.11 (dd, J = 13.7, 7.0 Hz, 1H), 2.81 (dd, J = 14.7, 4.3 Hz, 1H), 2.69 (t, J = 7.4 Hz, 2H), 2.63 (dd, J = 14.7, 9.3 Hz, 1H), 1.89-1.76 (m, 2H), 1.68-1.61 (m, 2H).	170.3, 167.6, 135.6, 135.2, 133.8, 133.7, 132.6, 130.8, 128.3, 127.9, 127.6, 127.2 (4), 127.1, 126.2, 125.8, 125.3, 124.8, 120.1, 116.9, 52.3, 49.0, 40.6, 40.3, 38.5, 28.2, 22.2.	[M+H]⁺ : C₂₉H₃₃N₄O₂ 468.259 8 469.258 3
12b	11	169.9-171.2	8.88-8.87 (m, 1H), 8.52-8.51 (m, 1H), 7.97-7.95 (m, 1H), 7.85-7.77 (m, 5H), 7.70-7.67 (m, 1H), 7.61-7.57 (m, 1H), 7.49-7.47 (m, 1H), 7.45-7.39 (m, 2H), 4.8-4.76 (m, 1H), 3.87-3.85 (m, 1H), 3.22-3.19 (m, 1H), 3.13-3.09 (m, 1H), 2.86-2.79 (m, 3H), 2.71-2.66 (m, 1H), 1.92-1.79 (m, 2H), 1.73-1.68 (m, 2H).	170.6, 167.8, 144.2, 135.2, 133.6, 132.5, 132.4, 128.5, 128.3, 127.9, 127.7, 127.4 (3), 127.3, 127.2 (3), 125.8, 125.3, 124.5, 52.3, 48.9, 40.7, 40.3, 38.5, 28.3, 22.4.	[M+H]⁺ : C₂₈H₃₂N₅O₂ 470.255 1 470.253 8

Compd.

Yield /%

mp /℃

¹H NMR
(600 MHz, CD₃OD) δ

¹³C NMR
(150 MHz, CD₃OD) δ

HR-ESI-MS
m/s
Formula
Calcd.
Found

78.8-80.8

8.14-8.13 (m, 1H), 7.81 (t, J = 8.1 Hz, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.48-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.34-7.30 (m, 4H), 7.25-7.22 (m, 1H), 4.70-4.64 (m, 1H), 3.79 (t, J = 6.1 Hz, 1H), 3.00 (dd, J = 13.7, 7.0 Hz, 1H), 2.92 (dd, J = 13.7, 7.1 Hz, 1H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.88-1.73 (m, 2H), 1.69-1.60 (m, 2H).

170.3, 168.1, 137.7, 135.7, 133.8, 130.8, 129.1 (2), 128.3, 128.2 (2), 127.2, 127.1, 126.4, 126.2, 124.8, 120.1, 116.8, 52.4, 48.9, 40.4, 40.2, 38.5, 28.4, 22.3.

[M+H]⁺ :
C₂₅H₃₁N₄O₂
419.244 2
419.242 2

112.2-114.0

8.18 (m, 1H), 7.80-7.75 (m, 3H), 7.55 (dd, J = 8.8, 2.1 Hz, 1H), 7.46-7.43 (m, 1H), 7.40-7.37 (m, 1H), 7.35-7.33 (m, 2H), 7.23-7.22 (m, 2H), 4.66-4.61 (m, 1H), 2.95-2.87 (m, 2H), 2.74-2.65 (m, 3H), 2.61 (dd, J = 14.5, 8.8 Hz, 1H), 1.69-1.48 (m, 5H), 1.28 (s, 9H).

161.8, 161.6, 135.8, 134.8, 133.8, 128.9 (2), 128.1, 127.2, 127.1, 126.1, 125.0 (2), 124.7, 120.0, 117.8, 116.5, 115.9, 53.8, 48.5, 48.2, 40.8, 38.9, 33.8, 31.3, 30.4 (3), 23.6.

[M+H]⁺ :
C₂₉H₃₉N₄O₂
475.306 8
475.305 2

67.2-69.1

8.12 (m, 1H), 7.80 (t, J = 8.7 Hz, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.41-7.39 (m, 3H), 7.37-7.34 (m, 2H), 7.31-7.28 (m, 1H), 7.23-7.20 (m, 2H), 6.95-6.94 (m, 2H), 5.02 (s, 2H), 4.64-4.61 (m, 1H), 3.88-3.86 (m, 1H), 2.94 (dd, J = 13.8, 6.7 Hz, 1H), 2.85 (dd, J = 13.8, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.58-2.54 (m, 1H), 1.91-1.78 (m, 2H), 1.70-1.64 (m, 2H).

170.3, 167.5, 157.8, 137.4, 135.7, 133.8, 130.8, 130.1 (2), 129.8, 128.3, 128.1 (2), 127.5, 127.3, 127.1 (3), 126.2, 124.8, 120.1, 116.8, 114.7 (2), 69.6, 52.3, 49.1, 40.5, 39.5, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₃₂H₃₇N₄O₃
525.286 0
525.286 1

85.9-87.9

8.14-8.13 (m, 1H), 7.81 (t, J = 8.8 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.69-7.68 (m, 2H), 7.53-7.49 (m, 3H), 7.48-7.45 (m, 1H), 7.43-7.37 (m, 1H), 4.75-4, 69 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 3.05 (dd, J = 7.0, 3.1 Hz, 2H), 2.77 (dd, J = 14.7, 4.4 Hz, 1H), 2.74-2.69 (m, 2H), 2.59 (dd, J = 14.7, 9.4 Hz, 1H), 1.91-1.76 (m, 2H), 1.69-1.62 (m, 2H).

169.9, 167.6, 143.7, 135.6, 133.8, 132.0 (2), 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 120.0, 118.3, 116.8, 110.3, 52.2, 40.6, 40.2, 38.5, 28.1, 27.6, 22.2.

[M+H]⁺ :
C₂₆H₃₀N₅O₂
444.239 4
444.237 6

78.4-80.4

8.12 (m, 1H), 7.81 (t, J = 7.9 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.51 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.19-7.12 (m, 4H), 4.67-4.61 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 2.96 (dd, J = 13.7, 6.8 Hz, 1H), 2.87 (dd, J = 13.7, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.55 (dd, J = 14.7, 9.5 Hz, 1H), 2.29 (s, 3H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).

170.3, 167.5, 136.1, 135.6, 134.5, 133.8, 130.8, 129.0 (2), 128.8 (2), 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 52.3, 49.0, 40.4, 39.9, 38.5, 28.1, 22.2, 19.7.

[M+H]⁺ :
C₂₆H₃₃N₄O₂
433.259 8
433.258 1

46.7-48.7

8.13 (m, 1H), 7.82-7.79 (m, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.32-7.30 (m, 2H), 7.06-7.02 (m, 2H), 4.66-4.63 (m, 1H), 3.86 (t, J = 6.1 Hz, 1H), 2.95 (qd, J = 13.9, 7.0 Hz, 2H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.89-1.79 (m, 2H), 1.69-1.64 (m, 2H).

170.2, 167.6, 161.9, 135.6, 133.8, 133.6, 130.8, 130.8, 130.7, 128.3, 127.3, 127.1, 126.2, 124.8, 120.1, 116.8, 114.9, 114.8, 52.3, 48.9, 40.5, 39.3, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₅H₃₀FN₄O₂
437.234 7
437.233 3

72.8-74.8

8.21-8.18 (m, 2H), 8.13 (m, 1H), 7.81 (t, J = 9.3 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.56-7.51 (m, 3H), 7.48-7.45 (m, 1H), 7.42-7.39 (m, 1H), 4.78-4.73 (m, 1H), 3.87-3.85 (m, 1H), 3.14-3.06 (m, 2H), 2.82-2.72 (m, 3H), 2.64-2.60 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).

169.9, 167.6, 147.0, 145.7, 135.6, 133.8, 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 123.2 (2), 120.0, 116.8, 52.2, 48.5, 40.7, 39.9, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₅H₃₀N₅O₄
464.229 2
464.227 8

67.3-68.6

8.13 (s, 1H), 7.82-7.79 (m, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.22-7.20 (m, 2H), 6.88-6.86 (m, 2H), 4.64-4.60 (m, 1H), 3.87-3.86 (m, 1H), 3.75 (m, 3H), 2.93 (dd, J = 13.9, 6.9 Hz, 1H), 2.87-2.83 (m, 1H), 2.75-2.70 (m, 3H), 2.58-2.53 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).

170.4, 167.5, 158.7, 135.7, 133.8, 130.8, 130.0 (2), 129.5, 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 113.6 (2), 54.2, 52.3, 49.1, 40.4, 39.4, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₆H₃₃N₄O₃
449.254 7
449.253 3

56.0-58.0

8.97-8.96 (m, 1H), 8.63-8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.89 (m, 1H), 7.72-7.69 (m, 1H), 7.63-7.60 (m, 1H), 7.32-7.29 (m, 4H), 7.23-7.20 (m, 1H), 4.68-4.66 (m, 1H), 3.89-3.87 (m, 1H), 3.03 (dd, J = 13.8, 7.1 Hz, 1H), 2.94 (dd, J = 13.7, 7.1 Hz, 1H), 2.86-2.83 (m, 2H), 2.78 (dd, J = 14.9, 4.6 Hz, 1H), 2.68-2.64 (m, 1H), 1.95-1.90 (m, 1H), 1.86-1.80 (m, 1H), 1.76-1.71 (m, 2H).

170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₃₀N₅O₂
420.239 4
420.238 3

98.5-100.5

170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₂₉N₆O₄
465.224 5
465.222 7

84.8-85.7

8.99 (s, 1H), 8.64 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.91-7.90 (m, 1H), 7.73-7.70 (m, 1H), 7.64-7.61 (m, 1H), 7.30 (m, 4H), 4.68-4.63 (m, 1H), 3.88 (s, 1H), 3.00-2.93 (m, 2H), 2.85- 2.77 (m, 3H), 2.65 (dd, J = 14.3, 8.6 Hz, 1H), 1.92 (s, 1H), 1.84-1.73 (m, 3H).

170.5, 167.8, 144.2, 144.1, 136.4, 132.5, 132.3, 130.7 (2), 128.5, 128.3 (3), 127.5 (2), 127.3, 124.5, 52.3, 48.7, 40.5, 39.3, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₂₉ClN₅O₂
454.200 4
454.199 3

68.3-69.9

8.97-8.96 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.69 (m, 1H), 7.62-7.60 (m, 1H), 7.37-7.28 (m, 5H), 7.23-7.19 (m, 2H), 6.92 (d, J = 7.4 Hz, 2H), 4.98 (s, 2H), 4.65-4.60 (m, 1H), 3.90-3.88 (m, 1H), 2.96 (dd, J = 13.8, 6.8 Hz, 1H), 2.87-2.81 (m, 3H), 2.76 (dd, J = 14.5, 5.2 Hz, 1H), 2.62 (dd, J = 14.8, 8.7 Hz, 1H), 1.95-1.80 (m, 2H), 1.76-1.71 (m, 2H).

170.7, 167.7, 157.8, 144.2, 144.1, 137.3, 132.5, 130.1 (2), 129.8, 128.5, 128.3, 128.1 (2), 127.5(3), 127.3, 127.1 (2), 124.5, 114.6 (2), 69.6, 52.3, 49.0, 40.4, 39.4, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₃₁H₃₆N₅O₃
526.281 3
526.280 3

91.5-92.8

8.97 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.70 (m, 1H), 7.63-7.60 (m, 1H), 7.34 (d, J = 7.6 Hz, 2H), 7.24-7.22 (m, 2H), 4.68-4.63 (m, 1H), 3.91-3.89 (m, 1H), 3.00 (dd, J = 13.7, 6.5Hz, 1H), 2.90-2.84 (m, 3H), 2.77 (dd, J =14.9, 4.8 Hz, 1H), 2.62 (dd, J = 14.8, 8.8 Hz, 1H), 1.96-1.84 (m, 2H), 1.77-1.73 (m, 2H), 1.26 (s, 9H).

170.7, 167.7, 149.4, 144.2, 144.1, 134.4, 132.5, 128.8 (2), 128.5, 128.3, 127.5, 127.4, 127.3, 125.1 (2), 124.4, 52.3, 48.8, 40.3, 39.6, 38.6, 33.8, 30.4 (3), 28.2, 22.4.

[M+H]⁺ :
C₂₈H₃₈N₅O₂
476.302 0
476.301 7

12a

162.3-164.2

8.11 (m, 1H), 7.85-7.79 (m, 5H), 7.77-7.75 (m, 2H), 7.51-7.43 (m, 5H), 7.42-7.39 (m, 1H), 4.89-4.76 (m, 1H), 3.84 (t, J = 6.1 Hz, 1H), 3.18 (dd, J = 13.7, 7.2 Hz, 1H), 3.11 (dd, J = 13.7, 7.0 Hz, 1H), 2.81 (dd, J = 14.7, 4.3 Hz, 1H), 2.69 (t, J = 7.4 Hz, 2H), 2.63 (dd, J = 14.7, 9.3 Hz, 1H), 1.89-1.76 (m, 2H), 1.68-1.61 (m, 2H).

170.3, 167.6, 135.6, 135.2, 133.8, 133.7, 132.6, 130.8, 128.3, 127.9, 127.6, 127.2 (4), 127.1, 126.2, 125.8, 125.3, 124.8, 120.1, 116.9, 52.3, 49.0, 40.6, 40.3, 38.5, 28.2, 22.2.

[M+H]⁺ :
C₂₉H₃₃N₄O₂
468.259 8
469.258 3

12b

169.9-171.2

8.88-8.87 (m, 1H), 8.52-8.51 (m, 1H), 7.97-7.95 (m, 1H), 7.85-7.77 (m, 5H), 7.70-7.67 (m, 1H), 7.61-7.57 (m, 1H), 7.49-7.47 (m, 1H), 7.45-7.39 (m, 2H), 4.8-4.76 (m, 1H), 3.87-3.85 (m, 1H), 3.22-3.19 (m, 1H), 3.13-3.09 (m, 1H), 2.86-2.79 (m, 3H), 2.71-2.66 (m, 1H), 1.92-1.79 (m, 2H), 1.73-1.68 (m, 2H).

170.6, 167.8, 144.2, 135.2, 133.6, 132.5, 132.4, 128.5, 128.3, 127.9, 127.7, 127.4 (3), 127.3, 127.2 (3), 125.8, 125.3, 124.5, 52.3, 48.9, 40.7, 40.3, 38.5, 28.3, 22.4.

[M+H]⁺ :
C₂₈H₃₂N₅O₂
470.255 1
470.253 8

Compd.	Yield /%	mp /℃	¹H NMR (600 MHz, CD₃OD) δ	¹³C NMR (150 MHz, CD₃OD) δ	HR-ESI-MS m/s Formula Calcd. Found
7a	38	78.8-80.8	8.14-8.13 (m, 1H), 7.81 (t, J = 8.1 Hz, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.48-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.34-7.30 (m, 4H), 7.25-7.22 (m, 1H), 4.70-4.64 (m, 1H), 3.79 (t, J = 6.1 Hz, 1H), 3.00 (dd, J = 13.7, 7.0 Hz, 1H), 2.92 (dd, J = 13.7, 7.1 Hz, 1H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.88-1.73 (m, 2H), 1.69-1.60 (m, 2H).	170.3, 168.1, 137.7, 135.7, 133.8, 130.8, 129.1 (2), 128.3, 128.2 (2), 127.2, 127.1, 126.4, 126.2, 124.8, 120.1, 116.8, 52.4, 48.9, 40.4, 40.2, 38.5, 28.4, 22.3.	[M+H]⁺ : C₂₅H₃₁N₄O₂ 419.244 2 419.242 2
7b	12	112.2-114.0	8.18 (m, 1H), 7.80-7.75 (m, 3H), 7.55 (dd, J = 8.8, 2.1 Hz, 1H), 7.46-7.43 (m, 1H), 7.40-7.37 (m, 1H), 7.35-7.33 (m, 2H), 7.23-7.22 (m, 2H), 4.66-4.61 (m, 1H), 2.95-2.87 (m, 2H), 2.74-2.65 (m, 3H), 2.61 (dd, J = 14.5, 8.8 Hz, 1H), 1.69-1.48 (m, 5H), 1.28 (s, 9H).	161.8, 161.6, 135.8, 134.8, 133.8, 128.9 (2), 128.1, 127.2, 127.1, 126.1, 125.0 (2), 124.7, 120.0, 117.8, 116.5, 115.9, 53.8, 48.5, 48.2, 40.8, 38.9, 33.8, 31.3, 30.4 (3), 23.6.	[M+H]⁺ : C₂₉H₃₉N₄O₂ 475.306 8 475.305 2
7c	24	67.2-69.1	8.12 (m, 1H), 7.80 (t, J = 8.7 Hz, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.41-7.39 (m, 3H), 7.37-7.34 (m, 2H), 7.31-7.28 (m, 1H), 7.23-7.20 (m, 2H), 6.95-6.94 (m, 2H), 5.02 (s, 2H), 4.64-4.61 (m, 1H), 3.88-3.86 (m, 1H), 2.94 (dd, J = 13.8, 6.7 Hz, 1H), 2.85 (dd, J = 13.8, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.58-2.54 (m, 1H), 1.91-1.78 (m, 2H), 1.70-1.64 (m, 2H).	170.3, 167.5, 157.8, 137.4, 135.7, 133.8, 130.8, 130.1 (2), 129.8, 128.3, 128.1 (2), 127.5, 127.3, 127.1 (3), 126.2, 124.8, 120.1, 116.8, 114.7 (2), 69.6, 52.3, 49.1, 40.5, 39.5, 38.5, 28.1, 22.2.	[M+H]⁺ : C₃₂H₃₇N₄O₃ 525.286 0 525.286 1
7d	21	85.9-87.9	8.14-8.13 (m, 1H), 7.81 (t, J = 8.8 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.69-7.68 (m, 2H), 7.53-7.49 (m, 3H), 7.48-7.45 (m, 1H), 7.43-7.37 (m, 1H), 4.75-4, 69 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 3.05 (dd, J = 7.0, 3.1 Hz, 2H), 2.77 (dd, J = 14.7, 4.4 Hz, 1H), 2.74-2.69 (m, 2H), 2.59 (dd, J = 14.7, 9.4 Hz, 1H), 1.91-1.76 (m, 2H), 1.69-1.62 (m, 2H).	169.9, 167.6, 143.7, 135.6, 133.8, 132.0 (2), 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 120.0, 118.3, 116.8, 110.3, 52.2, 40.6, 40.2, 38.5, 28.1, 27.6, 22.2.	[M+H]⁺ : C₂₆H₃₀N₅O₂ 444.239 4 444.237 6
7e	34	78.4-80.4	8.12 (m, 1H), 7.81 (t, J = 7.9 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.51 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.19-7.12 (m, 4H), 4.67-4.61 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 2.96 (dd, J = 13.7, 6.8 Hz, 1H), 2.87 (dd, J = 13.7, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.55 (dd, J = 14.7, 9.5 Hz, 1H), 2.29 (s, 3H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	170.3, 167.5, 136.1, 135.6, 134.5, 133.8, 130.8, 129.0 (2), 128.8 (2), 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 52.3, 49.0, 40.4, 39.9, 38.5, 28.1, 22.2, 19.7.	[M+H]⁺ : C₂₆H₃₃N₄O₂ 433.259 8 433.258 1
7f	21	46.7-48.7	8.13 (m, 1H), 7.82-7.79 (m, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.32-7.30 (m, 2H), 7.06-7.02 (m, 2H), 4.66-4.63 (m, 1H), 3.86 (t, J = 6.1 Hz, 1H), 2.95 (qd, J = 13.9, 7.0 Hz, 2H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.89-1.79 (m, 2H), 1.69-1.64 (m, 2H).	170.2, 167.6, 161.9, 135.6, 133.8, 133.6, 130.8, 130.8, 130.7, 128.3, 127.3, 127.1, 126.2, 124.8, 120.1, 116.8, 114.9, 114.8, 52.3, 48.9, 40.5, 39.3, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₅H₃₀FN₄O₂ 437.234 7 437.233 3
7g	8	72.8-74.8	8.21-8.18 (m, 2H), 8.13 (m, 1H), 7.81 (t, J = 9.3 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.56-7.51 (m, 3H), 7.48-7.45 (m, 1H), 7.42-7.39 (m, 1H), 4.78-4.73 (m, 1H), 3.87-3.85 (m, 1H), 3.14-3.06 (m, 2H), 2.82-2.72 (m, 3H), 2.64-2.60 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	169.9, 167.6, 147.0, 145.7, 135.6, 133.8, 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 123.2 (2), 120.0, 116.8, 52.2, 48.5, 40.7, 39.9, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₅H₃₀N₅O₄ 464.229 2 464.227 8
7h	11	67.3-68.6	8.13 (s, 1H), 7.82-7.79 (m, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.22-7.20 (m, 2H), 6.88-6.86 (m, 2H), 4.64-4.60 (m, 1H), 3.87-3.86 (m, 1H), 3.75 (m, 3H), 2.93 (dd, J = 13.9, 6.9 Hz, 1H), 2.87-2.83 (m, 1H), 2.75-2.70 (m, 3H), 2.58-2.53 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	170.4, 167.5, 158.7, 135.7, 133.8, 130.8, 130.0 (2), 129.5, 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 113.6 (2), 54.2, 52.3, 49.1, 40.4, 39.4, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₆H₃₃N₄O₃ 449.254 7 449.253 3
7i	4	56.0-58.0	8.97-8.96 (m, 1H), 8.63-8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.89 (m, 1H), 7.72-7.69 (m, 1H), 7.63-7.60 (m, 1H), 7.32-7.29 (m, 4H), 7.23-7.20 (m, 1H), 4.68-4.66 (m, 1H), 3.89-3.87 (m, 1H), 3.03 (dd, J = 13.8, 7.1 Hz, 1H), 2.94 (dd, J = 13.7, 7.1 Hz, 1H), 2.86-2.83 (m, 2H), 2.78 (dd, J = 14.9, 4.6 Hz, 1H), 2.68-2.64 (m, 1H), 1.95-1.90 (m, 1H), 1.86-1.80 (m, 1H), 1.76-1.71 (m, 2H).	170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₃₀N₅O₂ 420.239 4 420.238 3
7j	33	98.5-100.5	8.97-8.96 (m, 1H), 8.63-8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.89 (m, 1H), 7.72-7.69 (m, 1H), 7.63-7.60 (m, 1H), 7.32-7.29 (m, 4H), 7.23-7.20 (m, 1H), 4.68-4.66 (m, 1H), 3.89-3.87 (m, 1H), 3.03 (dd, J = 13.8, 7.1 Hz, 1H), 2.94 (dd, J = 13.7, 7.1 Hz, 1H), 2.86-2.83 (m, 2H), 2.78 (dd, J = 14.9, 4.6 Hz, 1H), 2.68-2.64 (m, 1H), 1.95-1.90 (m, 1H), 1.86-1.80 (m, 1H), 1.76-1.71 (m, 2H).	170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₂₉N₆O₄ 465.224 5 465.222 7
7k	33	84.8-85.7	8.99 (s, 1H), 8.64 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.91-7.90 (m, 1H), 7.73-7.70 (m, 1H), 7.64-7.61 (m, 1H), 7.30 (m, 4H), 4.68-4.63 (m, 1H), 3.88 (s, 1H), 3.00-2.93 (m, 2H), 2.85- 2.77 (m, 3H), 2.65 (dd, J = 14.3, 8.6 Hz, 1H), 1.92 (s, 1H), 1.84-1.73 (m, 3H).	170.5, 167.8, 144.2, 144.1, 136.4, 132.5, 132.3, 130.7 (2), 128.5, 128.3 (3), 127.5 (2), 127.3, 124.5, 52.3, 48.7, 40.5, 39.3, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₂₉ClN₅O₂ 454.200 4 454.199 3
7l	7	68.3-69.9	8.97-8.96 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.69 (m, 1H), 7.62-7.60 (m, 1H), 7.37-7.28 (m, 5H), 7.23-7.19 (m, 2H), 6.92 (d, J = 7.4 Hz, 2H), 4.98 (s, 2H), 4.65-4.60 (m, 1H), 3.90-3.88 (m, 1H), 2.96 (dd, J = 13.8, 6.8 Hz, 1H), 2.87-2.81 (m, 3H), 2.76 (dd, J = 14.5, 5.2 Hz, 1H), 2.62 (dd, J = 14.8, 8.7 Hz, 1H), 1.95-1.80 (m, 2H), 1.76-1.71 (m, 2H).	170.7, 167.7, 157.8, 144.2, 144.1, 137.3, 132.5, 130.1 (2), 129.8, 128.5, 128.3, 128.1 (2), 127.5(3), 127.3, 127.1 (2), 124.5, 114.6 (2), 69.6, 52.3, 49.0, 40.4, 39.4, 38.6, 28.2, 22.4.	[M+H]⁺ : C₃₁H₃₆N₅O₃ 526.281 3 526.280 3
7m	33	91.5-92.8	8.97 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.70 (m, 1H), 7.63-7.60 (m, 1H), 7.34 (d, J = 7.6 Hz, 2H), 7.24-7.22 (m, 2H), 4.68-4.63 (m, 1H), 3.91-3.89 (m, 1H), 3.00 (dd, J = 13.7, 6.5Hz, 1H), 2.90-2.84 (m, 3H), 2.77 (dd, J =14.9, 4.8 Hz, 1H), 2.62 (dd, J = 14.8, 8.8 Hz, 1H), 1.96-1.84 (m, 2H), 1.77-1.73 (m, 2H), 1.26 (s, 9H).	170.7, 167.7, 149.4, 144.2, 144.1, 134.4, 132.5, 128.8 (2), 128.5, 128.3, 127.5, 127.4, 127.3, 125.1 (2), 124.4, 52.3, 48.8, 40.3, 39.6, 38.6, 33.8, 30.4 (3), 28.2, 22.4.	[M+H]⁺ : C₂₈H₃₈N₅O₂ 476.302 0 476.301 7
12a	11	162.3-164.2	8.11 (m, 1H), 7.85-7.79 (m, 5H), 7.77-7.75 (m, 2H), 7.51-7.43 (m, 5H), 7.42-7.39 (m, 1H), 4.89-4.76 (m, 1H), 3.84 (t, J = 6.1 Hz, 1H), 3.18 (dd, J = 13.7, 7.2 Hz, 1H), 3.11 (dd, J = 13.7, 7.0 Hz, 1H), 2.81 (dd, J = 14.7, 4.3 Hz, 1H), 2.69 (t, J = 7.4 Hz, 2H), 2.63 (dd, J = 14.7, 9.3 Hz, 1H), 1.89-1.76 (m, 2H), 1.68-1.61 (m, 2H).	170.3, 167.6, 135.6, 135.2, 133.8, 133.7, 132.6, 130.8, 128.3, 127.9, 127.6, 127.2 (4), 127.1, 126.2, 125.8, 125.3, 124.8, 120.1, 116.9, 52.3, 49.0, 40.6, 40.3, 38.5, 28.2, 22.2.	[M+H]⁺ : C₂₉H₃₃N₄O₂ 468.259 8 469.258 3
12b	11	169.9-171.2	8.88-8.87 (m, 1H), 8.52-8.51 (m, 1H), 7.97-7.95 (m, 1H), 7.85-7.77 (m, 5H), 7.70-7.67 (m, 1H), 7.61-7.57 (m, 1H), 7.49-7.47 (m, 1H), 7.45-7.39 (m, 2H), 4.8-4.76 (m, 1H), 3.87-3.85 (m, 1H), 3.22-3.19 (m, 1H), 3.13-3.09 (m, 1H), 2.86-2.79 (m, 3H), 2.71-2.66 (m, 1H), 1.92-1.79 (m, 2H), 1.73-1.68 (m, 2H).	170.6, 167.8, 144.2, 135.2, 133.6, 132.5, 132.4, 128.5, 128.3, 127.9, 127.7, 127.4 (3), 127.3, 127.2 (3), 125.8, 125.3, 124.5, 52.3, 48.9, 40.7, 40.3, 38.5, 28.3, 22.4.	[M+H]⁺ : C₂₈H₃₂N₅O₂ 470.255 1 470.253 8

Compd.

Yield /%

mp /℃

¹H NMR
(600 MHz, CD₃OD) δ

¹³C NMR
(150 MHz, CD₃OD) δ

HR-ESI-MS
m/s
Formula
Calcd.
Found

78.8-80.8

170.3, 168.1, 137.7, 135.7, 133.8, 130.8, 129.1 (2), 128.3, 128.2 (2), 127.2, 127.1, 126.4, 126.2, 124.8, 120.1, 116.8, 52.4, 48.9, 40.4, 40.2, 38.5, 28.4, 22.3.

[M+H]⁺ :
C₂₅H₃₁N₄O₂
419.244 2
419.242 2

112.2-114.0

161.8, 161.6, 135.8, 134.8, 133.8, 128.9 (2), 128.1, 127.2, 127.1, 126.1, 125.0 (2), 124.7, 120.0, 117.8, 116.5, 115.9, 53.8, 48.5, 48.2, 40.8, 38.9, 33.8, 31.3, 30.4 (3), 23.6.

[M+H]⁺ :
C₂₉H₃₉N₄O₂
475.306 8
475.305 2

67.2-69.1

170.3, 167.5, 157.8, 137.4, 135.7, 133.8, 130.8, 130.1 (2), 129.8, 128.3, 128.1 (2), 127.5, 127.3, 127.1 (3), 126.2, 124.8, 120.1, 116.8, 114.7 (2), 69.6, 52.3, 49.1, 40.5, 39.5, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₃₂H₃₇N₄O₃
525.286 0
525.286 1

85.9-87.9

169.9, 167.6, 143.7, 135.6, 133.8, 132.0 (2), 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 120.0, 118.3, 116.8, 110.3, 52.2, 40.6, 40.2, 38.5, 28.1, 27.6, 22.2.

[M+H]⁺ :
C₂₆H₃₀N₅O₂
444.239 4
444.237 6

78.4-80.4

170.3, 167.5, 136.1, 135.6, 134.5, 133.8, 130.8, 129.0 (2), 128.8 (2), 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 52.3, 49.0, 40.4, 39.9, 38.5, 28.1, 22.2, 19.7.

[M+H]⁺ :
C₂₆H₃₃N₄O₂
433.259 8
433.258 1

46.7-48.7

170.2, 167.6, 161.9, 135.6, 133.8, 133.6, 130.8, 130.8, 130.7, 128.3, 127.3, 127.1, 126.2, 124.8, 120.1, 116.8, 114.9, 114.8, 52.3, 48.9, 40.5, 39.3, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₅H₃₀FN₄O₂
437.234 7
437.233 3

72.8-74.8

169.9, 167.6, 147.0, 145.7, 135.6, 133.8, 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 123.2 (2), 120.0, 116.8, 52.2, 48.5, 40.7, 39.9, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₅H₃₀N₅O₄
464.229 2
464.227 8

67.3-68.6

170.4, 167.5, 158.7, 135.7, 133.8, 130.8, 130.0 (2), 129.5, 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 113.6 (2), 54.2, 52.3, 49.1, 40.4, 39.4, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₆H₃₃N₄O₃
449.254 7
449.253 3

56.0-58.0

170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₃₀N₅O₂
420.239 4
420.238 3

98.5-100.5

170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₂₉N₆O₄
465.224 5
465.222 7

84.8-85.7

170.5, 167.8, 144.2, 144.1, 136.4, 132.5, 132.3, 130.7 (2), 128.5, 128.3 (3), 127.5 (2), 127.3, 124.5, 52.3, 48.7, 40.5, 39.3, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₂₉ClN₅O₂
454.200 4
454.199 3

68.3-69.9

170.7, 167.7, 157.8, 144.2, 144.1, 137.3, 132.5, 130.1 (2), 129.8, 128.5, 128.3, 128.1 (2), 127.5(3), 127.3, 127.1 (2), 124.5, 114.6 (2), 69.6, 52.3, 49.0, 40.4, 39.4, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₃₁H₃₆N₅O₃
526.281 3
526.280 3

91.5-92.8

170.7, 167.7, 149.4, 144.2, 144.1, 134.4, 132.5, 128.8 (2), 128.5, 128.3, 127.5, 127.4, 127.3, 125.1 (2), 124.4, 52.3, 48.8, 40.3, 39.6, 38.6, 33.8, 30.4 (3), 28.2, 22.4.

[M+H]⁺ :
C₂₈H₃₈N₅O₂
476.302 0
476.301 7

12a

162.3-164.2

170.3, 167.6, 135.6, 135.2, 133.8, 133.7, 132.6, 130.8, 128.3, 127.9, 127.6, 127.2 (4), 127.1, 126.2, 125.8, 125.3, 124.8, 120.1, 116.9, 52.3, 49.0, 40.6, 40.3, 38.5, 28.2, 22.2.

[M+H]⁺ :
C₂₉H₃₃N₄O₂
468.259 8
469.258 3

12b

169.9-171.2

170.6, 167.8, 144.2, 135.2, 133.6, 132.5, 132.4, 128.5, 128.3, 127.9, 127.7, 127.4 (3), 127.3, 127.2 (3), 125.8, 125.3, 124.5, 52.3, 48.9, 40.7, 40.3, 38.5, 28.3, 22.4.

[M+H]⁺ :
C₂₈H₃₂N₅O₂
470.255 1
470.253 8


7a	-	C	> 128	2	64	1	< 1
7b	t-Bu	C	32	2	16	0.125	0.562 5
7c	OBn	C	32	2	16	0.25	0.625
7d	CN	C	> 128	2	128	1	< 1.5
7e	CH₃	C	128	2	64	1	1
7f	F	C	> 128	2	64	1	< 1
7g	NO₂	C	> 128	2	16	1	< 0.625
7h	OCH₃	C	> 128	2	32	0.5	< 0.5
7i	-	N	> 128	2	64	1	< 1
7j	NO₂	N	> 128	2	64	1	< 1
7k	Cl	N	> 128	2	8	1	< 0.562 5
7l	OBn	N	128	2	64	0.5	0.75
7m	t-Bu	N	128	2	64	0.5	0.75
12a	-	C	64	2	32	0.25	0.625
12b	-	N	128	2	16	0.5	0.375
PAβN	-	-	> 128	2	128	0.5	< 1.25

No.

Alone

Combined

FICI

MIC^a

MIC^AZT

MIC^a

MIC^AZT

> 128

< 1

t-Bu

0.125

0.562 5

OBn

0.25

0.625

> 128

128

< 1.5

CH₃

128

> 128

< 1

NO₂

> 128

< 0.625

OCH₃

> 128

0.5

< 0.5

> 128

< 1

NO₂

> 128

< 1

> 128

< 0.562 5

OBn

128

0.5

0.75

t-Bu

128

0.5

0.75

12a

0.25

0.625

12b

128

0.5

0.375

PAβN

> 128

128

0.5

< 1.25


7a	-	C	> 128	2	64	1	< 1
7b	t-Bu	C	32	2	16	0.125	0.562 5
7c	OBn	C	32	2	16	0.25	0.625
7d	CN	C	> 128	2	128	1	< 1.5
7e	CH₃	C	128	2	64	1	1
7f	F	C	> 128	2	64	1	< 1
7g	NO₂	C	> 128	2	16	1	< 0.625
7h	OCH₃	C	> 128	2	32	0.5	< 0.5
7i	-	N	> 128	2	64	1	< 1
7j	NO₂	N	> 128	2	64	1	< 1
7k	Cl	N	> 128	2	8	1	< 0.562 5
7l	OBn	N	128	2	64	0.5	0.75
7m	t-Bu	N	128	2	64	0.5	0.75
12a	-	C	64	2	32	0.25	0.625
12b	-	N	128	2	16	0.5	0.375
PAβN	-	-	> 128	2	128	0.5	< 1.25

No.

Alone

Combined

FICI

MIC^a

MIC^AZT

MIC^a

MIC^AZT

> 128

< 1

t-Bu

0.125

0.562 5

OBn

0.25

0.625

> 128

128

< 1.5

CH₃

128

> 128

< 1

NO₂

> 128

< 0.625

OCH₃

> 128

0.5

< 0.5

> 128

< 1

NO₂

> 128

< 1

> 128

< 0.562 5

OBn

128

0.5

0.75

t-Bu

128

0.5

0.75

12a

0.25

0.625

12b

128

0.5

0.375

PAβN

> 128

128

0.5

< 1.25

Strain	Compd.	Alone		Combined	FICI
26	7h	128	8		32	1	0.375
12b	128	8		32	0.5	0.312 5
PAβN	> 128	16		16	2	< 0.25

Strain

Compd.

Alone

Combined

FICI

MIC^a

MIC^AZT

MIC^a

MIC^AZT

128

0.375

12b

128

0.5

0.312 5

PAβN

> 128

< 0.25

Strain	Compd.	Alone		Combined	FICI
26	7h	128	8		32	1	0.375
12b	128	8		32	0.5	0.312 5
PAβN	> 128	16		16	2	< 0.25

Strain

Compd.

Alone

Combined

FICI

MIC^a

MIC^AZT

MIC^a

MIC^AZT

128

0.375

12b

128

0.5

0.312 5

PAβN

> 128

< 0.25

Strain	Compd.	Alone		Combined	FICI
181245	AZT	8	> 512	> 512	2	128	2	128	< 0.5	< 0.5
	CLR	256	> 512	> 512	0.5	128	0.5	128	< 0.25	< 0.25
	CAZ	64	> 512	> 512	2	128	16	128	< 0.28	< 0.5
	MEM	32	> 512	> 512	8	128	8	128	< 0.5	< 0.5
181643	AZT	4	> 512	512	1	128	1	128	< 0.5	0.5
	CLR	256	512	512	32	128	32	128	0.375	0.375
	CAZ	2	512	512	0.5	128	0.5	128	0.5	0.5

Strain

Compd.

Alone

Combined

FICI

MIC^a

MIC^12b

MIC^PAβN

MIC^a

MIC^12b

MIC^a

MIC^PAβN

12b

PAβN

181245

AZT

> 512

128

< 0.5

CLR

256

> 512

0.5

128

0.5

128

< 0.25

CAZ

> 512

128

< 0.28

< 0.5

MEM

> 512

128

< 0.5

181643

AZT

> 512

512

128

< 0.5

0.5

CLR

256

512

128

0.375

CAZ

512

0.5

128

0.5

128

0.5

Strain	Compd.	Alone		Combined	FICI
181245	AZT	8	> 512	> 512	2	128	2	128	< 0.5	< 0.5
	CLR	256	> 512	> 512	0.5	128	0.5	128	< 0.25	< 0.25
	CAZ	64	> 512	> 512	2	128	16	128	< 0.28	< 0.5
	MEM	32	> 512	> 512	8	128	8	128	< 0.5	< 0.5
181643	AZT	4	> 512	512	1	128	1	128	< 0.5	0.5
	CLR	256	512	512	32	128	32	128	0.375	0.375
	CAZ	2	512	512	0.5	128	0.5	128	0.5	0.5

Strain

Compd.

Alone

Combined

FICI

MIC^a

MIC^12b

MIC^PAβN

MIC^a

MIC^12b

MIC^a

MIC^PAβN

12b

PAβN

181245

AZT

> 512

128

< 0.5

CLR

256

> 512

0.5

128

0.5

128

< 0.25

CAZ

> 512

128

< 0.28

< 0.5

MEM

> 512

128

< 0.5

181643

AZT

> 512

512

128

< 0.5

0.5

CLR

256

512

128

0.375

CAZ

512

0.5

128

0.5

128

0.5

Compd.	Absn	CYP	TOX	ADMET
12b	4.014	0	0.6	4.014

Compd.

Absn

CYP

TOX

ADMET

12b

4.014

0.6

4.014

Compd.	Absn	CYP	TOX	ADMET
12b	4.014	0	0.6	4.014

Compd.

Absn

CYP

TOX

ADMET

12b

4.014

0.6

4.014

鸟氨酸拟肽类外排泵抑制剂合成及增效抗生素对抗铜绿假单胞菌活性研究

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朱喜 , 马西灿 , 张昕彤 , 刘忆霜 , 何宁 , 解云英 , 宋丹青 ^*

药学学报 | 研究论文 2024,59(6): 1720-1729

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药学学报 | 研究论文 2024, 59(6): 1720-1729

鸟氨酸拟肽类外排泵抑制剂合成及增效抗生素对抗铜绿假单胞菌活性研究

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朱喜, 马西灿, 张昕彤, 刘忆霜, 何宁, 解云英, 宋丹青^*

作者信息

中国医学科学院、北京协和医学院医药生物技术研究所, 北京 100050

通讯作者:

^*宋丹青, Tel/Fax: 86-10-63165268, E-mail: songdanqingsdq@sina.com

Synthesis of ornithine peptidomimetic efflux pump inhibitors and synergistic antibiotic activity against Pseudomonas aeruginosa

Xi ZHU, Xi-can MA, Xin-tong ZHANG, Yi-shuang LIU, Ning HE, Yun-ying XIE, Dan-qing SONG^*

Affiliations

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2024-06-12 doi: 10.16438/j.0513-4870.2024-0104

文章导航

摘要

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为解决铜绿假单胞菌(P.aeruginosa) 对多类抗生素耐药难题, 寻找P.aeruginosa外排泵抑制剂是一条有效途径。本研究以二肽类外排泵抑制剂PAβN为先导物, 通过改变天然氨基酸侧链结构, 设计合成15个全新的鸟氨酸拟肽类衍生物, 评价了其协同单环β-内酰胺类抗生素氨曲南对抗P.aeruginosa活性。其中, 化合物12b不仅可增强β-内酰胺类抗生素, 包括氨曲南、头孢他啶、美罗培南的抗菌活性, 还可显著增强大环内酯类抗生素克拉霉素的抗菌作用, 显示出广谱的协同增敏作用。此外, 化合物12b还具有良好的安全性。初步机制显示, 12b直接靶向外排转运体MexB发挥作用。研究结果为发展一类新型的对抗MDR P.aeruginosa的外排泵抑制剂提供了新先导化合物。

关键词

外排泵抑制剂 / 合成 / 铜绿假单胞菌 / 协同活性 / MexAB-OprM

Abstract

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In order to solve the problem of resistance of Pseudomonas aeruginosa to multiple antibiotics, it is an effective way to find inhibitors of P.aeruginosa efflux pump. In this study, 15 new ornithine peptidomimetic derivatives were designed and synthesized by changing the side chain structure of natural amino acids with PAβN, a dipeptide efflux pump inhibitor, and their synergic activity with aztreonam, a monocyclic β-lactam antibiotic, against P.aeruginosa was evaluated. Among them, the representative compound 12b not only enhanced the antibacterial activity of β-lactam antibiotics aztreonam, ceftazidime and meropenem, but also significantly enhanced the antibacterial action of macrolide antibiotics clarithromycin, showing a broad-spectrum synergic sensitization effect. In addition, compound 12b also has a good safety. Preliminary mechanisms suggest that 12b works by directly targeting the efflux transporter MexB. These results provide a new lead compound for the development of a new class of efflux pump inhibitors against P.aeruginosa.

Key words

efflux pump inhibitor / synthesis / Pseudomonas aeruginosa / synergistic activity / MexAB-OprM

引用本文

朱喜, 马西灿, 张昕彤, 刘忆霜, 何宁, 解云英, 宋丹青. 鸟氨酸拟肽类外排泵抑制剂合成及增效抗生素对抗铜绿假单胞菌活性研究. 药学学报, 2024 , 59 (6) : 1720 -1729 . DOI: 10.16438/j.0513-4870.2024-0104

Xi ZHU, Xi-can MA, Xin-tong ZHANG, Yi-shuang LIU, Ning HE, Yun-ying XIE, Dan-qing SONG. Synthesis of ornithine peptidomimetic efflux pump inhibitors and synergistic antibiotic activity against Pseudomonas aeruginosa[J]. Acta Pharmaceutica Sinica, 2024 , 59 (6) : 1720 -1729 . DOI: 10.16438/j.0513-4870.2024-0104

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多药耐药(MDR) 细菌在全球范围内的广泛传播对全球公共卫生健康造成巨大威胁^[1-5]。世界卫生组织公布了一份迫切需要开发新的抗微生物药物的病原体清单, 其中包括绿假单胞菌^[6,7] (P.aeruginosa)。P.aeruginosa是一种革兰阴性病原体, 可造成重症和免疫功能低下的人群感染^[8]。药物外排泵的表达以及外膜通透性的降低, 是造成P.aeruginosa对抗生素耐药并引起广泛传播的主要原因^[9]。P.aeruginosa存在多种药物外排泵, 其中MexAB-OprM是与抗生素耐药性有关的主要外排系统^[10-12]。因此, 寻找靶向关键外排泵的药物可能为恢复病原体的药物敏感性提供新的策略。目前, 靶向P.aeruginosa的外排泵抑制剂(EPI) 主要有3类: 第一类是基于吡啶并嘧啶结构的EPI, 代表性化合物ABI-PP^[13] (图 1) 与P.aeruginosa外排系统中MexB发生特异性结合, 从而抑制部分药物外排, 但不抑制其他外排系统如MexY^[14], 限制其发展为治疗MDR P.aeruginosa药物; 第二类EPI代表性化合物TXA09155 (图 1) 可抑制多种药物外排系统, 增强野生型与MDR P.aeruginosa中抗生素的敏感性, 发挥广谱外排抑制作用, 但其在低浓度下抑制hERG引发关注^[15], 还需进一步优化; 最后一类是临床研究较广泛的二肽类化合物苯丙氨酸-精氨酸-β-萘酰胺(PAβN, 图 1), 是P.aeruginosa中多种外排系统的底物, 可以竞争性抑制药物外排。PAβN是第一个广谱的二肽类EPI, 可以恢复MDR P.aeruginosa对抗生素的敏感性^[16]。但其稳定性差, 且存在潜在的肾毒性, 限制了在临床进一步应用^[17]。综上, 由于多种原因, 目前尚无EPI作为抗生素佐剂被批准用于临床, 急需开发新的EPI。

据此, 本课题以PAβN为先导化合物, 首先将PAβN的A部分侧链胍基替换为氨基, 使A侧链的精氨酸结构变换为鸟氨酸结构; 其次, 将侧链B部分的苯丙氨酸结构替换为β-氨基-苯丁酸和β-氨基-萘丁酸, 以解决PAβN中含有天然氨基酸结构引起的代谢不稳定, 提高先导化合物的活性并改善其成药性, 同时在苯环的对位引入各种吸电子与给电子基团, 观察各类取代基对化合物活性的影响; 最后还根据生物电子等排原理将C部分的2-萘胺替换为3-氨基喹啉, 从而设计合成了一类新型的鸟氨酸拟肽类衍生物。

继而, 采用棋盘法评价该类化合物协同抗生素对抗P.aeruginosa的活性, 同时对化合物进行了安全性评价, 并初步探讨了鸟氨酸拟肽类结构类型的化合物对P.aeruginosa外排泵的影响。

结果与讨论

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1 化学合成

目标化合物的合成见合成路线1, 将缩合剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI) 与1-羟基苯并三唑(HOBT) 加入起始原料2a~2m的超干乙酸乙酯溶液^[18] (加热时, DCM沸点较低溶剂损耗较多, 而DMF在反应完成后难以旋蒸除去, 影响产物纯度, 因此最终采用乙酸乙酯作为缩合溶剂) 中, 50 ℃搅拌30 min, 然后加入1a和1b, 50 ℃搅拌8 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体3a~3m; 将反应中间体3a~3m加入装有超干二氯甲烷的烧瓶中, 再加入三氟乙酸(TFA), 室温搅拌2 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体4a~4m。将HOBT和EDCI加入到搅拌中的5a的超干乙酸乙酯溶液中, 在室温下搅拌30 min, 然后加入中间体4a~4m, 室温搅拌8 h, TLC检测反应完全, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体6a~6m; 将反应中间体加入装有超干二氯甲烷的烧瓶中, 加入TFA, 室温搅拌2 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到目标终产物7a~7m, 总产率为4%~38%。

将EDCI与HOBT加入起始原料8a的超干乙酸乙酯溶液中, 50 ℃搅拌30 min, 然后加入1a和1b, 50 ℃继续搅拌8 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体9a和9b; 将9a和9b加入装有超干二氯甲烷的烧瓶中, 再加入TFA, 室温搅拌2 h, TLC检测反应完成, 有机相浓缩, 残余物继续用硅胶快速柱色谱法纯化, 得到反应中间体10a和10b。将HOBT和EDCI加入到搅拌中的5a的超干乙酸乙酯溶液中, 在室温下搅拌30 min, 然后加入中间体10a和10b, 室温搅拌8 h, TLC检测反应完全, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体11a和11b; 将反应中间体加入装有超干二氯甲烷的烧瓶中, 加入TFA, 室温搅拌2 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到目标终产物12a和12b, 总产率为11%。

采用高斯计算方法, 在B3LYP/6-311G (d, p) 水平, 基于时间依赖性密度泛函理论(TDDFT) 在0.5 eV的半频带宽度下得到代表性化合物12b的理论圆二色谱(ECD), 见合成路线1, 与实验得到的ECD光谱高度相似, 表明此类化合物的构型(S, S) 即为预测构型(S, S)。

所有化合物结构经¹H NMR、¹³C NMR及HR-MS分析确证, 目标化合物的收率、理化参数和波谱数据见表 1。

2 协同抗生素抗铜绿假单胞菌的活性评价

本课题共设计合成了15个全新的鸟氨酸拟肽类衍生物, 其协同单环β-内酰胺类抗生素氨曲南(AZT) 对抗铜绿假单胞菌的活性及结构见表 2。所有化合物活性的测定均以二肽类外排泵抑制剂PAβN作为对照药物。活性数据结果显示, 引入氨基使苯丙氨酸结构变为β-氨基-苯丁酸^[16], 并在β-氨基-苯丁酸对位引入不同的吸电子与给电子基团包括叔丁基、苄氧基、氰基、硝基、甲氧基等时, 部分化合物对AZT显示出一定程度的增敏作用, 并且引入给电子基团时, 衍生物增敏AZT的作用优于吸电子基团的引入, 特别是叔丁基的引入使化合物7h将AZT的MIC由2 μg·mL^-1降低至0.5 μg·mL^-1, FICI值(fractional inhibitory concentration index) 小于0.5, 对AZT有显著协同作用, 而当3-氨基喹啉替代2-萘胺时, 活性无显著变化; 接着将β-氨基-苯丁酸替换为β-氨基-萘丁酸, 同时将2-萘胺替换为3-氨基喹啉时, 衍生物12b的协同增敏活性进一步增强, FICI值为0.375, 活性显著优于对照药物PAβN。

为了进一步验证该类化合物对抗生素的协同增敏作用, 选取初步具有协同活性的化合物7h与12b, 与AZT联用对抗单一表达外排泵MexAB-OprM的P.aeruginosa 26菌株(用质粒pAK1900-MexAB-OprM转化)。结果如表 3所示, 代表性化合物7h与12b对AZT仍表现出较强的协同增敏活性, 并且化合物12b的活性更优于7h。

基于此, 进一步评价12b联用多种抗生素, 包括AZT、美罗培南(MEM)、头孢他啶(CAZ) 和克拉霉素(CLR) 对抗临床分离的MDR P.aeruginosa的协同增敏活性。结果显示(表 4), 化合物12b对上述4种抗生素均表现出明显的协同增敏活性。提示化合物12b对抗生素具有广谱的协同作用, 可作为抗生素佐剂, 调节抗生素在亚MIC浓度下发挥抗菌作用。

3 安全性与类药性评价

3.1 细胞毒性的测定

进一步评价代表性化合物12b在不同细胞系的安全性, 包括人胚胎肾细胞(HEK293细胞) 与非洲绿猴肾细胞(Vero细胞)。以PAβN为对照化合物, 采用MTT法测定了化合物与细胞共培养24 h的半数细胞毒性浓度(CC₅₀)。结果表明, 在HEK293细胞和Vero细胞中, 12b的CC₅₀值均大于100 μmol·L^-1 (图 2a), 表明化合物12b在高浓度时(100 μmol·L^-1), 对HEK293和Vero细胞均无细胞毒性, 具有良好的安全性。

3.2 hERG心脏毒性的测定

进一步评估化合物是否具有潜在的心脏毒性, 采用电生理手动膜片钳方法测试了代表性化合物12b对hERG钾通道(human Ether-a-go-go related gene potassium channel) 电流的影响。结果显示(图 2b), 化合物12b对hERG钾通道电流的半数抑制浓度值(IC₅₀) 大于40 μmol·L^-1, 这表明化合物12b的心脏毒性较小, 具有良好的安全性。

3.3 代表性化合物12b类药性预测

使用ADMET Predictor 9.5软件对化合物12b的吸收、代谢和毒理性质进行类药性预测并打分, 包括吸收风险系数(Absn risk)、代谢性质风险系数(CYP risk)、毒性风险系数(TOX risk) 及ADMET risk几个方面对12b的吸收、代谢及毒性性质进行全方面的类药性预测评估(表 5)。结果显示, 12b的多数风险系数预测分值在成药性范围内, 提示12b具有一定类药性特点。

4 生物学验证与分子对接

4.1 外排机制的验证

为验证化合物是否靶向细菌外排泵发挥作用, 评估了在P.aeruginosa 26菌株中, 化合物12b抑制荧光底物Hoechst 33342 (RND外排泵底物) 外排的潜在能力。通过比较化合物12b与PAβN处理与未处理过的细菌最终荧光计算得到相对最终荧光(RFF)值^[19], 见图 3。结果表明, 12b表现出剂量依赖性的外排抑制能力, 在低浓度16 μg·mL^-1时, 具有明显优于PAβN的抑制外排能力; 而在高浓度时, 与PAβN抑制外排的活性相当。表明化合物12b具有对外排泵MexAB-OprM的潜在调节活性, 通过抑制细菌外排泵恢复细菌对抗生素的敏感性。

4.2 Schrodinger分子对接

鉴于MexB是RND外排系统中结合抗生素的主要外排转运体^[20], 采用Schrodinger软件对化合物12b与MexB (PDB ID: 3W9I) 进行分子对接, 并通过Docking score来评估配体在受体腔内的结合能力。结果显示, 12b对接打分为-6.792, PAβN对接打分为-5.906, 化合物12b打分结果更优于PAβN, 表明化合物12b与PAβN相比, 对外排转运体MexB可能有较高的亲和力。

采用Schrodinger将对接结果进行可视化分析。结果如图 4a、b所示, 化合物12b可与MexB活性腔的ASP-85、ARG-185、GLN-273、ASP-274、GLN-773等氨基酸残基形成较强的氢键相互作用, 同时与ASP-274形成盐桥; 而PAβN与MexB活性腔的氨基酸残基GLN-273、ASP-274、TRP-753、SER-755、GLN-773形成氢键相互作用, 并未形成盐桥(图 4c、d), 这可能是12b对MexB结合力更强的原因。

综上所述, 代表性化合物12b和MexB的结合方式与PAβN相似, 且亲和力更强, 提示12b直接靶向外排转运体MexB发挥抑制外排作用, 从而恢复细菌对抗生素的敏感性。

结论

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本研究设计合成了15个全新的鸟氨酸拟肽类衍生物, 通过棋盘法评价了其协同单环β-内酰胺类抗生素AZT对抗P.aeruginosa敏感菌株活性。其中化合物12b表现出最好的协同抗菌活性, 进一步研究显示, 12b不仅增强β-内酰胺类抗生素AZT、CAZ、MEM, 而且还增强大环内酯类抗生素CLR对抗MDR P.aeruginosa的活性。初步作用机制显示, 此类化合物主要通过靶向外排泵MexAB-OprM发挥作用, 与先导物PAβN一致。另外, 化合物12b具有合成简单、安全性高的特点, 是一个理想的先导物, 值得进一步修饰探索。

实验部分

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所用化学试剂及原料均购自泰坦公司、毕得公司、伊诺凯公司和通广等公司的分析纯试剂, 溶剂未经纯化直接使用。抗生素标准品购自中国食品药品检定研究院。MHB培养基、LB培养基购自海博生物技术有限公司。

化合物熔点采用MP90型精密熔点仪(Mettler Toledo, Switzerland) 测得, 温度未校正; ¹H NMR和 ¹³C NMR采用Bruker Avance Ⅲ 600 MHz核磁共振仪(Varian, USA) 测得, 溶剂均为CD₃OD, 内标为TMS; HR-MS采用Autospec Ultima-TOF质谱测定仪(Micromass UK Ltd., UK) 测得, Flash柱分离纯化采用Combi flash Rf 200快速制备液相(Teledyne, USA); 薄层色谱(TLC) 采用Merck 60 F₂₅₄薄层色谱硅胶板, 紫外检测用ZF-7A手提紫外检测灯(上海宝山顾村电光仪器厂); 吸光度采用酶标仪(Multiskan FC, Thermo, USA) 测定。

1 化学合成

鸟氨酸拟肽类衍生物7a~7m的合成 将缩合剂EDCI与HOBT加入起始原料2a~2m的超干乙酸乙酯溶液中, 50 ℃搅拌30 min, 然后加入1a与1b, 50 ℃搅拌8 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体3a~3m; 将反应中间体3a~3m加入装有超干二氯甲烷的烧瓶中, 再加入三氟乙酸TFA, 室温搅拌2 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体4a~4m。将HOBT和EDCI加入到搅拌中的5a的超干乙酸乙酯溶液中, 在室温下搅拌30 min, 然后加入中间体4a~4m, 室温搅拌8 h, TLC检测反应完全, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体6a~6m; 将反应中间体加入装有超干二氯甲烷的烧瓶中, 加入TFA, 室温搅拌2 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到目标终产物7a~7m。

鸟氨酸拟肽类衍生物12a、12b的合成 将EDCI与HOBT加入起始原料8a的超干乙酸乙酯溶液中, 50 ℃搅拌30 min, 然后加入1a和1b, 50 ℃搅拌8 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体9a和9b; 将9a和9b加入装有超干二氯甲烷的烧瓶中, 再加入TFA, 室温搅拌2 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体10a和10b。将HOBT和EDCI加入搅拌中5a的超干乙酸乙酯溶液中, 在室温下搅拌30 min, 然后加入中间体10a和10b, 室温搅拌8 h, TLC检测反应完全, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到反应中间体11a和11b; 将反应中间体加入装有超干二氯甲烷的烧瓶中, 加入TFA, 室温搅拌2 h, TLC检测反应完成, 有机相浓缩, 残余物用硅胶快速柱色谱法纯化, 得到目标终产物12a和12b。

2 棋盘法测定协同活性

本研究按照CLSI标准, 采用微量肉汤稀释法进行抑菌活性测定和棋盘法试验。化合物和PAβN用阳离子调节的Mueller-Hinton肉汤(CAMH肉汤, 含有20~25 mg·mL^-1 Mg²⁺, 10~12.5 mg·mL^-1 Ca²⁺) 在96孔板上沿纵坐标2倍稀释, 抗生素以同样方式沿横坐标稀释, 每孔终体积为90 μL。所有测试菌株均在37 ℃的Luria-Bertani肉汤(LB肉汤) 中培养过夜。将培养物调至0.5 McFarland (1.5×10⁸ CFU·mL^-1), 并用CAMH肉汤以1∶30稀释至5×10⁶ CFU·mL^-1。然后将10 μL细菌悬浮液加入各孔中, 使最终细菌浊度为5×10⁵ CFU·mL^-1。将96孔板置于37 ℃恒温孵育18 h。观察菌株生长情况, 测定化合物与抗生素的最低抑菌浓度并计算FICI值, FICI = MIC (A in combination with B)/MIC (A alone) + MIC (B in combination with A)/MIC (B alone), synergy was defined as FICI^[17] ≤ 0.5。

3 MTT法测定细胞毒性

取对数期生长期的Vero细胞及HEK293细胞, 在96孔板中每孔接种1×10⁴个数量的细胞, 与药物共培养24 h。将20 μL MTT (5 mg·mL^-1) 溶液加入到每孔中, 并于37 ℃、5% CO₂的培养箱中孵育4 h。去除培养基并加入150 μL DMSO以完全溶解结晶物后, 测定其在570 nm处的吸光度值。

4 电生理手动膜片钳方法测定hERG心脏毒性

稳定表达hERG的CHO细胞置于37 ℃、5% CO₂的培养箱培养, 每48 h按1∶5比例进行传代。试验当天, 吸走细胞培养液, 用细胞外液淋洗1遍后加入0.25% Trypsin-EDTA (Invitrogen) 溶液, 在室温下消化5 min, 吸走消化液, 用细胞外液重悬后将细胞转移到实验皿中备用。阳性对照化合物采用西沙比利(cisapride), 最终测试浓度为300 nmol·L^-1。设置化合物的测试浓度, 每个浓度至少测试2个细胞(n ≥ 2)。数据分析处理采用pClamp、GraphPad Prism 8和Excel软件。不同化合物浓度对hERG钾电流的抑制程度用公式计算: Inhibition % = [1- (I/Io)] × 100%。

5 Schrodinger Maestro Version 12.8 117分子对接

对接过程: 使用软件自带模块处理MexB蛋白(UniProt数据库, ID: 3W9I), pH设置为7.4 ± 0.05; 配体小分子化合物按软件默认设置处理; 根据sitemap预测位点生成对接盒子, 其余选项均为默认设置。

6 测定化合物12b和PAβN对Hoechst 33342的外排抑制

通过比较EPI处理与未处理细胞的最终荧光计算RFF值, 实验具体操作参考文献^[20]。RFF指数计算公式: RFF = (RF_{45 min处理} - RF_{45 min未处理})/RF_{45 min未处理}。

致谢: 感谢中国医学科学院医药生物技术研究所分析测试中心测定目标化合物的核磁共振氢谱、碳谱及高分辨质谱。

作者贡献: 朱喜负责目标化合物合成与构型确定、耐药菌株活性测定、分子对接以及初稿撰写; 马西灿负责化合物的设计与指导; 张昕彤负责细胞毒性的测定; 刘忆霜副研究员负责敏感菌株活性初筛与机制研究; 何宁老师负责耐药菌株的管理; 解云英研究员负责菌株的管理指导; 本文通讯作者宋丹青研究员负责实验的设计与把关, 以及稿件修改等工作。

利益冲突: 所有作者均声明不存在利益冲突。

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国家自然科学基金资助项目(32141003)
中国医学科学院医学科学创新基金项目(2021-12M-1-070)

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Mills JP, Marchaim D. Multidrug-resistant Gram-negative bacteria: infection prevention and control update [J]. Infect Dis Clin North Am, 2021, 35: 969-994.

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Li Z, Guo Z, Lu X, et al. Evolution and development of potent monobactam sulfonate candidate IMBZ18g as a dual inhibitor against MDR Gram-negative bacteria producing ESBLs [J]. Acta Pharm Sin B, 2023, 13: 3067-3079.

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Murray CJL, Ikuta KS, Sharara F, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis [J]. Lancet, 2022, 399: 629-655.

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De Oliveira DMP, Forde BM, Kidd TJ, et al. Antimicrobial resistance in ESKAPE pathogens [J]. Clin Microbiol Rev, 2020, 33: e00181-19.

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Hwang W, Yoon SS. Virulence characteristics and an action mode of antibiotic resistance in multidrug-resistant Pseudomonas aeruginosa [J]. Sci Rep, 2019, 9: 487.

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Behzadi P, Baráth Z, Gajdács M. It's not easy being green: a narrative review on the microbiology, virulence and therapeutic prospects of multidrug-resistant Pseudomonas aeruginosa [J]. Antibiotics, 2021, 10: 42.

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Krishnamoorthy G, Leus IV, Weeks JW, et al. Synergy between active efflux and outer membrane diffusion defines rules of antibiotic permeation into Gram-negative bacteria [J]. mBio, 2017, 8: e01172-17.

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Piddock LJV. Multidrug-resistance efflux pumps? Not just for resistance [J]. Nat Rev Microbiol, 2006, 4: 629-636.

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Puzari M, Chetia P. RND efflux pump mediated antibiotic resistance in Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa: a major issue worldwide [J]. World J Microbiol Biotechnol, 2017, 33: 24.

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Renau TE, Léger R, Flamme EM, et al. Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin [J]. J Med Chem, 1999, 42: 4928-4931.

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Yoshida KI, Nakayama K, Ohtsuka M, et al. MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate [J]. Bioorg Med Chem, 2007, 15: 7087-7097.

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Nakashima R, Sakurai K, Yamasaki S, et al. Structural basis for the inhibition of bacterial multidrug exporters [J]. Nature, 2013, 500: 102-106.

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Compagne N, Cruz AVD, Müller RT, et al. Update on the discovery of efflux pump inhibitors against critical priority Gram-negative bacteria [J]. Antibiotics, 2023, 12: 180.

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Ma X, Guo W, Zhu X, et al. Synthesis of peptidomimetics as antibiotic adjuvants for combination with aztreonam to combat MDR Pseudomonas aeruginosa [J]. Eur J Med Chem, 2023, 260: 115778.

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Wright GD. Antibiotic adjuvants: rescuing antibiotics from resistance [J]. Trends Microbiol, 2016, 24: 862-871.

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Renau TE, Léger R, Filonova L, et al. Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa [J]. Bioorg Med Chem Lett, 2003, 13: 2755-2758.

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Tambat R, Mahey N, Chandal N, et al. A microbe-derived efflux pump inhibitor of the resistance-nodulation-cell division protein restores antibiotic susceptibility in Escherichia coli and Pseudomonas aeruginosa [J]. ACS Infect Dis, 2022, 8: 255-270.

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Verchère A, Dezi M, Adrien V, et al. In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa [J]. Nat Commun, 2015, 6: 6890.

2024年第59卷第6期

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doi: 10.16438/j.0513-4870.2024-0104

接收时间：2024-02-01
首发时间：2025-11-26
出版时间：2024-06-12

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收稿日期：2024-02-01
修回日期：2024-03-15

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国家自然科学基金资助项目(32141003)

中国医学科学院医学科学创新基金项目(2021-12M-1-070)

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中国医学科学院、北京协和医学院医药生物技术研究所, 北京 100050

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^*宋丹青, Tel/Fax: 86-10-63165268, E-mail: songdanqingsdq@sina.com

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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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Compd.	Yield /%	mp /℃	¹H NMR (600 MHz, CD₃OD) δ	¹³C NMR (150 MHz, CD₃OD) δ	HR-ESI-MS m/s Formula Calcd. Found
7a	38	78.8-80.8	8.14-8.13 (m, 1H), 7.81 (t, J = 8.1 Hz, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.48-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.34-7.30 (m, 4H), 7.25-7.22 (m, 1H), 4.70-4.64 (m, 1H), 3.79 (t, J = 6.1 Hz, 1H), 3.00 (dd, J = 13.7, 7.0 Hz, 1H), 2.92 (dd, J = 13.7, 7.1 Hz, 1H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.88-1.73 (m, 2H), 1.69-1.60 (m, 2H).	170.3, 168.1, 137.7, 135.7, 133.8, 130.8, 129.1 (2), 128.3, 128.2 (2), 127.2, 127.1, 126.4, 126.2, 124.8, 120.1, 116.8, 52.4, 48.9, 40.4, 40.2, 38.5, 28.4, 22.3.	[M+H]⁺ : C₂₅H₃₁N₄O₂ 419.244 2 419.242 2
7b	12	112.2-114.0	8.18 (m, 1H), 7.80-7.75 (m, 3H), 7.55 (dd, J = 8.8, 2.1 Hz, 1H), 7.46-7.43 (m, 1H), 7.40-7.37 (m, 1H), 7.35-7.33 (m, 2H), 7.23-7.22 (m, 2H), 4.66-4.61 (m, 1H), 2.95-2.87 (m, 2H), 2.74-2.65 (m, 3H), 2.61 (dd, J = 14.5, 8.8 Hz, 1H), 1.69-1.48 (m, 5H), 1.28 (s, 9H).	161.8, 161.6, 135.8, 134.8, 133.8, 128.9 (2), 128.1, 127.2, 127.1, 126.1, 125.0 (2), 124.7, 120.0, 117.8, 116.5, 115.9, 53.8, 48.5, 48.2, 40.8, 38.9, 33.8, 31.3, 30.4 (3), 23.6.	[M+H]⁺ : C₂₉H₃₉N₄O₂ 475.306 8 475.305 2
7c	24	67.2-69.1	8.12 (m, 1H), 7.80 (t, J = 8.7 Hz, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.41-7.39 (m, 3H), 7.37-7.34 (m, 2H), 7.31-7.28 (m, 1H), 7.23-7.20 (m, 2H), 6.95-6.94 (m, 2H), 5.02 (s, 2H), 4.64-4.61 (m, 1H), 3.88-3.86 (m, 1H), 2.94 (dd, J = 13.8, 6.7 Hz, 1H), 2.85 (dd, J = 13.8, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.58-2.54 (m, 1H), 1.91-1.78 (m, 2H), 1.70-1.64 (m, 2H).	170.3, 167.5, 157.8, 137.4, 135.7, 133.8, 130.8, 130.1 (2), 129.8, 128.3, 128.1 (2), 127.5, 127.3, 127.1 (3), 126.2, 124.8, 120.1, 116.8, 114.7 (2), 69.6, 52.3, 49.1, 40.5, 39.5, 38.5, 28.1, 22.2.	[M+H]⁺ : C₃₂H₃₇N₄O₃ 525.286 0 525.286 1
7d	21	85.9-87.9	8.14-8.13 (m, 1H), 7.81 (t, J = 8.8 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.69-7.68 (m, 2H), 7.53-7.49 (m, 3H), 7.48-7.45 (m, 1H), 7.43-7.37 (m, 1H), 4.75-4, 69 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 3.05 (dd, J = 7.0, 3.1 Hz, 2H), 2.77 (dd, J = 14.7, 4.4 Hz, 1H), 2.74-2.69 (m, 2H), 2.59 (dd, J = 14.7, 9.4 Hz, 1H), 1.91-1.76 (m, 2H), 1.69-1.62 (m, 2H).	169.9, 167.6, 143.7, 135.6, 133.8, 132.0 (2), 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 120.0, 118.3, 116.8, 110.3, 52.2, 40.6, 40.2, 38.5, 28.1, 27.6, 22.2.	[M+H]⁺ : C₂₆H₃₀N₅O₂ 444.239 4 444.237 6
7e	34	78.4-80.4	8.12 (m, 1H), 7.81 (t, J = 7.9 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.51 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.19-7.12 (m, 4H), 4.67-4.61 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 2.96 (dd, J = 13.7, 6.8 Hz, 1H), 2.87 (dd, J = 13.7, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.55 (dd, J = 14.7, 9.5 Hz, 1H), 2.29 (s, 3H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	170.3, 167.5, 136.1, 135.6, 134.5, 133.8, 130.8, 129.0 (2), 128.8 (2), 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 52.3, 49.0, 40.4, 39.9, 38.5, 28.1, 22.2, 19.7.	[M+H]⁺ : C₂₆H₃₃N₄O₂ 433.259 8 433.258 1
7f	21	46.7-48.7	8.13 (m, 1H), 7.82-7.79 (m, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.32-7.30 (m, 2H), 7.06-7.02 (m, 2H), 4.66-4.63 (m, 1H), 3.86 (t, J = 6.1 Hz, 1H), 2.95 (qd, J = 13.9, 7.0 Hz, 2H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.89-1.79 (m, 2H), 1.69-1.64 (m, 2H).	170.2, 167.6, 161.9, 135.6, 133.8, 133.6, 130.8, 130.8, 130.7, 128.3, 127.3, 127.1, 126.2, 124.8, 120.1, 116.8, 114.9, 114.8, 52.3, 48.9, 40.5, 39.3, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₅H₃₀FN₄O₂ 437.234 7 437.233 3
7g	8	72.8-74.8	8.21-8.18 (m, 2H), 8.13 (m, 1H), 7.81 (t, J = 9.3 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.56-7.51 (m, 3H), 7.48-7.45 (m, 1H), 7.42-7.39 (m, 1H), 4.78-4.73 (m, 1H), 3.87-3.85 (m, 1H), 3.14-3.06 (m, 2H), 2.82-2.72 (m, 3H), 2.64-2.60 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	169.9, 167.6, 147.0, 145.7, 135.6, 133.8, 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 123.2 (2), 120.0, 116.8, 52.2, 48.5, 40.7, 39.9, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₅H₃₀N₅O₄ 464.229 2 464.227 8
7h	11	67.3-68.6	8.13 (s, 1H), 7.82-7.79 (m, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.22-7.20 (m, 2H), 6.88-6.86 (m, 2H), 4.64-4.60 (m, 1H), 3.87-3.86 (m, 1H), 3.75 (m, 3H), 2.93 (dd, J = 13.9, 6.9 Hz, 1H), 2.87-2.83 (m, 1H), 2.75-2.70 (m, 3H), 2.58-2.53 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).	170.4, 167.5, 158.7, 135.7, 133.8, 130.8, 130.0 (2), 129.5, 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 113.6 (2), 54.2, 52.3, 49.1, 40.4, 39.4, 38.5, 28.1, 22.2.	[M+H]⁺ : C₂₆H₃₃N₄O₃ 449.254 7 449.253 3
7i	4	56.0-58.0	8.97-8.96 (m, 1H), 8.63-8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.89 (m, 1H), 7.72-7.69 (m, 1H), 7.63-7.60 (m, 1H), 7.32-7.29 (m, 4H), 7.23-7.20 (m, 1H), 4.68-4.66 (m, 1H), 3.89-3.87 (m, 1H), 3.03 (dd, J = 13.8, 7.1 Hz, 1H), 2.94 (dd, J = 13.7, 7.1 Hz, 1H), 2.86-2.83 (m, 2H), 2.78 (dd, J = 14.9, 4.6 Hz, 1H), 2.68-2.64 (m, 1H), 1.95-1.90 (m, 1H), 1.86-1.80 (m, 1H), 1.76-1.71 (m, 2H).	170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₃₀N₅O₂ 420.239 4 420.238 3
7j	33	98.5-100.5	8.97-8.96 (m, 1H), 8.63-8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.89 (m, 1H), 7.72-7.69 (m, 1H), 7.63-7.60 (m, 1H), 7.32-7.29 (m, 4H), 7.23-7.20 (m, 1H), 4.68-4.66 (m, 1H), 3.89-3.87 (m, 1H), 3.03 (dd, J = 13.8, 7.1 Hz, 1H), 2.94 (dd, J = 13.7, 7.1 Hz, 1H), 2.86-2.83 (m, 2H), 2.78 (dd, J = 14.9, 4.6 Hz, 1H), 2.68-2.64 (m, 1H), 1.95-1.90 (m, 1H), 1.86-1.80 (m, 1H), 1.76-1.71 (m, 2H).	170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₂₉N₆O₄ 465.224 5 465.222 7
7k	33	84.8-85.7	8.99 (s, 1H), 8.64 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.91-7.90 (m, 1H), 7.73-7.70 (m, 1H), 7.64-7.61 (m, 1H), 7.30 (m, 4H), 4.68-4.63 (m, 1H), 3.88 (s, 1H), 3.00-2.93 (m, 2H), 2.85- 2.77 (m, 3H), 2.65 (dd, J = 14.3, 8.6 Hz, 1H), 1.92 (s, 1H), 1.84-1.73 (m, 3H).	170.5, 167.8, 144.2, 144.1, 136.4, 132.5, 132.3, 130.7 (2), 128.5, 128.3 (3), 127.5 (2), 127.3, 124.5, 52.3, 48.7, 40.5, 39.3, 38.6, 28.2, 22.4.	[M+H]⁺ : C₂₄H₂₉ClN₅O₂ 454.200 4 454.199 3
7l	7	68.3-69.9	8.97-8.96 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.69 (m, 1H), 7.62-7.60 (m, 1H), 7.37-7.28 (m, 5H), 7.23-7.19 (m, 2H), 6.92 (d, J = 7.4 Hz, 2H), 4.98 (s, 2H), 4.65-4.60 (m, 1H), 3.90-3.88 (m, 1H), 2.96 (dd, J = 13.8, 6.8 Hz, 1H), 2.87-2.81 (m, 3H), 2.76 (dd, J = 14.5, 5.2 Hz, 1H), 2.62 (dd, J = 14.8, 8.7 Hz, 1H), 1.95-1.80 (m, 2H), 1.76-1.71 (m, 2H).	170.7, 167.7, 157.8, 144.2, 144.1, 137.3, 132.5, 130.1 (2), 129.8, 128.5, 128.3, 128.1 (2), 127.5(3), 127.3, 127.1 (2), 124.5, 114.6 (2), 69.6, 52.3, 49.0, 40.4, 39.4, 38.6, 28.2, 22.4.	[M+H]⁺ : C₃₁H₃₆N₅O₃ 526.281 3 526.280 3
7m	33	91.5-92.8	8.97 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.70 (m, 1H), 7.63-7.60 (m, 1H), 7.34 (d, J = 7.6 Hz, 2H), 7.24-7.22 (m, 2H), 4.68-4.63 (m, 1H), 3.91-3.89 (m, 1H), 3.00 (dd, J = 13.7, 6.5Hz, 1H), 2.90-2.84 (m, 3H), 2.77 (dd, J =14.9, 4.8 Hz, 1H), 2.62 (dd, J = 14.8, 8.8 Hz, 1H), 1.96-1.84 (m, 2H), 1.77-1.73 (m, 2H), 1.26 (s, 9H).	170.7, 167.7, 149.4, 144.2, 144.1, 134.4, 132.5, 128.8 (2), 128.5, 128.3, 127.5, 127.4, 127.3, 125.1 (2), 124.4, 52.3, 48.8, 40.3, 39.6, 38.6, 33.8, 30.4 (3), 28.2, 22.4.	[M+H]⁺ : C₂₈H₃₈N₅O₂ 476.302 0 476.301 7
12a	11	162.3-164.2	8.11 (m, 1H), 7.85-7.79 (m, 5H), 7.77-7.75 (m, 2H), 7.51-7.43 (m, 5H), 7.42-7.39 (m, 1H), 4.89-4.76 (m, 1H), 3.84 (t, J = 6.1 Hz, 1H), 3.18 (dd, J = 13.7, 7.2 Hz, 1H), 3.11 (dd, J = 13.7, 7.0 Hz, 1H), 2.81 (dd, J = 14.7, 4.3 Hz, 1H), 2.69 (t, J = 7.4 Hz, 2H), 2.63 (dd, J = 14.7, 9.3 Hz, 1H), 1.89-1.76 (m, 2H), 1.68-1.61 (m, 2H).	170.3, 167.6, 135.6, 135.2, 133.8, 133.7, 132.6, 130.8, 128.3, 127.9, 127.6, 127.2 (4), 127.1, 126.2, 125.8, 125.3, 124.8, 120.1, 116.9, 52.3, 49.0, 40.6, 40.3, 38.5, 28.2, 22.2.	[M+H]⁺ : C₂₉H₃₃N₄O₂ 468.259 8 469.258 3
12b	11	169.9-171.2	8.88-8.87 (m, 1H), 8.52-8.51 (m, 1H), 7.97-7.95 (m, 1H), 7.85-7.77 (m, 5H), 7.70-7.67 (m, 1H), 7.61-7.57 (m, 1H), 7.49-7.47 (m, 1H), 7.45-7.39 (m, 2H), 4.8-4.76 (m, 1H), 3.87-3.85 (m, 1H), 3.22-3.19 (m, 1H), 3.13-3.09 (m, 1H), 2.86-2.79 (m, 3H), 2.71-2.66 (m, 1H), 1.92-1.79 (m, 2H), 1.73-1.68 (m, 2H).	170.6, 167.8, 144.2, 135.2, 133.6, 132.5, 132.4, 128.5, 128.3, 127.9, 127.7, 127.4 (3), 127.3, 127.2 (3), 125.8, 125.3, 124.5, 52.3, 48.9, 40.7, 40.3, 38.5, 28.3, 22.4.	[M+H]⁺ : C₂₈H₃₂N₅O₂ 470.255 1 470.253 8

Compd.

Yield /%

mp /℃

¹H NMR
(600 MHz, CD₃OD) δ

¹³C NMR
(150 MHz, CD₃OD) δ

HR-ESI-MS
m/s
Formula
Calcd.
Found

78.8-80.8

170.3, 168.1, 137.7, 135.7, 133.8, 130.8, 129.1 (2), 128.3, 128.2 (2), 127.2, 127.1, 126.4, 126.2, 124.8, 120.1, 116.8, 52.4, 48.9, 40.4, 40.2, 38.5, 28.4, 22.3.

[M+H]⁺ :
C₂₅H₃₁N₄O₂
419.244 2
419.242 2

112.2-114.0

161.8, 161.6, 135.8, 134.8, 133.8, 128.9 (2), 128.1, 127.2, 127.1, 126.1, 125.0 (2), 124.7, 120.0, 117.8, 116.5, 115.9, 53.8, 48.5, 48.2, 40.8, 38.9, 33.8, 31.3, 30.4 (3), 23.6.

[M+H]⁺ :
C₂₉H₃₉N₄O₂
475.306 8
475.305 2

67.2-69.1

170.3, 167.5, 157.8, 137.4, 135.7, 133.8, 130.8, 130.1 (2), 129.8, 128.3, 128.1 (2), 127.5, 127.3, 127.1 (3), 126.2, 124.8, 120.1, 116.8, 114.7 (2), 69.6, 52.3, 49.1, 40.5, 39.5, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₃₂H₃₇N₄O₃
525.286 0
525.286 1

85.9-87.9

169.9, 167.6, 143.7, 135.6, 133.8, 132.0 (2), 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 120.0, 118.3, 116.8, 110.3, 52.2, 40.6, 40.2, 38.5, 28.1, 27.6, 22.2.

[M+H]⁺ :
C₂₆H₃₀N₅O₂
444.239 4
444.237 6

78.4-80.4

170.3, 167.5, 136.1, 135.6, 134.5, 133.8, 130.8, 129.0 (2), 128.8 (2), 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 52.3, 49.0, 40.4, 39.9, 38.5, 28.1, 22.2, 19.7.

[M+H]⁺ :
C₂₆H₃₃N₄O₂
433.259 8
433.258 1

46.7-48.7

170.2, 167.6, 161.9, 135.6, 133.8, 133.6, 130.8, 130.8, 130.7, 128.3, 127.3, 127.1, 126.2, 124.8, 120.1, 116.8, 114.9, 114.8, 52.3, 48.9, 40.5, 39.3, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₅H₃₀FN₄O₂
437.234 7
437.233 3

72.8-74.8

169.9, 167.6, 147.0, 145.7, 135.6, 133.8, 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 123.2 (2), 120.0, 116.8, 52.2, 48.5, 40.7, 39.9, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₅H₃₀N₅O₄
464.229 2
464.227 8

67.3-68.6

170.4, 167.5, 158.7, 135.7, 133.8, 130.8, 130.0 (2), 129.5, 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 113.6 (2), 54.2, 52.3, 49.1, 40.4, 39.4, 38.5, 28.1, 22.2.

[M+H]⁺ :
C₂₆H₃₃N₄O₃
449.254 7
449.253 3

56.0-58.0

170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₃₀N₅O₂
420.239 4
420.238 3

98.5-100.5

170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₂₉N₆O₄
465.224 5
465.222 7

84.8-85.7

170.5, 167.8, 144.2, 144.1, 136.4, 132.5, 132.3, 130.7 (2), 128.5, 128.3 (3), 127.5 (2), 127.3, 124.5, 52.3, 48.7, 40.5, 39.3, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₂₄H₂₉ClN₅O₂
454.200 4
454.199 3

68.3-69.9

170.7, 167.7, 157.8, 144.2, 144.1, 137.3, 132.5, 130.1 (2), 129.8, 128.5, 128.3, 128.1 (2), 127.5(3), 127.3, 127.1 (2), 124.5, 114.6 (2), 69.6, 52.3, 49.0, 40.4, 39.4, 38.6, 28.2, 22.4.

[M+H]⁺ :
C₃₁H₃₆N₅O₃
526.281 3
526.280 3

91.5-92.8

170.7, 167.7, 149.4, 144.2, 144.1, 134.4, 132.5, 128.8 (2), 128.5, 128.3, 127.5, 127.4, 127.3, 125.1 (2), 124.4, 52.3, 48.8, 40.3, 39.6, 38.6, 33.8, 30.4 (3), 28.2, 22.4.

[M+H]⁺ :
C₂₈H₃₈N₅O₂
476.302 0
476.301 7

12a

162.3-164.2

170.3, 167.6, 135.6, 135.2, 133.8, 133.7, 132.6, 130.8, 128.3, 127.9, 127.6, 127.2 (4), 127.1, 126.2, 125.8, 125.3, 124.8, 120.1, 116.9, 52.3, 49.0, 40.6, 40.3, 38.5, 28.2, 22.2.

[M+H]⁺ :
C₂₉H₃₃N₄O₂
468.259 8
469.258 3

12b

169.9-171.2

170.6, 167.8, 144.2, 135.2, 133.6, 132.5, 132.4, 128.5, 128.3, 127.9, 127.7, 127.4 (3), 127.3, 127.2 (3), 125.8, 125.3, 124.5, 52.3, 48.9, 40.7, 40.3, 38.5, 28.3, 22.4.

[M+H]⁺ :
C₂₈H₃₂N₅O₂
470.255 1
470.253 8


7a	-	C	> 128	2	64	1	< 1
7b	t-Bu	C	32	2	16	0.125	0.562 5
7c	OBn	C	32	2	16	0.25	0.625
7d	CN	C	> 128	2	128	1	< 1.5
7e	CH₃	C	128	2	64	1	1
7f	F	C	> 128	2	64	1	< 1
7g	NO₂	C	> 128	2	16	1	< 0.625
7h	OCH₃	C	> 128	2	32	0.5	< 0.5
7i	-	N	> 128	2	64	1	< 1
7j	NO₂	N	> 128	2	64	1	< 1
7k	Cl	N	> 128	2	8	1	< 0.562 5
7l	OBn	N	128	2	64	0.5	0.75
7m	t-Bu	N	128	2	64	0.5	0.75
12a	-	C	64	2	32	0.25	0.625
12b	-	N	128	2	16	0.5	0.375
PAβN	-	-	> 128	2	128	0.5	< 1.25

No.

Alone

Combined

FICI

MIC^a

MIC^AZT

MIC^a

MIC^AZT

> 128

< 1

t-Bu

0.125

0.562 5

OBn

0.25

0.625

> 128

128

< 1.5

CH₃

128

> 128

< 1

NO₂

> 128

< 0.625

OCH₃

> 128

0.5

< 0.5

> 128

< 1

NO₂

> 128

< 1

> 128

< 0.562 5

OBn

128

0.5

0.75

t-Bu

128

0.5

0.75

12a

0.25

0.625

12b

128

0.5

0.375

PAβN

> 128

128

0.5

< 1.25

Strain	Compd.	Alone		Combined	FICI
26	7h	128	8		32	1	0.375
12b	128	8		32	0.5	0.312 5
PAβN	> 128	16		16	2	< 0.25

Strain

Compd.

Alone

Combined

FICI

MIC^a

MIC^AZT

MIC^a

MIC^AZT

128

0.375

12b

128

0.5

0.312 5

PAβN

> 128

< 0.25

Strain	Compd.	Alone		Combined	FICI
181245	AZT	8	> 512	> 512	2	128	2	128	< 0.5	< 0.5
	CLR	256	> 512	> 512	0.5	128	0.5	128	< 0.25	< 0.25
	CAZ	64	> 512	> 512	2	128	16	128	< 0.28	< 0.5
	MEM	32	> 512	> 512	8	128	8	128	< 0.5	< 0.5
181643	AZT	4	> 512	512	1	128	1	128	< 0.5	0.5
	CLR	256	512	512	32	128	32	128	0.375	0.375
	CAZ	2	512	512	0.5	128	0.5	128	0.5	0.5

Strain

Compd.

Alone

Combined

FICI

MIC^a

MIC^12b

MIC^PAβN

MIC^a

MIC^12b

MIC^a

MIC^PAβN

12b

PAβN

181245

AZT

> 512

128

< 0.5

CLR

256

> 512

0.5

128

0.5

128

< 0.25

CAZ

> 512

128

< 0.28

< 0.5

MEM

> 512

128

< 0.5

181643

AZT

> 512

512

128

< 0.5

0.5

CLR

256

512

128

0.375

CAZ

512

0.5

128

0.5

128

0.5

Compd.	Absn	CYP	TOX	ADMET
12b	4.014	0	0.6	4.014

Compd.

Absn

CYP

TOX

ADMET

12b

4.014

0.6

4.014