第四章 實驗部分
秤取 1 克(0.0028 莫耳)之化合物 6，加入 100 毫升脫水 後之二氯甲烷，緩慢加入 5 毫升(0.042 莫耳)之二氯亞硫醯
13C-NMR (CDCl3, 50 MHz)δ(ppm) (圖 9f-4)︰
13.54 (C-21) 19.91 (C-20) 31.57 (C-19) 38.73 (C-18) 53.31 (C-22) 55.47 (C-30) 99.99 (C-13) 108.65 (C-4) 110.75 (C-16) 115.54 (C-3) 119.05 (C-15) 121.63 (C-12) 126.78 (C-24, 28) 127.73 (C-26) 128.59 (C-25, 27) 134.31 (C-8) 136.04 (C-11) 136.29 (C-23) 146.94 (C-5) 149.48 (C-2) 155.41 (C-14) 158.23 (C-6)
V-7 5-(1-Benzyl-5-methoxy-1H-indazol-3-yl)-N-hydroxyfuramide (9g) 之合成
秤取 1 克(0.0028 莫耳)之化合物 6，加入 100 毫升脫水 後之二氯甲烷，緩慢加入 5 毫升(0.042 莫耳)之二氯亞硫醯 以進行氯化反應，升溫至迴流，反應 16 小時，降至室溫，
反應液減壓濃縮至無溶劑蒸出，於室溫下，加入 50 毫升之 甲苯，緩慢加至 20 毫升(0.303 莫耳)之羥氨(hydroxylamine, solution 50 % in water)中，反應 1 小時，固體析出，取固體 以 50 %乙醇做再結晶，可得化合物 9g (0.11 克)，產率 10.0
%。
化合物 9g:為淺黃色細小柱狀結晶，Rf 值(乙酸乙酯)為 0.54，熔點 196.1–198.2 ℃。
質 譜 (EIMS)(圖 9g-1): m/z 363
UV 光譜 :λmax (MeOH) nm (logε): 344.0 (4.43)，227.8 (4.54)，
207.8 (4.60)
IR 光譜(圖 9g-2)︰ν(KBr) cm-1︰3185.3 (-NH)，1642.4 (C=O)，
1561.0 (C=N) 元 素 分 析:以 C20H17N3O4計算
C% H% N%
計算值 66.11 4.72 11.56 實驗值 66.14 4.75 11.52
1H-NMR (DMSO-d6, 200 MHz)δ(ppm) (圖 9g-3)︰
3.87 (3H, s, H-27) 5.69 (2H, s, H-19)
7.10 (1H, d, J = 3.5 Hz, H-4) 7.15 (1H, d, J = 2.2 Hz, H-15) 7.22 (1H, d, J = 3.5 Hz, H-3)
7.25-7.31 (5H, m, H-21, 22, 23, 24, 25) 7.53 (1H, d, J = 2.2 Hz, H-13)
7.68 (1H, d, J = 9.2 Hz, H-16) 9.12 (1H, s, H-17)
11.20 (1H, s, H-18)
13C-NMR (DMSO-d6, 50 MHz)δ(ppm) (圖 9g-4)︰
52.50 (C-19) 55.87 (C-27) 100.99 (C-13) 108.26 (C-4) 111.75 (C-16) 115.15 (C-3) 119.07 (C-15) 121.13 (C-12) 127.48 (C-21, 25) 127.85 (C-23) 128.85 (C-22, 24) 134.03 (C-8) 136.52 (C-11) 137.42 (C-20) 145.50 (C-5) 150.17 (C-2) 155.49 (C-14) 156.86 (C-6)
V-8. 5-(1-Benzyl-5-methoxy-1H-indazol-3-yl)-N-hydroxyethyl-2- furamide (9h)之合成
取 10 毫升(0.164 莫耳)乙醇氨(ethanolamine)依照化合物(9g) 之步驟，固體析出，取固體以 50%乙醇做再結晶，可得化合物 9h (0.02 克)，產率 2.3 %。
化合物 9h:為黃色細小針狀結晶，Rf值(乙酸乙酯)為 0.41，熔點 83.1–
85.4 ℃。
質 譜 (EIMS)(圖 9h-1): m/z 391
UV 光譜 :λmax (MeOH) nm (logε): 344.0 (4.49)， 226.6 (4.61)，
208.2 (4.68)
IR 光譜(圖 9h-2)︰ν(KBr) cm-1︰3361.2 (-NH)，1657.7 (C=O)，
1586.4 (C=N) 元 素 分 析:以 C22H21N3O4計算
C% H% N%
計算值 67.51 5.41 10.74 實驗值 67.53 5.43 10.76
1H-NMR (DMSO-d6, 200 MHz)δ(ppm) (圖 9h-3)︰
3.51 (4H, m, H-19, 18) 3.86 (3H, s, H-29)
4.79 (1H, t, J = 5.4 Hz, H-20) 5.70 (2H, s, H-21)
7.12 (1H, d, J = 2.2 Hz, H-15) 7.13 (1H, d, J = 3.5 Hz, H-4)
7.24-7.34 (6H, m, H-3, 23, 24, 25, 26, 27) 7.50 (1H, d, J = 2.2 Hz, H-13)
7.69 (1H, d, J = 7.1 Hz, H-15) 8.30 (1H, d, J = 5.6 Hz, H-17)
13C-NMR (DMSO-d6, 50 MHz)δ(ppm) (圖 9h-4)︰
41.68 (C-19) 52.49 (C-18) 55.80 (C-21) 59.98 (C-29) 100.73 (C-13) 108.57 (C-4) 111.80 (C-16) 115.27 (C-3) 119.19 (C-15) 121.15 (C-12) 127.44 (C-23, 27) 127.86 (C-25) 128.85 (C-24, 26) 134.05 (C-8) 136.53 (C-11) 137.42 (C-22) 147.20 (C-5) 149.88 (C-2) 155.50 (C-14) 158.08 (C-6)
VI. Methyl-substituted-{[5-(1-benzyl-5-methoxy-1H-indazol-3-yl) furoyl]amino} acetate 類化合物(10a–10f)之合成
VI-1. Methyl {[5-(1-benzyl-5-methoxy-1H-indazol-3-yl)furoyl]amino}
acetate (10a)之合成
(a) 秤取 1 克(0.0028 莫耳)之化合物 6，加入 100 毫升之二氯甲烷，
緩慢加入 5 毫升(0.042 莫耳)之二氯亞硫醯以進行氯化反應，
升溫至迴流，反應 16 小時，降至室溫，反應液減壓濃縮至無 溶劑蒸出，於室溫下，加入 50 毫升之甲苯。
(b) 秤取 2 克(0.016 莫耳)之甘胺酸甲酯鹽酸鹽(glycine methyl ester hydrochloride)，於冰浴下，加至 20 毫升之 3.5 %氫氧化鈉水 溶液，後緩慢滴入(a)之溶液，反應 1 小時，有機層以二氯甲 烷萃取，水洗滌至中性，並以無水硫酸鎂乾燥，濾液減壓濃 縮至無溶劑蒸出，粗產物以管柱層析分離純化(正己烷:乙酸乙 酯=1:1/矽膠)，以乙醇做再結晶，可得化合物 10a (0.41 克)，
產率 35.1 %。
化合物 10a:為白色細長針狀結晶，Rf值(正己烷:乙酸乙酯=1:1)為 0.29
，熔點 173.5-174.1 ℃。
質 譜 (EIMS) (圖 10a-1): m/z 419
UV 光譜 :λmax (MeOH) nm (logε): 344.0 (4.66)， 226.8 (4.77)，
209.0 (4.83)
IR 光譜(圖 10a-2):ν(KBr) cm-1︰3314.6 (-NH)，1757.5 (C=O)，
1637.7 (C=N) 元素分析:以 C23H21N3O5計算
C% H% N%
計算值 65.86 5.05 10.02 實驗值 65.88 5.09 10.05
1H-NMR (CDCl3-d1, 200 MHz)δ(ppm) (圖 10a-3):
3.66 (3H, s, H-31) 3.87 (3H, s, H-29)
4.03 (2H, d, J = 5.9 Hz, H-18) 5.70 (1H, s, H-21)
7.13 (1H, dd, J = 9.1 Hz, 2.2 Hz, H-15) 7.17 (1H, d, J = 3.6 Hz, H-4)
7.19-7.32 (5H, m, H-23, 24, 25, 26, 27) 7.34 (1H, d, J = 3.6 Hz, H-3)
7.52 (1H, d, J = 2.2 Hz, H-13) 7.70 (1H, d, J = 9.1Hz, H-16) 8.83 (1H, t, J = 5.9Hz, H-17)
13C-NMR (CDCl3-d1, 50 MHz)δ(ppm) (圖 10a-4):
40.77 (C-18) 52.04 (C-31) 52.52 (C-21) 55.86 (C-29) 100.81 (C-13) 108.72 (C-4) 111.83 (C-16) 116.14 (C-3) 119.16 (C-15) 121.18 (C-12) 127.44 (C-23, 27) 127.86 (C-25) 128.86 (C-24, 26) 133.92 (C-8) 136.55 (C-11) 137.39 (C-22) 146.59 (C-5) 150.32 (C-2)
155.56 (C-14) 158.27 (C-6) 170.54 (C-19)
VI-2 .Methyl 2-{[5-(1-benzyl-5-methoxy-1H-indazol-3-yl)furoyl]
amino}propanoate (10b)之合成
秤取 2 克(0.014 莫耳)之 L-丙胺酸甲酯鹽酸鹽(L-alanine methyl ester hydrochloride)及 20 毫升之 2.9 %氫氧化鈉水溶液 依照化合物 10a 步驟，粗產物以管柱層析分離純化(正己烷:
乙酸乙酯=3:2/矽膠)，以正己烷做再結晶，可得化合物 10b (0.35 克)，產率 29.5 %。
化合物 10b:為白色細長針狀結晶，Rf值(正己烷:乙酸乙酯=3:2)為 0.31
，熔點 108.5-109.7 ℃。
質 譜 (EIMS) (圖 10b-1): m/z 433
UV 光譜 :λmax (MeOH) nm (logε): 344.8 (4.52)， 227.0 (4.61)，
208.0 (4.68)
IR 光譜(圖 10b-2):ν(KBr) cm-1︰3314.6 (-NH)，1754.5 (C=O)，
1637.3 (C=N) 元 素 分 析:以 C24H23N3O5計算
C% H% N%
計算值 66.50 5.35 9.69 實驗值 66.48 5.37 9.72
1H-NMR (CDCl3, 200 MHz)δ(ppm) (圖 10b-3):
1.56 (3H, d, J = 7.1 Hz, H-19) 3.79 (3H, s, H-32)
3.95 (3H, s, H-28)
4.78-4.85 (1H, m, H-18) 5.62 (2H, s, H-20)
6.95 (1H, d, J = 3.6 Hz, H-4)
7.06 (1H, dd, J = 9.1 Hz, 2.2 Hz, H-15)
7.18-7.32 (8H, m, H-3, 16, 17, 22, 23, 24, 25, 26)
7.40 (1H, d, J = 2.2 Hz, H-13)
13C-NMR (CDCl3, 50 MHz)δ(ppm) (圖 10b-4):
18.48 (C-19) 47.67(C-18) 52.30 (C-32) 53.36 (C-20) 55.51 (C-28) 99.92 (C-13) 108.18 (C-4) 110.70 (C-16) 116.35 (C-3) 119.29 (C-15) 121.69 (C-12) 126.83 (C-22, 26) 127.72 (C-24) 128.59 (C-23, 25) 134.24 (C-8) 136.06 (C-11) 136.26 (C-21) 146.16 (C-5) 150.59 (C-2) 155.56 (C-14) 157.55 (C-6) 173.16 (C-29)
VI-3. Methyl 2-{[5-(1-benzyl-5-methoxy-1H-indazol-3-yl)furoyl]
amino}-3-phenylpropanoate (10c)之合成
秤 取 2 克 (0.009 莫 耳 )之 L- 苯 丙 胺 酸 甲 酯 鹽 酸 鹽 (L-phenylalanine methyl ester hydrochloride)及 20 毫升之 1.85 % 氫氧化鈉水溶液依照化合物 10a 之步驟，粗產物以管柱層析純 化(正己烷:乙酸乙酯=2:1/矽膠)，以乙醇/正己烷做再結晶，可 得化合物 10c (0.33 克)，產率 23.8 %。
化合物 10c:為白色細小針狀結晶，Rf 值 (正己烷:乙酸乙酯=2:1)為 0.38，熔點 81.0-83.1 ℃。
質 譜 (EIMS) (圖 10c-1): m/z 509
UV 光譜 :λmax (MeOH) nm (logε): 342.6 (4.77)， 224.5 (4.91)，
212.3 (4.93)
IR 光譜(圖 10c-2):ν(KBr) cm-1︰3314.6 (-NH)，1744.2 (C=O)，
1651.0 (C=N) 元 素 分 析:以 C30H27N3O5計算
C% H% N%
計算值 70.71 5.34 8.25 實驗值 70.73 5.38 8.24
1H-NMR (DMSO-d6, 200 MHz)δ(ppm) (圖 10c-3):
3.13-3.19 (2H, m, H-19) 3.65 (3H, s, H-38)
3.85 (3H, s, H-34)
4.72-4.74 (1H, m, H-18) 5.69 (2H, s, H-26)
7.12 (1H, d, J = 3.7 Hz, H-4)
7.13 (1H, dd, J = 9.1 Hz, 2.1 Hz, H-15)
7.20-7.32 (10H, m, H-21, 22, 23, 24, 25, 28, 29, 30, 31, 32) 7.34 (1H, d, J = 3.7 Hz, H-3)
7.51 (1H, d, J = 2.1 Hz, H-13) 7.68 (1H, d, J = 9.1 Hz, H-16) 8.80 (2H, d, J = 8.0 Hz, H-17)
13C-NMR (DMSO-d6, 50 MHz)δ(ppm) (圖 10c-4):
36.42 (C-19) 52.27 (C-18) 52.54 (C-38) 53.76 (C-26) 55.79 (C-34) 100.79 (C-13) 108.48 (C-4) 111.80 (C-16) 116.25 (C-3)
119.25 (C-15) 121.17 (C-12) 126.76-129.24 (C-21, 22, 23, 24, 25, 28, 29, 30, 31, 32) 133.95 (C-8)
136.54 (C-11) 137.37 (C-27) 137.79 (C-20) 146.40 (C-5) 150.51 (C-2) 155.54 (C-14) 157.95 (C-6) 172.22 (C-35)
VI-4. Methyl 3-methyl-2-{[5-(1-benzyl-5-methoxy-1H-indazol-3-yl) furoyl]amino}butanoate (10d)之合成
秤取 2 克 (0.012 莫耳 )之 L-纈胺酸甲酯鹽酸鹽(L-valine methyl ester hydrochloride)及 20 毫升之 2.4%氫氧化鈉水溶液依 照化合物 10a 之步驟，粗產物以管柱層析分離純化(正己烷:乙酸 乙酯=2:1/矽膠)，以乙酸乙酯/正己烷做再結晶，可得化合物 10d (0.26 克)，產率 20.9 %。
化合物 10d: 為白色細小針狀結晶，Rf 值(正己烷:乙酸乙酯=2:1)為 0.41，熔點 121.0-123.1 ℃。
質 譜 (EIMS) (圖 10d-1): m/z 461
UV 光譜 :λmax (MeOH) nm (logε): 345.1 (4.72)， 221.5 (4.89)，
202.6 (4.75)
IR 光譜(圖 10d-2):ν(KBr) cm-1︰3455.2 (-NH)，1744.2 (C=O)，
1637.3 (C=N) 元 素 分 析:以 C26H27N3O5計算
C% H% N%
計算值 67.66 5.90 9.10 實驗值 67.65 5.92 9.13
1H-NMR (CDCl3, 200 MHz)δ(ppm) (圖 10d-3):
1.00-1.06 (6H, d, J = 5.1 Hz, H-32, 20) 2.29 (1H, m, H-19)
3.78 (3H, s, H-34) 3.93 (3H, s, H-29)
4.79 (1H, dd, J = 8.8 Hz, 4.9 Hz, H-18) 5.62 (2H, s, H-21)
6.97 (1H, d, J = 3.6 Hz, H-4)
7.06 (1H, dd, J = 9.1 Hz, 2.2 Hz, H-15)
7.13-7.31 (8H, m, H-3, 16, 17, 23, 24, 25, 26, 27) 7.43 (1H, d, J = 2.2 Hz, H-13)
13C-NMR (CDCl3, 50 MHz)δ(ppm) (圖 10d-4):
17.67 (C-32) 18.74 (C-20) 31.46 (C-19) 51.99 (C-18) 53.36 (C-34) 55.43 (C-21) 56.56 (C-29) 99.78 (C-13) 108.07 (C-4) 110.71 (C-16) 116.47 (C-3) 119.39 (C-15) 121.69 (C-12) 126.82 (C-23, 27) 127.71 (C-25) 128.58 (C-24, 26) 134.27 (C-8) 136.06 (C-11) 136.23 (C-22) 146.13 (C-5) 150.85 (C-2) 155.554 (C-14) 157.94 (C-6) 172.19 (C-30)
VI-5. Methyl 2-{[5-(1-benzyl-5-methoxy-1H-indazol-3-yl)furoyl]
amino}-4-methyl pentanoate (10e)之合成
秤取 2克(0.012莫耳)之 L-亮胺酸甲酯鹽酸鹽(L-leucine methyl ester hydrochloride)及 20 毫升之 2.4 %氫氧化鈉水溶 液依照化合物 10a 之步驟，粗產物以管柱層析分離純化(正 己烷:乙酸乙酯=2:1/矽膠)，以乙酸乙酯/正己烷做再結晶，
可得化合物 10e (0.15 克)，產率 23.2 %。
化合物 10e:為白色細小針狀結晶，Rf 值 (正己烷:乙酸乙酯=2:1)為 0.42，熔點 108.0-110.1 ℃。
質 譜 (EIMS) (圖 10e-1): m/z 475
UV 光譜 :λmax (MeOH) nm (logε): 341.7 (4.71)， 222.5 (4.67)，
210.2 (4.90)
IR 光譜(圖 10e-2):ν(KBr) cm-1︰3294.6 (-NH)，1749.4 (C=O)，
1639.6 (C=N)
元 素 分 析:以 C27H29N3O5計算 C% H% N%
計算值 68.19 6.15 8.84 實驗值 68.20 6.17 8.82
1H-NMR (DMSO-d6, 200 MHz)δ(ppm) (圖 10e-3):
0.86-0.92 (6H, d, J = 5.9 Hz, H-33, 21) 1.55-1.88 (3H, m, H-20, 19)
3.65 (3H, s, H-35) 3.87 (3H, s, H-30)
4.46-4.55 (1H, m, H-18) 5.62 (2H, s, H-21)
5.70 (2H, s, H-22)
7.11 (1H, d, J = 1.4 Hz, H-15) 7.13 (1H, d, J = 3.3 Hz, H-4)
7.20-7.34 (5H, m, H-24, 25, 26, 27, 28) 7.37 (1H, d, J = 3.3 Hz, H-3)
7.55 (1H, d, J = 1.4 Hz, H-13) 7.69 (1H, d, J = 9.1 Hz, H-16) 8.75 (1H, d, J = 7.9 Hz, H-17)
13C-NMR (DMSO-d6, 50 MHz)δ(ppm) (圖 10e-4):
21.27 (C-33) 23.09 (C-21) 24.64 (C-20) 40.24 (C-19) 50.50 (C-18) 52.19 (C-35) 52.52 (C-22) 55.63 (C-30) 100.71 (C-13) 108.37 (C-4) 111.80 (C-16) 116.17 (C-3) 119.35 (C-15) 121.16 (C-12) 127.44 (C-24, 28) 127.85 (C-26) 128.84 (C-25, 27) 134.01 (C-8) 136.53 (C-11) 137.40 (C-23) 146.52 (C-5) 150.50 (C-2) 155.48 (C-14) 158.18 (C-6) 173.18 (C-31)
VI-6. Methyl 2-{[5-(1-benzyl-5-methoxy-1H-indazol-3-yl)furoyl]
amino}-3-methyl pentanoate (10f)之合成
秤取 2克(0.011 莫耳)之 L-異亮胺酸甲酯鹽酸鹽(L-isoleucine methyl ester hydrochloride)及 20 毫升之 2.2%氫氧化鈉水溶液依照化合物 10a 之步驟，粗產物以管柱層析分離純化(正己烷:乙酸乙酯=2:1/矽 膠)，以乙酸乙酯/正己烷做再結晶，可得化合物 10f (0.62 克)，產率 47.9 %。
化合物 10f: 為白色細小針狀結晶，Rf 值(正己烷:乙酸乙酯=2:1)為
0.48，熔點 127.6-129.3 ℃。
質 譜 (EIMS) (圖 10f-1): m/z 475
UV 光譜 :λmax (MeOH) nm (logε): 343.6 (4.74)， 227.6 (4.98)，
217.5 (4.99)
IR 光譜(圖 10f-2):ν(KBr) cm-1︰3286.9 (-NH)，1744.3 (C=O)，
1637.3 (C=N) 元 素 分 析:以 C27H29N3O5計算
C% H% N%
計算值 68.19 6.15 8.84 實驗值 68.22 6.15 8.80
1H-NMR (DMSO-d6, 200 MHz)δ(ppm) (圖 10f-3):
0.85 (3H, t, J = 5.4 Hz, H-21) 0.90 (3H, t, J = 5.9 Hz, H-33) 1.19-1.55 (2H, m, H-20) 1.96-2.02 (1H, m, H-19) 3.66 (3H, s, H-35)
3.87 (3H, s, H-30)
4.37 (1H, t, J = 7.5 Hz, H-18) 5.70 (2H, s, H-22)
7.11 (1H, dd, J = 8.7 Hz, 1.5, H-15) 7.12 (1H, d, J = 3.5 Hz, H-4)
7.21-7.33 (5H, m, H-24, 25, 26, 27, 28) 7.44 (1H, d, J = 3.5 Hz, H-3)
7.56 (1H, d, J = 1.5 Hz, H-13) 7.68 (1H, d, J = 8.7 Hz, H-16) 8.56 (1H, d, J = 7.9 Hz, H-17)
13C-NMR (DMSO-d6, 50 MHz)δ(ppm) (圖 10f-4):
11.04 (C-21) 15.67 (C-20) 25.34 (C-33) 35.91 (C-19) 51.94 (C-35) 52.52 (C-22) 55.65 (C-30) 56.89 (C-18) 100.65 (C-13) 108.25 (C-4) 111.80 (C-16) 116.28 (C-3) 119.34 (C-15) 121.17 (C-12) 127.45 (C-24, 28) 127.84 (C-26) 128.83 (C-25, 27) 134.03 (C-8) 136.52 (C-11) 137.39 (C-23) 146.37 (C-5) 150.60 (C-2) 155.48 (C-14) 158.23 (C-6)
172.38 (C-31)
第四節 藥理試驗方法
I. 抗血小板凝集活性試驗 (一)血小板凝集引發劑之製備
(1) Thrombin–購自 Park Davis Co. USA，溶解於 50 % (v/v) glycerol 中，製備成 100 NIH units/ml 之 stock solution。
(2) Arachidonic acid (AA)–購自 Sigma Chem. Co. USA，以去離 子水溶解之。
(3) Collagen (Type I bovine Achilles tendon)–購自 Sigma Chem.
Co. USA，4 ℃下於 25 毫升醋酸中研磨均勻，以去離子水稀 釋成 1 mg/ml，儲存於-70 ℃下。
(4) Platelet-activation factor (PAF)–購自 Sigma Chem. Co. USA，
溶於氯仿，儲存於-20 ℃，使用前以 0.9 % NaCl 稀釋之。
(5) Adenosine diphosphate (ADP)–以去離子水溶解備用。
(二)血小板懸浮液(Platelet suspension)之製備
將 EDTA 與兔耳靜脈抽出血混合後，使 EDTA 之最終濃度 為 6 mM，在室溫下即以 90 × g 離心 10 分鐘，取出上層富含血 小板之血漿(platelet-rich plasma)，再將其以 500 × g 離心 10 分鐘，
除去血漿後，將下層血小板以含有 EDTA (2 mM)及 Bovine serum albumin (3.5 mg/ml)的 Tyrode's solution (calcium free)清洗之，在
於相同轉速(500 × g)下離心 10 分鐘，所得之血小板以不含有 EDTA 之 Tyrode's solution 清洗之，再於相同之條件下離心後，取 血小板層，將其懸浮於 Tyrode's solution，其組成如下(mM)：NaCl (136.8)、KCl (2.8)、NaHCO3 (11.9)、MgCl2 (1.1)、NaH2PO4 (0.33)、
CaCl2 (1.0) and glucose (11.2)，並以 Coulter counter (Model ZM)計 數，調整血小板數約為 4.5 × 108 platelet/ml 左右，最後加 1 mM 鈣離子(Ca+2)放置 30 分鐘後，進行實驗。
(三)血小板凝集(platelet aggregation)之試驗
利用混濁度法(turbidimetric method)之原理來測定凝集程度 55，並以 Lumi-aggregometer (Model 1020, PayLon, Canada)測定 之。將血小板懸浮液 0.4 毫升加至經 silicone 包衣的小玻璃管之 中，並以小磁棒做每分鐘 900 轉(900 rpm)的攪拌，若未特別說明，
均在加入樣品三分鐘後，再加入凝集引發劑，六分鐘後觀察結果。
為了排除溶媒(DMSO)影響，在血小板溶液的濃度為 0.5 %，全部 反應過程皆在 37 ℃下進行，凝集程度的表示方法如下 56：
凝集(%)=[(A1-A2) ÷ (A1-Ab)] × 100%
A1 =加引發劑前的吸光度 A2 =加引發劑後的吸光度 Ab = Tyrode's solution 的吸光度 II. 抗血管增生之活性試驗
MTT 分析法
MTT assay 是 一 種 應 用 常 見 於 分 析 細 胞 增 殖 (cell proliferation) 、 細 胞 存 活 率 (percent of viable cells) 和 細 胞 毒 性 (cytotoxicity)的分析方法。此種分析方式是利用檢測細胞之粒腺體 內的酵素(succinate-tetrazolium reductase)活性來測定細胞的生長狀 態，進而得知相對的細胞比例。只有正常存活的細胞可將 MTT 代 謝成水不溶性的 formazan，其反應式如下：
首先將細胞分到平底 96 孔細胞培養盤中(細胞數目約 6 × 103 cells/wells)經過 24 小時待細胞適應環境後，以無血清之培養液將細 胞同步化 24 小時。將培養液置換成含 15 %胎牛血清之培養液，並 分別加入不同濃度的藥物，最後總體積為 100 µl。另外，還需一組 不含藥物之正對照組和不含血清及藥物的負對照組。過 20 小時 後，於避光的環境中加入 10 µl/wells 的 MTT (添加量為培養液總體 積的 1/10)，再經過 4 小時後加入 100 µl/wells 的 solubilization solution 終止反應並溶解 formazan 鹽類。再放於培養箱中經過隔夜 (overnight)反應，以酵素免疫分析儀在 OD 590 nm 下測定其吸光值。
◎MTT 試劑：(避光 4 ℃儲存)
組 成 最終濃度 初濃度 體積/重量
MTT 5 mg/ml - 250 mg
PBS (pH7.4) - - 50 ml
總 體 積 50 ml
◎Solubilization solution：(室溫儲存)
組 成 最終濃度 初濃度 體積/重量
SDS 10 % - 10 g
HCl 0.01 M 1 M 1 ml
加 DDW 到 總 體 積 100 ml
III. 細胞致毒活性試驗
人類前骨髓性血癌細胞(HL-60 cells)增殖作用(proliferation)實驗 1. 細胞培養與前處理：
Indazole 類緣化合物用 DMSO 溶解；並將一系列不同濃度之化 合物儲放於-20 ℃冰箱，待加藥時再解凍。DSMO 之最後濃度需控 制在 0.1 %以下，以避免 MDSO 本身對 HL-60 cells 之影響 57。HL-60 cells (1 × 105/ mL) 培 養 於 24-well 之 培 養 皿 中 使 總 體 基 為 1 mL/well；並加入各種不同濃度之化合物於溫度 37 ℃、溼度 95 %、
5 % CO2之培養箱中培養 72 小時之後，分別取出作 MTT-proliferation assay 實驗。
2. MTT-proliferation assay
首先自每個 well 取出已去培養基之 50μ L 細胞培養液置入 96-well plate 中，加入 10μL MTT solution 於 37 ℃培養箱培養 4 小 時；取出後再使用 DMSO (150 μL/well)溶解細胞之紫黑色顆粒，
最後以 ELISA reader 於波長 570 nm 之條件測得 OD 570 值 58,59。 Proliferation (%) = Sample OD 570 / Cont. OD 570 × 100 %
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