標的化合物 ITP 之預估與細胞致毒活性相對應性
由此現象不難發現化合物 57 與 68 在細胞週期的表現是不盡相同的，這是一個相當 有趣的試驗結果，因為兩者結構相同，唯化合物 68 是化合物 57 的 tromethamine 鹽類，
由此現象不難發現化合物 57 與 68 在細胞週期的表現是不盡相同的，這是一個相當 有趣的試驗結果，因為兩者結構相同，唯化合物 68 是化合物 57 的 tromethamine 鹽類，
或許可能是因為化合物 in vitro 試驗時，在培養基的酸鹼環境造成兩者活性表現不同，
或是兩者之 IC50濃度不同而影響細胞週期測試的結果。而確實的影響仍須做更深入的機 轉探討才可得知其差異的原因。
綜合以上之結果，化合物 57 與 68 對 PC-3、HL60 細胞週期的影響，皆主要 是作用在 G2/M phase arrest。
Table 12. The cell cycle effect of HL-60 cell line
Data was presented as mean ± SD from three separate experiments.
* p < 0.05 compared with control.
** p < 0.01 compared with control.
*** p < 0.001 compared with control.
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參、嗜中性白血球超氧自由基形成之抑制活性
嗜中性白血球在個體防禦病菌感染時扮演重要的角色。在受到適當的刺激時會產生超 氧自由基來殺死病原菌，但細胞過度活化會產生過量的超氧自由基，反而對周圍正 常的組織細胞有損傷。這些細胞損傷可能與許多疾病的致病原有關，包括肺氣腫、
急性呼吸困難、動脈硬化、類風濕性關節炎等疾病的發生與惡化。藥物若能抑制過 多的超氧自由基產生，則有助於減少對組織的傷害。
如 Table 13-15 所示，對於 fMLP 誘導老鼠嗜中性白血球超氧自由基生成作用的抑 制試驗中，2-phenyl-4-quinolone 類化合物中以第三位 COOEt 的水解為 COOH 後，酸基 取代之化合物的抑制活性顯然較強，而酯類中間產物中唯有 35 與 36 有明顯之抑制，
其 IC50依序為 16.3 及 17.6 µM。
標的化合物中以化合物 57 對 fMLP 誘導老鼠嗜中性白血球過氧化物形成的抑制 (IC50 = 8.1µM)最強，與 trifluoperazine (IC50 = 6.2 µM)的活性相當；化合物 51 (IC50 = 10.8 µM)、52 (IC50 = 20.9 µM)、54 (IC50 = 18.9 µM)、55 (IC50 = 17.2 µM)、56 (IC50 = 18.9 µM)、
58 (IC50 = 13.9 µM)對 fMLP 誘導老鼠嗜中性白血球超氧自由基形成的抑制活性也分別 具有明顯之活性，其餘的化合物抑制活性皆不明顯。
在鹽類部分則以化合物 62 (IC50 = 23.2 µM)、66 (IC50 = 15.2 µM)、69 (IC50 = 20.7 µM) 具有較明顯之活性。第 6 位是 F、第 3’位為 OCH3取代的酸基與 tromethamine 鹽類化合 物(36, 52)兩者皆對 fMLP 誘導老鼠嗜中性白血球超氧自由基形成的抑制有作用外，其 餘的酸基取代與 tromethamine 鹽類活性大小的差異都較大，有些是酸基取代如化合物 52、54-56、58 有活性而 tromethamine 鹽類的化合物(63, 65-67, 69 )無活性；有些則是 tromethamine 鹽類如化合物 66、69 有活性而酸基取代的化合物(55, 58)無活性。
綜合以上，對 fMLP 誘導老鼠嗜中性白血球超氧自由基形成的抑制結果可歸納下列 三點：
（一） 當 2-PQ 類化合物在第三位為酸基取代時抑制活性較酯類或 tromethamine 鹽類強。
（二）2-PQ 類化合物第三位酸基取代與 tromethamine 鹽類活性大小的差異都較 大，所以這兩類化合物作用機制或許不同。
（三）當 2-PQ 類化合物在第三位為酸基取代時，以第 6 位是 methoxy、第 3’位是 fluoro 取代時活性最強，效價與 trifluoperazine 相當。
Table 13. The inhibitory effects of compounds 34-44 on neutrophil superoxide formation (in vitro)
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Table 14. The inhibitory effects of compounds 50-60 on neutrophil superoxide formation (in vitro)
N
30 3.49±0.18 40.2±1.4** 3.40±0.42 30.0±10.9 TFP 3 4.41±0.08 24.3±1.9* 4.49±0.24 8.6±2.0
10 1.14±0.17 78.1±5.4** 0.94±0.09 80.2±2.0**
30 0.03±0.12 99.1±3.3** 0.39±0.03 90.9±0.4**
IC50 (µM) 6.2±0.3 7.6±0.3
* P < 0.05, ** P < 0.01; N = 3;
Trifluoperazine (TFP): positive control
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Table 15. The inhibitory effects of compounds 61-71 on neutrophil superoxide formation (in vitro)
The neutrophil suspension was preincubated at 37℃ with 0.5 % DMSO or tested compound for 3 min. Fifteen minutes after the addition of PMA, the absorbance was determined at 550 nm. Values are presented as mean ± S.E., n = 3, * P < 0.05, ** P < 0.01 Trifluoperazine (TFP):
positive control
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肆、嗜中性白血球脫顆粒反應之抑制活性
2-phenyl-4-quinolone 類標的化合物對 fMLP 所誘發老鼠嗜中性白血球脫顆粒 反應之抑制活性，如 Table16-18 所示。此類化合物之 IC50都大於 30?µM，抑制活 性相當弱。
伍、肥胖細胞脫顆粒反應之抑制活性
如 Table 19-21 所示，以 compound 48/80 誘發老鼠肥胖細胞脫顆粒反應之抑 制試驗中，2-phenyl-4-quinolone 類化合物中只有第三位為酯基取代的化合物 38
（第 6 位是 chloro，第 3'位是 fluoro）與化合物 35（第 6 位是 fluoro，第 3'位是 chloro）在濃度為 30?µM 均有約 38%的抑制活性外，其餘化合物無論是酯類、酸 基或 tromethamine 鹽類取代之 IC50都大於 30?µM，抑制活性相當不明顯。
陸、一氧化氮蓄積之抑制活性
2-phenyl-4-quinolone 類化合物對於 RAW 264.7 細胞中一氧化氮蓄積與 LPS
＋IFN-γ刺激 N9 細胞中一氧化氮蓄積之抑制活性，如 Table 22-24 所示。此類化 合物之 IC50都大於 30?µM，抑制活性相當弱。
柒、TNF-a 形成之抑制活性
關於 2-phenyl-4-quinolone 類化合物對 LPS 刺激末梢巨噬細胞(peripheral macrophage, RW 264.7cells)及之抑制活性，如 Table 25-27 所示。化合物 38、44、
55 在 30?µM 濃度下對 LPS 刺激 RW 264.7 細胞中 TNF-a 形成，均有大於 30%的 抑制活性。化 合物 34、68 在 30?µM 濃度下，對 LPS＋IFN-γ刺激 N9 細胞中 TNF-a 形成呈現較明顯的抑制，抑制百分比分別為 39.3 %及 32.7 %。
從 Table 25-27 也可明顯看出化合物 35、36、39、43、56、57、59、61、66 及 68 在 30?µM 有細胞致毒活性，其中化合物 57、59 甚至在 10?µM 的低濃度下仍 具細胞致毒活性。
Table 16. The inhibitory effects of compounds 34-44 on neutrophil degranulation (in vitro)
The neutrophil suspension was preincubated at 37℃ with 0.5 % DMSO or tested compound for 3 min in the presence of cytochalasin B 5 µg/ml. Forty- five minutes after the addition of fMLP (1µM), ß-glucuronidase and lysozyme in the supernatant was determined. Values are presented as mean ± S.E., n = 3, * P < 0.05, ** P < 0.01
TFP (Trifluoperazine): positive control
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Table 17. The inhibitory effects of compounds 50-60 on neutrophil degranulation (in vitro)
N
The neutrophil suspension was preincubated at 37℃ with 0.5 % DMSO or tested compound for 3 min in the presence of cytochalasin B 5 µg/ml. Forty- five minutes after the addition of fMLP (1µM), ß-glucuronidase and lysozyme in the supernatant was determined. Values are presented as mean ± S.E., n = 3, * P < 0.05, ** P < 0.01
TFP (Trifluoperazine): positive control
Table 18. The inhibitory effects of compounds 61-71 on neutrophil degranulation (in vitro)
The neutrophil suspension was preincubated at 37℃ with 0.5 % DMSO or tested compound for 3 min in the presence of cytochalasin B 5 µg/ml. Forty- five minutes after the addition of fMLP (1µM), ß-glucuronidase and lysozyme in the supernatant was determined. Values are presented as mean ± S.E., n = 3, * P < 0.05, ** P < 0.01
TFP (Trifluoperazine): positive control
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Table 19. The inhibitory effects of compounds 34-44 on mast cell degranulation (in vitro)
The mast cell suspension was preincubated at 37℃ with 0.5 % DMSO or tested compound for 3 min. Fifteen minutes after the addition of compound 48/80 (10 µg/ml), ß-glucuronidase and histamine in the supernatant was determined. Values are presented as mean ± S.E., n = 3,
* P < 0.05, ** P < 0.01 Mepacrine: positive control
Table 20. The inhibitory effects of compounds 50-60 on mast cell degranulation (in vitro)
The mast cell suspension was preincubated at 37℃ with 0.5 % DMSO or tested compound for 3 min. Fifteen minutes after the addition of compound 48/80 (10 µg/ml), ß-glucuronidase and histamine in the supernatant was determined. Values are presented as mean ± S.E., n = 3,
* P < 0.05, ** P < 0.01 Mepacrine: positive control
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Table 21. The inhibitory effects of compounds 61-71 on mast cell degranulation (in vitro)
N
The mast cell suspension was preincubated at 37℃ with 0.5 % DMSO or tested compound for 3 min. Fifteen minutes after the addition of compound 48/80 (10 µg/ml), ß-glucuronidase and histamine in the supernatant was determined. Values are presented as mean ± S.E., n = 3,
* P < 0.05, ** P < 0.01 Mepacrine: positive control
Table 22. The inhibitory effects of compounds 34-44 on accumulation of nitrite in medium
1400W: N-(3-Aminomethyl)benzylacetamidine as positive control
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Table 23. The inhibitory effects of compounds 50-60 on accumulation of nitrite in medium
N
1400W: N-(3-Aminomethyl)benzylacetamidine as positive control
Table 24. The inhibitory effects of compounds 61-71 on accumulation of nitrite in medium
1400W: N-(3-Aminomethyl)benzylacetamidine as positive control
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Table 25. The inhibitory effects of compounds34-44 on TNF-α formation in medium
N
R1 COOC2H5
O
R2 H
Cell line: Raw 264.7 cells Inducer: LPS (1 µg/ml)
Cell line: N9 cells Inducer: LPS (10 ng/ml)+ IFN-γ (10 U/ml)
Conc. TNF-α formation (ng/ml)
Drugs
R1
R2
(µM)
---
RAW
% inhibition
N9
% inhibition
Control 75.1±4.9 2.65±0.05
34 F F 30 60.1±9.1 19.6±12.5 1.78±0.05 32.7±2.9*
35 F Cl 10 -- 2.60±0.09 1.9±2.7
30 57.1±2.8 22.9±8.2 Cytotoxic 36 F OCH3 10 -- 2.44±0.12 7.8±3.0
30 66.4±6.3 10.9±10.0 Cytotoxic 38 Cl F 30 44.0±9.3 42.5±8.4** 2.13±0.25 19.9±8.3 39 Cl Cl 10 -- 3.02±0.04 -14.2±2.6
30 58.1±9.2 23.5±6.9 Cytotoxic 40 Cl OCH3 30 53.7±6.6 31.9±3.1* 1.98±0.06 24.9±2.3 41 OCH3 F 30 66.2±2.3 10.9±7.2 2.14±0.17 19.2±5.5 42 OCH3 Cl 30 82.0±5.0 -10.3±10.6 2.37±0.25 10.6±7.7 43 OCH3 OCH3 10 -- 3.01±0.15 -13.4±4.4
30 79.6±4.1 -7.0±9.7 Cytotoxic 44 OCH3 H 30 46.4±8.9 39.2±7.4** 2.48±0.16 6.5±4.2 Dexamethasone 0.01 58.1±3.9 22.4±4.8 1.82±0.01 31.1±0.9*
0.1 41.8±2.9 42.5±2.0** 1.12±0.04 55.6±2.4**
1 33.6±6.0 55.4±9.5** 0.69±0.02 72.9±0.9**
IC50(µM) 0.42±0.12 0.074±0.01 N = 3; * P < 0.05, ** P < 0.01; --, not determined
Dexamethasone: positive control
Table 26. The inhibitory effects of compounds 50-60 on TNF-α formation in medium
N
R1 COOH
O
R2 H
Cell line: Raw 264.7 cells Inducer: LPS (1 µg/ml)
Cell line: N9 cells Inducer: LPS (10 ng/ml)+ IFN-γ (10 U/ml)
Conc. TNF-α formation (ng/ml)
Drugs