芳基丙烯酰胺类化合物的合成及其抗锥体虫半胱氨酸蛋白酶活性
作者:王千里 程卯生 王庆河 宋红岩 潘莉 沈建民
单位:王千里(沈阳药科大学合成二室,沈阳 110015);程卯生(沈阳药科大学合成二室,沈阳 110015);王庆河(沈阳药科大学合成二室,沈阳 110015);宋红岩(沈阳药科大学合成二室,沈阳 110015);潘莉(沈阳药科大学合成二室,沈阳 110015);沈建民(沈阳药科大学合成二室,沈阳 110015)
关键词:合成;芳基丙烯酰胺;半胱氨酸蛋白酶
中国药物化学杂志000108 摘 要:设计合成了18个芳基丙烯酰胺类化合物,并对其进行了抗锥体虫半胱氨酸蛋白酶的药理活性筛选.实验表明化合物(4),(8),(10),(11),(12),(14),(15),(16)和(17)的活性较强.
Synthesis and Their Anticruzain Activities of Arylacrylamide Compounds
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Wang Qianli Cheng Maosheng Wang Qinghe Song Hongyan Pan Li Shen Jianmin
(No.2 Lab.of Drug Synthesis,Shenyang Pharmaceutical University,Shenyang 110015)
Abstract:Eighteen arylacrylamide compounds were synthesized and their activities against cruzain in vitro were tested.The results showed that compounds (4),(8),(10),(11),(12),(14),(15),(16) and (17) had good anticruzain activities.
Key words:synthesis;arylacrylamide;cysteine protease▲
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卡格氏病(Chagas′ disease)是多发于南美洲的一种寄生虫病,也称南美洲锥体虫病.其主要临床症状是一些器官的肿大,如肝、脾肿大,严重的可导致心脏衰竭.据报道,世界上约2 400万人已感染此病.目前治疗这种疾病的药物较少,毒性较大,且易引起耐药性.研究人员对克鲁斯氏锥体虫进行了详细的生物化学基础研究,分离鉴定了锥体虫体内的许多蛋白酶,发现其中活性较强的是一种半胱氨酸蛋白酶〔1〕.进一步研究证实,它可降解宿主免疫系统中的免疫球蛋白G(IgG),保护锥体虫不被宿主免疫系统破坏〔2〕,并且对锥体虫在宿主细胞内的复制和变异过程起重要作用,如参与细胞内代谢、锥体虫和巨噬细胞的相互作用、入侵宿主细胞以及向靶细胞的粘附等〔3〕. 研究人员以半胱氨酸蛋白酶为药物作用靶点,利用计算机辅助药物分子模型模拟设计出半胱氨酸蛋白酶抑制剂的先导化合物N2,N′2-二(2-羟基-1-萘亚甲基)乙二酰肼(Ⅰ)〔4〕,通过对其进行结构修饰,合成了数百个酰肼类化合物,其中有些活性较强〔5~7〕.构效关系研究表明:缩短先导化合物中芳香环之间的连接链,使其成为不对称合成的单酰肼类化合物,抗卡格氏病活性增强;改变酰肼化合物芳香环的大小且引入杂原子其水溶性增强,与半胱氨酸蛋白酶活性位点的氢键作用增强,活性增强;以取代苯环代替酰肼化合物中的萘环、喹啉和异喹啉等,可更好地满足酶对抑制剂在大小和电性等方面的要求,达到提高化合物活性和治疗指数的目的.
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1 合成路线
曾报道过苯基烯丙基硫醚、亚砜和砜类化合物的合成及其抗锥体虫半胱氨酸蛋白酶活性,在此基础上,本文运用生物电子等排代换原理,保持结构中连桥链的长度进行结构修饰,设计合成了18个芳基丙烯酰胺类化合物(见图1),并对这类化合物进行了抗锥体虫半胱氨酸蛋白酶活性.
Fig.1 Lead optimization by chemical modification
综合文献报道,采用图2所示的路线合成目标化合物〔8~12〕.
Fig.2 The synthetic route of the target compounds
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2 合成实验
化合物测定所用仪器:Bruker ARX-300核磁共振仪,VG 7070 E质谱仪,Bruker IFS-55红外测试仪.熔点用毛细管法测定,温度计读数未经校正.中间体5-硝基-2-噻吩甲醛购于Aldrich公司.5-硝基-2-呋喃丙烯酸的合成是以2-呋喃甲醛为原料,先与丙二酸反应生成2-呋喃丙烯酸,再用硝酸硝化制得〔9〕.
2.1 2-呋喃丙烯酸的制备
将19.2 g(0.2 mol)新蒸呋喃甲醛,20.8 g(0.2 mol)丙二酸,10 mL(0.12 mol)吡啶和10滴哌啶加入100 mL圆底瓶中,维持100℃反应2 h,冷却,加入20 mL水稀释,加入浓氨水使溶液呈碱性,过滤,水洗,滤液冷却下用浓盐酸酸化析出白色固体,过滤,水洗至中性,乙醇重结晶得无色针状结晶24.8 g,收率:90%,mp 140~142℃(文献〔8〕收率:91%~92%,mp 141℃).
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2.2 5-硝基-2-呋喃丙烯酸的制备
将30 mL 1,2-二氯乙烷,40 mL乙酸酐加入反应瓶中,搅拌下冷至-15℃,滴加12 mL(0.29 mol)发烟硝酸,控制滴加温度不超过-10℃,加毕,再冷至-20℃,分批加入16.6 g(0.12 mol)呋喃丙烯酸,加料温度控制-15℃以下.加毕,继续搅拌1 h.抽滤,滤饼水洗至中性,干燥得黄色固体15.0 g,收率:68%,mp 230℃(分解)〔文献〔9〕收率:62.5%,mp 236℃〕.
2.3 N-2′-氯苯基-5-硝基-2-呋喃丙烯酰胺(1)的制备
将0.5 g(2.7 mmol)5-硝基-2-呋喃丙烯酸,0.75 g(3.6 mmol)五氯化磷和10 mL 1,2-二氯乙烷加入反应瓶中,加热回流直至固体溶解.所得的5-硝基-2-呋喃丙烯酰氯的1,2-二氯乙烷溶液冰浴冷却至-5℃,缓慢滴加0.56 g(4.4 mmol)2-氯苯胺的1,2-二氯乙烷溶液5 mL,加毕,搅拌反应1 h,抽滤,水洗,得黄色固体.乙醇重结晶得黄色絮状物0.40 g,收率:50.3%,mp 189~191℃.
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同法制得化合物(2)~(8)和(18),其物理性质及光谱数据见表1.
Tab.1 Physical and spectral data of the compounds Compd.
X
R1
R2
R3
R4
R5
Form
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Solvent
for
recrystn.
mp/℃
Yield/%
1H-NMR δ
1
O
NO2
Cl
H
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H
H
pale
yellow
needles
ethanol
189~191
50.3
3155,1678,1630,1590,1568,1513,1478,1441,1350,1238,1172,983,761
(CDCl3),6.77~6.78(d,1H,furan-3H),6.83~6.88(d,1H,=CH,J=15 Hz),7.06~7.11(m,1H,4′-H),7.29~7.34(m,1H,5′-H),7.36~7.37(d,1H,furan-4-H),7.40~7.42(d,1H,3′-H),7.51~7.56(d,1H,=CH
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,J=15 Hz),7.88(s,1H,—NH),8.49~8.51(d,1H,6′-H)
2
O
NO2
H
H
H
H
yellow
needles
ethanol
196
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45.7
3364,3129,1675,1630,1601,1536,1501,1473,1353,1238,1165,971,756,689
(CDCl3),6.72~6.73(d,1H,furan-3H),6.80~6.85(d,1H,=CH,J=15 Hz),7.13~7.18(m,1H,4′-H),7.34~7.39(m,3H,3′-H and furan-4-H),7.49~7.54(d,1H,=CH,J=15 Hz),7.57(s,1H,—NH),7.61~7.64(d,2H,2′-H)
3
O
NO2
H
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H
CH3
H
yellow
needles
acetic
acid
203~205
52.0
3371,3119,1675,1630,1597,1566,1531,1474,1356,1241,981,815
(CDCl3),2.33(s,3H,—CH3),6.72~6.73(d,1H,furan-3-H),6.78~6.83(d,1H,=CH,J=15 Hz),7.15~7.17(d,2H,3′-H),7.34~7.35(d,1H,furan-4-H),7.47~7.52(br,4H,— CH ,J=15 Hz,—NH and 2′-H)
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4
O
NO2
H
H
Cl
F
yellow
powder
acetic
acid
243~245
41.4
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3390,3140,1690,1633,1603,1565,1534,1502,1467,1360,1241,971,808
(d6-DMSO),6.89~6.94(d,1H, CH ,J=15 Hz),7.20~7.21(d,1H,furan-3-H),7.37~7.43(t,1H,2′-H),7.47~7.52(d,2H,=CH,J=15 Hz and 5′-H),7.75~7.76(d,1H,furan-4-H),8.01~8.03(d,1H,6′-H),10.65(s,1H,—NH)
5
O
NO2
H
H
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Br
H
yellow
needles
ethanol
221~222
46.3
3375,1669,1631,1593,1527,1471,1355,1235,966,815
(CDCl3),6.75~6.76(d,1H,furan-3-H),6.76~6.81(d,1H,=CH,J=15 Hz),7.35~7.36(d,1H,furan-4-H),7.37(s,1H,—NH),7.46~7.54(m,5H,— CH ,J=15 Hz,2′-H and 3′-H)
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6
O
NO2
H
H
Cl
H
yellow
tabletes
ethanol
213~214
45.1
3372,3112,1669,1633,1595,1530,1474,1398,1355,1236,963,816
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(CDCl3),6.75~6.76(d,1H,furan-3-H),6.76~6.81(d,1H,=CH,J=15 Hz),7.32~7.35(d,2H,3′-H),7.35~7.36(d,1H,furan-4-H),7.39(s,1H,—NH),7.49~7.54(d,1H,— CH ,J=15 Hz),7.56~7.59(d,2H,2′-H)
7
O
NO2
H
H
OCH3
H
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red
needles
acetic
acid
182~184
52.0
3357,3139,1677,1630,1601,1536,1510,1358,1242,957
(CDCl3),3.81(s,3H,—OCH3),6.72~6.73(d,1H,furan-3-H),6.76~6.81(d,1H,=CH,J=15 Hz),6.88~6.91(d,2H,3′-H),7.34~7.35(d,2H,furan-4-H and —NH),7.47~7.54(m,3H,— CH ,J=15 Hz and 2′-H)
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8
O
NO2
H
H
COC2H5
H
yellow
powder
ethanol
214~215
38.4
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3366,1674,1632,1593,1564,1526,1469,1406,1354,1238,1177,957,802
(d6-DMSO),1.07(t,3H,—CH3),2.98(q,2H,—CH2—),6.97~7.02(d,1H,=CH,J=15 Hz),7.20~7.21(d,1H,furan-3-H),7.49~7.54(d,1H,— CH ,J=15 Hz),7.75~7.76(d,1H,furan-4-H),7.79~7.82(d,2H,2′-H),7.95~7.98(d,2H,3′-H),10.74(s,1H,—NH)
9
S
NO2
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H
H
H
orange
powder
ethanol
218~219
44.2
3364,1668,1621,1600,1544,1495,1440,1332,1165,964,754,689
(CDCl3),6.51~6.56(d,1H,=CH,J=15 Hz),7.17~7.18(d,2H,furan-3-H and 1 Ph-H),7.32~7.40(m,3H,Ph-H),7.60(br,2H,—NH and 1 Ph-H),7.75~7.80(d,1H,— CH ,J=15 Hz),7.85~7.86(d,1H,furan-4-H)
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10
S
NO2
Cl
H
H
H
yellow
needles
ethanol
188~190
41.9
3361,1677,1619,1589,1530,1434,1331,1157
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(CDCl3),6.55~6.60(d,1H,=CH,J=15 Hz),7.08~7.12(t,1H,4′-H),7.21~7.22(d,1H,furan-3-H),7.28~7.32(t,1H,5′-H),7.40~7.43(d,1H,3′-H),7.76~7.81(d,2H,— CH ,J=15 Hz and —NH),7.86~7.87(d,1H,furan-4-H),8.48~8.51(d,1H,6′-H)
11
S
NO2
H
H
Cl
H
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yellow
needles
ethanol
244~246
42.6
3358,1668,1622,1596,535,1492,1330,1163,964,824
(d6-DMSO),6.81
~6.86(d,1H,=CH,J=15 Hz),7.39~7.42(d,2H,3′-H),7.56~7.57(d,1H,furan-3-H),7.70~7.73(d,2H,2′-H),7.75~7.80(d,1H,=CH,J=15 Hz),8.13~8.14(d,1H,furan-4-H),10.48(s,1H,—NH)
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12
S
NO2
H
H
Br
H
yellow
powder
ethanol
238~239
40.0
3361,1667,1622,1595,1534,1489,1323,1164,965,821
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(d6-DMSO),6.81~
6.86(d,1H,=CH,J=15 Hz),7.52~7.55(d,2H,3′-H),7.56~7.57(d,1H,furan-3-H),7.65~7.68(d,2H,2′-H),7.75~7.80(d,1H,=CH,J=15 Hz),8.13~8.14(d,1H,furan-4-H),10.48(s,1H,—NH)
13
S
NO2
H
H
CH3
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H
orange
powder
acetic
acid
243~245
48.5
3365,1666,1623,1599,1537,1491,1328,1164,964,819
(d6-DMSO),2.27(s,3H,—CH3),6.83~6.88(d,1H,=CH,J=15 Hz),7.13~7.16(d,2H,3′-H),7.54~7.59(t,3H,2′-H and furan-3-H),7.72~7.77(d,1H,=CH,J=15 Hz),8.13~8.14(d,1H,furan-4-H),10.26(s,1H,—NH)
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14
S
NO2
H
F
Cl
H
yellow
powder
acetic
acid
241~243
39.0
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3354,1681,1622,1543,1497,1426,1339,1162,962,810
(d6-DMSO),6.78
~6.83(d,1H,=CH,J=15 Hz),7.41~7.44(t,1H,2′-H),7.53(br,1H,5′-H),7.57~7.58(d,1H,furan-3-H),7.77~7.82(d,1H, CH ,J=15 Hz),8.02~8.04(dd,1H,6′-H),8.13~8.14(d,1H,furan-4-H),10.56(s,1H,—NH)
15
S
NO2
H
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H
OCH3
H
deep red
powder
acetic
acid
234~236
52.7
3360,3099,1677,1621,1546,1510,1485,1425,1326,1163,950,840
(d6-DMSO),3.68(s,3H,—OCH3),6.75~6.80(d,1H, =CH,J=15 Hz),6.85~6.88(d,2H,3′-H),7.47~7.48(d,1H,furan-3-H),7.53~7.56(d,2H,2′-H),7.64~7.69(d,1H,=CH,J=15 Hz),8.06~8.07(d,1H,furan-4-H),10.16(s,1H,—NH)
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16
S
NO2
H
H
COC2H5
H
yellow
needles
acetic
acid
245~247
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36.3
3360,3090,1676,1622,1596,1532,1487,1424,1366,1322,1225,1160,950,814
(d6-DMSO),1.08(t,3H,—CH3),2.99(q,2H,—CH2—),6.85~6.90(d,1H, CH ,J=15 Hz),7.57~7.58(d,1H,furan-3-H),7.76~7.84(m,3H, CH ,J=15 Hz and 2′-H),7.96~7.99(d,2H,3′-H),8.13~8.14(d,1H,furan-4-H),10.65(s,1H,—NH)
17
S
NO2
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OCH3
H
H
H
yellow
powder
ethanol
160~162
51.0
3342,3102,1672,1616,1534,1495,1461,1433,1332,1255,1159,989,820,747
(CDCl3),3.94(s,3H,—OCH3),6.56~6.61(d,1H,=CH,J=15 Hz),6.90~6.93(d,1H,3′-H),6.98~7.03(t,1H,4′-H or 5′-H),7.07~7.12(t,1H,4′-H or 5′-H),7.17~7.18(d,1H,furan-3-H),7.73~7.78(d,1H, CH ,J=15 Hz),7.85~7.86(d,1H,furan-4-H),7.99(s,1H,—NH),8.46~8.49(d,1H,6′-H)
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18
O
NO2
OCH3
H
H
H
yellow
needles
ethanol
184~185
44.0
3416,3149,1676,1633,1600,1570,1521,1481,1432,1348,1236,1159,979,807,755
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(CDCl3),3.93(s,3H,—OCH3),6.73~6.74(d,1H,furan-3-H),6.83~6.88(d,1H,=CH ,J=15 Hz),6.90~6.93(d,1H,3′-H),6.96~7.02(t,1H,4′-H or 5′-H),7.07~7.12(t,1H,4′-H or 5′-H),7.35~7.36(d,1H,furan-4H),7.47~7.52(d,1H,— CH ,J=15 Hz),8.10(s,1H,—NH),8.47~8.50(d,1H,6′-H)
2.4 5-硝基-2-噻吩丙烯酸的制备
将1.5 g(9.5 mmol)5-硝基-2-噻吩甲醛,1.05 g(10 mmol)丙二酸,5 mL吡啶和5滴哌啶加入50 mL圆底瓶中,维持100℃反应2 h,然后升温至130℃反应0.5 h.冷却,加入10 mL水稀释,加入浓氨水使溶液呈碱性,过滤,水洗,滤液冷却下用浓盐酸酸化析出白色固体,过滤,水洗至中性,干燥,得目标物1.4 g,收率:73.7%,mp 249℃.
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2.5 N-2′-氯苯基-5-硝基-2-噻吩丙烯酰胺(10)的制备
将0.2 g(1 mmol)5-硝基-2-噻吩丙烯酸,0.25 g(1.2 mmol)五氯化磷和5 mL 1,2-二氯乙烷加入反应瓶中,加热回流直至固体溶解.所得的5-硝基-2-噻吩丙烯酰氯的1,2-二氯乙烷溶液冰浴冷却至-5℃,缓慢滴加0.22 g(1.7 mmol)2-氯苯胺的1,2-二氯乙烷溶液3 mL,加毕,搅拌反应1 h,抽滤,水洗,得黄色固体.乙醇重结晶得黄色针状结晶0.13 g,收率:41.9%,mp 188~190℃.
同法制得化合物(9),(11)~(17),其物理性质及光谱数据见表1.
3 药理实验
抗锥体虫半胱氨酸蛋白酶体外活性是通过标记缩氨酸底物中释放的荧光离去基团——7-氨基-4-甲基香豆素而测定的.具体操作为:于25℃下,用0.1 mol/L醋酸钠培养该酶,保持pH 5.5,培养基内含5 mmol/L二硫苏糖醇.采用微量滴定板测试,用自动荧光分光计检测最高通过量.以二甲基亚砜为溶剂,所测化合物浓度从20 μmol/L开始,运用连续稀释法测定.每次测定采用三个对照组:仅有酶的体系,酶和二甲基亚砜体系,酶、二甲基亚砜和化合物(Ⅱ)体系.目标化合物抗锥体虫半胱氨酸蛋白酶活性见表2.
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Tab.2 Anticruzain activities of the compounds in vitro μmol/L Compd.
IC50
Compd.
IC50
1
10
11
3
2
10
12
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6
3
10
13
10
4
6
14
1
5
10
15
6
6
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10
16
3
7
10
17
6
8
6
18
10
9
10
Ⅱ①
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1
10
6
① control compound
从表2中数据可以看出,这一类型的化合物(10)~(12)可不同程度地抑制锥体虫半胱氨酸蛋白酶的活性,其中化合物(4),(8),(14)~(17)的活性较强,特别是(14)的活性与阳性对照药(Ⅱ)相同.此类型其它化合物的合成及药理活性研究仍在进行中.
致谢:核磁共振光谱由沈阳药科大学分析测试中心代测,质谱由中国科学院沈阳应用生态研究所有机质谱实验室代测,药理活性由美国加州大学旧金山分校药学院Dr.McKerrow JH所在实验室人员测试.
参考文献:
[1]Bontempi E,Franke de cazzulo BM,Ruiz AM,et al.Purification and some properties of an acidic protease from epimastigotes of Trypanasoma cruzi.Comp Biochem Physiol,1994,77B,599~604
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[2]Hans-Hartwig Otto,Tanja schirmeister.Cysteine proteases and their inh]ibitors.Chem Rev,1997,97(1):133~171
[3]Mckerrow JH,Sun E,Rosenthal PJ,et al.The proteases and patho-genicity of parasitic protozoa.Annu Rev Microbial,1993,47:821~853
[4]Ring CS,Sun E,Mckerrow JH,et al.Structure-based inhibitor design by using pro]tein models for the development of antiparasitic agents.Proc Natl Acad Sci USA,1993,90:3583~3587
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[5]Li Z,Chen X,Davidson E,et al.Antimalarial drug development using models of enzyme structure.Chem Biol,1994,(1):31~37
[6]Li R,Kenyon GL,Cohen FE,et al.In vitro antimalarial activity of chalcones and heir derivatives.J Med Chem,1995,38(26):5031~5037
[7]Li Z,Chen X,Gong B,et al.Structure-based design of parasitic protease inhibitor.Bioorganic and Medicinal Chemistry,1994,4(9):1421~1427
[8]Rajagopalan S,Johnson JR.Furylacrylic acid.Org Synth,Coll Ⅲ:425~427
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[9]叶松林,钱夏莉,陈福祥,等.全国原料药工艺汇编.国家医药管理局,1980.286~288
[10]Carrara G,Ettorre R,Fava F,et al.4-Nitrocinnamic and β-(5-nitro-2-thienyl)-acrylic derivatives.J Am Chem Soc,1954,76(17):4391~4395
[11]池田政男.Chemical and chemotherapeutical studies on the furan derivatives.Ⅹ Ⅶ Bacteriostatic action of 2-(5-nitro)-furanacrylic acid derivatives.J Pharm Soc Japan,1955,75:628~632
[12]Yoichi Taniguchi,Hidefumi Kato.Studies on thiophene derivatives.Ⅸ Decarboxylation of 3-(5-substituted 2-thienyl)malonic acids.Chem Pharm Bull,1973,21(9):2070~2073
收稿日期:1999-06-07, 百拇医药
单位:王千里(沈阳药科大学合成二室,沈阳 110015);程卯生(沈阳药科大学合成二室,沈阳 110015);王庆河(沈阳药科大学合成二室,沈阳 110015);宋红岩(沈阳药科大学合成二室,沈阳 110015);潘莉(沈阳药科大学合成二室,沈阳 110015);沈建民(沈阳药科大学合成二室,沈阳 110015)
关键词:合成;芳基丙烯酰胺;半胱氨酸蛋白酶
中国药物化学杂志000108 摘 要:设计合成了18个芳基丙烯酰胺类化合物,并对其进行了抗锥体虫半胱氨酸蛋白酶的药理活性筛选.实验表明化合物(4),(8),(10),(11),(12),(14),(15),(16)和(17)的活性较强.
Synthesis and Their Anticruzain Activities of Arylacrylamide Compounds
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Wang Qianli Cheng Maosheng Wang Qinghe Song Hongyan Pan Li Shen Jianmin
(No.2 Lab.of Drug Synthesis,Shenyang Pharmaceutical University,Shenyang 110015)
Abstract:Eighteen arylacrylamide compounds were synthesized and their activities against cruzain in vitro were tested.The results showed that compounds (4),(8),(10),(11),(12),(14),(15),(16) and (17) had good anticruzain activities.
Key words:synthesis;arylacrylamide;cysteine protease▲
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卡格氏病(Chagas′ disease)是多发于南美洲的一种寄生虫病,也称南美洲锥体虫病.其主要临床症状是一些器官的肿大,如肝、脾肿大,严重的可导致心脏衰竭.据报道,世界上约2 400万人已感染此病.目前治疗这种疾病的药物较少,毒性较大,且易引起耐药性.研究人员对克鲁斯氏锥体虫进行了详细的生物化学基础研究,分离鉴定了锥体虫体内的许多蛋白酶,发现其中活性较强的是一种半胱氨酸蛋白酶〔1〕.进一步研究证实,它可降解宿主免疫系统中的免疫球蛋白G(IgG),保护锥体虫不被宿主免疫系统破坏〔2〕,并且对锥体虫在宿主细胞内的复制和变异过程起重要作用,如参与细胞内代谢、锥体虫和巨噬细胞的相互作用、入侵宿主细胞以及向靶细胞的粘附等〔3〕. 研究人员以半胱氨酸蛋白酶为药物作用靶点,利用计算机辅助药物分子模型模拟设计出半胱氨酸蛋白酶抑制剂的先导化合物N2,N′2-二(2-羟基-1-萘亚甲基)乙二酰肼(Ⅰ)〔4〕,通过对其进行结构修饰,合成了数百个酰肼类化合物,其中有些活性较强〔5~7〕.构效关系研究表明:缩短先导化合物中芳香环之间的连接链,使其成为不对称合成的单酰肼类化合物,抗卡格氏病活性增强;改变酰肼化合物芳香环的大小且引入杂原子其水溶性增强,与半胱氨酸蛋白酶活性位点的氢键作用增强,活性增强;以取代苯环代替酰肼化合物中的萘环、喹啉和异喹啉等,可更好地满足酶对抑制剂在大小和电性等方面的要求,达到提高化合物活性和治疗指数的目的.
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1 合成路线
曾报道过苯基烯丙基硫醚、亚砜和砜类化合物的合成及其抗锥体虫半胱氨酸蛋白酶活性,在此基础上,本文运用生物电子等排代换原理,保持结构中连桥链的长度进行结构修饰,设计合成了18个芳基丙烯酰胺类化合物(见图1),并对这类化合物进行了抗锥体虫半胱氨酸蛋白酶活性.
Fig.1 Lead optimization by chemical modification
综合文献报道,采用图2所示的路线合成目标化合物〔8~12〕.
Fig.2 The synthetic route of the target compounds
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2 合成实验
化合物测定所用仪器:Bruker ARX-300核磁共振仪,VG 7070 E质谱仪,Bruker IFS-55红外测试仪.熔点用毛细管法测定,温度计读数未经校正.中间体5-硝基-2-噻吩甲醛购于Aldrich公司.5-硝基-2-呋喃丙烯酸的合成是以2-呋喃甲醛为原料,先与丙二酸反应生成2-呋喃丙烯酸,再用硝酸硝化制得〔9〕.
2.1 2-呋喃丙烯酸的制备
将19.2 g(0.2 mol)新蒸呋喃甲醛,20.8 g(0.2 mol)丙二酸,10 mL(0.12 mol)吡啶和10滴哌啶加入100 mL圆底瓶中,维持100℃反应2 h,冷却,加入20 mL水稀释,加入浓氨水使溶液呈碱性,过滤,水洗,滤液冷却下用浓盐酸酸化析出白色固体,过滤,水洗至中性,乙醇重结晶得无色针状结晶24.8 g,收率:90%,mp 140~142℃(文献〔8〕收率:91%~92%,mp 141℃).
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2.2 5-硝基-2-呋喃丙烯酸的制备
将30 mL 1,2-二氯乙烷,40 mL乙酸酐加入反应瓶中,搅拌下冷至-15℃,滴加12 mL(0.29 mol)发烟硝酸,控制滴加温度不超过-10℃,加毕,再冷至-20℃,分批加入16.6 g(0.12 mol)呋喃丙烯酸,加料温度控制-15℃以下.加毕,继续搅拌1 h.抽滤,滤饼水洗至中性,干燥得黄色固体15.0 g,收率:68%,mp 230℃(分解)〔文献〔9〕收率:62.5%,mp 236℃〕.
2.3 N-2′-氯苯基-5-硝基-2-呋喃丙烯酰胺(1)的制备
将0.5 g(2.7 mmol)5-硝基-2-呋喃丙烯酸,0.75 g(3.6 mmol)五氯化磷和10 mL 1,2-二氯乙烷加入反应瓶中,加热回流直至固体溶解.所得的5-硝基-2-呋喃丙烯酰氯的1,2-二氯乙烷溶液冰浴冷却至-5℃,缓慢滴加0.56 g(4.4 mmol)2-氯苯胺的1,2-二氯乙烷溶液5 mL,加毕,搅拌反应1 h,抽滤,水洗,得黄色固体.乙醇重结晶得黄色絮状物0.40 g,收率:50.3%,mp 189~191℃.
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同法制得化合物(2)~(8)和(18),其物理性质及光谱数据见表1.
Tab.1 Physical and spectral data of the compounds Compd.
X
R1
R2
R3
R4
R5
Form
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Solvent
for
recrystn.
mp/℃
Yield/%
1H-NMR δ
1
O
NO2
Cl
H
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H
H
pale
yellow
needles
ethanol
189~191
50.3
3155,1678,1630,1590,1568,1513,1478,1441,1350,1238,1172,983,761
(CDCl3),6.77~6.78(d,1H,furan-3H),6.83~6.88(d,1H,=CH,J=15 Hz),7.06~7.11(m,1H,4′-H),7.29~7.34(m,1H,5′-H),7.36~7.37(d,1H,furan-4-H),7.40~7.42(d,1H,3′-H),7.51~7.56(d,1H,=CH
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,J=15 Hz),7.88(s,1H,—NH),8.49~8.51(d,1H,6′-H)
2
O
NO2
H
H
H
H
yellow
needles
ethanol
196
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45.7
3364,3129,1675,1630,1601,1536,1501,1473,1353,1238,1165,971,756,689
(CDCl3),6.72~6.73(d,1H,furan-3H),6.80~6.85(d,1H,=CH,J=15 Hz),7.13~7.18(m,1H,4′-H),7.34~7.39(m,3H,3′-H and furan-4-H),7.49~7.54(d,1H,=CH,J=15 Hz),7.57(s,1H,—NH),7.61~7.64(d,2H,2′-H)
3
O
NO2
H
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H
CH3
H
yellow
needles
acetic
acid
203~205
52.0
3371,3119,1675,1630,1597,1566,1531,1474,1356,1241,981,815
(CDCl3),2.33(s,3H,—CH3),6.72~6.73(d,1H,furan-3-H),6.78~6.83(d,1H,=CH,J=15 Hz),7.15~7.17(d,2H,3′-H),7.34~7.35(d,1H,furan-4-H),7.47~7.52(br,4H,— CH ,J=15 Hz,—NH and 2′-H)
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4
O
NO2
H
H
Cl
F
yellow
powder
acetic
acid
243~245
41.4
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3390,3140,1690,1633,1603,1565,1534,1502,1467,1360,1241,971,808
(d6-DMSO),6.89~6.94(d,1H, CH ,J=15 Hz),7.20~7.21(d,1H,furan-3-H),7.37~7.43(t,1H,2′-H),7.47~7.52(d,2H,=CH,J=15 Hz and 5′-H),7.75~7.76(d,1H,furan-4-H),8.01~8.03(d,1H,6′-H),10.65(s,1H,—NH)
5
O
NO2
H
H
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Br
H
yellow
needles
ethanol
221~222
46.3
3375,1669,1631,1593,1527,1471,1355,1235,966,815
(CDCl3),6.75~6.76(d,1H,furan-3-H),6.76~6.81(d,1H,=CH,J=15 Hz),7.35~7.36(d,1H,furan-4-H),7.37(s,1H,—NH),7.46~7.54(m,5H,— CH ,J=15 Hz,2′-H and 3′-H)
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6
O
NO2
H
H
Cl
H
yellow
tabletes
ethanol
213~214
45.1
3372,3112,1669,1633,1595,1530,1474,1398,1355,1236,963,816
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(CDCl3),6.75~6.76(d,1H,furan-3-H),6.76~6.81(d,1H,=CH,J=15 Hz),7.32~7.35(d,2H,3′-H),7.35~7.36(d,1H,furan-4-H),7.39(s,1H,—NH),7.49~7.54(d,1H,— CH ,J=15 Hz),7.56~7.59(d,2H,2′-H)
7
O
NO2
H
H
OCH3
H
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red
needles
acetic
acid
182~184
52.0
3357,3139,1677,1630,1601,1536,1510,1358,1242,957
(CDCl3),3.81(s,3H,—OCH3),6.72~6.73(d,1H,furan-3-H),6.76~6.81(d,1H,=CH,J=15 Hz),6.88~6.91(d,2H,3′-H),7.34~7.35(d,2H,furan-4-H and —NH),7.47~7.54(m,3H,— CH ,J=15 Hz and 2′-H)
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8
O
NO2
H
H
COC2H5
H
yellow
powder
ethanol
214~215
38.4
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3366,1674,1632,1593,1564,1526,1469,1406,1354,1238,1177,957,802
(d6-DMSO),1.07(t,3H,—CH3),2.98(q,2H,—CH2—),6.97~7.02(d,1H,=CH,J=15 Hz),7.20~7.21(d,1H,furan-3-H),7.49~7.54(d,1H,— CH ,J=15 Hz),7.75~7.76(d,1H,furan-4-H),7.79~7.82(d,2H,2′-H),7.95~7.98(d,2H,3′-H),10.74(s,1H,—NH)
9
S
NO2
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H
H
H
orange
powder
ethanol
218~219
44.2
3364,1668,1621,1600,1544,1495,1440,1332,1165,964,754,689
(CDCl3),6.51~6.56(d,1H,=CH,J=15 Hz),7.17~7.18(d,2H,furan-3-H and 1 Ph-H),7.32~7.40(m,3H,Ph-H),7.60(br,2H,—NH and 1 Ph-H),7.75~7.80(d,1H,— CH ,J=15 Hz),7.85~7.86(d,1H,furan-4-H)
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10
S
NO2
Cl
H
H
H
yellow
needles
ethanol
188~190
41.9
3361,1677,1619,1589,1530,1434,1331,1157
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(CDCl3),6.55~6.60(d,1H,=CH,J=15 Hz),7.08~7.12(t,1H,4′-H),7.21~7.22(d,1H,furan-3-H),7.28~7.32(t,1H,5′-H),7.40~7.43(d,1H,3′-H),7.76~7.81(d,2H,— CH ,J=15 Hz and —NH),7.86~7.87(d,1H,furan-4-H),8.48~8.51(d,1H,6′-H)
11
S
NO2
H
H
Cl
H
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yellow
needles
ethanol
244~246
42.6
3358,1668,1622,1596,535,1492,1330,1163,964,824
(d6-DMSO),6.81
~6.86(d,1H,=CH,J=15 Hz),7.39~7.42(d,2H,3′-H),7.56~7.57(d,1H,furan-3-H),7.70~7.73(d,2H,2′-H),7.75~7.80(d,1H,=CH,J=15 Hz),8.13~8.14(d,1H,furan-4-H),10.48(s,1H,—NH)
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12
S
NO2
H
H
Br
H
yellow
powder
ethanol
238~239
40.0
3361,1667,1622,1595,1534,1489,1323,1164,965,821
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(d6-DMSO),6.81~
6.86(d,1H,=CH,J=15 Hz),7.52~7.55(d,2H,3′-H),7.56~7.57(d,1H,furan-3-H),7.65~7.68(d,2H,2′-H),7.75~7.80(d,1H,=CH,J=15 Hz),8.13~8.14(d,1H,furan-4-H),10.48(s,1H,—NH)
13
S
NO2
H
H
CH3
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H
orange
powder
acetic
acid
243~245
48.5
3365,1666,1623,1599,1537,1491,1328,1164,964,819
(d6-DMSO),2.27(s,3H,—CH3),6.83~6.88(d,1H,=CH,J=15 Hz),7.13~7.16(d,2H,3′-H),7.54~7.59(t,3H,2′-H and furan-3-H),7.72~7.77(d,1H,=CH,J=15 Hz),8.13~8.14(d,1H,furan-4-H),10.26(s,1H,—NH)
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14
S
NO2
H
F
Cl
H
yellow
powder
acetic
acid
241~243
39.0
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3354,1681,1622,1543,1497,1426,1339,1162,962,810
(d6-DMSO),6.78
~6.83(d,1H,=CH,J=15 Hz),7.41~7.44(t,1H,2′-H),7.53(br,1H,5′-H),7.57~7.58(d,1H,furan-3-H),7.77~7.82(d,1H, CH ,J=15 Hz),8.02~8.04(dd,1H,6′-H),8.13~8.14(d,1H,furan-4-H),10.56(s,1H,—NH)
15
S
NO2
H
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H
OCH3
H
deep red
powder
acetic
acid
234~236
52.7
3360,3099,1677,1621,1546,1510,1485,1425,1326,1163,950,840
(d6-DMSO),3.68(s,3H,—OCH3),6.75~6.80(d,1H, =CH,J=15 Hz),6.85~6.88(d,2H,3′-H),7.47~7.48(d,1H,furan-3-H),7.53~7.56(d,2H,2′-H),7.64~7.69(d,1H,=CH,J=15 Hz),8.06~8.07(d,1H,furan-4-H),10.16(s,1H,—NH)
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16
S
NO2
H
H
COC2H5
H
yellow
needles
acetic
acid
245~247
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36.3
3360,3090,1676,1622,1596,1532,1487,1424,1366,1322,1225,1160,950,814
(d6-DMSO),1.08(t,3H,—CH3),2.99(q,2H,—CH2—),6.85~6.90(d,1H, CH ,J=15 Hz),7.57~7.58(d,1H,furan-3-H),7.76~7.84(m,3H, CH ,J=15 Hz and 2′-H),7.96~7.99(d,2H,3′-H),8.13~8.14(d,1H,furan-4-H),10.65(s,1H,—NH)
17
S
NO2
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OCH3
H
H
H
yellow
powder
ethanol
160~162
51.0
3342,3102,1672,1616,1534,1495,1461,1433,1332,1255,1159,989,820,747
(CDCl3),3.94(s,3H,—OCH3),6.56~6.61(d,1H,=CH,J=15 Hz),6.90~6.93(d,1H,3′-H),6.98~7.03(t,1H,4′-H or 5′-H),7.07~7.12(t,1H,4′-H or 5′-H),7.17~7.18(d,1H,furan-3-H),7.73~7.78(d,1H, CH ,J=15 Hz),7.85~7.86(d,1H,furan-4-H),7.99(s,1H,—NH),8.46~8.49(d,1H,6′-H)
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18
O
NO2
OCH3
H
H
H
yellow
needles
ethanol
184~185
44.0
3416,3149,1676,1633,1600,1570,1521,1481,1432,1348,1236,1159,979,807,755
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(CDCl3),3.93(s,3H,—OCH3),6.73~6.74(d,1H,furan-3-H),6.83~6.88(d,1H,=CH ,J=15 Hz),6.90~6.93(d,1H,3′-H),6.96~7.02(t,1H,4′-H or 5′-H),7.07~7.12(t,1H,4′-H or 5′-H),7.35~7.36(d,1H,furan-4H),7.47~7.52(d,1H,— CH ,J=15 Hz),8.10(s,1H,—NH),8.47~8.50(d,1H,6′-H)
2.4 5-硝基-2-噻吩丙烯酸的制备
将1.5 g(9.5 mmol)5-硝基-2-噻吩甲醛,1.05 g(10 mmol)丙二酸,5 mL吡啶和5滴哌啶加入50 mL圆底瓶中,维持100℃反应2 h,然后升温至130℃反应0.5 h.冷却,加入10 mL水稀释,加入浓氨水使溶液呈碱性,过滤,水洗,滤液冷却下用浓盐酸酸化析出白色固体,过滤,水洗至中性,干燥,得目标物1.4 g,收率:73.7%,mp 249℃.
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2.5 N-2′-氯苯基-5-硝基-2-噻吩丙烯酰胺(10)的制备
将0.2 g(1 mmol)5-硝基-2-噻吩丙烯酸,0.25 g(1.2 mmol)五氯化磷和5 mL 1,2-二氯乙烷加入反应瓶中,加热回流直至固体溶解.所得的5-硝基-2-噻吩丙烯酰氯的1,2-二氯乙烷溶液冰浴冷却至-5℃,缓慢滴加0.22 g(1.7 mmol)2-氯苯胺的1,2-二氯乙烷溶液3 mL,加毕,搅拌反应1 h,抽滤,水洗,得黄色固体.乙醇重结晶得黄色针状结晶0.13 g,收率:41.9%,mp 188~190℃.
同法制得化合物(9),(11)~(17),其物理性质及光谱数据见表1.
3 药理实验
抗锥体虫半胱氨酸蛋白酶体外活性是通过标记缩氨酸底物中释放的荧光离去基团——7-氨基-4-甲基香豆素而测定的.具体操作为:于25℃下,用0.1 mol/L醋酸钠培养该酶,保持pH 5.5,培养基内含5 mmol/L二硫苏糖醇.采用微量滴定板测试,用自动荧光分光计检测最高通过量.以二甲基亚砜为溶剂,所测化合物浓度从20 μmol/L开始,运用连续稀释法测定.每次测定采用三个对照组:仅有酶的体系,酶和二甲基亚砜体系,酶、二甲基亚砜和化合物(Ⅱ)体系.目标化合物抗锥体虫半胱氨酸蛋白酶活性见表2.
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Tab.2 Anticruzain activities of the compounds in vitro μmol/L Compd.
IC50
Compd.
IC50
1
10
11
3
2
10
12
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6
3
10
13
10
4
6
14
1
5
10
15
6
6
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10
16
3
7
10
17
6
8
6
18
10
9
10
Ⅱ①
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1
10
6
① control compound
从表2中数据可以看出,这一类型的化合物(10)~(12)可不同程度地抑制锥体虫半胱氨酸蛋白酶的活性,其中化合物(4),(8),(14)~(17)的活性较强,特别是(14)的活性与阳性对照药(Ⅱ)相同.此类型其它化合物的合成及药理活性研究仍在进行中.
致谢:核磁共振光谱由沈阳药科大学分析测试中心代测,质谱由中国科学院沈阳应用生态研究所有机质谱实验室代测,药理活性由美国加州大学旧金山分校药学院Dr.McKerrow JH所在实验室人员测试.
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收稿日期:1999-06-07, 百拇医药