CN112094319A - Glu-Asp-Gly修饰的甲氨蝶呤,其合成,抗转移活性和应用 - Google Patents

Glu-Asp-Gly修饰的甲氨蝶呤,其合成,抗转移活性和应用 Download PDF

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CN112094319A
CN112094319A CN201910527897.6A CN201910527897A CN112094319A CN 112094319 A CN112094319 A CN 112094319A CN 201910527897 A CN201910527897 A CN 201910527897A CN 112094319 A CN112094319 A CN 112094319A
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赵明
彭师奇
梁梦
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Abstract

本发明公开了下面通式的Glu‑Asp‑Gly修饰的甲氨蝶呤(式中R1为Glu‑Asp‑Gly时R2为OH,R1为OH时R2为Glu‑Asp‑Gly,以及R1和R2同时为Glu‑Asp‑Gly),公开了它们的制备方法,公开了它们对肿瘤转移的抑制作用。因而本发明公开了它们在制备抗肿瘤转移药物中的应用。
Figure DDA0002098805630000011

Description

Glu-Asp-Gly修饰的甲氨蝶呤,其合成,抗转移活性和应用
技术领域
本发明涉及Glu-Asp-Gly修饰的甲氨蝶呤,涉及它们的制备方法,涉及它们的抗肿瘤转移作用。因而本发明涉及它们在制备抗肿瘤转移药物中的应用。本发明属于生物医药领域。
背景技术
癌症是指细胞不受控制以及不正常的增殖,并有机会通过机体血液系统或者淋巴系统向其它部位扩散转移的的一类疾病,是世界性的重大医学难关。据国家癌症中心2019年初发布的我国2015年各地区恶性肿瘤发病以及死亡数据结果显示,白血病位列死亡率最高的恶性肿瘤前十位。作为最早用于治疗急性白血病的药物之一,甲氨蝶呤已有70余年临床应用史。可是,骨髓毒性,肝毒性,肾毒性,口腔粘膜副作用以及耐药性严重限制了甲氨蝶呤的应用及疗效。发明人在一份申请书中描述了用Glu-Asp-Gly修饰甲氨蝶呤得到的下面通式的Glu-Asp-Gly-甲氨蝶呤(式中R1为Glu-Asp-Gly时R2为OH,R1为OH时R2为Glu-Asp-Gly,以及R1和R2同时为Glu-Asp-Gly)可克服这些问题的发明。本案进一步描述Glu-Asp-Gly-甲氨蝶呤意外地具有抗肿瘤转移活性。根据这个意外的发现，发明人提出了本发明。
Figure BDA0002098805610000011
发明内容
本发明的第一个内容是提供下面通式的Glu-Asp-Gly修饰的甲氨蝶呤(式中R1为Glu-Asp-Gly时R2为OH,R1为OH时R2为Glu-Asp-Gly,以及R1和R2同时为Glu-Asp-Gly)。
Figure BDA0002098805610000012
本发明的第二个内容是提供Glu-Asp-Gly修饰的甲氨蝶呤的制备方法,该方法包括：
1采用二环己基碳二亚胺为缩合剂,N-羟基苯并三唑为催化剂,液相缩合合成Boc-Glu(OBzl)-Asp(OBzl)-Gly-OBzl；
2脱除Boc合成Glu(OBzl)-Asp(OBzl)-Gly-OBzl；
3采用二环己基碳二亚胺为缩合剂,N-羟基苯并三唑为催化剂,将甲氨蝶呤与Glu(OBzl)-Asp(OBzl)-Gly-OBzl偶联生成如下通式的Glu(OBzl)-Asp(OBzl)-Gly-OBzl修饰的甲氨蝶呤,式中R1’为Glu(OBzl)-Asp(OBzl)-Gly-OBzl时R2’为OH,R1为OH时R2’为Glu(OBzl)-Asp(OBzl)-Gly-OBzl,以及R1’和R2’同时为Glu(OBzl)-Asp(OBzl)-Gly-OBzl；
Figure BDA0002098805610000021
4在碱性条件下脱除保护基生成下面通式的Glu-Asp-Gly修饰的甲氨蝶呤(式中R1为Glu-Asp-Gly时R2为OH,R1为OH时R2为Glu-Asp-Gly,以及R1和R2同时为Glu-Asp-Gly)。
Figure BDA0002098805610000022
本发明的第三个内容是评价上面通式的Glu-Asp-Gly修饰的甲氨蝶呤的抑制肿瘤转移活性。
附图说明
图1Glu-Asp-Gly修饰的甲氨蝶呤的合成路线.(ⅰ)无水四氢呋喃,二环己基碳二亚胺,N-羟基苯并三氮唑,N-甲基吗啉；(ⅱ)氯化氢的乙酸乙酯溶液；(ⅲ)无水N,N-二甲基甲酰胺,二环己基碳二亚胺,N-羟基苯并三氮唑,N-甲基吗啉；(ⅳ)甲醇/N,N-二甲基甲酰胺,2MNaOH。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1制备Boc-Asp(OBzl)-Gly-OBzl(1)
用60mL无水四氢呋喃溶解1.42g(4.40mmol)Boc-Asp(OBzl)，得到1号溶液。0℃与搅拌下,将1.36g(6.60mmol)二环己基碳二亚胺和0.59g(4.40mmol)N-羟基苯并三氮唑的无水四氢呋喃溶液加入到1号溶液中搅拌30分钟。随后,加入1.10g(3.26mmol)Tos·Gly-OBzl,用N-甲基吗啉调节反应液pH至8-9,移去冰浴,在室温下充分搅拌18小时后TLC(二氯甲烷/甲醇＝30/1)显示反应完成。滤除反应液中的无色固体,将滤液浓缩,残留物用100mL乙酸乙酯溶解,溶液依次分别用饱和碳酸氢钠水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3),5％硫酸氢钾水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3),饱和碳酸氢钠水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3),随后将乙酸乙酯相用无水硫酸钠干燥12小时,过滤,滤液浓缩,得到1.53g(100％)目标化合物,为黄色油状物。ESI-MS(m/e):471[M+1]+。
实施例2制备Asp(OBzl)-Gly-OBzl(2)
将1.34g(2.85mmol)Boc-Asp(OBzl)-Gly-OBzl(1)用无水乙酸乙酯溶解，0℃与搅拌下,加入20mL氯化氢的乙酸乙酯溶液(4M),搅拌7小时后TLC(二氯甲烷/甲醇＝30/1)显示反应完成,温水浴条件下,反复减压浓缩反应液,随后用无水乙酸乙酯溶解浓缩物,重复减压浓缩反应液(3次),再用无水乙醚反复磨洗反应物,得到1.05g(100％)目标化合物,为黄色粘稠油状物。ESI-MS(m/e):371[M+1]+。
实施例3制备Boc-Glu(OBzl)-Asp(OBzl)-Gly-OBzl(3)
采用实施例1的方法，从0.83g(2.47mmol)Boc-Glu(OBzl)和1.05g(2.84mmol)化合物(2)得到1.31g(77％)目标化合物,为无色粉末。ESI-MS(m/e):690[M+H]+,1H NMR(300MHz,DMSO-d6):δ/ppm＝8.32(t,J＝5.7Hz,1H),8.24(d,J＝8.1Hz,1H),7.39-7.30(m,15H),7.03(d,J＝7.8Hz,1H),5.10-5.06(m,6H),4.71(m,1H),3.90(m,3H),2.81(dd,J＝8.4Hz,5.1Hz,2H),2.52(m,2H),1.83(m,2H),1.36(s,9H)。
实施例4制备Glu(OBzl)-Asp(OBzl)-Gly-OBzl(4)
采用实施例2的方法,从1.24g(1.80mmol)化合物(3)得到1.06g(100％)目标化合物,为无色粉末。ESI-MS(m/e):590[M+1]+。
实施例5制备Glu(OBzl)-Asp(OBzl)-Gly(OBzl)修饰的甲氨蝶呤(1a,1b,1c)
用30mL无水N,N-二甲基甲酰胺溶解0.68g(1.50mmol)甲氨蝶呤,得到1号溶液。0℃与搅拌下，将0.37g(1.80mmol))二环己基碳二亚胺和0.20g(1.50mmol)N-羟基苯并三唑的无水N,N-二甲基甲酰胺溶液加入到1号溶液中,搅拌30分钟。随后加入1.01g(1.61mmol)化合物(4),用N-甲基吗啉调节反应液pH至9,室温充分搅拌14小时后TLC(乙酸乙酯/水/冰醋酸＝5/1/1)显示反应完成。反应液过滤,滤液减压浓缩,得到的酒红色油状物用硅胶柱层析纯化(二氯甲烷/甲醇＝8/1,二氯甲烷/甲醇＝30/1及二氯甲烷/甲醇＝6/1三个梯度)分别得到0.19g(11％)化合物1a,0.17g(13％)化合物1b和0.15g(9％)化合物1c。它们的结构式如下：
Figure BDA0002098805610000041
1a为橘黄色粉末,ESI-MS(m/e):1026[M+H]+,1H NMR(300MHz,DMSO-d6):δ/ppm＝12.12(s,1H),8.55(d,1H),8.40-7.80(m,4H),7.74-7.69(m,3H),7.34-7.31(m,15H),6.80-6.76(m,J＝2.1Hz,2H),6.65(s,1H),5.07-5.05(m，6H),4.78(s,2H),4.73-4.66(m,2H),4.37-4.24(m,1H),3.90-3.65(m,3H),3.19(d,J＝6.0Hz,3H),2.93-2.61(m,2H),2.41-2.39(m,2H),2.35-2.25(m,2H),2.04-1.78(m,4H)；13CNMR(75MHz,DMSO-d6):δ/ppm＝174.58,173.29,172.78,172.68,172.42,171.37,171.12,171.05,170.41,170.30,169.79,169.60,167.10,163.18,163.03,151.58,151.49,149.59,146.69,136.65,136.44,136.30,129.49,128.82,128.49,128.45,128.36,128.29,128.24,121.93,121.66,121.27,111.51,66.32,66.27,66.17,55.34,53.53,49.89,49.80,41.39,40.39,36.50,31.03,30.94,27.37,27.12,26.90。
1b为橘黄色粉末,ESI-MS(m/e):1597[M+H]+,1H NMR(300MHz,DMSO-d6):δ/ppm＝8.59(d,J＝3.6Hz,1H),8.34-8.25(m,6H),8.09-8.00(m,3H),7.75-7.69(m,2H),7.33-7.27(m,30H),6.79-6.74(m,2H),5.08-5.05(m,12H),4.80(s,2H),4.71-4.67(m,4H),4.30-4.24(m,1H),3.89-3.83(m,6H),3.19(d,J＝5.1Hz,3H),2.80-2.68(m,4H),2.38-2.34(m,4H),2.27-2.24(m,2H),1.97-1.82(m,6H)；13C NMR(125MHz,DMSO-d6):δ/ppm＝173.40,172.81,172.74,172.70,172.68,172.50,171.70,171.43,171.22,171.17,171.14,171.05,170.42,170.36,170.30,169.85,169.84,166.91,163.14,149.33,149.29,136.63,136.61,136.40,136.38,136.25,136.27,129.53,129.50,128.86,128.82,128.52,128.48,128.40,128.36,128.32,128.31,128.27,128.24,122.27,121.74,121.24,111.48,111.44,66.34,66.28,66.19,65.89,65.86,64.02,55.29,55.24,54.25,53.73,53.13,50.25,49.66,41.33,40.58,39.56,36.47,32.34,30.42,30.29,27.51,27.49,27.44。
1c为橘黄色粉末,ESI-MS(m/e):1026[M+H]+,1H NMR(300MHz,DMSO-d6):δ/ppm＝12.50(s,1H),8.65(d,J＝5.1Hz,1H),8.37-8.26(m,2H),8.20(d,J＝4.8Hz,1H),8.11(d,J＝4.8Hz,1H),7.75(d,J＝8.7Hz,2H),7.34-7.33(m,15H),6.81(d,J＝8.7Hz,2H),5.09-5.06(m,6H),4.84(s,2H),4.71-4.66(m,2H),4.36-4.24(m,1H),3.99-3.78(m,3H),3.23(s,3H),2.86-2.60(m,2H),2.41-2.36(m,2H),2.26-2.24(m,2H),2.08-1.74(m,4H)；13C NMR(75MHz,DMSO-d6):δ/ppm＝174.21,174.13,174.12,172.76,172.68,172.61,171.60,171.18,170.55,170.38,169.82,169.70,166.72,166.64,163.16,158.55,151.28,151.23,150.08,149.35,136.66,136.41,136.29,129.43,128.85,128.50,128.38,128.30,122.41,121.90,121.70,111.57,66.74,66.34,66.19,56.32,53.04,52.20,49.69,41.36,36.50,32.32,30.43,27.42,27.06。
实施例6制备结构式如下的Glu-Asp-Gly修饰的甲氨蝶呤(2a)
Figure BDA0002098805610000051
用3mL甲醇溶解0.05g(0.049mmol)化合物1a,0℃与搅拌下,滴加2M NaOH,调节反应液pH＝12。搅拌6小时后TLC(乙酸乙酯/水/冰醋酸＝3:1:1)点板显示反应完成,终止反应。0℃下,用饱和硫酸氢钾调节反应液pH＝7,浓缩,加入5mL水,调节溶液pH＝6,过滤,Sephadex除去杂质盐,收集洗脱液,-80℃冰箱低温预冻,冷冻干燥机冻干样品。得到0.019g(49％)化合物2a,为橘黄色粉末。ESI-MS(m/e):756[M+H]+,1H NMR(300MHz,D2O):δ/ppm＝8.55(s,1H),7.66(d，J＝8.4Hz，2H),6.84(d，J＝9.0Hz，2H),4.78(s，2H),4.47-4.26(m，2H),3.68-3.45(m，2H),3.11(s,3H),2.76-2.51(m,2H),2.29-2.25(m,2H),2.18-2.08(m，2H),2.05-2.19(m,4H)；13C NMR(125MHz,DMSO-d6):δ/ppm＝175.47,175.28,174.82,173.37,172.23,171.88,171.25,166.85,163.30,163.16,155.63,149.65,146.48,129.48,129.40,121.88,121.75,121.58,111.54,55.30,53.36,51.27,50.82,43.86,38.69,31.66,31.44,27.88,27.64。
实施例7制备结构如下的Glu-Asp-Gly修饰甲氨蝶呤(2b)
Figure BDA0002098805610000052
用2mL无水N,N-二甲基甲酰胺溶解0.054g(0.034mmol)化合物1b。0℃与搅拌下,滴加2M NaOH调节反应液pH＝12。充分搅拌5小时后TLC(乙酸乙酯/水/冰醋酸＝2/1/1)显示反应完成。0℃下,用饱和硫酸氢钾调节反应液pH＝7,浓缩,加入5mL水,调节溶液pH＝5,过滤,Sephadex除去杂质盐,收集洗脱液,在-80℃冰箱低温预冻,冷冻干燥机冻干样品。得到0.018g(50％)化合物2b,为橘黄色粉末。ESI-MS(m/e):1055[M-H]-,1H NMR(300MHz,D2O):δ/ppm＝8.58-8.52(m,1H),7.51(m，2H),6.65-6.62(m,2H),4.57-4.26(m,2H),4.13-4.01(m,2H),3.73-3.61(m,2H),3.11(s,3H),2.80-2.71(m,2H),2.42-2.28(m,5H),2.12-2.02(m,4H),1.85(m,2H)；13C NMR(125MHz,D2O):δ/ppm＝176.95,176.03,174.66,174.27,173.52,173.36,172.63,172.56,172.53,169.54,169.25,163.03,156.60,151.78,149.34,129.07,123.42,122.05,120.90,119.75,119.38,118.37,115.85,111.71,111.65,54.71,54.39,54.03,53.94,53.87,51.27,51.03,43.31,38.87,38.76,37.98,31.98,31.68,27.05,26.87,26.81。
实施例8制备结构是如下的Glu-Asp-Gly修饰甲氨蝶呤(2c)
Figure BDA0002098805610000061
用3mL甲醇溶解0.080g化合物1c,0℃与搅拌下,滴加2M NaOH,调节反应液pH＝12。充分搅拌反应5小时后TLC(乙酸乙酯/水/冰醋酸＝3/1/1)显示反应完成,终止反应。在冰浴条件下,用饱和硫酸氢钾调节反应液pH＝7,浓缩,加入5mL水,调节溶液pH＝6,过滤,C18柱层析纯化,收集洗脱液,将收集液在-80℃冰箱低温预冻,冷冻干燥机冻干样品,得到0.0086g(15％)化合物2c,为橘黄色粉末,ESI-MS(m/e):754[M+H]-,1H NMR(300MHz,D2O):δ/ppm＝8.41(s,1H),8.42(d,J＝8.4Hz,2H),6.51(d,J＝8.7Hz,2H),4.58(s,2H),4.29-4.26(m,2H),3.89-3.84(m,1H),3.64-3.62(m,2H),3.01(s,3H),2.67-2.48(m,2H),2.34-2.26(m,2H),2.14-1.97(m,4H),1.76-1.72(m,1H),1.56-1.52(m,1H)；13C NMR(125MHz,D2O):δ/ppm＝178.65,178.37,176.80,176.12,175.97,173.43,172.52,172.45,168.39,162.89,156.28,151.56,151.36,149.15,145.55,128.70,121.94,120.00,111.53,57.40,53.76,50.96,48.84,43.19,37.67,32.52,31.53,26.89,26.55。
实验例1测定化合物2a-c的抗肿瘤细胞迁移活性
1)化合物2a-c用无胎牛血清培养基配制成所需浓度。
2)肿瘤细胞为A549(人非小细胞肺癌细胞)。
3)将生长状态良好,处于对数生长期的A549细胞按照5×106个/mL的密度使用无血清的培养基接种在Transwell的上室,每室加入100μL,加入化合物2a-c(终浓度分别为1,1,5μM)。同时在下室加入600μL含有10％胎牛血清的培养基,将Transwell小室放入24孔培养板中,于37℃的5％二氧化碳培养箱中孵育7小时。用棉签拭去上室的细胞,吸弃下室的培养基,用4％多聚甲醛固定液固定细胞30分钟,弃固定液,用PBS洗2次,用结晶紫染色15分钟,清水洗去浮色,显微镜观察。随机选取6个不同的视野观察细胞并计算迁移数。结果见表1,数据经t检验。可以确认,在IC10浓度下化合物2a-c有效地抑制肿瘤细胞迁移。此外,2a-c的抗迁移活性与20μM浓度下Arg-Gly-Asp-Ser(RGDS)无显著性差异。这是本发明的突出的技术效果。
表1化合物2a-c抑制A549细胞迁移活性
Figure BDA0002098805610000071
a)与PBS组比P<0.01；b)与PBS比P<0.01,与RGDS比P>0.05；n＝6。
实验例2测定化合物2a-c抑制肿瘤细胞侵袭活性
1)化合物2a-c用无胎牛血清培养基配制成所需浓度。
2)肿瘤细胞为A549(人非小细胞肺癌细胞)。
3)标准型基底膜胶Matrigel用无血清的培养基稀释10倍,37℃沉降12小时,弃上清液。
4)将生长状态良好,处于对数生长期的A549细胞按照1×107个/mL的密度使用无血清的培养基接种在Transwell的上室,每室加入100μL,加入化合物2a-c(终浓度分别为1,1,5μM)，同时在下室加入600μL含有10％胎牛血清的培养基,将Transwell小室放入24孔培养板中,37℃的5％二氧化碳培养箱中孵育12小时。用棉签拭去上室的细胞,弃下室的培养基,用4％多聚甲醛固定液固定细胞30分钟,弃固定液,用PBS洗2次,用结晶紫染色15分钟，清水洗去浮色,用显微镜观察。随机选取6个不同的视野观察细胞并计算侵袭数。结果见表2,数据经t检验。可以看到,在IC10浓度下,化合物2a-c有效地抑制肿瘤细胞侵袭。此外,2a-c的抗侵袭活性与20μM浓度下Arg-Gly-Asp-Ser(RGDS)的活性无显著性差异。这是本发明的突出的技术效果。
表2化合物2a-c抑制A549细胞侵袭活性
Figure BDA0002098805610000072
Figure BDA0002098805610000081
a)与PBS比P<0.01；b)与PBS比P<0.05,与RGDS比P>0.05；c)与PBS比P<0.01,与RGDS比P>0.05；n＝6。
实验例3化合物2a-c抑制肿瘤肺转移活性
将本化合物2a-c溶于生理盐水。Lewis小鼠肺癌细胞(LLC,购自ATCC)用含有10％FBS和1×105U·L-1青霉素和100mg·L-1链霉素的DMEM培养基培养。每天传代一次,富集细胞。待细胞处于对数生长期且生长状态良好时消化细胞。用生理盐水调整细胞密度至2×107个/mL。
体重为20±2g的近交系C57BL/6雄性小鼠用左手固定,用75％乙醇涂在小鼠右前肢腋窝皮肤处消毒,右手以1mL无菌注射器于消毒皮下处注射肿瘤细胞悬液,每只注射0.2mL,取接种10天生长状态良好的Lewis肺癌荷瘤小鼠,乙醚麻醉后颈椎脱臼处死。用75％乙醇浸泡消毒10min,在超净台上操作剥离瘤体,选择生长良好的肿瘤组织,在无菌培养皿中剪碎,放置于玻璃组织匀浆器中研磨。研磨时按照瘤块重量(g)/生理盐水体积(mL)为1/3的比例加4℃预冷的生理盐水。研磨制得的细胞悬液用200目的尼龙网过滤,收集的细胞用生理盐水调浓度为2×107个/mL。取体重为20±2g的近交系C57BL/6雄性小鼠,左手固定小鼠,用75％乙醇涂在小鼠右前肢腋窝皮肤处消毒,右手以1mL无菌注射器于消毒皮下处注射肿瘤细胞悬液,每只注射0.2mL,接种后10天可以长成绿豆大小的肿瘤。测量肿瘤体积,肿瘤直径为4-6mm的小鼠随机分组。化合物2a-c组小鼠连续腹腔注射给药10天，剂量为0.033μmol/kg/天。以Arg-Gly-Asp-Ser(RGDS,剂量为20μmol/kg/天)作阳性对照。空白组小鼠每日腹腔注射0.2mL/只/天生理盐水。给药第11天称小鼠体重，乙醚麻醉，剖取各组小鼠的肺计算转移的瘤节数。结果见表3,数据经t检验。可以看到,当2a-c的给药剂量降至Glu-Asp-Gly的1％时，2a(抑制率为63.69％)和2c(抑制率为72.67％)仍表现出与Glu-Asp-Gly(抑制率为63.80％)和RGDS(抑制率为72.18％)无统计学差异的体内抗肿瘤转移活性,具有良好的体内抗Lewis肺癌向小鼠肺部转移活性,可见本发明化合物2a,2c具有显著的技术效果。而当2b给药剂量降低为0.033μmol/kg/天时,不具有体内抗Lewis肺癌向小鼠肺部转移的活性。
表3化合物2a-c抑制肿瘤肺转移活性
Figure BDA0002098805610000091
a)与生理盐水组相比P<0.01；b)与生理盐水组相比P<0.01,与RGDS组相比P>0.05,与Glu-Asp-Gly组相比P>0.05；n＝11。

Claims (3)

1.下式的Glu-Asp-Gly修饰的甲氨蝶呤,式中R1为Glu-Asp-Gly时R2为OH,R1为OH时R2为Glu-Asp-Gly,以及R1和R2同时为Glu-Asp-Gly,
Figure FDA0002098805600000011
2.权利要求1的Glu-Asp-Gly修饰的甲氨蝶呤的制备方法,该方法包括:
2.1 采用二环己基碳二亚胺为缩合剂,N-羟基苯并三唑为催化剂,液相缩合合成Boc-Glu(OBzl)-Asp(OBzl)-Gly-OBzl；
2.2 脱除Boc合成Glu(OBzl)-Asp(OBzl)-Gly-OBzl；
2.3 采用二环己基碳二亚胺为缩合剂,N-羟基苯并三唑为催化剂,将甲氨蝶呤与Glu(OBzl)-Asp(OBzl)-Gly-OBzl偶联生成如下通式的Glu(OBzl)-Asp(OBzl)-Gly-OBzl修饰的甲氨蝶呤；
2.4 在碱性条件下脱除保护基生成权利要求书1的Glu-Asp-Gly修饰的甲氨蝶呤。
3.权利要求1的Glu-Asp-Gly修饰的甲氨蝶呤在制备抗肿瘤转移药物中的应用。
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