CN107474109A - 4’-氧乙酰-apak-5-羟基-7-氧乙酰-rgdv-异黄酮,其合成,活性和应用 - Google Patents
4’-氧乙酰-apak-5-羟基-7-氧乙酰-rgdv-异黄酮,其合成,活性和应用 Download PDFInfo
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- CN107474109A CN107474109A CN201610404649.9A CN201610404649A CN107474109A CN 107474109 A CN107474109 A CN 107474109A CN 201610404649 A CN201610404649 A CN 201610404649A CN 107474109 A CN107474109 A CN 107474109A
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- China
- Prior art keywords
- ala
- oxygen acetyl
- isoflavones
- lys
- pro
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- Granted
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1019—Tetrapeptides with the first amino acid being basic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了4’‑氧乙酰‑Ala‑Pro‑Ala‑Lys‑5‑羟基‑7‑氧乙酰‑Arg‑Gly‑Asp‑Val‑异黄酮,公开了它的制备方法,公开了它的抗血栓活性,公开了它的溶血栓活性,公开了它的治疗脑血栓活性,公开了它的清除自由基活性,因而本发明公开了它在制备抗血栓药物,溶血栓药物,治疗脑血栓药物和清除自由基药物中的应用。
Description
技术领域
本发明涉及4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮,涉及它的抗血栓活性及涉及它的溶血栓活性,涉及它的治疗脑血栓活性,涉及它清除自由基活性,因而本发明涉及它在制备抗血栓药物,溶血栓药物,治疗脑血栓药物和清除自由基药物中的应用。本发明属于生物医药领域。
背景技术
缺血性中风是一类较常见且危害严重的脑血管疾病,特点是发病率高、病死率高、致残率高和复发率高。目前临床治疗缺血性中风面临没有有效药物的现实,尤其中风面4h以上的患者非死即残。发明对中风面4小时以上的患者有效的药物是临床的重要需求。本发明设定的基本目标之一就是为缺血性中风发明新型化合物,突破缺血性中风4小时黄金治疗时间窗口,帮助缺血性中风的临床治疗走出困境。为了达到这个目标,发明人经历了艰苦的发明过程。发明人的第一个发明是涉及P6A相关的溶血栓化合物,它们大约于1996年获得国家专利授权。因为溶血栓活性不等于治疗缺血性中风活性,所以这个专利中与P6A相关的溶血栓化合物没有评价出治疗缺血性中风功能。发明人的第二个发明涉及RGD-四肽与P6A相关的溶血栓化合物缀合形成抗血栓化合物。尽管在2.5μmol/kg剂量下RGDF具有抗血栓活性,但是在2.5μmol/kg剂量下P6A-RGDF和QP6A-RGDF却没有抗血栓活性。尽管在5μmol/kg剂量下没有抗血栓活性的RGDS和RGDV与P6A和QP6A的缀合物P6A-RGDS、P6A-RGDV、QP6A-RGDS和QP6A-RGDV在2.5μmol/kg剂量下却有抗血栓活性。尽管10μmol/kg P6A和QP6A都有溶血栓活性,但是P6A-RGDF、QP6A-RGDF、P6A-RGDS、P6A-RGDV、QP6A-RGDS和QP6A-RGDV实质上都没有溶血栓活性。可见,药效团组合并不是总能导致期望的发明。发明人的第三个发明是将PAK序列(ARPAK、GRPAK、QRPAK)与1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉缀合,创造具有清除自由基和溶血栓双重功能的缀合物。在10μmol/kg剂量下1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉-ARPAK、1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉-GRPAK和1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉-QRPAK显示溶血栓活性。它们的活性明显低于剂量为20000IU/kg的UK的溶血栓活性,并只与ARPAK、GRPAK和QRPAK的活性相当。1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉-ARPAK、1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉 -GRPAK和1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉-QRPAK在ESR仪(即顺磁共振仪)上可以测到清除O·自由基、·OH自由基和NO·自由基活性,但是在国际公认的评价模型(对乙酰胆碱舒张的大鼠主动脉条的抑制实验)上它们不能拮抗乙酰胆碱诱导的大鼠主动脉条舒张。1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉-ARPAK、1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉-GRPAK和1,3-二氧基-2-[(4-氧乙酸)苯基]-4,4,5,5-四甲基咪唑啉-QRPAK最不理想的性质是没有治疗缺血性中风的作用。发明人曾经公开式II的咪唑啉化合物在中风面24h的大鼠缺血性中风模型上,显示优秀疗效。即连续静脉注射6天式II的咪唑啉化合物,每天1次,首次剂量为5μmol/kg,后5次的剂量为2μmol/kg具有优秀疗效。式中aa1和aa2可为同时存在,aa1存在但aa2不存在,或同时不存在;当aa1和aa2同时存在时,aa1为R(Arg),且aa2为G(Gly),A(Ala)或Q(Gln);当aa1存在但aa2不存在时,aa1为R(Arg);aa3可为S(Ser),V(Val)或F(Phe)。由于式II的咪唑啉化合物的2-位是4-氧乙酰-Lys。而该Lys的侧链氨基和主链羧基分别与RGD抗血栓四肽及ARPAK溶栓肽相连接,所以结构比较复杂需要简化。
发明人经过3年实验研究,发现异黄酮的4’位用氧乙酰-Ala-Pro-Ala-Lys取代和7位用氧乙酰-Arg-Gly-Asp-Val取代可以获得治疗缺血性中风的有效药物。对于异黄酮的结构修饰来说,这是意想不到的技术效果。按照这个发现,发明人提出了本发明。
发明内容
本发明的第一个内容是提供4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮。
本发明的第二个内容是提供4’-氧乙酰APAK-5-羟基-7-氧乙酰RGDV-异黄酮的合成方法,该方法包括:
(1)在K2CO3的催化下按标准方法制备4’,7-二氧乙酸甲酯-异黄酮(1);
(2)在2N NaOH作用下4’,7-二氧乙酸甲酯-异黄酮(1)皂化为4’,7-二氧乙酸-异黄酮(2);
(3)4’,7-二氧乙酸-异黄酮(2)与Ala-Pro-Ala-Lys(Z)-OBzl反应制备4’-氧乙酰-APAK(Z)-OBzl-5-羟基-7-氧乙酸-异黄酮(3);
(4)4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酸-异黄酮(3)与HCl·Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl反应制备4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酰-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl-异黄酮(4);
(5)4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酰-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl-异黄酮(4)在三氟醋酸,三氟甲磺酸作用下制备4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)。
本发明的第三个内容是评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮抗动脉血栓作用。
本发明的第四个内容是评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮的抗深静脉血栓的活性。
本发明的第五个内容是评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮溶血栓作用。
本发明的第六个内容是评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮治疗脑血栓作用。
本发明的第七个内容是评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮清除自由基作用。
附图说明
图1 为4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮的合成路线.i)BrCH2COOC2H5,K2CO3,THF ii)2N NaOH,MeOH iii)DCC,DMAP,DMF,NMM iv)DCC,DMAP,DMF,NMM v)TFA,TFMSA。
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4’,7-二氧乙酸甲酯-异黄酮(1)
将11.69g(43.3mmol)染料木素溶于200mL四氢呋喃(THF),向反应液中加入46.92g(340mmol)碳酸钾,活化20分钟后加入14mL(87mmol)溴乙酸乙酯,在45℃油浴下加热 反应5天。TLC(二氯甲烷:甲醇=20:1)监测反应进程。反应完全后,将反应液沉降过滤,滤液减压旋除THF,加入大量石油醚磨洗以除去过量的溴乙酸乙酯,得到的固体加入甲醇进行溶解,发现微溶,于60℃加热,趁热过滤,固体晾干,滤液进行冷热重结晶,过滤,收集白色固体,得到11.70g(61.1%)标题化合物。ESI--MS(m/e):413[M-H]-。
实施例2制备4’,7-二氧乙酸-异黄酮(2)
取5.17g(12.5mmol)4’,7-二氧乙酸甲酯-异黄酮(1)加入100mL甲醇,微溶,向反应液中滴加2N NaOH水溶液,调节溶液pH值为12,固体逐渐溶解,室温反应4小时后,TLC(二氯甲烷:甲醇:冰醋酸=20:1:2滴)监测反应进程,原料点消失,加入饱和硫酸氢钾溶液调节溶液pH值至1-2后析出大量白色固体,加入大量水溶解盐,而后过滤,并且滤饼反复用水冲洗,白色固体晾干收集,得到4.10g(84.2%)标题化合物。ESI--MS(m/e):385[M-H]-。
实施例3制备4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酸-异黄酮(3)
将1.09g(2.8mmol)4’,7-二氧乙酸-异黄酮(2)溶于80mL无水DMF,分别加入80mg(0.66mmol)DMAP和0.64g(3.1mmol)DCC,活化20分钟后,用40mL无水DMF溶解2.13g(2.8mmol)HCl·Ala-Pro-Ala-Lys(Z)-OBzl,冰浴下用NMM调节pH值至8-9,加入到反应液中,并且用NMM调节pH值至8-9,反应18小时后,吹干DMF,二氯甲烷溶解,过滤,滤液进行柱层析分离纯化(二氯甲烷/甲醇),得到1.71g(59.7%)标题化合物。ESI--MS(m/e):976[M-H]-。
实施例4制备4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酰-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl-异黄酮(4)
将1.59g(4.1mmol)4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酸-异黄酮(3)溶于80mL无水DMF,分别加入100mg(0.82mmol)DMAP和1.02g(1.79mmol)DCC,活化20分钟后,用40mL无水DMF溶解1.06g(1.79mmol)HCl·R(NO2)-G-D(OBzl)-V-OBzl,冰浴下用NMM调节pH值至8-9,加入到反应液中,并且用NMM调节pH值至8-9,反应36小时后,吹干DMF,二氯甲烷溶解,过滤,滤液进行柱层析分离纯化(二氯甲烷/甲醇),得到1.43g(52.9%)标题化合物。ESI+-MS(m/e):1630[M+H]+。
实施例5制备4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)
将398mg(0.24mmol)4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酰-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl-异黄酮(4)加入100mL反应瓶中,冰浴下向反应瓶中加入4mL三氟醋酸使其溶解,而后加入1mL三氟甲磺酸,反应30分钟,向反应瓶中加入大量乙醚,搅拌20分钟,而后静置15分钟,倾倒出上清液,再加入大量乙醚搅拌,重复上述操作3遍,最后将剩余的少量乙醚抽干成粉末。加入10mL三蒸水溶解粉末,使用氨水调节溶 液pH值至7,3000转/min离心10分钟,取上清液通过Sephadex G15除盐,水溶液进行冷冻干燥,得到的冻干粉末经过制备液相分离,纯化得12mg(4.3%)标题化合物。ESI--MS(m/e):1179[M-H]-;Mp:192.5~193.5℃;[α]D 25=-20.0(c=0.12,H2O);IR(cm-1):3300,3076,2942,2867,1643,1512,1444,1373,1243,1162,1029,878,835,637;1H-NMR(800MHz,DMSO-d6):δ/ppm=8.764~8.073(m,5H),7.994(s,1H),7.846~7.654(m,4H),7.535(s,2H),7.281(d,J=8.8Hz,2H),6.786(d,J=8.0Hz,2H),4.798(s,2H),4.648(s,2H),4.484(m,2H),4.369(m,2H),4.268(m,1H),4.250(m,1H),4.109(m,2H),3.788(m,2H),2.176~1.449(m,18H),1.288(d,J=5.6Hz,6H),1.107(m,2H),0.818(d,J=5.6Hz,6H)。
实施例6评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的抗动脉血栓活性
1)将聚乙烯管拉成一端为斜口的细管,定长为10.0cm,分别为右颈静脉(管径较粗)及左颈动脉(管径较细)插管;中段聚乙烯管定长为8.0cm,血栓线压在颈动脉插管方向,插管前需在管中充满肝素。
2)将体重200±20g雄性大鼠口服167μmol/kg阿司匹林或3mL/kg生理盐水30分钟后,腹腔注射20%的乌拉坦麻醉(7mL/kg)。仰卧位将大鼠固定于鼠板上,剪开颈部皮肤,分离右颈总动脉及左颈静脉,血管下压线,结扎远心端,静脉靠远心端处剪一小口,进行静脉端插管,注射肝素,而后取下注射肝素的注射器,换上装有4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的注射器,注射剂量为50nmol/kg,系线固定,再用动脉夹夹住动脉近心端,靠近远心端方向剪一小口,进行动脉端结扎,系线固定后松开动脉夹,建立体外循环旁路。循环15分钟后先剪断静脉端观察血液循环是否正常,若正常从动脉端取出血栓线,在纸上沾干浮血后称重,以栓重表示化合物的活性,数据列入表1。结果表明,4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)治疗大鼠的血栓重明显小于生理盐水治疗大鼠的血栓重,显示确切的抗血栓活性。有效剂量为50nmol/kg,获得了意想不到的技术效果。
表1 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的抗动脉血栓活性
n=8;a)与生理盐水比p<0.01.
实施例7评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的抗深静脉血栓活性
将体重250±20g雄性大鼠口服1.5mg/kg华法林或3mL/kg生理盐水,30分钟后,腹腔注射20%的乌拉坦进行麻醉(7mL/kg)。鼠尾静脉给50nmol/kg的4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5),用20%乌拉坦的生理盐水溶液进行麻醉,之后进行手术。固定大鼠,沿腹白线切开进腹,并将切口周围用由生理盐水润湿的纱布盖住,漏出切口,将大鼠腹内肠及相关脏器拉出置于纱布上,可见下腔静脉,将下腔静脉上的组织钝性分开,暴露出完整地静脉,分离下腔静脉,于左肾静脉与下腔静脉交汇处用手术线结扎,将脏器归位并缝合腹壁,4小时后剪开腹壁,分别分离下腔静脉的三到四根分支静脉,用手术线结扎,并用动脉夹夹住结扎处稍后部分,夹闭血管,用弯剪于结扎处和动脉夹夹住处中间部位剪断全部分支血管,用止血钳夹住下腔静脉远心端分支处,用止血钳夹住下腔静脉近心端,提起结扎线,用弯剪贴着止血钳剪断下腔静脉近心端,慢慢向远心端剪,避免剪破下腔静脉,直至远心端分支处,沿远心端止血钳剪断下腔静脉血管,用弯镊由下腔静脉近心端向远心端挤出血栓,称量血栓湿重并记录,统计每组的血栓湿重(均值±SD),用统计学方法作t检验,以生理盐水(3mL/kg)作空白对照,以华法林(1.5mg/kg,3mL/kg)作阳性对照。以栓重表示化合物的活性,数据列入表2。结果表明,4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)治疗大鼠的血栓重明显小于生理盐水治疗大鼠的血栓重,显示确切的抗深静脉血栓活性。有效剂量为50nmol/kg,获得了意想不到的技术效果。
表2 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的抗深静脉血栓的活性
n=8;a)与生理盐水相比,p<0.01.
实施例8评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的溶栓活性
将大鼠静息饲养一天,随机分组,每组10只,禁食不禁水。
1)将体重250±20g雄性SD大鼠用20%乌拉坦溶液进行麻醉(7mL/kg腹腔)。麻醉后的大鼠固定于鼠板上,分离右侧颈总动脉,于近心端夹动脉夹,近心端和远心端分别传入手术 线,在远心端插取血管,松开动脉夹取约1mL动脉血。迅速将动脉血注射入垂直的造栓管(长16mm,内径2.5mm,外径5mm,管底用1mL EP管底座)中,(注意不可有气泡)每个造栓管中注入0.1mL大鼠动脉血液,往管内迅速插入血栓固定螺栓(长20mm),血液凝固40min,用针灸针取出血栓,称取血栓重量。
2)旁路插管由三部分组成,其中中段为聚乙烯胶管,长60mm,内径3.5mm,两端为相同的聚乙烯管,长100mm,内径1mm,外景2mm,该管的一段拉成尖管,另一端的外部套一段长7mm,外径3.5mm的聚乙烯管。三段管的内壁均为硅烷化。
3)将血栓包裹的血栓固定螺栓置于中段的聚乙烯胶管内,胶管的两端分别与两根聚乙烯的加粗端相套,用注射器通过尖管端注满肝素生理盐水溶液。将充满肝素钠的生理盐水溶液一端插入左侧静脉,另一端用注射器加入准确量的肝素钠抗凝后,拔掉肝素钠的注射器,插入动脉端。用头皮针将生理盐水(3mL/kg)或者尿激酶的生理盐水溶液(20000IU/kg)或4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的生理盐水溶液(剂量为50nmol/kg)通过旁路管的中段,刺入远离血栓固定螺栓的近静脉处,打开动脉夹,使血流通过旁路管道从动脉流入静脉的时刻为循环起始时间,缓慢的将注射器中的液体注入到血液中(约6min),使生理盐水,尿激酶,4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)通过血液循环,按静脉-心脏-动脉的顺序作用到血栓上。60min后取出附有血栓的螺栓,蘸去浮血,记录重量。以血栓减重代表溶栓活性。数据列入表3。结果表明,4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)治疗大鼠的血栓减重明显大于生理盐水治疗大鼠的血栓减重,显示确切的溶血栓活性。有效剂量为50nmol/kg,获得了意想不到的技术效果。
表3 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的溶栓活性
n=8;a)与生理盐水比p<0.01.
实施例9评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的治疗脑血栓活性
SD雄性大鼠(300±20g)用10%水合氯醛的生理盐水溶液(400mg/kg)腹腔注射麻醉,从颈部正中稍偏右部竖直剪一小口,沿胸锁乳突肌内侧缘分离到右颈总动脉、颈外动脉 及颈内动脉。将事先准备好的血栓加入1mL生理盐水用钢铲把血液凝块捣成大小均一的细小血栓块,然后把细小血栓块混悬液转移至1mL注射器内备用。在松开大鼠颈内动脉夹的同时,将1mL注射器内的血栓块混悬液缓慢从大鼠颈外动脉向近心端途经颈内动脉注入到大鼠大脑,然后结扎颈外动脉的近心端,依次打开颈内动脉和颈总动脉处的动脉夹,恢复血流,缝合伤口。给予青霉素(40mg/10mL)1mL苏醒24h后,将大鼠进行神经生物学评分后分组,保证每组中有不同评分的大鼠,然后鼠尾静脉给4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5),连续给药六天。
所有实验用大鼠全部做脑缺血手术,24小时后将存活的大鼠评分,并进行分组。按照Zealonga方法评定神经功能缺损程度。0分表示无任何神经功能缺失体征,1分表示未损伤侧前肢不能伸展,2分表示向未损伤侧行走,3分表示向未损伤侧转圈成追尾状行走,4分表示意识障碍无自主行走,5分表示死亡。将不同评分的大鼠平均分配到各组中,构成缺血程度不同的样本组,每组至少10只,连续7天对各组大鼠进行评分,结果见表4-表7.
生理盐水组治疗前4分的大鼠有1只,占总比例的7.69%;治疗前3分的大鼠有1只,占总比例的7.69%;2分的大鼠有3只,占总比例的23.08%;1分的大鼠有8只,占总比例的61.54%,连续治疗7天之后意识障碍无自主行走的大鼠有1只,评分为4分,占总比例的7.69%,侧转圈成追尾状行走的大鼠有2只,评分为3分,占总比例的15.38%;侧行走的大鼠有7只,评分为2分,占总比例的53.85%;自助行走恢复的大鼠有3只,评分为1分,占总比例的23.08%。脑缺血整体状况变坏。
4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)(50nmol/kg)治疗前4分的大鼠有1只,占总比例的9.09%;治疗前3分的大鼠有1只,占总比例的9.09%;2分的大鼠有5只,占总比例的45.45%;1分的大鼠有4只,占总比例的36.36%;连续治疗7天之后侧行走的大鼠有1只,评分为2分,占总比例的9.09%;90.91%的大鼠自助行走恢复,1分的大鼠有2只,占总比例的18.18%,0分的大鼠有8只,占总比例的72.73%。脑缺血整体状况变好。获得了意想不到的技术效果。
4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)(5nmol/kg)治疗前4分的大鼠有1只,占总比例的10.00%;治疗前3分的大鼠有2只,占总比例的20.00%;2分的大鼠有5只,占总比例的50.00%;1分的大鼠有2只,占总比例的20.00%;连续治疗7天之后侧行走的大鼠有3只,评分为2分,占总比例的33.33%;66.67%的大鼠自助行走恢复,1分的大鼠有4只,占总比例的44.44%,0分的大鼠有2只,占总比例的22.22%。
4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)(0.5nmol/kg)治疗前4分的大鼠有1只,占总比例的10.00%;治疗前3分的大鼠有1只,占总比例的10.00%; 2分的大鼠有6只,占总比例的60.00%;1分的大鼠有2只,占总比例的20.00%;连续治疗7天之后侧转圈成追尾状行走的大鼠有1只,评分为3分,占总比例的11.11%;侧行走的大鼠有5只,评分为2分,占总比例的55.56%;33.33%的大鼠自助行走恢复,评分为1分。
表4生理盐水连续治疗6天脑缺血24小时大鼠的神经生物学评分
n=13,剂量:3mL/kg
表5 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)连续治疗6天脑缺血24小时大鼠神经生物学评分
n=11,剂量:50nmol/kg
表6 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)连续治疗6天脑缺血24小时大鼠神经生物学评分
n=10,剂量:5nmol/kg
表7 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)连续治疗6天脑缺血24小时大鼠神经生物学评分
n=10,剂量:0.5nmol/kg
按照Zealonga方法评定神经功能缺损程度之后,用乌拉坦麻醉后断头取脑,将脑组织置于-20℃冰箱2小时后,使大脑完全冻住,从前额极开始行约2mm冠状连续切片,共6片,然后置于2%的2,3,5—氯化三苯基四氮唑(TTC)溶液中于37℃避光孵育30min后,观察脑切片的颜色变化,正常组织会被TTC染成红色,而缺血部分组织呈白色。用数码相机照相,记录原始数据,经SPSS统计软件处理,计算冠状切片中梗死体积和正常组织的面积,统计各组大鼠大脑的梗死体积百分比值,并做t检验。以脑梗死体积(%)表示化合物的活性,数据列入表8。结果表明,4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)治疗大鼠的脑梗死体积(%)明显小于生理盐水治疗大鼠的脑梗死体积(%),脑梗死脑体积比显著下降。获得了意想不到的技术效果。且4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的活性随着浓度的降低而降低,因此具有剂量和效应的依赖关系。
表8 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)治疗6天缺血24小时大鼠的脑梗死体积比
n=8;a)与生理盐水相比p<0.01;b)与生理盐水相比p<0.01,且与50nmol/kg剂量下的5相比,p<0.05;c)与生理盐水相比,p>0.05.
实施例10评价4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮的体外清除自由基活性
1)体外清除OH·自由基
配制二甲基吡啶N-氧化物(DMPO)溶液:1.1mg DMPO溶解在1mL蒸馏水中。
配制FeSO4溶液:2.8mg FeSO4·7H2O溶解于1mL蒸馏水中。
配制H2O2溶液:医用30%H2O2溶液稀释到1%。
测定方法:5μL FeSO4溶液+5μL DMPO溶液+5μL 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)溶液或水+5μL H2O2,依次加入。先测定OH·自由基基础峰高度,作为空白对照,再测定加入4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰 -Arg-Gly-Asp-Val-异黄酮(5)溶液后的峰高度。
清除率=(基础·OH信号峰高度-加入4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)后·OH信号峰高度)/基础·OH信号峰高度×100%
2)体外清除NO·自由基活性
配制N-甲基-葡萄糖胺二硫代甲酸(MGD)溶液:7.3mg MGD溶解在1mL蒸馏水中。
配制FeSO4溶液:3.5mg FeSO4·7H2O溶解与1mL蒸馏水中。
配制亚硝基乙酰青霉胺(SNAP)溶液:2.5mg SNAP溶于1mL蒸馏水,稀释100倍。
测定方法:5.0μL MGD+5.0μL FeSO4溶液+5μL 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)溶液或水+5.0μL SNAP溶液。先测定NO·自由基基础峰高度,作为空白高度,再测定加入4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)后的峰高度。
清除率=(基础NO·信号峰高度-加入4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)后NO·信号峰高度)/基础NO·信号峰高度×100%。计算半数有效清除率(EC50).
表9 4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)的清除OH·、NO·自由基活性
Claims (7)
1.下式的4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮
2.制备权利要求1的4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮的方法,该方法包括:
(1)在K2CO3的催化下按标准方法制备4’,7-二氧乙酸甲酯-异黄酮(1);
(2)在2N NaOH作用下4’,7-二氧乙酸甲酯-异黄酮(1)皂化为4’,7-二氧乙酸-异黄酮(2);
(3)4’,7-二氧乙酸-异黄酮(2)与Ala-Pro-Ala-Lys(Z)-OBzl反应制备4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酸-异黄酮(3);
(4)4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酸-异黄酮(3)与HCl·Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl反应制备4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酰-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl-异黄酮(4);
(5)4’-氧乙酰-Ala-Pro-Ala-Lys(Z)-OBzl-5-羟基-7-氧乙酰-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl-异黄酮(4)在三氟醋酸,三氟甲磺酸作用下制备4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮(5)。
3.权利要求1的4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮在制备抗栓药物中的应用。
4.权利要求1的4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮在制备抗深静脉血栓药物中的应用。
5.权利要求1的4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮在制备溶栓药物中的应用。
6.权利要求1的4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮在制备治疗脑血栓药物中的应用。
7.权利要求1的4’-氧乙酰-Ala-Pro-Ala-Lys-5-羟基-7-氧乙酰-Arg-Gly-Asp-Val-异黄酮在制备清除自由基药物中的应用。
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