CN117466929A - 一种新的半胱氨酸衍生物及其在制备环肽中的应用 - Google Patents
一种新的半胱氨酸衍生物及其在制备环肽中的应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/0803—Compounds with Si-C or Si-Si linkages
- C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/0803—Compounds with Si-C or Si-Si linkages
- C07F7/0825—Preparations of compounds not comprising Si-Si or Si-cyano linkages
- C07F7/083—Syntheses without formation of a Si-C bond
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及医药合成领域,公开了一种新的半胱氨酸衍生物及其在制备环肽中的应用。本发明所述新的半胱氨酸衍生物,用于制备构象更稳定的环肽。
Description
技术领域
本发明涉及一种新的半胱氨酸衍生物及其在制备环肽中的应用。
背景技术
订书肽是一种化学修饰环肽,是基于多肽需形成α-螺旋通过细胞膜进入细胞的需求上发展起来的。生物体内的多种生命进程调节都是通过蛋白质与蛋白质之间的相互作用来实现的。例如病毒的自组装,细胞的生长,分裂,分化等过程。而通常蛋白-蛋白相互作用的界面太大,从而使小分子药物很难对其进行靶向定位,达到高效特异性地阻断这种相互作用,展现良好的治疗效果。蛋白类药物因为很难顺利通过细胞膜所以也达不到直接靶向细胞内相互作用的效果,因此,研究者们开始寻求一种能够克服这两种药物缺点的既能够进入细胞膜又能特异性靶向蛋白-蛋白相互作用的新的药物分子。
研究表明,具有α-螺旋结构和富含正电荷的多肽可以穿过细胞膜。因此,人们开发了利用二硫键与分子内酰胺键作为支架的α-螺旋结构,但是,这些支架在生理环境下均不能稳定存在。2000年,Verdine等发展了一种用碳碳键作为支架来稳定多肽α-螺旋结构的方法,由此方法得到的多肽成为订书肽(Stapled peptides)。订书肽有更高的α-螺旋程度,亲和力更强,能通过细胞膜,难被蛋白酶水解,在生物体内半衰期长等优点。
订书肽常规合成策略为在固相合成肽链过程中引入两个含有α-甲基,α-烯基的非天然氨基酸,然后两个非天然氨基酸之间发生烯烃复分解反应(RCM)环化构成稳定α-螺旋结构构象的全碳支架,从而合成订书肽。由于烯烃复分解反应收率低,同时需要采用氢化加氢工艺,所以严重影响了产品收率。
本发明提供了一种新的半胱氨酸衍生物,为订书肽提供了一种新的解决方案,同时可以大幅度提高环肽的收率,更适合规模化生产。
发明内容
本发明首先提供了一种新的半胱氨酸衍生物。
结构I中的半胱氨酸为L-半胱氨酸,或为D-半胱氨酸。
结构I中的m1为2至10的整数;
结构I中的m2为2至10的整数;
结构I中的R为Teoc,或为Troc;
上述的氨基酸衍生物,用于制备稳定构象的环肽。
具体实施方式
本发明公开了一种新的氨基酸衍生物及其在制备环肽中的应用,本领域技术人员可以借鉴本文内容,适当改进相关参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的的化合物和制备方法进行改动或适当变更与组合,来实现和应用本发明技术。
本发明中涉及的英文缩写所对应的中文名称见下表所示:
英文缩写 | 中文名称 | 英文缩写 | 中文名称 |
Fmoc | 9-芴甲氧羰基 | OAll | 丙烯氧基 |
tBu | 叔丁基 | Boc | 叔丁氧羰酰基 |
Teoc | 2-(三甲基甲硅烷基)乙氧基羰基 | Troc | 2,2,2-三氯乙氧基羰基 |
实施例1化合物1的制备
化学反应式如下:
1、中间体1的制备
取0.8mol原料1和1mol原料2,用适量DMF溶解,加入1mol吡啶,60℃搅拌反应过夜,蒸干DMF,残留物用2L 10%TFA/DCM溶液溶解并搅拌反应1小时,蒸干TFA/DCM溶液,残留物用乙酸乙酯溶解,滤过去残渣,滤液并用饱和食盐水洗涤6次,蒸干滤液得中间体1。
2、中间体2的制备
中间体1用60%二氧六烷水溶液溶解,加入1molNaHCO3的水溶液,搅拌下加入1mol的Teoc-OSu,室温搅拌反应6小时,蒸干溶剂,残留物用乙酸乙酯提取,用饱和食盐水洗涤5次,有机相用无水硫酸钠干燥,过滤收集有机相,蒸干溶剂得中间体2。
3、中间体3的制备
中间体2用乙酸乙酯溶解,滤过去残渣,滤液并用饱和食盐水和10%碳酸钠溶液交替洗涤6次,蒸干滤液得中间体3。
4、中间体4的制备
中间体3用DCM溶解,加入取1mol溴代乙酸和1mol DIC,搅拌反应6小时,蒸干溶剂,再加入DCM溶解溶解后再蒸干,重复3次,得中间体4。
5、中间体5的制备
中间体4用50%四氢呋喃水溶液溶解,搅拌下加入1mol半胱氨酸,搅拌下加入碳酸钠溶液,维持PH8.0,继续搅拌反应3小时,用10%盐酸溶液调PH4.0,蒸干溶剂,残渣用乙酸乙酯溶解,抱和食盐水洗涤6次,蒸干拥挤得中间体5。
6、化合物1的制备
中间体5用50%四氢呋喃水溶液溶解,加入1molNaHCO3的水溶液,搅拌下加入1mol的Fmoc-OSu,室温搅拌反应6小时,用1%盐水调PH=3.5,过滤的粗品,再用正己烷-乙酸乙酯重结晶的化合物1。
实施例2其他化合物制备
用上述方法制备了以下化合物:
实施例3环肽AOD243907的制备
制备方法,包括:采用固相多肽合成法制备肽树脂,肽树脂再经酸解得到粗品,最后粗品经过纯化得到纯品。
1、肽树脂的合成
使用Rink Amide BHHA树脂为载体树脂,通过去Fmoc保护和偶联反应,依次与多肽序列对应的保护氨基酸偶联,制得肽树脂。
(1)接入主链第1个保护氨基酸
取0.03mol第1个保护氨基酸和0.03mol HOBt,用适量DMF溶解;另取0.03mol DIC,搅拌下慢慢加入至保护氨基酸DMF溶液中,于室温环境中搅拌反应30分钟,得到活化后的保护氨基酸溶液,备用。
取0.01mol的Rink amide MBHA树脂(取代值约0.4mmol/g),采用20%PIP/DMF溶液去保护25分钟,洗涤过滤得到去Fmoc的树脂。
将活化后的第1个保护氨基酸溶液加入到已去Fmoc的树脂中,偶联反应60~300分钟,过滤洗涤,得含1个保护氨基酸的树脂。
(2)接入主链保护氨基酸
采用上述接入主链第1个保护氨基酸同样方法,依次接入相应多肽序列对应的保护氨基酸,得含主链氨基酸的树脂。
第1位Tyr对应的保护氨基酸为Boc-Tyr(tBu)。
第16位Cys对应的保护氨基酸为Fmoc-Cys(S-乙酸烯丙酯)。
第20位Cys对应的保护氨基酸为化合物1。
(3)环化
取5mmol四三苯基膦钯和50mmol苯硅烷,用适量二氯甲烷溶解,将含主链氨基酸的树脂去Alloc和All保护基8小时,过滤洗涤,得到去Alloc和All的树脂备用。
取0.03mol HOBt,用适量DMF溶解;另取0.03mol DIC,用适量DMF溶解;搅拌下加入到已去Alloc和All的树脂,偶联反应60~300分钟,过滤洗涤,得到环化的树脂。
(4)接入侧链保护氨基酸或单保护脂肪酸
取上述环化的树脂,采用1mol/L四丁基氟化胺/四氢呋喃溶液去保护36小时,洗涤过滤得到去Teoc的树脂。
采用上述接入主链第1个保护氨基酸同样方法,依次接入侧链对应的保护氨基酸和单保护脂肪酸,得到肽树脂。
2、粗品的制备
取上述肽树脂,加入体积比为TFA︰水︰EDT=95︰5︰5的裂解试剂(裂解试剂10mL/克树脂),搅拌均匀,室温搅拌反应3小时,反应混合物使用砂芯漏斗过滤,收集滤液,树脂再用少量TFA洗涤3次,合并滤液后减压浓缩,加入无水乙醚沉淀,再用无水乙醚洗沉淀3次,抽干得类白色粉末即为粗品。
3、纯品的制备
取上述粗品,加水搅拌,用氨水调pH8.0至完全溶解,溶液用0.45μm混合微孔滤膜过滤,纯化备用;
采用高效液相色谱法进行纯化,纯化用色谱填料为10μm的反相C18,流动相系统为0.1%TFA/水溶液-0.1%TFA/乙腈溶液,30mm*250mm的色谱柱流速为20mL/min,采用梯度系统洗脱,循环进样纯化,取粗品溶液上样于色谱柱中,启动流动相洗脱,收集主峰蒸去乙腈后,得纯化中间体浓缩液。
纯化中间体浓缩液用0.45μm滤膜滤过备用,采用高效液相色谱法进行换盐,流动相系统为1%醋酸/水溶液-乙腈,纯化用色谱填料为10μm的反相C18,30mm*250mm的色谱柱流速为20mL/min(可根据不同规格的色谱柱,调整相应的流速);采用梯度洗脱,循环上样方法,上样于色谱柱中,启动流动相洗脱,采集图谱,观测吸收度的变化,收集换盐主峰并用分析液相检测纯度,合并换盐主峰溶液,减压浓缩,得到纯品醋酸水溶液,冷冻干燥得纯肽。
实施例4环肽AOD243907GLP-1活性的测定
1、测定方法
GLP-1R在其特异性的激动剂的刺激下,能激活细胞内腺苷酸环化酶通路,升高cAMP水平,最终导致胰岛素的生成和释放。通过待测物刺激稳定转染了GLP-1R的细胞株,使细胞胞内cAMP水平迅速升高,通过化学发光方法测定各剂量刺激细胞后的相对光单位(RLU),进而计算出激动剂的EC50,该活性测定方法是目前国内外通用的GLP-1受体激动剂活性检测方法。
采用稳定表达GLP-1R的CHO-K1细胞株,用不同浓度的激动剂刺激稳转细胞,通过测定各剂量刺激后细胞后的相对光单位,计算得到激动剂的EC50值。
2、测定结果
测定结果见下表:
化合物 | GLP-1活性【EC50(pmol)】 |
Tirzepatide | 140.1 |
AOD243907 | 55.7 |
实施例3环肽AOD243907GIP活性的测定
1、测定方法
GIPR在其特异性的激动剂的刺激下,能激活细胞内腺苷酸环化酶通路,升高cAMP水平,最终导致胰岛素的生成和释放。通过待测物刺激稳定转染了GIPR的细胞株,使细胞胞内cAMP水平迅速升高,通过化学发光方法测定各剂量刺激细胞后的相对光单位(RLU),进而计算出激动剂的EC50,该活性测定方法是目前国内外通用的GIP受体激动剂活性检测方法。
采用稳定表达GIPR的CHO-K1细胞株,用不同浓度的激动剂刺激稳转细胞,通过测定各剂量刺激后细胞后的相对光单位,计算得到激动剂的EC50值。
2、测定结果
测定结果见下表:
化合物 | GIP活性【EC50(pmol)】 |
Tirzepatide | 45.0 |
AOD243907 | 39.8 |
Claims (2)
1.一种具有结构式Ⅰ的一种新的半胱氨酸衍生物。
结构I中的半胱氨酸为L-半胱氨酸,或为D-半胱氨酸。
结构I中的m1为2至10的整数;
结构I中的m2为2至10的整数;
结构I中的R为Teoc,或为Troc。
2.根据权利要求1所述的一种新的半胱氨酸衍生物,用于制备稳定构象的环肽。
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