CN114191564A - 一种非天然鹅膏毒肽类抗体偶联物 - Google Patents
一种非天然鹅膏毒肽类抗体偶联物 Download PDFInfo
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- CN114191564A CN114191564A CN202111502809.0A CN202111502809A CN114191564A CN 114191564 A CN114191564 A CN 114191564A CN 202111502809 A CN202111502809 A CN 202111502809A CN 114191564 A CN114191564 A CN 114191564A
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Abstract
本发明公开了一种非天然鹅膏毒肽类抗体偶联物,本发明将毒性与天然鹅膏毒肽类似的非天然鹅膏毒肽通过生物药学上可接受的连接结构与能与靶标结合的生物分子偶联,获得了在血浆中稳定,在细胞中能高效杀死肿瘤细胞的化合物。
Description
本申请是申请号为2018109463219、申请日为2018年8月18日、发明名称为“非天然鹅膏毒肽类抗体偶联物”的分案申请。
技术领域
本发明涉及一种二环八肽类衍生物,该类化合物以特殊的化学结构与相应的靶结合基团偶联,其结构在血浆中稳定而在特定的生物环境中裂解成为活性成份的药物,以达到对靶细胞杀伤性的最大化和对非靶细胞毒害副作用的最小化,可用于多种恶性肿瘤的治疗。
背景技术
鹅膏毒肽(amanitin)是由剧毒蘑菇中分离出来的鹅膏肽类毒素中的一种,为8个氨基酸组成的双环肽,已分离纯化的天然鹅膏毒肽有9种,分别是α-鹅膏毒肽、β-鹅膏毒肽、γ-鹅膏毒肽、ε-鹅膏毒肽、鹅膏素(amanin)、三羟毒伞肽酰胺(amaninamide)、鹅膏无毒环肽(amanullin)、一羟鹅膏毒肽羧酸(amanullinic acid)和前鹅膏无毒环肽(proamanullin),其中α-鹅膏毒肽和β-鹅膏毒肽是导致死亡的主要毒素。鹅膏毒肽是一类慢作用毒素,可抑制真核RNA聚合酶II和RNA聚合酶III的转录,导致蛋白质缺失和细胞死亡。该类毒素对RNA聚合酶II的抑制性极高,KD可达3nM,在肠胃道中因肠肝循环而反复在体内被吸收,对人体的肝肾心肺等器官能产生持续性的严重损害。
将鹅膏毒肽与大的生物分子载体(如抗体分子)偶联后,其毒性大大降低甚至相对无毒,仅在特定生理环境中生物分子载体被切除之后,鹅膏毒肽才会显现其细胞毒性。
根据Theodor Wieland团队的研究,当把天然鹅膏毒肽中的亚砜结构改为硫元素后,形成的非天然鹅膏毒肽对细胞的毒性变化不大。德国海德堡医药有限责任公司将天然鹅膏毒肽分子与单抗偶联,获得了具有抗肿瘤活性的药物分子。
本发明将毒性与天然鹅膏毒肽类似的非天然鹅膏毒肽通过生物药学上可接受的连接结构与能与靶标结合的生物分子偶联,获得了在血浆中稳定,在细胞中能高效杀死肿瘤细胞的化合物。
定义
按照本领域的标准惯例,杂(原子、烷基、芳基、环基),是指含有除碳原子以外的其它原子的相应化学结构。
发明内容
本发明提供了一种双环八肽类鹅膏毒肽衍生物和生物大分子的偶合物,它们在血液循环系统内稳定,被目标细胞内吞后被剪切,释放出作为RNA聚合酶抑制剂的鹅膏毒肽衍生物,并通过对真核生物mRNA合成的专一抑制产生对细胞的强烈毒性,具体为结构式(I)的毒素偶联物:
其中:
R1为H、-OH或-O-L-A;
R2为H、-OH;
R3为OC1~6烷基;
R4为H或-L-A;
R5为-NH2、-OH、-NH-L-A或-O-L-A。
其中O为氧原子,N为氮原子,H为氢原子。
A为与靶点结合的生物大分子部分,作为优选方式,所述L包含下列结构:
-L1-[L2]m-[AA]n-L3-
其中,L1为接头,连接生物大分子A;L2为间隔物,L2为连接L1和AA之间的片段,m为1-6,且m为整数;L3连接结构式(I)所示毒素;AA为含1~6个氨基酸组成的片段,n为0或1。
作为优选方式,所述A为与靶点结合的生物大分子部分,包括抗体或其抗原结合片段、抗体样蛋白、核酸配适体等。
作为优选方式,所述靶点结合的生物大分子为抗体或其抗原结合片段,选自嵌合抗体、去免疫抗体、人源化抗体、人类抗体、双体、四体和纳米抗体。
进一步优选,所述抗原结合片段选自自由Fab、F(ab’)、Fd、Fv、单链Fv和二硫键连接的Fv(dsFv)组成的组。
作为优选方式,所述R1、R4、R5中有且仅有一个含有-L-A结构。
作为优选方式,所述L1为接头,连接生物大分子A,选自:
作为优选方式,所述L2为间隔物,选自取代或未取代C1-C6烷基、取代或未取代C3-C20的环烷基、取代或未取代C3-C20的杂环烷基、取代或未取代C5-C20芳基、取代或未取代C5-C20杂芳基、-(CH2CH2O)a-(其中a为1~20,且为整数)等中一种或多种相同或不同的组合,且间隔物与间隔物之间通过任意的化学键相连。
进一步优选,所述取代基选自羟基、巯基、卤素、羧基、氨基、磷酸基、硝基、氰基、磺酸基、取代或未取代C1-C6烷基等中一种或多种相同或不同的组合。
进一步优选,所述取代基选自羟基、巯基、卤素、羧基、氨基、磷酸基、硝基、氰基、磺酸基等中一种或多种相同或不同的组合。
作为优选方式,AA为1~6个氨基酸组成的片段,所述氨基酸为L-氨基酸,选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸等。
进一步优选苯丙氨酸、瓜氨酸、缬氨酸、赖氨酸、丝氨酸、谷氨酸、天冬氨酸、甘氨酸。
作为优选方式,所述L3选自
其中,波浪线连接AA,星号连接结构式(I)所示毒素。
进一步优选,所述L4选自羰基或单键。
进一步优选,所述R6、R7相互独立的选自氢、C1-C6烷基等。
进一步优选,其特征在于所述L5为C2-C12烷基。
作为优选方式,所述药物,其含有以上任一项所述的含结构式(I)的毒素偶联物或其盐。
作为优选方式,以上任一项所述的一种结构式(I)的毒素偶联物或其盐在制备抗肿瘤药物或抗癌药物中的用途。
作为优选方式,所述抗肿瘤药物或抗癌药物为抗肺癌药物、抗肾癌药物、抗尿道癌药物、抗结直肠癌药物、抗前列腺癌药物、抗胶质细胞瘤药物、抗卵巢癌药物、抗胰腺癌药物、抗乳腺癌药物、抗黑色素瘤药物、抗肝癌药物、抗膀胱癌药物、抗恶性淋巴瘤药物、抗白血病药物、抗胃癌药物或抗食道癌药物。
通用缩写和符号:
g:克
mg:毫克
min:分钟
mL:毫升
mol:摩尔
℃:摄氏度
Boc:叔丁氧羰基
PyBOP:1H-苯并三唑-1-基氧三吡咯烷基鏻六氟磷酸盐
Cit:瓜氨酸
CO2:二氧化碳
DCM:二氯甲烷
DIPEA:二异丙基乙胺
DMF:二甲基甲酰胺
DMSO:二甲基亚砜
DPBS:杜氏磷酸盐缓冲液
DTPA:二乙基三胺五乙酸
DTT:二硫苏糖醇
EA:乙酸乙酯
EDTA:乙二胺四乙酸
FBS:胎牛血清
HATU:O-(7-氮苯并三氮唑)-N,N,N,N-四甲基脲六氟磷酸酯
H2O:水
HOBt:1-羟基苯并三唑
mAb:单克隆抗体
MEM:最低基础培养基
MTS:3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲酯基)-2-(4-磺苯基)-2H-四唑,内盐
MTT:3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐
PAB:对氨基苯甲氧基
PBS:磷酸盐缓冲液
Sodium Pyruvate:丙酮酸钠
THF:四氢呋喃
TLC:薄层色谱法
Tris:三羟甲基氨基甲烷
Val:缬氨酸
Trt-Cl:三苯基氯甲烷
TBTU:O-(1H-苯并三唑-1-基)-N,N,N’,N’-四甲基异脲四氟化硼
HOBT:1-羟基苯并三唑
TFA:三氟醋酸
TBS-Cl:叔丁基二甲基氯硅烷
HOSu:N-羟基丁二酰亚胺
Na2CO3:碳酸钠
EDCI:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐
CuBr:溴化亚酮
具体实施方式
下面结合具体实施例,进一步阐述本发明。这些实施例仅用于说明本发明而不是用于限制本发明的范围。除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1小分子payload ama-0301的合成
1)固相合成中间体08
以N-芴甲氧羰基-O-叔丁基-L-羟脯氨酸预装树脂为起始原料,经20%哌啶(1g树脂加入20ml 20%哌啶)脱去保护基Fmoc以后,DMF洗涤5次,至PH中性,然后以DMF为溶液(20ml/g),依次加入Fmoc-N-丙炔基-L-天冬酰胺(Fmoc-Asn(Trt)-OH)(3eq),TBTU(2.5eq),HOBT(1.8eq),DIPEA(6eq),于室温(28℃)下反应2h后,用DMF(每次1g树脂20mlDMF)洗涤3次,再按前面的操作连接后面的氨基酸,最后连接完全以后,用1%TFA二氯甲烷溶液(每次1g树脂20ml,1%TFA 5min,重复三次)从树脂上切割下来,旋去溶液,用甲基叔丁基醚搅拌析晶,就得到化合物08,总收率约为43%,高效纯度为81.3%。MS:[M+H]+1244.6521
2)化合物09合成
取8g化合物08粗品,用TFA(10ml/g)溶解后,于室温下搅拌反应5h,50℃减压除去TFA,制备液相色谱纯化,得纯品化合物09约3.8g,收率为71%,纯度为96.4%。MS:[M+H]+828.3241
3)化合物14的合成
于250ml单口瓶中加入(4S)-羟基异亮氨酸2.94g,1,4-二氧六环40ml,饱和碳酸钠溶液ml,搅拌均匀,并分批加入Fmoc-OSu,10min后,继续于室温下搅拌反应12h,原料反应完全,向反应液中加入50ml水,并用5%的柠檬酸溶液调PH至4左右,乙酸乙酯萃取3次(每次50ml),收集有机层,饱和食盐水50ml洗涤一次,无水硫酸钠干燥,浓缩得淡黄色油状物。无需纯化直接投下一步。收率>100%。
4)化合物15的合成
将上述化合物13的粗品用40mlDMF溶解后,加入咪唑2.68g(2eq),分批加入TBS-Cl,加毕,于室温下搅拌12h,原料反应完全,加入水50ml,乙酸乙酯50ml,搅拌,分出有机层,水层再用乙酸乙酯萃取2次(每次50ml),收集有机层,无水硫酸钠干燥,过滤浓缩得淡黄色油状物,硅胶柱层析(洗脱液:PE:EA=5:1),得油状物4.5g。两步收率约为46.6%。
5)化合物10的合成
于250ml单口瓶中依次加入化合物14,HOSu(1.23g,1.15eq),DCC(2.23g,1.15eq),THF50ml,氮气保护下,室温搅拌6h,反应完全后,加水50ml,乙酸乙酯50ml,搅拌10min后,分出有机层,水层再用乙酸乙酯萃取2次,每次50ml,合并有机层,无水硫酸钠干燥,过滤,浓缩得淡黄色油状物,制备液相色谱纯化得白色泡沫状固体约3.24g,收率60%。1H-NMR(400MHz,DMSO-d6):0.08(s,6H),0.86(s,9H),0.98(d,3H,J=8.0Hz),1.06(d,3H,J=5.6),1.95(t,J=10.8),2.83(s,4H),4.21(dd,1H,J=16.8Hz,8.0Hz),4.34(dd,1H,J=12Hz,4Hz),4.67~4.73(m,1H),7.31(d,2H,J=8.0Hz),7.34~7.46(m,2H),7.70~7.76(m,2H),7.89(t,2H,J=12.0),8.24(d,1H,J=8.8Hz);MS:581.34[M+H]
6)化合物11的合成
将0.5g化合物09用干燥的DMF1.5ml溶解后,加入化合物10(701mg,2eq),DIPEA调pH至8~9氮气保护下,于室温反应5h,HPLC监控反应,原料09基本反应完全。无需后处理直接投下一步。
7)化合物12的合成
向上述反应液中加入哌啶0.3ml(20%),继续于室温下搅拌反应2h,HPLC检测原料反应完全,停止反应,用制备液相色谱纯化(中性,乙腈/纯水体系),收集目标峰,减压除去乙腈以后,冻干得到白色粉末状固体277mg,两步收率约为42.8%,MS:[M+H]+1071.5120.
8)化合物13的合成
将270mg化合物12用干燥的DMF溶解后,加入EDCI(96.6mg,2eq),HOBT(170mg,5eq),DIPEA(0.22ml,5eq),于室温下搅拌4h后,HPLC检测反应完全后,制备液相色谱纯化(中性,乙腈/纯水体系),收集目标峰,减压除去乙腈以后,冻干得到白色粉末状固体约136.4mg,收率约51.4%,MS:[M+H]+1053.4908.
9)化合物16的合成
将1g 6-(马来酰亚胺基)己酸琥珀酰亚胺酯用10ml四氢呋喃溶解后,加入0.678g2-[2-(2-叠氮乙氧基)乙氧基]乙胺(1.2eq),氮气保护下,室温搅拌4h,TLC监测反应完全后,加入水,EA萃取3次,每次10ml,合并有机层,干燥浓缩,柱层析得目标产品约0.8g,收率84%。
10)化合物17的合成
将80mg化合物16用DMSO 5ml溶解后,加入化合物13(114.7mg,0.5eq),溴化亚酮44.7mg(1.5eq),纯净水0.2ml,氮气保护下,于室温下搅拌3h后,HPLC监测化合物13反应完全后,制备液相纯化,收集目标峰,旋去有机溶剂后,冻干得到白色固体约50.5mg,收率32.6%。MS:[M+H]+1420.7031
11)化合物ama-0301的合成
取45mg化合物17,加入1ml 5%TFA/MeOH溶清,氮气保护,室温反应1h,HPLC检测原料17反应完全,氮气吹干溶剂后,制备液相纯化后,浓缩除去有机溶剂后,冻干得白色固体15.4mg,收率37.2%。MS:[M+H]+1306.6013
实施例2小分子payload ama-0302的合成
1)固相合成关键中间体24
参考化合物08的合成,甲基叔丁基醚析晶以后,得到棕黄色固体约10.6g,高效纯度约79.8%。
2)化合物25的合成
参考化合物09的合成,将化合物24的粗品用50mlTFA溶解后,氮气保护下,于室温下搅拌5h后,高效检测,原料反应完全,主点明显,制备液相纯化,收集目标峰,减压除去有机溶剂以后,冻干得白色固体2.38g,MS:[M+H]+831.4251
3)化合物26的合成
参考化合物11的合成,原料25投料1.0g,冻干得白色固体约820mg,收率52.5%,MS:[M+H]+1296.6431
4)化合物27的合成
参考化合物12的合成,原料26投料800mg,冻干得白色固体约308.8mg,收率约46.6%,MS:[M+H]+1074.5184
5)化合物28的合成
参考化合物13的合成,原料27投料300mg,冻干得白色固体约208.4mg,收率约70.6%。MS:[M+H]+1056.5243
6)化合物29的合成
将100mg化合物28用5%TFA的甲醇溶液1ml溶解后,氮气保护下,室温搅拌反应2h,高效HPLC监测原料反应完全,制备液相纯化后,收集目标峰,冻干得淡黄色固体约69.5mg,收率77.9%,MS:[M+H]+942.4571
7)化合物30的合成
将50mg化合物29加入到10ml反应瓶中,氮气保护下,加入四(三苯基膦钯)67.5mg(1.1eq),氮气置换空气,用针筒向反应瓶中加入5ml干燥的四氢呋喃,0.1ml干燥的吗啉,原料溶解,继续于室温下搅拌12h,HPLC检测原料反应完全,制备液相纯化,收集目标峰,冻干得淡黄色固体约35.2mg,收率73.5%。MS:[M+H]+902.4123
8)化合物31的合成
取Fmoc-L-缬氨酸(20g)、HoSu(7.46g,1.1eq),用200ml的THF将其溶解后,将反应瓶置于冰盐浴中降至0℃,缓慢加入DCC缩合剂(14.6g,1.1eq),控制反应温度在0-5℃,3小时加毕。移出冰浴,室温搅拌反应12h。TLC检测Fmoc-L-缬氨酸反应完全,停止反应。减压抽滤,并用100ml的THF洗涤滤饼,滤液旋干,再在其残余物中加入100ml的DCM搅拌溶解(35℃下搅拌溶解),有机滤膜滤掉少许不溶物,再将其置于35℃的油浴锅中,搅拌加入100ml的石油醚,自然降温析晶1小时,再将其置于冰盐浴中降温析晶2小时,抽滤,并用石油醚洗涤固体,固体置于40℃的真空干燥箱中烘干得21.86g的白色粉末状固体,收率约85%。
9)化合物32的合成
将20g的化合物31溶于200mL的溶剂THF中,加入9.64g(1.2eq)L-瓜氨酸,加入1M碳酸钠调pH至8~9,反应未溶清,室温搅拌反应48h,TLC检测反应完全。于冰浴搅拌下,用柠檬酸水溶液调节反应液pH为3-4,用异丙醇:EA=1:5(40ml异丙醇+200ml EA)萃取3次,有机相合并,无水硫酸钠干燥,过滤旋干,再加入200ml的甲叔醚搅拌,搅拌2小时后抽滤,收集滤饼,并置于45℃真空干燥箱中干燥得18.6g白色固体产物,收率约81.7%。
10)化合物33的合成
取18g化合物32加入到500mL的反应瓶中,用200mLDMF溶解后,依次加入8g(1.0eq)4-[(N-叔丁氧羰基)氨甲基]苯胺,HATU20g(1.5eq),DIPEA(18ml,3eq),于室温下搅拌24h,TLC检测原料反应完全后,加入水200mL,二氯甲烷200mL,搅拌10min后,分出有机层,再用水洗涤两次,每次50mL,收集有机层,无水硫酸钠干燥后,过滤浓缩,得棕色油状物,向油状物中加入甲基叔丁基醚200ml,搅拌30min后,有固体洗出,过滤,滤饼用甲基叔丁基醚洗涤2次,每次50ml,收集滤饼,50℃真空干燥箱中干燥得褐色固体18.9g,收率74.4%。
11)化合物34的合成
取15g化合物33,加入到250ml反应瓶中,加入20%哌啶的DMF溶液75ml,室温搅拌1h后,TLC检测原料反应完全后,油泵减压蒸去溶剂后,加入甲基叔丁基醚200ml,室温搅拌2h,有褐色固体析出,过滤,滤饼用甲基叔丁基醚洗涤2次,每次50ml,收集滤饼,50℃真空干燥箱中干燥得褐色固体6.8g,收率66.4%。
12)化合物35的合成
取1g化合物34用10mlDMF溶解后,加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯773mg(1.2eq),于室温下搅拌5h,TLC检测,原料反应完全,向反应液中加入水10ml,乙酸乙酯20ml,搅拌10min后,分出有机层,无水硫酸钠干燥,过滤,浓缩,柱层析(石油醚:乙酸乙酯50:1)纯化后,得淡黄色泡沫状固体956mg,收率68.3%。
13)化合物36的合成
取30mg化合物35,用20%TFA的二氯甲烷溶液溶解后,于室温下搅拌反应2h,TLC检测反应完全后,减压旋去溶剂,得化合物36的粗品,备用。无需纯化直接投下一步。
14)ama-0302的合成
将上一步所得的化合物36(1.2eq)的粗品用1mlDMF溶解后,加入32.5mg化合物30,HATU20.5mg(1.5eq),用DIPEA调pH至8~9,氮气保护下,室温搅拌6h,HPLC检测原料30反应完全,制备液相纯化,收集目标峰,冻干后,得淡黄色固体15.3mg,收率29.1%,MS:[M+H]+1455.7011.
实施例3小分子payload ama-0303的合成
1)化合物37的合成
参考文献J.Am.Chem.Soc.2018,140,6513-6517的合成方法。
2)化合物38的合成
参考化合物11的合成,冻干后得到目标化合物约751.5mg,收率56.8%,MS:[M+H]+1426.6751
3)化合物39的合成
参考化合物12的合成,化合物38投料700mg,冻干得白色固体约482.3mg,收率为81.6%。MS:[M+H]+1205.6124
4)化合物40的合成
参考化合物13的合成,化合物39投料450mg,制备纯化后,冻干得白色固体约384.2mg,收率86.7%,[M+H]+1186.6012
5)化合物41的合成
参考化合物29的合成,化合物40投料100mg,制备纯化后,冻干得白色固体约54.2mg,收率约67.2%。[M+H]+958.4250.
6)化合物42的合成
参考化合物30的合成,化合物41投料50mg,制备纯化,冻干目标峰得白色固体约28.6mg,收率59.7%。[M+H]+918.4413
7)化合物ama-0303的合成
参考化合物ama-0302的合成,化合物42投料25mg,制备纯化后,冻干得淡黄色固体约15.2mg,收率38%。[M+H]+1471.6820
实施例4小分子payload ama-0304的合成
1)化合物49的合成
合成方法参考化合物08的合成,拿到化合物49的粗品约2.4g,纯度大约在86.2%。无需纯化,直接投下一步。
2)化合物50的合成
参考化合物09的合成,制备纯化后,得到目标化合物1.1g。[M+H]+790.4126
3)化合物51的合成
参考化合物11的合成,化合物50投料500mg,制备纯化后,得目标化合物341.5mg,收率43%。[M+H]+1255.6195
4)化合物52的合成
参考化合物12的合成,化合物51投料300mg,制备纯化后,冻干目标峰,得白色固体约186.4mg,收率75.5%,[M+H]+1033.5013
5)化合物53的合成
参考化合物13的合成,化合物52投料150mg,制备纯化后,冻干目标峰,得白色固体约86.5mg,收率58.7%。[M+H]+1015.5121
6)化合物54的合成
取80mg化合物53,用干燥的DMSO溶解后,加入叔丁基N-(4-溴丁基)氨基甲酸159mg(8eq),叔丁醇钾88mg(10eq),室温下搅拌反应12h,再加入叔丁基N-(4-溴丁基)氨基甲酸159mg(8eq),叔丁醇钾88mg(10eq)再次室温下搅拌反应24h,HPLC监测,原料消失,制备液相纯化,收集目标峰得18.2mg,收率19.5%,[M+H]+1186.6137.
7)化合物55的合成
将上述所得18.2mg化合物54用0.1mL三氟醋酸溶解后,室温下搅拌30min,加入2mL二氯甲烷,搅拌均匀后,减压旋去溶剂,备用。
8)化合物ama-0304的合成
将上述所得化合物55的粗品用1ml DMF溶解后,加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯9.5mg(2eq),用DIPEA调pH至8~9,氮气保护下,于室温下搅拌5h,HPLC监测,原料反应完全,制备液相纯化,收集目标峰,旋去有机溶剂后,冻干得类白色固体10.4mg,收率53%,[M+H]+1279.6537.
实施例5小分子payload ama-0305的合成
1)化合物56的合成
参考化合物11的合成,原料50投料200mg,冻干得白色固体约211.3mg,收率60.2%,[M+H]+1385.6713.
2)化合物57的合成
参考化合物12的合成,原料56投料200mg,冻干得白色固体约114.8mg,收率68.3%,[M+H]+1163.5793.
3)化合物58的合成
参考化合物13的合成,原料57投料110mg,冻干得类白色固体约72.5mg,收率66.9%,[M+H]+1145.5901
4)化合物59的合成
取上述所得化合物58约70mg,用0.5mL TFA的甲醇溶液溶解后,于室温下搅拌反应1h,HPLC监测原料反应完全,制备液相纯化,收集目标峰,旋去有机溶剂,冻干得类白色固体约45.7mg,收率81.6%,[M+H]+917.4013
5)化合物60的合成
参考专利wo2012041504的方法
6)化合物61的合成
取原料59约42mg,用1ml干燥的DMF溶解后,加入22mg(2eq)化合物60,58mg二丁基二月桂酸锡,加毕,氮气保护下,室温搅拌24h后,补加22mg(2eq)化合物60,继续于室温下搅拌52h,HPLC监测,原料59消失,向上述反应液中加入0.2ml甲醇终止反应,制备液相纯化,收集目标峰,旋去有机溶剂,冻干得淡黄色固体约9.6mg,收率18.1%,[M+H]+1159.5703.
7)化合物62的合成
将上述所得9.6mg化合物61,用0.1ml三氟醋酸溶解后,室温下搅拌30min,加入2ml二氯甲烷搅拌均匀后,减压旋去溶剂,备用,无需纯化,直接投下一步反应。
8)化合物ama-0305的合成
将上述所得的化合物62用干燥的DMF1ml溶解后,加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯5.1mg(2eq),氮气保护下,室温搅拌5h,HPLC监测原料反应完全,制备液相纯化,收集主峰,氮气
吹干,得类白色固体约4.6mg,收率44.4%,[M+H]+1252.6091.
实施例6制备抗体-药物偶联物
1)通用偶联方法:将通过初步的纯化后单体率大于95%的抗体分子,使用超滤离心管换液至含有EDTA的磷酸盐缓冲液中,浓度10mg/ml。加入10倍于抗体摩尔分子数的TCEP,室温下反应2h,。使用超滤离心管换液至pH6.5的磷酸缓冲液中,再加入10倍于抗体摩尔分子数的DHAA,室温下反应2h。然后加入3倍于抗体摩尔分子数的payload,室温下反应4h。反应结束后,使用截留分子量为30KDa的超滤离心管换液至PBS中,并去除未偶联的payload。
2)抗体-药物偶联DAR的检测
单体率检测条件:
样品14000rpm离心5分钟,取上清液进样分析。
仪器:Waters e2695(2489UV/Vis)
色谱柱:TSKgel G3000SWXL(7.8×300mm,5μm)
流动相:A:50mM PB,300mM NaCl,200mM Arg,5%IPA,pH6.5
流动相A等度洗脱30min,流速:0.714ml/min,柱温25℃,检测波长:280nm。
DAR检测条件:
样品14000rpm离心5分钟,取上清液进样分析。
仪器:Waters H-class(TUV)
色谱柱:Proteomix HIC Butyl-NP5(4.6×35mm,5μm)
流动相:A:1.5M硫酸铵,0.025M无水磷酸钠,pH7.0
B:0.025M无水磷酸钠,25%IPA,pH7.0
流动相A平衡色谱柱,流动相A和B梯度洗脱,流速0.8ml/min;柱温25℃,检测波长:214nm。
3)结果
4)结论
将ama-0301/ama-0302/ama-0303/ama-0304/ama-0305与Trastuzumab偶联以后,偶联效率较高,单体率较好。
实施例7血浆稳定性
1)操作
取一定量的ADC样品,加入到已去除人IgG的人血浆中,每种ADC重复三管,放置37℃水浴中孵育,分别孵育72h、144h后,取出ADC样品,每管加入ProteinA resin(MabSelectSuReTM LX Lot:#10221479GE,用取PBS洗涤过的)100ul,垂直混合仪晃动吸附2h,经过洗涤洗脱步骤,获得孵育后的ADC.对孵育特定时间的ADC样品进行RP-HPLC检测.
2)结果
3)结论
在人血浆中,各抗体-药物偶联物在3天和6天时,几乎无降解,稳定性较好。
实施例8体外活性测试
1)实验材料
细胞:来源于中国科学院细胞库
肿瘤细胞培养基:Gibco
FBS:BIOWEST
2)培养基的配制
生长培养基(with 10%FBS,Penicillin/streptomycin(100U/ml)
检测培养基(with 1%FBS,Penicillin/streptomycin(100U/ml)
3)操作
提前30min开启生物安全柜紫外灯照射,后开通风3min。将生长培养基、检测培养基、D-PBS和胰酶放入37℃恒温水浴锅里预热,之后用酒精对表面进行消毒,放入生物安全柜中。选择汇合率在80%左右的细胞,放于生物安全柜中,吸掉旧培养基,用D-PBS润洗,吸弃,用胰酶消化2~3min,后加入生长培养基中和,离心1200rpm,3min。吸去离心上清,用4ml检测培养基混匀,取100ul计数(其中取出50ul细胞液,加入50ul Trypan Blue Stain并混匀,混匀后计数)。按照之前定好的细胞数铺板,80ul/孔铺于96孔板中,孔E11、F11、G11只加80ul检测培养基,边缘孔加入,150ul的DPBS。抗体溶液的稀释:用检测培养基在V型96孔板第一列中配制起始浓度为5uM,300ul的供试品品溶液,后面第2到10列分别加入210ul的检测培养基,从混匀的第一列中取30ul加入到第二列,用排枪上下混匀10次,弃枪头,后面7个浓度依次操作。铺板24h后加入稀释抗体,每孔20ul和设置对照,第11列只加入20ul的检测培养基,每个浓度设置2个复孔,加入后于细胞涡旋震荡器上混匀,550rpm,3min。
4)检测
4天后取出MTS试剂,常温避光解冻后,充分涡旋混匀后,在生物安全柜中,沿孔侧壁按每100μL细胞培养体积加入20μL CellTiterOne Solution Reagen MTS试剂,轻轻拍动板面,使MTS溶液混合均匀后,放于细胞培养箱中避光静置孵育2h。反应结束后,取出96孔板,于酶标仪中检测OD490nm吸光值,并进行数据记录、整理、存储。
5)结果
Claims (19)
2.根据权利要求1所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述L包含下列结构
-L1-[L2]m-[AA]n-L3-
其中,L1为接头,连接生物大分子A;L2为间隔物,L2为连接L1和AA之间的片段,m选自1-6,且m为整数;L3连接为结构式(I)所示毒素的片段;AA为含1~6个氨基酸组成的片段,n为0或1。
3.根据权利要求1所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述A为与靶点结合的生物大分子部分,包括抗体、其抗原结合片段、抗体样蛋白或核酸配适体。
4.根据权利要求3所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述靶点结合的生物大分子为抗体或其抗原结合片段,选自嵌合抗体、去免疫抗体、人源化抗体、人类抗体、双体、四体或纳米抗体。
5.根据权利要求3或4所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述抗原结合片段选自自由Fab、F(ab’)、Fd、Fv、单链Fv或二硫键连接的Fv(dsFv)组成的组。
6.根据权利要求1所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,R1、R4、R5中有且仅有一个含有-L-A结构。
8.根据权利要求2所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述L2为间隔物,选自取代或未取代C1-C6烷基、取代或未取代C3-C20的环烷基、取代或未取代C3-C20的杂环烷基、取代或未取代C5-C20芳基、取代或未取代C5-C20杂芳基、-(CH2CH2O)a-,其中a选自1~20,且为整数,中一种或多种,且间隔物与间隔物之间通过合理的化学键相连。
9.根据权利要求8所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述取代基选自羟基、巯基、卤素、羧基、氨基、磷酸基、硝基、氰基、磺酸基、取代或未取代C1-C6烷基中一种或多种。
10.根据权利要求9所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述取代基选自羟基、巯基、卤素、羧基、氨基、磷酸基、硝基、氰基或磺酸基中一种或多种。
11.根据权利要求2所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,AA为1~6个氨基酸组成的片段,所述氨基酸为L-氨基酸,选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸。
12.根据权利要求11所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述氨基酸选自苯丙氨酸、瓜氨酸、缬氨酸、赖氨酸、丝氨酸、谷氨酸、天冬氨酸或甘氨酸。
14.根据权利要求13所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述L4选自羰基或单键。
15.根据权利要求13所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述R6、R7相互独立的选自氢或C1-C6烷基。
16.根据权利要求13所述的非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分,其特征在于,所述L5为C2-C12烷基。
17.一种包括权利要求1~16中任一项所述非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分的药物组合物。
18.一种权利要求1~16中任一项所述非天然鹅膏毒肽类抗体偶联物或其药学上可接受的盐,以及药学上可接受的制剂成分在制备抗肿瘤药物或抗癌药物中的用途。
19.根据权利要求17所述的药物组合物,其特征在于:所述药物组合物为抗肺癌药物、抗肾癌药物、抗尿道癌药物、抗结直肠癌药物、抗前列腺癌药物、抗胶质细胞瘤药物、抗卵巢癌药物、抗胰腺癌药物、抗乳腺癌药物、抗黑色素瘤药物、抗肝癌药物、抗膀胱癌药物、抗恶性淋巴瘤药物、抗白血病药物、抗胃癌药物或抗食道癌药物。
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