CN115557965B - 双重靶向降解雌激素受体α和芳构化酶的PROTACs化合物及其应用 - Google Patents
双重靶向降解雌激素受体α和芳构化酶的PROTACs化合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种双重靶向降解雌激素受体α和芳构化酶的PROTACs化合物及其应用,属于医药技术领域。本发明化合物具有如下的结构通式,通式中,X为CH或N;E3 ligand是具有泛素化功能的由VHL或CRBN基因等编码的蛋白酶小分子;Linker为连接基团,包括‑亚烷基、‑烷氧基。本发明的PROTAC化合物分子量更小,具有理想的ERα/芳构化酶双重靶向降解和抑制能力,并能有效抑制MCF‑7细胞的体外增殖和芳构化酶的体外活性,同时对正常细胞没有明显的细胞毒性,此类化合物在乳腺癌治疗中的具有应用前景。
Description
技术领域
本发明属于医药技术领域,涉及一种双重靶向降解雌激素受体α(ERα)和芳构化酶的 PROTACs化合物及其应用。
背景技术
靶向蛋白降解策略依赖于泛素-蛋白酶体系统完成从非药物靶标到药物靶标的靶向蛋白降解,在高选择性、解决耐药突变和靶向非药物靶点等方面具有极大的优势。尽管如此,目前的(蛋白靶向嵌合体,PROteolysis-TArgeting Chimera)PROTACs仍面临着明显的局限性,包括结合亲和力较弱、成药性较差、脱靶效应和潜在的毒性等。因此需要对经典的PROTACs 技术进行优化,从而促进这一技术向临床应用转化。对此人们也研究出一系列优化的PROTAC 策略,包括Homo-PROTACs、变构PROTACs、双靶点PROTACs以及与抗体结合作用的 PROTACs等,这些新型的PROTACs技术对传统的PROTACs做了进一步的调整和优化,为它们赋予新的生物功能的同时弥补其不足,相信积极开发的蛋白质降解新技术将进入临床,对细胞内外的蛋白质水平进行灵活的调节以治疗各种疾病。
然而,每一种方法都有其局限性。除耐药性外,单靶点药物的副作用和组织毒性往往导致患者疗效降低,生活质量下降。为克服单靶点药物的缺点,针对肿瘤组织中两种或多种不同信号通路的联合用药已成为公认的有效方法。受双靶点药物,尤其是双特异性抗体的启发,已经有研究人员设想通过结合PROTAC技术和双靶点药物的概念,设计双靶点PROTAC,即通过将两个独立配体和一个E3连接酶分子配体通过linker连接,可以降解两个目标蛋白,从而发挥多个信号通路或靶蛋白的调节作用,实现更优的生物活性。双靶点PROTACs的成功案例,大大拓宽PROTAC方法的应用范围,为药物发现开辟一个新的领域。然而即使双靶点 PROTACs具有优越的生物学特性,而分子量的增加会带来较差的成药性和药代动力学问题,需要进一步修饰优化,以提高其体内外抗肿瘤活性。
发明内容
本发明首要目的在于解决现有技术存在的问题提供一种双重靶向降解ERα和芳构化酶的 PROTAC化合物。
本发明的第二目的是提供所述的PROTAC化合物的制备方法。
本发明的第三目的是提供所述的PROTAC化合物的在治疗ER阳性乳腺癌方面的应用。所述的PROTAC化合物能够有效降解ERα蛋白和芳构化酶,进而抑制MCF-7细胞增殖,可以作为新的治疗乳腺癌药物进行开发,具有广泛的应用前景。
为了实现上述目的,本发明所采取的技术方案如下:
一种双重靶向降解ERα和芳构化酶的PROTAC化合物具有如下的结构通式:
通式中,X为CH或N;E3 ligand(E3连接酶配体)是具有泛素化功能的由VHL(希佩尔-林道,von Hippel-Lindau)或CRBN(Cereblon)基因等编码的蛋白酶小分子,包括(VHL配体)、(带甲基VHL配体)、(泊马度胺)、(4-羟基沙利度胺)和(来那度胺)。Linker为连接基团,包括-亚烷基、-烷氧基,所述的-亚烷基-(CH2)n1、其中n1表示1至11的自然数;所述的-烷氧基-(CH2CH2O)n2,其中n2表示1至4的自然数。
进一步地,所述的PROTAC化合物包括但不限于如下表1所示的化合物:
表1
所述的双重靶向降解ERα和芳构化酶的PROTAC类化合物的制备方法,包括如下步骤:不同长度酸侧链的OBHSA衍生物与E3连接酶配体在HATU、DIPEA条件下进行缩合反应,得到PROTAC化合物。所述的缩合反应优选在DMF(N,N-二甲基甲酰胺)中进行,反应的温度优选为15-35℃;OBHSA衍生物、E3连接酶配体、HATU和DIPEA的物质的量之比优选为1:1.05:1.0:3.0。其中,不同长度酸侧链的OBHSA衍生物的结构式如下:
不同长度酸侧链的OBHSA衍生物通过包括如下步骤的方法制备得到:化合物1和2经过亲核取代获得化合物3,化合物3再经过环合,BBr3脱甲基,DABAL-H还原,suzuki偶联反应得到化合物7a和7b。对甲氧基苯胺8与三氟乙酸酐发生酰胺反应,经BH3·SMe2还原和二氯乙烷磺酰氯得到亲二烯体化合物12,化合物12在BBr3条件下脱甲基,之后与不同烷基链的溴代乙酸乙酯发生亲核取代反应得到化合物15a-i,化合物15a-i与呋喃环衍生物7a-b发生Diels-Aldol反应,再经碱水解得到不同长度酸性侧链的OBHSA衍生物17a-j。
上述PROTACs化合物能够降解雌激素受体α和芳构化酶、抑制人乳腺癌细胞和芳构化酶,其可用于制备靶向降解雌激素受体α和芳构化酶的药物或抗乳腺癌药物、抑制芳构化酶活性的药物。
一种靶向降解雌激素受体α和芳构化酶的药物或抗乳腺癌药物或抑制芳构化酶活性的药物,包含上述PROTACs化合物,还包含一种或多种药学上可接受的载体或赋形剂。
本发明的优点和有益效果:本发明的PROTAC化合物的制备渐变、产率高,其相对于现有的PROTAC化合物分子量更小,其具有理想的ERα/芳构化酶双重靶向降解和抑制能力,并能有效抑制MCF-7细胞的体外增殖和芳构化酶的体外活性,同时对正常细胞没有明显的细胞毒性,此类化合物在乳腺癌治疗中的具有应用前景。
附图说明
图1目标化合物对ERα和芳构化酶的降解能力。(A)在1μM浓度下不同化合物处理的MCF-7细胞中ERα和芳构化酶的Western印迹分析。(B)不同时间点23c、Ful(氟维斯群) 对ERα降解能力。不同剂量的23a、23b(C)和23c(D)对ERα和芳构化酶降解。
具体实施方式
本发明的PROTACs化合物的制备包括以下步骤:
1、(4-(4-(1H-咪唑-1-基)苯基)呋喃-3-基)苯酚7a和4-(4-(4-(1H-1,2,4-三唑-1-基)苯基)呋喃-3-基)苯酚7b的合成
合成路线见下反应式,具体包括以下步骤:
反应条件:(a)Et3N,CH3CN,rt,3h;(b)NaH,DMSO,rt,3h;(c)BBr3,CH2Cl2,-20℃,12h;(d)DIBAL-H,THF,-78℃,8h;(e)1H-imidazole,CuI,Cs2CO3,DMF,120℃,40h;(f) 1,2,4H-triazole,CuI,Cs2CO3,DMF,120℃,40h。
(1)2-(4-甲氧基苯基)-2-羰基乙基-2-(4-溴苯基)乙酸酯3的合成
称取购买的2-溴-1-(4-溴苯基)乙烷-1-酮1(1.6g,6.94mmol)和对溴苯乙酸2(1.2g, 6.94mmol)于50mL的圆底烧瓶中,加入25mL的无水乙腈,缓慢滴加无水三乙胺(0.7mg,6.94mmol)后,室温继续反应12h后,TLC监测反应完全,反应结束之后减压浓缩除去乙腈和三乙胺,加入乙酸乙酯溶解,先后用稀盐酸(2M,30mL)、饱和碳酸氢钠(2×30mL)和饱和氯化钠(30mL)洗涤,有机层用无水硫酸钠干燥,过滤旋干得粗品,经柱层析纯化后得黄色固体化合物3,产率为88%。
(2)3,4-二(4-溴-苯基)呋喃-2-酮4的合成
将25mL的两口瓶、磁子在105℃下烘烤15min后,趁热装置,无水无氧操作,在通Ar下,称取化合物3(786.2mg,2.5mmol)其中,加入10mL的无水DMSO,缓慢滴加80%NaH(150.1mg,5.0mmol)后,25℃反应3h后,TLC监测反应完全,加入5mL 2N HCl淬灭反应,用乙酸乙酯(3×25mL)萃取,有机层无水Na2SO4干燥,减压脱溶得到粗产物,硅胶柱纯化 (石油醚/乙酸乙酯=9:1,v/v)得到475.9mg(产率64.3%)化合物4。
(3)3,4-二(4-羟基-苯基呋喃)-2-酮5的合成
将100mL的单口瓶、磁子在105℃下烘烤15min后,趁热装置,无水无氧操作,在通Ar下,称取化合物4(1.345g,4.56mmol)其中,加入25mL DCM,-20℃下加入BBr3(2.6mL,27.33mmol)反应12h后,加入10mL水淬灭反应,用乙酸乙酯(3×20mL)萃取,饱和NaHCO3溶液(15mL)洗涤,有机层无水Na2SO4干燥,减压脱溶得到粗产物,硅胶柱纯化(石油醚/ 乙酸乙酯=7:3)得到1.06g(产率86.7%)化合物5。
(4)3,4-二(4-羟基-苯基)呋喃6的合成
将50mL的单口瓶、磁子在105℃下烘烤15min后,趁热装置,无水无氧操作,在通Ar下,称取化合物5(560mg,1.98mmol)其中,-78℃下加入二异丁基氢化铝(DIBAL-H,8mL,7.93mmol)反应12h后,加入4%H2SO4淬灭反应,用乙酸乙酯(3×25mL)萃取,饱和NaCl 溶液(30mL)洗涤,有机层无水Na2SO4干燥,减压脱溶得到粗产物,硅胶柱纯化(石油醚/ 乙酸乙酯=6:4)得到203.1mg(产率40.7%)化合物6。1H NMR(400MHz,CDCl3):δ7.41(s,2H), 6.94(d,J=8.4Hz,2H),6.87(d,J=8.8Hz,2H)。
(5)(4-(4-(1H-咪唑-1-基)苯基)呋喃-3-基)苯酚7a或4-(4-(4-(1H-1,2,4-三唑-1-基) 苯基)呋喃-3-基)苯酚7b的合成
将50mL的单口瓶、磁子在105℃下烘烤15min后,加入化合物6(500mg,1.64mmol),1H-咪唑(129.6mg,1.80mmol)或1,2,4-三唑(129.6mg,1.80mmol),CuI(30.2mg,0.15mmol),Cs2CO3(723.66mg,2.22mmol)无水无氧操作,溶解于2mL的DMF中,120℃下反应40h,用乙酸乙酯(3×25mL)萃取,饱和NaCl溶液(30mL)洗涤,有机层无水NaSO4干燥,减压脱溶得到粗产物,硅胶柱纯化(二氯甲烷/甲醇=150:1)得到268.60mg(产率56%)化合物 7a或者276.20mg(产率58%)7b。
2、N-(4-羟基苯基)-N-(2,2,2-三氟乙基)乙烯磺酰胺13的合成
合成路线见下反应式,具体的合成是4-甲氧基苯胺8(500mg,4.06mmol)在室温下与三氟乙酸酐(895.38mg,4.26mmol)搅拌4h得到化合物9。化合物9(889.81mg,4.06mmol)与硼烷二甲硫醚(610.23mg,8.12mmol)在60℃下还原得到化合物10,化合物10(999.66mg,4.87mmol)与二氯乙烷磺酰氯(953.10mg,5.84mmol)、三乙胺(1479.03mg,14.61mmol) 在室温下反应得到化合物12,化合物12(1.0g,3.39mmol)与BBr3(1.70g,6.77mmol)在 -20℃下反应得到化合物13。
反应条件:(a)(CF3CO)2O,TEA,DCM,rt,3h;(b)BH3·SMe2,THF,64℃,12h;(c)TEA,DCM,0℃to rt,12h;(d)BBr3,DCM,-20℃,12h。
3、不同长度侧链的中间体17a-i、17j、19a-d、22a-d的合成
(1)在碱性条件下,化合物13(1.0equiv.)先与不同长度的溴代酸乙酯(1.2equiv.)(化合物14a-i,n=3-11)反应获得带有乙酯侧链的亲二烯体15a-i,随后,15a-i(1.2equiv.)和呋喃化合物7(1.2equiv.)通过Diels-Alder反应得到对应的OBHSA类中间体16a-i,最后再将16a-i经LiOH(1.2equiv.)碱性水解得到衍生物17a-i、17j,具体见下反应式。
反应条件:(a)K2CO3,DMF,80℃,3h;(b)THF,90℃,12h;(c)NaOH,EtOH,rt。
(2)带有对甲苯磺酰基的醚链酸叔丁醇酯19a-d的合成见下反应式:首先,将不同缩合程度的聚乙二醇化合物24a-d(1.0equiv.)在NaH(1.2equiv.)的作用下,与溴乙酸叔丁酯 (1.2equiv.)反应得到化合物25a-d。化合物25a-d(1.0equiv.)与对甲基苯磺酰氯(1.2equiv.) 于室温下反应获得化合物19a-d。
反应条件:(a)NaH,DMF,0℃-rt,12h;(b)p-TsCl,Et3N,DMAP,DCM,rt,12h。
(3)含酸性侧链的OBHSA衍生物22a-d合成通用路线见下反应式:化合物13(1.0equiv.) 与不同长度的对甲苯磺酰基保护的醚链酸叔丁醇酯19a-d(1.2equiv.)、无水的碳酸钾(2.0 equiv.)于DMF中溶解,反应过夜获得带有叔丁醇酯侧链的亲二烯体20a-d,随后,化合物 20a-d(1.0equiv.)和(4-(4-(1H-咪唑-1-基)苯基)呋喃-3-基)苯酚7a(1.0equiv.)通过 Diels-Alder反应到对应OBHSA类中间体21a-d,再用三氟乙酸(1.2equiv.)脱去Boc得到 OBHSA酸性侧链衍生物22a-d。
反应条件:(a)K2CO3,DMF,85℃,10h;(b)THF,90℃,12h;(c)TFA,DCM,1h。
4、VHL配体VIII的合成
VHL配体VIII的合成路线见下反应式:首先,在碱性条件下,用二碳酸二叔丁酯(1.0 equiv.)对(S)-(-)-1-(4-溴苯)乙胺I(1.2equiv.)的氨基进行Boc保护得到中间体化合物IV,然后化合物IV(1.0equiv.)与四甲基噻唑(1.2equiv.)于90℃下被醋酸钯(0.3equiv.)催化反应生成中间体V。V经过TFA(1.1equiv.)脱保护后,室温下与Boc-Hyp-OH(1.0equiv.) 进行酰胺缩合获得中间体VI,VI经过TFA脱保护后室温下与Boc-L-tert-Leu(1.05equiv.)进行酰胺缩合生成中间体VII,VII经过TFA(1.0equiv.)脱保护后生成化合物VIII。
反应条件:(a)(Boc)2O,NaHCO3,DCM/H2O(1:1);(b)Pd(OAc)2,KOAc,DMAC,90℃,12h; (c)TFA,rt,1h;(d)Boc-Hyp-OH,HATU,DIPEA,DCM,rt,1h;(e)TFA,rt,1h;Boc-L-tert-Leu, HATU,DIPEA,DCM,rt,1h;(f)TFA,rt,1h。
5、芳构化酶-ERα双重靶向PROTACs目标化合物的合成
在获得带不同长度酸侧链的OBHSA衍生物17a-j、22a-d后,将这11个中间体分别与VHL配体VIII进行缩合。合成路线见下反应式,在碱性条件下以HATU为缩合剂,DMF溶解反应物后室温反应,得到带不同长度和不同类型侧链的终产物23a-j、24a-d。
反应条件:HATU,DIPEA,DCM,rt,1h。
以下实施例用于进一步说明本发明,但不应理解为对本发明的限制。本发明的实施方式并不受这些实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
实施例1:(4R)-1-(1-(4-(4-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4- 羟基苯基)-N-(2,2,2-三氟乙基)-7-恶唑环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)丁胺基)-2,2-二甲基丙基)-4-羟基-N-(1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(23a) 的制备:
称取化合物VIII(1.1eq)、化合物17a(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23a。黄色固体,产率57.3%;m.p. 145-146℃;1H NMR(400MHz,Methanol-d4)δ8.86(s,1H),8.17(d,J=20.2Hz,1H),7.61– 7.56(m,1H),7.52(d,J=6.2Hz,1H),7.47(dd,J=6.5,2.3Hz,1H),7.42(s,6H),7.40–7.35(m,2H),7.32(dd,J=9.1,2.9Hz,6H),7.15(d,J=8.7Hz,3H),6.83(td,J=8.8,8.3,2.1Hz,1H),6.74(dd,J=8.8,2.6Hz,2H),5.57–5.53(m,1H),5.37(d,J=4.5Hz,1H),4.67–4.59(m,2H),4.45(d, J=6.1Hz,2H),3.98–3.88(m,3H),3.75(dq,J=11.5,4.1Hz,1H),3.59(dq,J=9.6,4.8Hz,1H),2.47(s,3H),2.25–2.16(m,2H),2.02(ddd,J=18.7,9.1,3.5Hz,4H),1.50(dq,J=6.9,2.6Hz, 3H),1.02(t,J=4.1Hz,9H).13C NMR(100MHz,Methanol-d4)δ171.82,170.84,158.91,151.49, 147.63,144.48,144.23,135.58,131.96,131.78,130.08,129.09,128.92,128.68,126.27,120.98,118.12,116.92(q,J=239.5Hz),115.72,115.44,114.81,84.24,82.89,69.60,67.00,61.69,59.22, 57.73,56.67,37.42,35.08,31.59,25.72,24.98,21.10,19.57,14.54,13.14.HRMS(ESI)calcd for C55H60F3N7O8S2[M+H]+,1068.3970;found 1068.3857.
实施例2:(4R)-1-(1-(5-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4-羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)五胺基)-2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(23b)的制备:
称取化合物VIII(1.1eq)、化合物17b(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23b。黄色固体,产率67.6%;m.p. 118-124℃;1H NMR(400MHz,Methanol-d4)δ8.89(s,1H),8.19(d,J=24.2Hz,1H),7.58– 7.55(m,J=8.7,3.0Hz,1H),7.51(dd,J=9.0,2.3Hz,1H),7.43(dd,J=8.7,2.6Hz,7H),7.39– 7.36(m,1H),7.33–7.28(m,1H),7.2–7.16(m,4H),6.84–6.72(m,4H),5.55(dd,J=4.3,2.6Hz,1H),5.39(d,J=4.5Hz,1H),5.03–4.99(m,1H),4.59(dd,J=9.2,7.4Hz,1H),4.45(d,J=8.3Hz,2H),3.94(t,J=6.2Hz,1H),3.91–3.86(m,2H),3.79–3.73(m,1H),3.63–3.53(m,1H),2.49(d,J=2.4Hz,3H),2.32–2.26(m,2H),2.25–2.16(m,2H),2.03–1.95(m,2H),1.78–1.74 (m,2H),1.68–1.61(m,2H),1.51(dd,J=7.2,2.3Hz,4H),1.07–1.02(m,12H).13C NMR(100 MHz,Methanol-d4)δ171.83,170.91,159.12,151.47,147.65,144.27,131.96,130.09,130.01, 129.09,128.56,128.11,126.22,121.96(q,J=188Hz),121.02,121.01,115.65,115.35,114.68,82.87,69.57,59.18,57.62,56.61,37.38,35.07,29.36,29.08,28.53,25.67,21.02,14.43.HRMS (ESI)calcd for C56H62F3N7O8S2[M+H]+,1082.4126;found1082.4150.
实施例3:(4R)-1-(1-(6-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4- 羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂双环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)己胺基)-2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(23c)的制备:
称取化合物VIII(1.1eq)、化合物17c(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23c。黄色固体,产率66.0%;m.p. 147-148℃;1H NMR(400MHz,Chloroform-d)δ8.68(s,1H),7.62–7.49(m,2H),7.41(d,J= 7.0Hz,5H),7.33–7.27(m,4H),7.25–7.18(m,4H),6.86–6.73(m,J=25.6,13.6,6.8Hz,4H),6.56(s,1H),5.56(d,J=22.5Hz,1H),5.37(d,J=4.6Hz,1H),5.10(d,J=7.2Hz,1H),4.70(t,J =8.0Hz,1H),4.63(d,J=8.7Hz,1H),4.51(s,1H),4.40–4.17(m,2H),4.09–4.00(m,2H),3.94–3.83(m,2H),3.67(t,J=9.4Hz,1H),3.52–3.36(m,1H),2.50(s,3H),2.45–2.29(m,2H),2.21 (s,2H),2.07(d,J=12.5Hz,1H),1.98(d,J=12.6Hz,1H),1.75–1.63(m,4H),1.48(d,J=6.8Hz,3H),1.04(d,J=3.7Hz,9H).13C NMR(100MHz,Chloroform-d)δ170.25,158.52,150.45, 135.16,131.66,130.58,129.42,128.20,127.88,126.44,121.46,120.77,118.07,117.24(q,J=167Hz),116.20,115.15,69.63,58.87,57.35,53.57,48.73,45.87,36.18,35.61,26.52,25.60,25.23, 22.22,16.03,9.02.HRMS(ESI)calcd forC57H64F3N7O8S2[M+H]+,1096.4283;found 1096.4318.
实施例4:(4R)-1-(1-(7-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4- 羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)庚胺基) -2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2- 甲酰胺(23d)的制备:
称取化合物VIII(1.1eq)、化合物17d(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23d。白色固体,产率54.9%;m.p. 148-150℃;1H NMR(400MHz,Methanol-d4)δ8.88(s,1H),8.18(d,J=22.7Hz,1H),7.64– 7.52(m,3H),7.43(d,J=1.6Hz,6H),7.40–7.34(m,2H),7.31(dd,J=9.0,2.4Hz,2H),7.20–7.16(m,2H),7.16–7.14(m,1H),6.96(dd,J=7.5,5.3Hz,1H),6.82(dd,J=9.0,2.7Hz,2H), 6.76–6.71(m,2H),5.54(d,J=5.6Hz,1H),5.38(d,J=4.1Hz,1H),5.04–5.00(m,1H),4.64(q,J=2.9,1.8Hz,1H),4.47–4.43(m,2H),3.91(d,J=10.8Hz,3H),3.78–3.74(m,1H),3.60– 3.56(m,1H),2.48(s,3H),2.38–2.31(m,2H),2.24–2.20(m,2H),2.11–2.03(m,1H),2.00–1.96(m,2H),1.85–1.79(m,2H),1.76(d,J=6.2Hz,3H),1.52–1.49(m,3H),1.04(q,J=5.8, 4.9Hz,12H).13C NMR(100MHz,Methanol-d4)δ170.91,159.01,157.91,151.48,147.63,144.25,131.96,130.05,129.09,129.03,128.59,126.24,121.01,117.03(q,J=221Hz),115.37,114.71,84.29,82.88,69.58,67.43,59.19,57.70,56.62,37.39,35.05,34.75,29.36,28.32,25.69,22.17, 21.05,14.44,7.85.HRMS(ESI)calcd for C58H66F3N7O8S2[M+Na]+,1132.4259;found 1132.4128.
实施例5:(4R)-1-(1-(8-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4- 羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂双环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)辛胺基)-2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(23e)的制备:
称取化合物VIII(1.1eq)、化合物17e(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23e。黄色固体,产率67.9%;m.p. 139-142℃;1H NMR(400MHz,Chloroform-d)δ8.69(s,1H),7.97–7.91(m,1H),7.58(d,J= 8.1Hz,1H),7.38(d,J=16.2Hz,6H),7.34–7.30(m,3H),7.24–7.21(m,4H),7.12(dd,J=8.9,4.7Hz,2H),6.85–6.80(m,2H),6.41(s,1H),5.58(d,J=15.7Hz,1H),5.38(d,J=4.7Hz,1H), 5.11(q,J=7.1Hz,1H),4.70(t,J=8.2Hz,1H),4.62(d,J=8.8Hz,1H),4.51(s,1H),4.38–4.36(m,1H),4.26–4.22(m,1H),4.10–4.01(m,2H),3.88(t,J=6.5Hz,2H),3.65(dd,J=7.8,3.7Hz, 1H),3.53–3.39(m,1H),2.51(s,3H),2.44–2.29(m,2H),2.21–2.14(m,2H),2.07(d,J=12.2Hz,2H),1.72(q,J=7.5,6.6Hz,2H),1.49(d,J=6.9Hz,3H),1.28(dd,J=17.3,4.3Hz,8H),1.05(d,J=2.4Hz,9H).13C NMR(100MHz,Chloroform-d)δ173.48,159.21,158.45,150.50,148.30, 143.31,135.25,131.67,130.69,130.52,129.50,128.86,126.48,121.55,118.12,117.26(q,J=176Hz),116.19,115.20,111.08,69.76,68.11,58.84,57.38,56.78,48.81,45.87,38.63,36.38,35.51, 29.07,28.96,26.52,25.77,22.21,16.05,8.85.HRMS(ESI)calcd for C59H68F3N7O8S2[M+H]+, 1124.4596;found1124.4589.
实施例6:(4R)-1-(1-(9-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4- 羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)壬胺基) -2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(23f)的制备:
称取化合物VIII(1.1eq)、化合物17f(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23f。黄色固体,产率54.4%;m.p. 130-134℃;1H NMR(400MHz,Chloroform-d)δ8.69(d,J=1.5Hz,1H),7.94(d,J=5.3Hz, 1H),7.58(t,J=7.2Hz,1H),7.32(d,J=7.1Hz,3H),7.28–7.20(m,4H),7.13(d,J=8.2Hz,2H),6.88–6.82(m,4H),6.48–6.37(m,1H),5.59(d,J=11.3Hz,1H),5.37(d,J=5.2Hz,1H),5.10(t, J=7.2Hz,1H),4.69(d,J=8.3Hz,1H),4.62(d,J=8.8Hz,1H),4.51(s,1H),4.37(t,J=7.9Hz,1H),4.29–4.18(m,1H),4.12–4.01(m,2H),3.93–3.86(m,2H),3.65(d,J=9.7Hz,1H),3.48–3.46(m,1H),2.52(d,J=2.3Hz,3H),2.47–2.35(m,2H),2.17(t,J=6.9Hz,2H),2.09(q,J=8.7,8.2Hz,2H),1.76–1.70(m,2H),1.49(d,J=6.9Hz,3H),1.28(p,J=5.3Hz,10H),1.05(s, 9H).13C NMR(100MHz,Chloroform-d)δ173.86,159.26,150.61,148.29,143.28,135.21,131.70, 130.73,130.57,129.54,128.89,128.34,126.53,121.60,118.21,117.30(q,J=188Hz),116.35,116.15,115.25,69.86,58.87,57.50,48.87,45.95,36.46,36.02,35.42,29.72,29.07,26.53,25.91, 25.61,22.18,16.03,8.65.HRMS(ESI)calcd forC60H70F3N7O8S2[M+H]+,1138.4752;found 1138.4739.
实施例7:(4R)-1-(1-(10-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4- 羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂双环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)癸胺基)-2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(23g)的制备:
称取化合物VIII(1.1eq)、化合物17g(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23g。黄色固体,产率55.0%;m.p. 123-130℃;1H NMR(400MHz,Chloroform-d)δ8.68(s,1H),7.99(s,1H),7.57(s,1H),7.39– 7.37,(m,5H),7.34–7.30(m,3H),7.23(q,J=5.2,4.4Hz,3H),7.12(d,J=8.4Hz,2H),6.90–6.79(m,4H),6.40(d,J=9.6Hz,1H),5.59(d,J=9.2Hz,1H),5.36(t,J=5.5Hz,1H),5.09(t,J=7.2Hz,1H),4.70(t,J=8.0Hz,1H),4.61(d,J=8.8Hz,1H),4.51(s,1H),4.37(dd,J=16.5,8.6Hz,1H),4.28–4.20(m,1H),4.03(d,J=11.6Hz,1H),3.89(t,J=6.7Hz,2H),3.65(d,J=8.9Hz, 1H),3.47–3.45(m,1H),2.51(s,3H),2.38(s,2H),2.17(d,J=7.0Hz,2H),1.76–1.71(m,2H),1.48(d,J=6.9Hz,3H),1.31–1.22(m,14H),1.04(s,9H).13C NMR(100MHz,Chloroform-d)δ 171.67,170.17,150.48,135.26,131.68,130.50,129.47,128.84,128.32,126.48,121.53,118.10,117.27(q,J=167Hz),116.24,115.22,69.73,58.91,57.36,56.80,48.79,45.96,38.61,36.47, 35.53,29.17,26.51,25.89,25.62,22.20,16.04,9.76,9.57.HRMS(ESI)calcd for C61H72F3N7O8S2[M+H]+,1152.4909;found1152.4924.
实施例8:(4R)-1-(1-(11-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4- 羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂双环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)十一酰胺基)-2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷 -2-甲酰胺(23h)的制备:
称取化合物VIII(1.1eq)、化合物17h(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23h。黄色固体,产率64.5%;m.p. 116-121℃;1H NMR(400MHz,Chloroform-d)δ8.68(s,1H),7.95(s,1H),7.57(d,J=5.3Hz, 1H),7.39–7.38(m,5H),7.33–7.30(m,2H),7.27–7.22(m,4H),7.13(d,J=8.4Hz,2H),6.89(dd,J=9.0,2.7Hz,2H),6.86–6.80(m,2H),6.39(d,J=8.9Hz,1H),5.60(d,J=7.9Hz,1H), 5.37(dd,J=8.7,4.2Hz,1H),5.11–5.07(m,1H),4.71(t,J=7.9Hz,1H),4.61(d,J=8.8Hz,1H),4.52(s,1H),4.38–4.36(m,1H),4.27–4.19(m,1H),4.05(s,1H),3.93–3.88(m,2H),3.67–3.63 (m,1H),3.47–3.45(m,1H),2.51(s,3H),2.42–2.40(m,2H),2.34–2.23(m,1H),2.19–2.15(m,2H),2.08(d,J=9.6Hz,1H),1.97(q,J=8.5,8.0Hz,1H),1.73(t,J=6.3Hz,2H),1.60–1.55(m, 2H),1.48(d,J=6.9Hz,3H),1.27–1.24(m,10H),1.04(s,9H).13C NMR(100MHz, Chloroform-d)δ173.50,171.83,170.08,159.26,150.43,148.32,143.29,135.24,131.66,131.13,130.50,129.50,128.82,128.33,126.46,124.03,123.63,121.55,121.41,118.10,117.25(q,J=170 Hz),116.40,116.21,115.20,111.06,69.74,57.36,56.69,48.80,45.86,38.62,36.51,35.92,35.48,29.42,29.32,29.22,29.05,26.52,25.92,25.63,22.23,16.06,8.83.HRMS(ESI)calcd for C62H74F3N7O8S2[M+K]+,1204.4624;found 1204.4684.
实施例9:(4R)-1-(1-(12-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4- 羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂双环[2.2.1]庚-5-烯)-2-磺胺基)苯氧基)十二酰胺)-2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(23i)的制备:
称取化合物VIII(1.1eq)、化合物17i(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23i。白色固体,产率76.4%;m.p. 121-123℃;1H NMR(400MHz,Chloroform-d)δ8.69(s,1H),7.98(s,1H),7.57(d,J=8.7Hz, 1H),7.38(d,J=5.9Hz,7H),7.32(d,J=6.3Hz,2H),7.28–7.23(m,3H),7.13(d,J=8.0Hz,2H),6.91–6.81(m,4H),6.42(s,1H),5.60(d,J=8.8Hz,1H),5.37(t,J=5.1Hz,1H),5.12–5.07(m,1H),4.70(s,1H),4.61(d,J=8.9Hz,1H),4.52(s,1H),4.36(d,J=8.7Hz,1H),4.28–4.22(m, 1H),4.06(d,J=11.0Hz,1H),3.90(d,J=5.9Hz,2H),3.66(d,J=3.3Hz,1H),3.52–3.43(m,1H),2.51(d,J=1.4Hz,3H),2.42–2.29(m,2H),2.21–2.09(m,4H),1.74(t,J=7.3Hz,2H), 1.48(d,J=6.9Hz,3H),1.26(dd,J=8.2,4.6Hz,16H),1.04(s,9H).13C NMR(100MHz, Chloroform-d)δ159.29,150.45,148.36,143.27,135.32,131.66,130.54,129.53,128.91,128.84,126.47,121.56,118.12,117.28(q,J=169Hz),116.43,116.25,115.21,111.18,69.80,58.76,57.40, 56.75,48.83,46.08,38.63,36.52,35.39,29.46,29.21,29.06,26.52,25.95,25.65,22.22,16.07,9.69,9.62.HRMS(ESI)calcd forC63H76F3N7O8S2[M+H]+,1180.5222;found 1180.5206.
实施例10:(4R)-1-(1-(6-(4-(((1R,4R)-6-(4-(1H-1,2,4-三唑-1-基)苯基)-5-(4-羟基苯基)-N- (2,2,2-三氟乙基)-7-氧杂双环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)六酰胺基)-2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(23j)的制备:
称取化合物VIII(1.1eq)、化合物17j(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌5小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物23j。黄色固体,产率60.8%;146-152℃;1H NMR(400MHz,Methanol-d4)δ9.11–9.02(m,1H),8.87(s,1H),8.17(d,J=13.4Hz,1H), 7.74(dd,J=22.5,8.2Hz,2H),7.47–7.37(m,7H),7.34–7.26(m,2H),7.22–7.12(m,3H),6.84–6.66(m,5H),5.54(s,1H),5.37(d,J=4.3Hz,1H),5.03–4.98(m,1H),4.64–4.57(m,2H),4.47–4.42(m,2H),3.97–3.86(m,3H),3.79–3.71(m,1H),3.63–3.56(m,1H),2.47(s,3H),2.32(m,2H),2.23(dd,J=9.9,5.1Hz,2H),2.02(m,2H),1.76(dd,J=11.2,4.6Hz,2H),1.66(d, J=7.5Hz,2H),1.52–1.49(m,3H),1.47(d,J=11.3Hz,2H),1.29(d,J=5.1Hz,2H),1.06– 1.02(m,9H).13C NMR(100MHz,Methanol-d4)δ171.87,171.01,159.05,151.61,144.16,130.04, 129.10,128.97,128.63,126.28,120.14(q,J=190Hz),120.04,119.76,115.44,114.76,82.86,69.65,67.67,59.27,57.75,53.50,37.33,35.06,28.49,25.73,25.31,21.09,14.51.HRMS(ESI) calcd for C56H63F3N8O8S2[M+H]+,1097.4235;found1097.4282.
实施例11:(4R)-1-(1-(2-(2-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5- (4-羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂环[2.2.1]庚-5-烯)-2-磺胺基苯氧基)乙氧基)乙酰胺)-2,2-二甲基丙基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)苯基)乙基)吡咯烷-2-甲酰胺(24a)的制备:
称取化合物VIII(1.1eq)、化合物22a(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),室温搅拌2-4小时,TLC监测反应,反应结束后,用乙酸乙酯/水洗涤三次,饱和氯化钠溶液洗涤,有机相无水Na2SO4干燥后,减压浓缩,硅胶柱层析(二氯甲烷:甲醇=80:1~30:1)纯化得产物24a。黄色固体,产率76.8%;m.p. 105-107℃;1H NMR(400MHz,Methanol-d4)δ8.89(d,J=2.4Hz,1H),8.65(d,J=4.7Hz,1H), 8.49(d,J=35.1Hz,1H),8.37–8.32(m,1H),7.77–7.68(m,1H),7.64–7.51(m,3H),7.46–7.41(m,6H),7.38–7.28(m,3H),7.25–7.15(m,4H),6.96–6.94(m,2H),6.75(dd,J=7.6,5.4 Hz,1H),5.55(s,1H),5.40(s,1H),4.73–4.69(m,1H),4.59(d,J=9.3Hz,1H),4.46(s,2H),4.20–4.10(m,5H),3.92(d,J=13.7Hz,2H),3.85(s,1H),3.77(t,J=5.4Hz,1H),3.62(s,1H),2.49 –2.47(m,3H),2.23(d,J=9.6Hz,2H),2.07–1.89(m,2H),1.54–1.46(m,3H),1.05–1.00(m, 9H).13C NMR(100MHz,Methanol-d4)δ204.03,151.52,131.19,129.09,127.97,126.25,120.38, 117.10(q,J=212Hz),115.39,114.99,69.60,59.25,56.73,37.42,25.59,14.44,7.82.HRMS(ESI)calcd for C55H60F3N7O9S2[M+H]+,1084.3919;found1084.4104.
实施例12:(4R)-1-(1-(2-(2-(2-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基) -5-(4-羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂环[2.2.1]庚-5-烯)-2-磺胺基苯氧基)乙氧基) 乙酰胺)-2,2-二甲基丙基)-4-羟基-N-(S)-1-(4-(4-甲基噻唑-5-苯基)乙基)吡咯烷(24b) 的制备:
称取化合物VIII(1.1eq)、化合物22b(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),在室温搅拌2-4小时左右,TLC确认反应完全后,加水淬灭,EA萃取,有机相经过饱和碳酸氢钠多次洗涤后浓缩旋干,经硅胶柱层析分离纯化得到终产物24b。减压脱溶,柱层析分离纯化(洗脱剂比例为v二氯甲烷/v甲醇=200:1-15:1),得到125mg黄色固体,产率68%。m.p.124-127℃;1H NMR(400MHz,Methanol-d4)δ8.84(d, J=2.3Hz,1H),8.21–8.10(m,1H),7.59–7.52(m,1H),7.51–7.47(m,1H),7.44(dd,J=5.8,2.5Hz,1H),7.38(dd,J=9.3,2.5Hz,7H),7.35–7.30(m,2H),7.22–7.12(m,4H),6.86(d,J=8.8Hz,1H),6.82–6.80(m,1H),6.74–6.72(m,2H),5.57–5.53(m,1H),5.38–5.36(m,1H),4.67 (q,J=3.9,3.0Hz,1H),4.64–4.56(m,1H),4.47–4.42(m,2H),4.12–4.11(m,2H),4.04–4.01(m,2H),3.87–3.82(m,2H),3.70(t,J=8.4Hz,6H),3.64–3.58(m,1H),2.44(d,J=2.2Hz,3H), 2.28–2.16(m,2H),2.05–1.93(m,2H),1.46(d,J=6.7Hz,3H),1.34–1.32(m,2H),1.00(dd,J=6.3,3.3Hz,9H).13C NMR(100MHz,Methanol-d4)δ151.50,147.62,144.26,131.96,130.20, 130.07,129.09,128.94,128.66,126.25,121.06,120.86,117.14(q,J=215Hz),115.68,115.36,114.89,70.92,70.29,69.56,69.42,59.23,56.79,56.71,37.40,35.66,25.60,21.07,14.45.HRMS (ESI)calcd for C57H64F3N7O10S2[M+H]+,1128.4181;found 1128.4254.
实施例13:(4R)-1-(1-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4-羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂双环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)-14,14-二甲基-11- 氧基-3,6,9-三氧基-12-氮杂十五烷-13-基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5-基)乙基) 苯基)吡咯烷(24c)的制备:
称取化合物VIII(1.1eq)、化合物22c(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),在室温搅拌2-4小时左右,TLC确认反应完全后,加水淬灭,EA萃取,有机相经过饱和碳酸氢钠多次洗涤后浓缩旋干,经硅胶柱层析分离纯化得到终产物24c减压脱溶,柱层析分离纯化(洗脱剂比例为v二氯甲烷/v甲醇=200:1-15:1),得到125mg黄色固体,产率74%。m.p.118-121℃;1H NMR(400MHz,Methanol-d4)δ8.84(d, J=2.7Hz,1H),8.18–8.05(m,1H),7.56(d,J=6.9Hz,1H),7.47(d,J=5.4Hz,1H),7.38(d,J=6.2Hz,8H),7.32(d,J=8.9Hz,2H),7.14(dd,J=9.5,5.4Hz,3H),6.86–6.79(m,2H),6.74(d,J =8.4Hz,2H),5.54(d,J=2.8Hz,1H),5.35(d,J=4.4Hz,1H),4.69(d,J=4.9Hz,1H),4.61(s,1H),4.44(d,J=5.8Hz,2H),4.14–4.00(m,6H),3.90–3.77(m,4H),3.70–3.63(m,10H),2.44(s,3H),2.23(dd,J=13.1,7.7Hz,2H),2.04–1.99(m,2H),1.47(d,J=7.0Hz,3H),1.03(d,J= 5.4Hz,9H).13C NMR(100MHz,Methanol-d4)δ171.71,170.68,170.28,158.77,151.56,147.61, 144.20,131.95,130.30,130.06,129.38,129.11,128.97,128.70,126.30,120.98,118.15,116.95(q,J=231Hz),115.52,114.95,70.87,70.28,70.09,69.65,69.29,67.47,59.27,56.81,48.78,37.40, 35.71,25.70,21.21,14.62.HRMS(ESI)calcd forC59H68F3N7O11S2[M+H]+,1172.4443;found 1172.4315.
实施例14:(4R)-1-(1-(4-((1R,4R)-6-(4-(1H-咪唑-1-基)苯基)-5-(4-羟基苯基)-N-(2,2,2-三氟乙基)-7-氧杂双环[2.2.1]庚-5-烯)-2-磺酰胺基)苯氧基)-17,17-二甲基-14-氧基-3,6,9,12-四氧基-15-氮杂十八烷-16-基)-4-羟基-N-((S)-1-(4-(4-甲基噻唑-5- 基)苯基)乙基)吡咯烷(24d)的制备:
称取化合物VIII(1.1eq)、化合物22d(1.1eq)置于50mL单口瓶中,加入DIPEA(1.2eq),搅拌数分钟后,加入HATU(3.0eq),在室温搅拌2-4小时左右,TLC确认反应完全后,加水淬灭,EA萃取,有机相经过饱和碳酸氢钠多次洗涤后浓缩旋干,经硅胶柱层析分离纯化得到终产物24d。减压脱溶,柱层析分离纯化(洗脱剂比例为v二氯甲烷/v甲醇=150:1-30:1),得到125mg淡黄色固体,产率65%。m.p.99-102℃;1H NMR(400MHz,Methanol-d4)δ8.88(s, 1H),8.19(d,J=25.1Hz,1H),7.67–7.46(m,4H),7.42(hept,J=3.9,3.2Hz,7H),7.34–7.31(m,1H),7.25–7.12(m,5H),6.85(d,J=9.0Hz,1H),6.81(d,J=8.3Hz,1H),6.74(d,J=8.2Hz,1H), 5.55–5.51(m,1H),5.38(d,J=4.3Hz,1H),4.71–4.68(m,1H),4.63–4.54(m,1H),4.47–4.42(m,2H),4.09–4.07(m,2H),4.03(p,J=2.4Hz,2H),3.90–3.79(m,3H),3.72–3.63(m,14H), 2.48(d,J=3.0Hz,3H),2.22(dd,J=12.8,8.1Hz,2H),2.06–1.90(m,2H),1.50(d,J=6.9Hz,2H),1.32(t,J=4.6Hz,2H),1.04(d,J=3.9Hz,9H).13C NMR(100MHz,Methanol-d4)δ171.87, 170.87,151.54,147.62,144.30,130.07,129.98,129.09,128.64,128.07,126.22,116.98(q,J=192 Hz),115.35,114.86,70.19,69.86,69.59,59.16,57.84,56.56,37.41,35.01,29.08,28.94,25.60,21.03,20.91,14.41,7.81.HRMS(ESI)calcd for C61H72F3N7O12S2[M+H]+,1216.4705;found 1216.4647.
实施例15:化合物的相对亲和力测定
目标化合物与ERα和ERβ的亲和力通过荧光偏振法进行测定,化合物的亲和力是内源性 E2亲和力的相对值,设定E2与受体亲和力的值RBA=100%。在384孔板中,加入20μL含0.8μM ERα或ERβ蛋白、150nM荧光示踪剂(coumestrol,Sigma-Aldrich,Mo)和2.4μg牛免疫球蛋白的磷酸钾缓冲液(磷酸二氢钾1.74g,磷酸氢二钾1.36g,定容至100mL,pH 7.4) 溶解,再加入20μL用磷酸钾缓冲液配制的化合物溶液,化合物浓度梯度为3.16×10-4M、1×10-4M、3.16×10-5M、1×10-5M、3.16×10-6M、1×10-6M、3.16×10-7M、1×10-7M、3.16×10-8M、1×10-8M、 3.16×10-9M。避光、室温下放置2小时后,在酶标仪上读板,选取485nm处波长为主波长, 528nm处波长为参照波长,分析实验结果,根据公式受体亲和力RBA=测试物Ki/雌二醇Ki×100%计算出每个化合物的RBA值,其中雌二醇的Ki=2.03nM(ERα),Ki=7.86nM(ERβ)。
表2目标化合物23a-j和目标化合物24a-d对ERα和ERβ的相对结合亲和力a
a相对结合亲和力是通过荧光偏振法测试,其表达为IC50雌二醇/IC50化合物×100±标准差 (雌二醇的结合亲和力设为100%)。OBHSA是(1R,4R)-5,6-双(4-羟基苯基)-N-(4-甲氧基苯基)-N-(2,2,2-三氟乙基)-7-氧杂二环[2.2.1]庚-5-烯-2-磺酰胺,为对照化合物,其结构式为
从表中可以看出,多数化合物对ERα均有较强的相对结合亲和力。首先,当linker为不同长度的烷基链时,该系列化合物对ERα的相对结合亲和力与母体化合物OBHSA(RBA=4.54)相比较弱,仅有当linker为7个碳原子的烷基链时,化合物23d的RBA值为10.2左右,强于OBHSA;而当linker为5个或12个碳原子的烷基链时,其RBA值分别为2.13和3.49,稍弱于OBHSA;该系列其余的化合物的RBA值均小于1。当咪唑环被三氮唑取代时,化合物23j对ERα的RBA值为8.71,强于OBHSA。但值得注意的是,该系列化合物整体上对 ERα具有较高的选择性(除了化合物23e和23h),其中化合物23c、23f、23g、23i对ERβ的RBA值小于0.01,显示出几乎对ERα完全的结合偏好。
然而,当linker为不同长度的醚链时,目标化合物对ERα的相对结合亲和力整体均强于 OBHSA或与之相当,其中化合物24b对ERα的RBA=9.76,强于母体化合物OBHSA,而该系列的其余化合物对ERα的RBA值均在2.62~3.46,与OBHSA相当。这些结果表明,在 OBHSA骨架上引入不同类型的linker,即使引入的位置相同,也会对化合物与其受体的结合亲和力和亚型选择性造成很大的影响。
实施例16:细胞活力测试
MCF-7、MCF-10A细胞在含10%胎牛血清(fetal bovine serum,FBS)的有酚红DMEM液体培养基中培养。细胞密度至80%~90%时,消化细胞,并用含10%FBS的无酚红DMEM 培养基将细胞悬浮液铺至96孔细胞培养板中。待细胞完全贴壁后,弃去原培养液,每孔加入100μL新鲜的用含10%FBS的DMEM培养基配制的化合物溶液,化合物浓度梯度为1×10-7.5M、1×10-7M、1×10-6.5M、1×10-6M、1×10-5.5M、1×10-5M、1×10-4.5M、1×10-4M。药物处理培养4天后,取出培养板,吸出培养液,每孔加入100μL CCK8工作液,置于37℃、5%CO2培养箱中孵育1.5-2小时。在酶标仪上读板,选取450nm处波长为参照波长,分析实验结果,并计算出IC50。
表3目标化合物23a-j和目标化合物24a-d对乳腺癌细胞MCF-7细胞活力影响
a IC50是至少三次独立实验的平均值±标准差。
表中可以看出,目标化合物均显示出较强的抗MCF-7增殖活性。具体来说,当linker为不同长度的烷基链时,含4个碳的烷基链化合物23a显示出对MCF-7细胞最强的抑制活性, IC50为0.13μM,强于阳性对照药物氟维司群;随着碳原子数增加,化合物对MCF-7的抑制活性减弱,化合物23b、23c对MCF-7的IC50值为0.60μM和0.54μM,与母体化合物OBHSA(IC50=0.99μM)相比较强,强于来曲唑而弱于氟维司群。随着烷基链中碳原子数的进一步增加,化合物23d-23f对MCF-7的抗增殖活性显著下降,但是当碳原子数增加至10时,其抗增殖活性又变强,化合物23g的IC50仅为0.33μM,优于母体化合物OBHSA(IC50=0.99μM) 及来曲唑(IC50=8.72μM),但是仍弱于氟维司群(IC50=0.16μM)。当碳链继续增长,化合物对MCF-7细胞的抑制活性又开始下降。值得注意的是,当化合物中咪唑环被三氮唑取代时,化合物23j对MCF-7的抑制活性也较强,IC50=0.6μM,稍强于OBHSA的抗增殖活性。
当linker为不同长度的醚链时,化合物24b、24c对MCF-7细胞的抑制能力较强,IC50分别为0.35μM、0.51μM,与母体化合物OBHSA活性相当。而化合物24a、24d的抑制活性仅有微摩尔水平。此外可以看出,这些化合物对正常细胞MCF-10A细胞的抑制能力几乎可以忽略(IC50>100μM),说明其对正常体细胞的毒性较弱,显示出较强的安全性。
实施例17:芳构化酶的抑制活性测定
芳香酶(CYP19A)抑制剂筛选试剂盒(荧光)试剂盒选自Biovision公司,通过用1mL芳香酶测定缓冲液重构制备重组人芳香酶原液(2×)。通过涡旋充分混合内容物以获得均匀溶液(溶液将具有略微不透明的乳状外观)并将溶液转移至15mL锥形管中。使用Aromatase Assay Buffer将体积增加至2450μL,加入50μL的NADPH生成系统(10×),最终总体积为 2.5mL。
准备含有测试化合物和相应的无抑制剂对照的溶液,背景对照溶液(不含荧光芳香酶底物)和5μM来曲唑溶液(5×的阳性抑制对照溶液,终浓度为1μM)。准备一小份芳香酶分析缓冲液,其中含有用于溶解最终浓度为5×的试验化合物的有机溶剂。
表4芳构化酶测试中各测试孔的溶液配制
无抑制剂(μL) | 测试化合物(μL) | 阴性对照(μL) | 阳性对照(μL) | |
芳构化酶原液(2×) | 50 | 50 | 50 | 50 |
测试化合物溶液(5×) | -- | 20 | -- | -- |
5μM来曲唑溶液(5×) | -- | -- | -- | 20 |
芳构化酶缓冲液(5×) | 20 | -- | 50 | -- |
将Corning 384孔板在37℃孵育至少10分钟,以使测试配体与芳香酶相互作用。根据作用机制,可以针对其他测试配体优化预孵育时间。
在孵育期间,通过添加6μL重构的1mM芳香酶制备芳香酶底物/NADP+混合物(3×)底物储备溶液和50μL重构的10mM的β-NADP+储备(100×)至1444μL芳香酶测定缓冲液,总体积为1.5mL。
通过使用多通道移液管向每个孔中添加30μl芳香酶底物/NADP+(3×)混合物(除了背景对照)开始反应,产生100μL/孔的最终反应体积。
立即(在1分钟内)在动力学模式下测量Ex/Em=488/527nm处的荧光60分钟。ΔF=(RFU2–RFU1),ΔT=(T2–T1)。R=(ΔF-ΔFBC)/ΔT。%Relative Inhibition=(RSC–RTC)/RSC×100%。
目标化合物23a-j和目标化合物24a-d对芳构化酶抑制活性测试(IC50,μM)
可以看出,当linker为烷基链时,短链的PROTAC分子似乎显示更高的芳构化酶抑制活性。其中化合物23a对芳构化酶的IC50值为1.17μM,而且在5μM浓度时可以完全抑制芳构化酶的活性;当linker增长至5个碳原子数时,化合物23b的IC50值为0.32μM,5μM浓度时可抑制82.63%的芳构化酶活性。化合物23c、23d的活性有所下降,对芳构化酶的IC50值均为5μM左右。而当碳链继续增长,化合物对芳构化酶的抑制活性完全丧失。值得注意的是,含三氮唑的化合物23j对芳构化酶也没有抑制活性。
而当linker为不同长度的醚链时,linker最长的化合物24d显示出较好的芳构化酶抑制活性,IC50值为0.64μM,5μM浓度时对芳构化酶的抑制率为75.1%.而化合物24a、24b对芳构化酶的IC50值均在微摩尔水平,5μM浓度时酶抑制率为57%~59%。而化合物24c对芳构化酶几乎没有抑制活性。而且所有化合物对芳构化酶的抑制能力均远远弱于来曲唑(IC50= 0.0018μM)。
实施例18:ER、芳构化酶降解活性测定
将MCF-7细胞与DMSO或化合物(5μM)一起孵育24小时。提取全蛋白质并通过蛋白质印迹分析ERα蛋白质水平。使用8%SDS-PAGE凝胶电泳分离来自细胞裂解物的蛋白质。然后将凝胶电印迹到聚偏二氟乙烯(PVDF)膜上,用5%脱脂乳封闭,与兔抗ERα抗体(1: 1000,CST)和小鼠抗GAPDH抗体(1:8000,Proteintech Group)或兔抗芳构化酶抗体(1: 1000,CST)一起孵育,4℃过夜。用含0.1%吐温-20的TBS洗涤膜,5分钟3次。然后将膜与山羊抗兔二抗一起室温孵育1小时,用含0.1%吐温-20的TBS洗涤膜,10分钟3次。拿至暗室用X光片显影,结果见图1。
从图1A中可以看出,在所有目标化合物中,部分化合物23b、23c、24b、24c和24d在1μM浓度处理24h时均显示出较明显的ERα和芳构化酶降解活性。选取其中对两个靶点降解最为明显的化合物23c进行ERα降解-时间和降解-浓度关系测定。图1B显示,当加入化合物23c后12h时,开始出现明显的ERα水平降低,而阳性对照药物氟维司群在加入后3h时即可明显降解ERα蛋白。且化合物23c在加入后24h达到与氟维司群相当的ERα降解效果。图1C的结果显示,在加入化合物24h后,23a对ERα和芳构化酶基本上没有降解活性;23b 在10μM到0.5μM的浓度梯度中可以显著降解ERα,在5μM和1μM时能降解芳构化酶。图1D的结果显示,23c在10到0.1μM的浓度梯度中对ERα均具有一定的降解活性,而在0.5μM 时达到最大降解程度;对芳构化酶的降解活性与之相似,于10到0.1μM时对芳构化酶均有一定的降解能力,并于0.5μM时达到Dmax。这些结果表明,化合物23b和23c具有较强的 ERα降解能力,且对目标蛋白的降解活性呈现时间和浓度依赖性。
Claims (4)
1. 一种双重靶向降解ERα和芳构化酶的PROTAC化合物,其特征在于:选自如下化合物:
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。
2.权利要求1所述的PROTAC化合物在制备抗乳腺癌药物中的应用。
3.一种药物,其特征在于:包含权利要求1所述的PROTAC化合物。
4.根据权利要求3所述的药物,其特征在于:还包含一种或多种药学上可接受的载体。
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