【作者】 李洋；

【导师】 刘捷；

【作者基本信息】 四川大学 ， 生物与医药， 2022， 博士

【摘要】 三阴性乳腺癌（Triple-negative breast cancer,TNBC）作为乳腺癌的一种特殊分子分型,占整体乳腺癌的15%左右,具有特殊的分子表达特征、生物学行为以及临床病理特征。目前临床上常用的TNBC治疗手段仍然是手术和常规的全身细胞毒化疗,但是密集型,高剂量的化疗会产生巨大的毒性,且肿瘤复发或转移后往往缺乏有效的药物,治疗效果不理想,预后依然很差。肿瘤血管和淋巴管生成在TNBC的发生和发展过程中起关键作用,诸多研究表明抗血管内皮生长因子受体（Vascular endothelial growth factor receptor,VEGFR）通路介导的血管和淋巴管生成是TNBC治疗领域富有潜力的策略。然而,VEGFR抑制剂的缺点也非常突出,包括抑制生理性血管生成而导致的严重不良反应（如出血、伤口愈合延迟、胃肠道穿孔、血栓栓塞并发症、蛋白尿等）、抗肿瘤效果持续时间短,血管生成代偿信号通路激活导致的治疗性耐药,这些都在一定程度上限制了该类抑制剂的临床应用。因此,设计和发现具有高效价、高选择性和低毒性的VEGFR抑制剂,不仅能够为抗TNBC的治疗药物研究提供科学策略,也能有助于进一步探索VEGFR通路在TNBC发生和发展中的具体机制和功能。本人在攻读博士学位期间,围绕VEGFR介导肿瘤血管和淋巴管生成,开展了新型VEGFR3选择性和VEGFR/PARP双靶标抑制剂的设计、合成和构效关系研究,并对优选化合物开展体内外抗TNBC药效学、作用机制和药代动力学性质研究。具体内容简介如下:一、选择性VEGFR3抑制剂的设计、合成及抗TNBC活性研究TNBC的转移是一个多步骤、多基因参与的过程,一旦患者发生远端转移常提示预后差,生存时间短,远端转移也是晚期TNBC的主要致死原因。淋巴管生成和重塑在TNBC的远端转移过程中发挥关键作用,原发肿瘤通过异常生成的瘤周淋巴管与淋巴结进行连接,促进肿瘤细胞进入淋巴结、全身循环和远端转移。诸多研究表明抗VEGFR通路介导的淋巴管生成是TNBC转移治疗领域富有潜力的策略。相对于同家族的VEGFR1/2,VEGFR3在TNBC转移相关的淋巴管生成过程中发挥着关键作用,是抗TNBC转移的一个潜力靶标。目前,国内外针对VEGFR3进行选择性抑制剂的药物设计还较少,尚无进入临床试验阶段的候选药物。SAR131675是唯一被报道的选择性VEGFR3抑制剂,体内研究表明其能够有效地抑制淋巴结侵袭和肺部转移,但其低体内药效和积蓄毒性,阻碍了它的进一步临床评估。研究发现SAR131675结构中的乙炔基团通过烷基化CYP450的合成血红素生色基团或关键残基,使肝脏CYP450酶失活,从而不可逆地抑制药物代谢系统,造成累积性毒性。因此,有必要研发具有更高亲和力、更高选择性,更好安全性的VEGFR3抑制剂。在本章中,我们通过对活性小分子库进行VEGFR3激酶抑制活性随机筛选,以此确定了具有中等抑制活性的苗头化合物1,其对VEGFR3酶水平抑制活性(IC₅₀)为3.5μM。紧接着,我们通过分子对接研究化合物1与VEGFR3的结合模式,以此对化合物1的结构进行优化和构效关系研究。第一步,通过对化合物1的噻吩并[2,3-d]嘧啶环骨架进行骨架跃迁,我们锁定噻吩并[2,3-d]嘧啶为优势骨架结构;第二步,我们对噻吩并[2,3-d]嘧啶的6位取代部分进行详细考察,找到N-甲基-4-（对苯基）哌嗪取代能够使生物活性显著提升;第三步,我们对噻吩并[2,3-d]嘧啶的2位取代部分进行探究,发现在该位置引入疏水基团可以提高抗增殖活性,但不利于VEGFR3抑制活性;第四步,我们对链接基团哌嗪进行结构修饰,以此考察对生物活性的影响。构效研究发现,对哌嗪基团的结构修饰会导致哌嗪发生不利于分子与蛋白结合的构象变化;最后,我们对头部基团进行优化,发现引入含有脲基的大位阻基团能够显著提高生物活性。整个研究过程共计合成52个化合物,最终发现优选化合物22k,该化合物对VEGFR3酶水平抑制活性为110.4 n M,并具有较优的抗TNBC细胞增殖活性(MDA-MB-231,IC₅₀=2.2μM;MDA-MB-436,IC₅₀=3.5μM)。另外,化合物22k对VEGFR3具有良好的选择性(VEGFR1,IC₅₀>10μM;VEGFR2,IC₅₀>10μM),优于阳性对照SAR131675(VEGFR1,IC₅₀=3100 n M;VEGFR2,IC₅₀=220 n M)。此外,化合物22k具有可接受的口服药代性质,口服生物利用度达到30.9%。随后,我们利用多种分子生物学实验对化合物22k的作用机制及体内药效进行深入研究。体外机制研究表明,在VEGFC诱导的人表皮淋巴管内皮细胞HDLEC中,22k通过靶向VEGFR3抑制细胞迁移和淋巴管路形成。同时,22k(IC₅₀=250 n M)表现出比阳性对照SAR131675(IC₅₀=307 n M)更优的抑制活性。在TNBC细胞MDA-MB-231和MDA-MB-436中,化合物22k通过靶向VEGFR3下调下游信号通路中STAT5、Akt和ERK1/2的磷酸化水平,诱导TNBC细胞凋亡和阻滞细胞周期于G1期。体内药效研究表明,化合物22k能以剂量依赖的方式显著抑制斑马鱼模型中血管的形成。在MDA-MB-231异种移植瘤模型中,化合物22k在50 mg/kg口服剂量下抑瘤率为61.9%,并且该化合物显示出比SAR131675更优的体内安全性。在MDA-MB-231肺部转移模型中,化合物22k能够有效抑制肺转移结节的形成。进一步体内机制研究表明,化合物22k通过选择性抑制VEGFR3及其下游信号通路蛋白达到体内抗肿瘤生长和抑制TNBC转移的效果。综上所述,我们获得了一个新型噻吩并[3,2-d]嘧啶类VEGFR3选择性抑制剂22k,显示了良好的体内外活性和抗转移活性。机制研究表明,化合物22k通过特异性靶向VEGFR3抑制TNBC肿瘤生长和转移,以上研究为发现新型小分子VEGFR3选择性抑制剂用于治疗TNBC提供研究基础,也为进一步研究VEGFR3的生物学功能提供工具分子。二、VEGFR/PARP双靶标抑制剂的设计、合成及抗TNBC活性研究为了增加VEGFR抑制剂的疗效,减少毒副作用,临床上常使VEGFR抑制剂联合化疗、放疗、手术、内分泌治疗等方案达到最大的协同增效作用。最新的研究表明,VEGFR抑制剂通过抑制血管生成而形成缺氧环境,致使关键同源重组修复相关因子（RAD51和BRCA1/2）表达下调,造成“BRCAness”影响DNA修复能力,在此环境中可以使未携带BRCA突变的肿瘤患者从聚腺苷二磷酸核糖聚合酶（Poly ADP-ribose polymerase,PARP）抑制剂中获益。在一项VEGFR抑制剂联合治疗策略的临床研究中,VEGFR抑制剂cediranib与PARP抑制剂olaparib联用,治疗复发性卵巢癌和TNBC患者（NCT02484404）,结果表明VEGFR抑制剂cediranib和PARP抑制剂olaparib联用不仅对携带BRCA基因突变的患者产生良好的疗效,也提高了非BRCA突变患者的无进展生存期。不仅如此,VEGFR抑制剂还可以在一定程度上逆转PARP抑制剂获得性耐药。另外,PARP抑制剂也可以提高VEGFR抑制剂的抗肿瘤血管生成活性。因此,通过研究双靶向VEGFR/PARP的小分子抑制剂为治疗TNBC提供了一个富有潜力的策略。基于以上背景,我们从VEGFR抑制剂pazopanib和PARP抑制剂veliparib结构出发,利用药效团融合策略,设计并合成了化合物4a和5a。具体而言,化合物4a和5a具有较好的VEGFR2（1μM时的抑制率分别为91.3%和86.6%）和PARP1（1μM时抑制率分别为70.1%和81.3%）抑制活性,并在低微摩尔浓度下抑制BRCA野生型TNBC细胞MDA-MB-231(IC₅₀值分别为12.1μM和7.7μM)和BRCA野生型原位ER阳性BC细胞MCF-7(IC₅₀值分别为10.5μM和6.4μM)的增殖。与阳性对照pazopanib(1μM时VEGFR2抑制率为97.7%;MDA-MB-231,IC₅₀=30.7μM;MCF-7,IC₅₀=34.6μM)和olaparib(1μM时PARP1抑制率为98.3%;MDA-MB-231,IC₅₀=35.4μM;MCF-7,IC₅₀=22.5μM)相比,化合物4a和5a的酶水平抑制活性较弱。为了提高靶标抑制活性,我们对化合物4a和5a进行了深入的构效关系研究和结构优化。第一步,为了评价2,3-二甲基-6-氨基-2H-吲唑片段对生物活性的影响,我们利用生物电子等排策略对化合物4a和5a中的2,3-二甲基-6-氨基-2H-吲唑片段进行结构优化,但未发现更理想的取代基团;第二步,我们对嘧啶母核进行优化,酶水平抑制活性表明嘧啶骨架的5位或6位取代及将嘧啶转换为噻吩并嘧啶结构都不利于分子与VEGFR2结合,进而导致分子的VEGFR2激酶活性下降甚至丧失;第三步,我们对链接区域的苯环进行了详细考察,发现在链接区域苯环的间位引入PARP药效团能够使生物活性显著提升,若同时在苯环对位引入甲基则更有利于生物活性;最后,我们对1H-苯并[d]咪唑-4-甲酰胺片段进行优化,但没有发现更为理想的取代基团。整个研究过程共计合成49个化合物,最终发现具有良好双靶向生物活性的化合物14b。具体而言,化合物14b具有较好的靶标抑制活性(VEGFR2,IC₅₀=190.6 n M;PARP1,IC₅₀=60.9 n M),和较优的BRCA野生型乳腺癌细胞增殖活性(MDA-MB-231,IC₅₀=4.1μM;MDA-MB-436,IC₅₀=3.9μM),同时具有一定的激酶选择性。另外,我们对化合物14b进行大鼠药代动力学评价,结果发现该化合物具有良好的口服药代性质,口服生物利用度达到60.1%。随后,我们利用多种分子生物学实验对化合物14b的作用机制及体内药效进行深入研究。体外机制研究表明,在MDA-MB-231和MCF-7中,化合物14b通过靶向PARP和VEGFR诱导细胞凋亡,抑制细胞周期进程,抑制DNA损伤修复而显示出良好的协同抗增殖作用。在VEGF165诱导的HUVEC细胞中,化合物14b(IC₅₀=2.2μM)具有比pazopanib与olaparib联用组(IC₅₀=4.6μM)更优的抗增殖活性。同时,化合物14b具有抗HUVEC细胞迁移和侵袭活性。在MDA-MB-231异种移植瘤模型中,化合物14b在50 mg/kg口服剂量下抑瘤率为72.1%,且该化合物显示出良好的体内安全性。在MDA-MB-231肺部转移模型中,化合物14b能够有效抑制肺转移结节的形成。进一步体内机制研究表明,化合物14b通过靶向VEGFR和PARP达到体内协同抗肿瘤生长和抑制TNBC肺转移的效果。以上研究都表明,VEGFR/PARP双靶抑制剂14b具有一定的治疗TNBC的潜力,为VEGFR抑制剂用于TNBC的治疗研究提供新方法与新思路。综上所述,本论文主要针对用于TNBC治疗的新型VEGFR3选择性抑制剂和VEGFR/PARP双靶抑制剂的设计、合成和构效关系进行研究,取得以下成果:（1）获得了一个新型噻吩并[3,2-d]嘧啶类VEGFR3选择性抑制剂22k,显示了良好的激酶选择性,体内安全性及体内外抗TNBC增殖和转移活性;（2）获得了一个新型VEGFR/PARP双靶标抑制剂14b,显示了良好的药代动力学特征和体内外抗TNBC增殖和转移活性。本论文为用于TNBC治疗的新型VEGFR抑制剂研发奠定了科学基础。更多还原

【Abstract】 Breast cancer（BC）is one of the most common female malignancies in the clinic.According to gene expression profiles,BC is classified into four major subtypes:Luminal A,luminal B,HER2-enriched,and triple-negative breast cancer（TNBC）.The TNBC subtype characteristically lacks expression of ER,PR,and HER-2,and accounts for 15%of all BC diagnoses.TNBC,as a heterogeneous disease,is characterized by diverse clinical courses,and pathological,molecular and genetic features.Additionally,it is associated with early age of onset,high degree of malignancy,high recurrence rate,early metastasis,and poor prognosis.Currently,the commonly used treatment method for TNBC is still surgery and conventional systemic cytotoxic chemotherapy.Anthracycline-and paclitaxel-based regimens have been widely used in postoperative adjuvant therapy for TNBC.However,traditional chemotherapeutic agents have the common drawback of high toxicity.Moreover,the prognosis of TNBC remains poor due to the lack of effective therapies and the high probability of tumor metastasis or recurrence after routine treatment.Angiogenesis and lymphangiogenesis are vital processes for the development and propagation of TNBC.Several studies have demonstrated that anti-angiogenic strategies that target vascular endothelial growth factor receptor（VEGFR）has shown tremendous potential in the field of effective TNBC therapy.However,VEGFR inhibitors also suffer from mechanism-related toxicities（including hypertension,fistula formation,and reversible posterior leukoencephalopathy）,short duration of efficacy,and acquired drug resistance.Therefore,it is demanding to identify novel and potent VEGFR inhibitors with low toxicity and high selectivity for the treatment of TNBC.This study also attempts to further enrich the knowledge regarding the potential function and molecular mechanism of VEGFR in TNBC and to provide a scientific basis for VEGFR inhibitors as a potential drug for the treatment of TNBC.During the period of my Ph.D.degree study,I carried out the design,synthesis and structure-activity relationship of novel VEGFR3-selective and VEGFR/PARP dual-target inhibitors to address the shortcomings of existing VEGFR inhibitors for the treatment of TNBC.The details are briefly described as follows:Part Ⅰ.Design,synthesis,and evaluation of highly selective VEGFR3 inhibitor for the potential treatment of metastatic TNBCIt is well known that TNBC metastasis is a multi-gene and multi-step process,and is often associated with poor prognosis and short survival time.In addition,metastasis is one of the leading lethal causes in TNBC.Although plenty of metastasis-related genes and the tumor-associated microenvironment have been deeply investigated,the specific regulatory mechanisms remains unclear.As established endocrine and targeted therapies are ineffective against TNBC,radio-and chemo-therapy remain the primary regimens for the treatment of TNBC.However,the application of these regimens is limited due to adverse side effects and rapidly developed drug resistance.Therefore,patients with TNBC have inferior disease free survival and overall survival as compared to non-TNBC patents.Lymphangiogenesis and lymphatic vessel remodeling play a critical role in TNBC distal metastasis.Specifically,the primary tumor connects to the lymph nodes through abnormally generated peritumoral lymphatics,facilitating the entry of tumor cells into the lymph nodes,systemic circulation and distal metastasis.Thus,anti-lymphatic angiogenesis is one of the promising strategies in the field of TNBC metastasis treatment.Inhibition of lymphovascular-mediated metastasis is the key to blocking the malignant progression of TNBC,which will effectively enhance the treatment effect,prolong patient survival and greatly improve the disease prognosis.VEGFR3 has significant biological functional differences from other family members,and plays an important role in lymphangiogenesis associated with TNBC metastasis.However,no VEGFR3 inhibitors for cancer therapy have yet been approved.Efforts in this area have been hampered by the lack of a VEGFR3 crystal structure.For this reason,structure-based design of selective inhibitors of VEGFR3 faced a considerable challenge.SAR131675 is the only reported selective VEGFR3 inhibitor that has been shown to be effective in inhibiting lymph node invasion and lung metastasis in in vivo studies,but its low in vivo potency and cumulative toxicity have prevented its further clinical evaluation.Therefore,a robust need exists to design and develop VEGFR3 inhibitors that are highly potent,selective and safe.In this chapter,hit compound 1 was identified by VEGFR3 random screen and its potency for VEGFR3(IC₅₀=3.5μM)was weak.Homology modeling was used to predict three-dimensional structure of VEGFR3 kinase.Furthermore,the binding mode of compound 1 to VEGFR3 was studied by molecular docking.Further optimization and SAR studies of compound 1 resulted in the identification of a novel series of VEGFR3 inhibitors with thieno[2,3-d]pyrimidine scaffold.Firstly,we locked the thieno[2,3-d]pyrimidine as the dominant essential scaffold by investigating the structure-activity relationship of compound 1;Secondly,we examined the 6-position substitution of thieno[2,3-d]pyrimidines in detail and finally found that the substitution of N-methyl-4-（p-phenyl）piperazine substitution could lead to a significant increase in efficacy;Thirdly,the 2-position substitution of thieno[2,3-d]pyrimidine scaffold was investigated in detail,and it found that the introduction of hydrophobic groups at this position improved antiproliferative activity but was detrimental to VEGFR3 inhibitory activity;Fourthly,we investigated the effect on biological activities through structurally modifying the linking group piperazine.It was found that structural modification of the piperazine moiety leads to conformational changes in piperazine that are detrimental to the binding of the molecule to the target protein;Finally,the head group was optimized.Interestingly,the introduction of a steric hindrance group containing a urea group could significantly improve the biological activities.A total of 52 molecules were synthesized,and compound 22k showed the most remarkable inhibitory activity against VEGFR3 with an IC₅₀value of 110.4 n M,and presented an acceptable kinase selectivity.In VEGFC-induced HDLEC cells,22k exhibited the most effective inhibitory effect(IC₅₀=250 n M)compared with that of positive control SAR131675(IC₅₀=307 n M).Functionally,22k was capable of suppressing lymphatic angiogenesis,and migration/invasion,and inducing cell apoptosis and cell cycle arrest in G1-phase of BC cells through inactivating the the VEGFR3 signaling pathway.In MDA-MB-231and MDA-MB-436 cells,compound 22k possessed highly anti-proliferative effect with IC₅₀values of 2.2 and 3.5μM,respectively.In vivo assays consistently indicated that compound 22k could significantly inhibit angiogenesis,metastasis and growth of xenografted TNBC in a dose-dependent manner.Moreover,this compound showed acceptable pharmacokinetic capabilities.Taken together,all these findings supported22k as a novel and selective VEGFR3 inhibitor for the treatment of TNBC,which is worthy of further research and development.Part Ⅱ.Design,synthesis,and evaluation of novel dual VEGFR/PARP inhibitors for the treatment of TNBCPresently,the identification of VEGFR/PARP dual-target inhibitors for the treatment of TNBC was implemented.The latest studies showed that VEGFR inhibitors create a hypoxic environment by inhibiting angiogenesis,resulting in downregulation of expression of key homologous recombination repair factors（RAD51,BRCA1/2）and reduced DNA repair capacity.Due to the reduced DNA repair potential,the sensitivity of poly ADP-ribose polymerase（PARP）inhibitors may be enhanced in a hypoxic environment,which could allow BRCA wild-type patients to benefit from PARP inhibitors.In a clinical study of a combination treatment strategy with VEGFR inhibitors,cediranib（VEGFR inhibitor）is combined with PARP inhibitor olaparib for the treatment of patients with recurrent ovarian and breast cancer（NCT02484404）.Further investigation indicated that the combination of cediranib and olaparib not only produced good efficacy in patients carrying BRCA mutations,but also improved progression-free survival in patients who did not carry BRCA mutations.Additionally,VEGFR inhibitors can also reverse the acquired resistance of PARP inhibitors to some extent.Similarly,PARP inhibitors can also enhance the anti-tumor angiogenic activity of VEGFR inhibitors.Furthermore,this study will explore the molecular mechanisms underlying the efficacy of VEGFR/PARP dual inhibitors,which would lay the foundation for the identification of novel drugs for the treatment of BRCA wild-type TNBC.In this chapter,we designed and synthesized a dual VEGFR/PARP inhibitors through rational drug design.Specifically,based on the pharmacophore fusion strategy,we selected the pharmacophore of VEGFR inhibitor pazopanib and the pharmacophore of PARP inhibitor veliparib to deign and obtain novel dual VEGFR/PARP inhibitors 4a and 5a.Compounds 4a and 5a display favourable efficacy against VEGFR2（inhibition rates of VEGFR2 at 1μM were 91.3%and86.6%,respectively）,PARP1（inhibition rates of PARP1 at 1μM were 70.1%and81.3%,respectively）,and BRCA wild-type BC cells MDA-MB-231 and MCF-7.Compared with the positive control pazopanib（inhibition rates of VEGFR2 at 1μM was 97.7%）and olaparib（inhibition rates of PARP1 at 1μM was 98.3%）,compounds 4a and 5a exhibited inferior potency against VEGFR2 and PARP1,but showed obviously higher efficacy against BRCA wild-type BC cells.To obtain an excellent compound with higher inhibitory activities against VEGFR2,PARP1,and BRCA wild-type BC cells,we served compounds 4a and 5a as the lead compounds for structure-activity relationship analysis and further structural optimization.Firstly,the substitution of 2,3-dimethyl-6-amino-2H-indazole group was explored in detail,but no more ideal substituent fragments were identification;Secondly,the pyrimidine scaffold was optimized.However,In vitro studies demonstrated that the substitution at the 5-or 6-position of the pyrimidine scaffold and the conversion of pyrimidines to thienopyrimidine are detrimental to the binding of the molecule to VEGFR2,resulting in a decrease or loss of the inhibitory activities against VEGFR2;Thirdly,to investigate the effect of the modification of the benzene ring in the linkage region,the substitution of the benzene ring was investigated in detail.Wonderfully,the introduction of PARP pharmacophore in the interposition of the benzene ring resulted in increased biological activity,while the introduction of methyl in the opposite position of the benzene ring was beneficial to the potency;Finally,the1H-benzo[d]imidazole-4-carboxamide moiety was optimized,but no more ideal substituent groups were found.A series of the structurally diverse pazopanib-and veliparib-based PARP1 and VEGFR dual inhibitors were obtained.Among them,compound 14 b showed superior inhibitory activities against VEGFR1,VEGFR2,VEGFR3,PAPR1 and PARP2 with IC50 values of 499.4 n M,190.6 n M,166.0 n M,60.9 n M and 78.4 n M,respectively.In MDA-MB-231 and MCF-7 cells,compound14 b possessed highly anti-proliferative effect with IC50 values of 4.1 μM and 3.9 μM,respectively.Mechanistic studies revealed that compound 14 b exerted favorable synergistic potency in BRCA wild-type BC cells through arresting cell cycle progression,inducing apoptosis,and suppressing DNA damage repair.Furthermore,compound 14 b possessed a higher efficiency against the VEGF165-induced HUVEC cells（IC50 = 3.9 μM）when compared with the combination of pazopanib and olaparib（IC50 = 4.6 μM）.In vivo assays consistently indicated that compound 14 b could significantly inhibit angiogenesis,metastasis and growth of xenografted TNBC in a dose-dependent manner.Moreover,the oral bioavailability of compound 14 b was determined to 60.1%,which showed the pharmacokinetic profile was favorable.The advantages of compound 14 b over olaparib and pazopanib demonstrate that dual VEGFR/PARP inhibitors are promising drugs for the treatment of BRCA wild-type TNBC.Collectively,this thesis focused on the design,synthesis,and structure-activity relationship of novel VEGFR inhibitors for TNBC therapy,with the following main results:（i）a novel thieno[3,2-d]pyrimidine VEGFR3-selective inhibitor 22 k was obtained,showing a good kinase selectivity,in vivo safety and anti-proliferative and anti-metastatic activities in vitro and in vivo;（ii）a novel VEGFR/PARP dual-target inhibitor 14 b,which showed good pharmacokinetic profile and anti-proliferative and anti-metastatic potency against BRCA wild-type TNBC cells in vitro and in vivo.This thesis has laid a good foundation and accumulated valuable experience for the development of novel VEGFR inhibitors for the treatment of TNBC.更多还原