蝶啶类表皮生长因子受体酪氨酸激酶抑制剂的设计、合成及其抗癌活性研究
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摘要
蛋白酪氨酸激酶在细胞的信号传导通路中占据了十分重要的地位，调节着细胞的生长、分化、凋亡等一系列生理生化过程。蛋白酪氨酸激酶功能的失调会引发生物体内的一系列疾病。特别是它们的异常表达不但导致细胞增殖调节发生紊乱，进而导致肿瘤发生，而且还与肿瘤的侵袭和转移，肿瘤新生血管的生成，肿瘤的放化疗抗性密切相关。有效抑蛋白制酪氨酸激酶，可以达到治疗肿瘤的目的。因此，蛋白酪氨酸激酶已成为抗肿瘤药物研究的新靶点。
表皮生长因子受体酪氨酸激酶(EGFR)是最早发现的蛋白酪氨酸激酶，EGFR的胞内区有ATP结合位点，EGFR抑制剂可以竞争性与ATP结合位点相结合，从而抑制EGFR的磷酸化过程，阻断下游信号的传导，进而抑制肿瘤细胞的生长、分化和转移。因为EGFR作为抗肿瘤靶点的生化过程正在被逐步阐明，其晶体结构和活性部位也已经比较清楚，以此为靶点的药物吉非替尼(Gefitinib)和埃罗替尼(Erlotinib)已经应用于临床，所以EGFR抑制剂的研究具有较好的前景。通过设计、合成新的化合物，然后进行筛选，我们希望从中能够发现活性好、选择性高的新型结构的先导化合物。
人们在以EGFR为靶点设计、合成EGFR抑制剂的探索中发现，苯胺基喹唑啉类化合物有较好的活性和选择性，并总结出了其构效关系。特别是PD0153035的发现，为进一有研究喹唑啉类EGFR抑制剂的构效关系起到了至关重要的作用。以后的许多化合物都是以PD 0153035为先导化合物进行结构改造和修饰的。然而喹唑啉环并非EGFR抑制剂所必须的，在随后的研究中，人们发现许多氮杂喹唑啉类也有很好的活性，如吡啶并[2，3-d]嘧啶类、吡啶并[3，2-d]嘧啶类、吡啶并[4，3-d]嘧啶类、吡啶并[3，4-d]嘧啶类、嘧啶并[5，4-d]嘧啶类，也就是在喹唑啉环的5,6,7,8-位都可以氮杂，且都有活性。另外五元环也可和嘧啶环稠和，如吡咯并[2，3-d]嘧啶类、吡唑并[3，4-d]嘧啶类也有较好的前景。
上述研究成果使我们想到了蝶啶环，因为蝶啶可看作是喹唑啉环的5,8-位氮杂环，而且取代基在6-位是天然蝶啶化合物的普通模式。鉴于此，根据该类化合物的构效关系，以PD 0153035为先导化合物，利用生物电子等排理论，设计了两类蝶啶类衍生物——6-烷氧基-4-芳胺基蝶啶类化合物Ⅰ和6,7-双烷氧基-4-芳胺基蝶啶类化合物Ⅲ，结构如下图所示：
其中，R_1，R_2=烷基，R=Cl，Br,F，CH_3等。
蝶啶类化合物在生物体内有着广泛的生理活性，从而为新药开发提供了新的母核，如在抗肿瘤药物、腺苷激酶抑制剂、一氧化氮合成酶抑制剂等中都有报道。目前，蝶啶类化合物国外研究较多，但国内研究较少，特别是烷氧基取代的蝶啶衍生物的合成很少见报道。
由于未检索到该类化合物的类似合成报道，我们对设计的目标化合物进行逆合成分析，经反复实验探索，不断对合成工艺进行修改和调整，最终找到了切实可行的合成路线。
在合成Ⅲ类化合物的过程中，我们意外发现N~4-芳基-4,5,6-三氨基嘧啶在和草酸环合时，具有区域选择性，最终导致了N~8-芳基-6-烷氧(胺)基-4-氨基-7(8H)蝶啶酮类化合物Ⅱ的发现(如上图所示)。
采用柔性分子对接方法对所设计的三类蝶啶化合物以及参考化合物Iressa与EGFR进行了分子对接研究，了解了设计化合物与EGFR的结合方式及相互作用机制，结果显示，设计的化合物能进入到ATP的结合区，并与ATP的结合位点有着较强的疏水作用和静电相互作用，为针对性设计或改善酪氨酸酶抑制剂提供了科学依据。
对Ⅰ类化合物的合成，我们用3-氨基吡嗪-2-甲酸经酯化、酯氨解、溴化、环合、烷氧基取代、氯化和芳胺取代7步反应而得到。总收率达39％以上。根据烷氧基和芳胺的不同，我们制备了20个未见文献报道的新化合物。
对Ⅱ类化合物的合成，我们以丙二酸二乙酯为原料，经环合、硝化、氯化、芳胺取代、氨取代、还原、草酸环合、氯代、烷氧基或胺取代9步反应而得到。总收率达22％以上。根据芳胺和侧链的不同，我们合成了13个未见文献报道的新化合物。
对Ⅲ类化合物的合成，我们用3-氨基吡嗪-2-甲酸经酯化、氯化、烷氧基取代、酯水解、内酯化、氨解环合、烷氧基取代、氯化和芳胺取代9步反应而得到。总收率达26％以上。根据芳胺的不同，我们制备了5个未见文献报道的新化合物。
上述所有化合物的结构均经~1H NMR、~(13)C NMR、ESI-MS和元素分析确证。在合成各类化合物的过程中，共得到未见文献报道的新型中间体50多个，并根据其结构特征，用相应的光谱对其结构进行确证。
在对Ⅱ类化合物的合成中，N~4-芳基-4,5,6-三氨基嘧啶在和草酸环合时，可能得到两种异构体，因为结构的相似性，用~1H NMR、~(13)C NMR、MS和元素分析不易确定，而其本身的溶解性较差，其氯化后的产品有较好的溶解性，我们对氯化产品培养了单晶，通过对单晶的分析，了解了环合的方式，发现了该环合反应的区域选择性。同时通过对脂肪族取代的三氨基嘧啶与草酸的环合反应的研究，发现两种环合方式都存在，这又说明了该环合反应的局限性，只能用于N~4-芳基-4,5,6-三氨基嘧啶与草酸的环合。
用MTT法对Ⅱ类化合物进行了体外培养的人肿瘤细胞的细胞增殖毒性试验，选取人早幼粒细胞白血病HL-60细胞、人胃腺癌SGC-7901细胞和人结肠癌SW-480细胞三种癌细胞为试验对象，用5-氟尿嘧啶为阳性对照。结果表明，所设计的Ⅱ类化合物对SGC-7901有较好的抑制作用，部分化合物对HL-60细胞也有明显的抑制作用，个别化合物在相似浓度下对SGC-7901和HL-60细胞的抑制作用和对照药物5-氟尿嘧啶相当。对SW-480细胞的抑制作用很弱。
Ⅰ和Ⅲ类化合物的药理实验正在进行中。
The protein tyrosine kinases (PTKs) play crucial roles in many of the signal transduction pathways regulating lots of cellular functions, such as cell growth, proliferation and apoptosis. Over-expression of the PTKs is thought to be related to cell proliferation, invasion, metastasis, resistance to chemoradiotherapy and poor prognosis of various human cancers. Targeted interference with PTKs activation and/or with intracellular PTK-activated signal transduction pathways represents a promising strategy for the development of novel and selective anticancer therapies.
Epidermal growth factor receptor tyrosine kinase (EGFR) is the first PTK to be examined seriously as a drug target. EGFR has its own ATP binding site in the intracellular domain. EGFR inhibitors can bind competitively to the ATP binding site and subsequently inhibit the phosphorylation of the EGFR as well as the downstream signaling pathways, then depress the growth, proliferation and metastasis of tumor. Two drugs, Gefitinib and Erlotinib, inhibiting the EGFR, have been launched for clinical use. Thus, EGFR has been identified as a promising target of tumor therapy.
During the research for drugs targeting the EGFR, anilinoquinazolines were found to inhibit EGFR potently and selectively by binding to the ATP binding site reversibly. PD 0153035, a quinazoline derivative, played an important role in establishing the structure-activity relationship(SAR) and many of the quinazoline derivatives were designed on the basis of PD 0153035. However, the research found that the quinazoline ring is not necessary for inhibiting EGFR. Many of the aza quinazolines have showed potent activity, such as pyrido[2,3-d]pyrimidine, pyrido [3,2-d]pyrimidine, pyrido[4,3-d]pyrimidine, pyrido[3,4-d]pyrimidine, and pyrimido [5,4-d]pyrimidine. Moreover, the compounds that the benzene in quinazoline ring was substituted by five-member rings, such as pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine, can also inhibit the EGFR and show bright prospects.
The studies stated above reminded us of the pteridine ring, as pteridine ring could be regarded as quinazoline ring that 5-C and 8-C are substituted by nitrogen atoms. Meanwhile the 6-substituted pteridines are the common forms in natural pteridine derivatives. In view of this, according to the SAR, we designed 2 series of pteridine derivatives : 6-alkoxy-4-arylaminopteridinesⅠand 6,7-dialkoxy-4- arylaminopteridinesⅢ, using the bioisoster theory and PD 0153035 as lead compound, the structures of the compounds we designed are shown as follow:
Pteridine-like compounds exhibit a wide range of physiological activity in human body and provide a novel template for new drugs, such as anticancer agents, adenosine kinase inhibitors and nitric oxide synthase inhibitors. At present, the study of pteridine derivatives is extensive abroad, but less in our country. Moreover, the preparation has seldom been reported for the alkoxy-substituted pteridines.
As no related reports about the synthesis of the designed compounds, we by ourselves designed the synthetic routes by means of retro-synthetic analysis. Through modifying and justifying the routes repeatedly, we eventually found the practical routes.
During the preparing of compoundsⅢ, we discovered unexpectedly that the cyclization of N~4-aryl-4,5,6-triaminopyrirnidine with oxalic acid is regioselective, which led to the discovery of a series of novel compounds: N~8-aryl-6-alkoxy (alkylamino)-4-amino-7(8H)pteridinonesⅡ(As shown above).
The flexible molecular docking method was applied to investigate the binding of the designed compounds to EGFR. The binding modes and the interaction mechanism between the designed compounds and EGFR were understood. The results, which are helpful for new drugs design, showed the designed compounds can be able to dock into the ATP binding domain by hydrophobic and electrostatic interactions.
CompoundsⅠstarted from 3-aminopyrazine-2-formic acid, from which 20 6-alkoxy-4-arylaminopteridine derivatives were obtained by esterification, amidation, bromination, cyclization, alkoxylation, chlorination and aromatic amination in a 7-step procedure with an overall yield of 39%. CompoundsⅡcommenced from diethyl malonate, from which 13 new pteridine derivatives were prepared in an overall yield of 22% in a 9-step procedure including cyclization, nitration, chlorination and arylamine substitution, amination, reduction, cyclization with oxalic acid, chlorination, alkoxylation or alkylamination. For compoundsⅢ, 3-aminopyrazine-2-formic acid was used as starting material, which underwent esterification, chlorination, alkoxylation, ester hydrolysis, lactonization, amidation and cyclization, alkoxylation, chlorination and aromatic amination to produce 5 new compounds in a total yield of 27% in a 9 steps procedure.
The structure of all compounds had been confirmed by ~1H NMR, ~(13)C NMR, ESI-MS and elemental analysis. More than 50 new intermediates were prepared through the experimental process, and elucidated from the corresponding spectra according to their structural characteristics.
Two possible isomers may occur in the cyclization of N~4-aryl-4,5,6-tri aminopyrimidine with oxalic acid. Their structural similarity makes it difficult to be confirmed by ~1H NMR, ~(13)C NMR, ESI-MS and elemental analysis. A single crystal of its chlorinated compound revealed its structure, and the cyclization pattern and regioselectivity were understood. However, the regioselectivity disappeared when aliphatic amino group replaced the arylamino group. Thus, the method can only be used to the cyclization of N~4-aryl-4,5,6-triaminopyrimidine with oxalic acid.
The toxicity of the compoundsⅡagainst human tumor cell proliferation was evaluated using MTT assay. Three kinds of cancer cell lines, including human promyelocytic leukemia HL-60 cells, human gastric adenocarcinoma SGC-7901 cells and human colon cancer cells SW-480, were selected to test the target compoundsⅡa-Ⅱm, using 5-FU as the contrast. The results suggested that the designed compounds II showed a certain inhibitory effect on SGC-7901 cells, some of them on HL-60 cells. At the similar concentration, the individual compounds showed equivalent inhibition against SGC-7901 and HL-60 cells to that of 5-FU. The designed compoundsⅡexhibited little effects on SW-480 cells.
The pharmacological experiments of compoundsⅠandⅢare in progress.
表皮生长因子受体酪氨酸激酶(EGFR)是最早发现的蛋白酪氨酸激酶，EGFR的胞内区有ATP结合位点，EGFR抑制剂可以竞争性与ATP结合位点相结合，从而抑制EGFR的磷酸化过程，阻断下游信号的传导，进而抑制肿瘤细胞的生长、分化和转移。因为EGFR作为抗肿瘤靶点的生化过程正在被逐步阐明，其晶体结构和活性部位也已经比较清楚，以此为靶点的药物吉非替尼(Gefitinib)和埃罗替尼(Erlotinib)已经应用于临床，所以EGFR抑制剂的研究具有较好的前景。通过设计、合成新的化合物，然后进行筛选，我们希望从中能够发现活性好、选择性高的新型结构的先导化合物。
人们在以EGFR为靶点设计、合成EGFR抑制剂的探索中发现，苯胺基喹唑啉类化合物有较好的活性和选择性，并总结出了其构效关系。特别是PD0153035的发现，为进一有研究喹唑啉类EGFR抑制剂的构效关系起到了至关重要的作用。以后的许多化合物都是以PD 0153035为先导化合物进行结构改造和修饰的。然而喹唑啉环并非EGFR抑制剂所必须的，在随后的研究中，人们发现许多氮杂喹唑啉类也有很好的活性，如吡啶并[2，3-d]嘧啶类、吡啶并[3，2-d]嘧啶类、吡啶并[4，3-d]嘧啶类、吡啶并[3，4-d]嘧啶类、嘧啶并[5，4-d]嘧啶类，也就是在喹唑啉环的5,6,7,8-位都可以氮杂，且都有活性。另外五元环也可和嘧啶环稠和，如吡咯并[2，3-d]嘧啶类、吡唑并[3，4-d]嘧啶类也有较好的前景。
上述研究成果使我们想到了蝶啶环，因为蝶啶可看作是喹唑啉环的5,8-位氮杂环，而且取代基在6-位是天然蝶啶化合物的普通模式。鉴于此，根据该类化合物的构效关系，以PD 0153035为先导化合物，利用生物电子等排理论，设计了两类蝶啶类衍生物——6-烷氧基-4-芳胺基蝶啶类化合物Ⅰ和6,7-双烷氧基-4-芳胺基蝶啶类化合物Ⅲ，结构如下图所示：
其中，R_1，R_2=烷基，R=Cl，Br,F，CH_3等。
蝶啶类化合物在生物体内有着广泛的生理活性，从而为新药开发提供了新的母核，如在抗肿瘤药物、腺苷激酶抑制剂、一氧化氮合成酶抑制剂等中都有报道。目前，蝶啶类化合物国外研究较多，但国内研究较少，特别是烷氧基取代的蝶啶衍生物的合成很少见报道。
由于未检索到该类化合物的类似合成报道，我们对设计的目标化合物进行逆合成分析，经反复实验探索，不断对合成工艺进行修改和调整，最终找到了切实可行的合成路线。
在合成Ⅲ类化合物的过程中，我们意外发现N~4-芳基-4,5,6-三氨基嘧啶在和草酸环合时，具有区域选择性，最终导致了N~8-芳基-6-烷氧(胺)基-4-氨基-7(8H)蝶啶酮类化合物Ⅱ的发现(如上图所示)。
采用柔性分子对接方法对所设计的三类蝶啶化合物以及参考化合物Iressa与EGFR进行了分子对接研究，了解了设计化合物与EGFR的结合方式及相互作用机制，结果显示，设计的化合物能进入到ATP的结合区，并与ATP的结合位点有着较强的疏水作用和静电相互作用，为针对性设计或改善酪氨酸酶抑制剂提供了科学依据。
对Ⅰ类化合物的合成，我们用3-氨基吡嗪-2-甲酸经酯化、酯氨解、溴化、环合、烷氧基取代、氯化和芳胺取代7步反应而得到。总收率达39％以上。根据烷氧基和芳胺的不同，我们制备了20个未见文献报道的新化合物。
对Ⅱ类化合物的合成，我们以丙二酸二乙酯为原料，经环合、硝化、氯化、芳胺取代、氨取代、还原、草酸环合、氯代、烷氧基或胺取代9步反应而得到。总收率达22％以上。根据芳胺和侧链的不同，我们合成了13个未见文献报道的新化合物。
对Ⅲ类化合物的合成，我们用3-氨基吡嗪-2-甲酸经酯化、氯化、烷氧基取代、酯水解、内酯化、氨解环合、烷氧基取代、氯化和芳胺取代9步反应而得到。总收率达26％以上。根据芳胺的不同，我们制备了5个未见文献报道的新化合物。
上述所有化合物的结构均经~1H NMR、~(13)C NMR、ESI-MS和元素分析确证。在合成各类化合物的过程中，共得到未见文献报道的新型中间体50多个，并根据其结构特征，用相应的光谱对其结构进行确证。
在对Ⅱ类化合物的合成中，N~4-芳基-4,5,6-三氨基嘧啶在和草酸环合时，可能得到两种异构体，因为结构的相似性，用~1H NMR、~(13)C NMR、MS和元素分析不易确定，而其本身的溶解性较差，其氯化后的产品有较好的溶解性，我们对氯化产品培养了单晶，通过对单晶的分析，了解了环合的方式，发现了该环合反应的区域选择性。同时通过对脂肪族取代的三氨基嘧啶与草酸的环合反应的研究，发现两种环合方式都存在，这又说明了该环合反应的局限性，只能用于N~4-芳基-4,5,6-三氨基嘧啶与草酸的环合。
用MTT法对Ⅱ类化合物进行了体外培养的人肿瘤细胞的细胞增殖毒性试验，选取人早幼粒细胞白血病HL-60细胞、人胃腺癌SGC-7901细胞和人结肠癌SW-480细胞三种癌细胞为试验对象，用5-氟尿嘧啶为阳性对照。结果表明，所设计的Ⅱ类化合物对SGC-7901有较好的抑制作用，部分化合物对HL-60细胞也有明显的抑制作用，个别化合物在相似浓度下对SGC-7901和HL-60细胞的抑制作用和对照药物5-氟尿嘧啶相当。对SW-480细胞的抑制作用很弱。
Ⅰ和Ⅲ类化合物的药理实验正在进行中。
The protein tyrosine kinases (PTKs) play crucial roles in many of the signal transduction pathways regulating lots of cellular functions, such as cell growth, proliferation and apoptosis. Over-expression of the PTKs is thought to be related to cell proliferation, invasion, metastasis, resistance to chemoradiotherapy and poor prognosis of various human cancers. Targeted interference with PTKs activation and/or with intracellular PTK-activated signal transduction pathways represents a promising strategy for the development of novel and selective anticancer therapies.
Epidermal growth factor receptor tyrosine kinase (EGFR) is the first PTK to be examined seriously as a drug target. EGFR has its own ATP binding site in the intracellular domain. EGFR inhibitors can bind competitively to the ATP binding site and subsequently inhibit the phosphorylation of the EGFR as well as the downstream signaling pathways, then depress the growth, proliferation and metastasis of tumor. Two drugs, Gefitinib and Erlotinib, inhibiting the EGFR, have been launched for clinical use. Thus, EGFR has been identified as a promising target of tumor therapy.
During the research for drugs targeting the EGFR, anilinoquinazolines were found to inhibit EGFR potently and selectively by binding to the ATP binding site reversibly. PD 0153035, a quinazoline derivative, played an important role in establishing the structure-activity relationship(SAR) and many of the quinazoline derivatives were designed on the basis of PD 0153035. However, the research found that the quinazoline ring is not necessary for inhibiting EGFR. Many of the aza quinazolines have showed potent activity, such as pyrido[2,3-d]pyrimidine, pyrido [3,2-d]pyrimidine, pyrido[4,3-d]pyrimidine, pyrido[3,4-d]pyrimidine, and pyrimido [5,4-d]pyrimidine. Moreover, the compounds that the benzene in quinazoline ring was substituted by five-member rings, such as pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine, can also inhibit the EGFR and show bright prospects.
The studies stated above reminded us of the pteridine ring, as pteridine ring could be regarded as quinazoline ring that 5-C and 8-C are substituted by nitrogen atoms. Meanwhile the 6-substituted pteridines are the common forms in natural pteridine derivatives. In view of this, according to the SAR, we designed 2 series of pteridine derivatives : 6-alkoxy-4-arylaminopteridinesⅠand 6,7-dialkoxy-4- arylaminopteridinesⅢ, using the bioisoster theory and PD 0153035 as lead compound, the structures of the compounds we designed are shown as follow:
Pteridine-like compounds exhibit a wide range of physiological activity in human body and provide a novel template for new drugs, such as anticancer agents, adenosine kinase inhibitors and nitric oxide synthase inhibitors. At present, the study of pteridine derivatives is extensive abroad, but less in our country. Moreover, the preparation has seldom been reported for the alkoxy-substituted pteridines.
As no related reports about the synthesis of the designed compounds, we by ourselves designed the synthetic routes by means of retro-synthetic analysis. Through modifying and justifying the routes repeatedly, we eventually found the practical routes.
During the preparing of compoundsⅢ, we discovered unexpectedly that the cyclization of N~4-aryl-4,5,6-triaminopyrirnidine with oxalic acid is regioselective, which led to the discovery of a series of novel compounds: N~8-aryl-6-alkoxy (alkylamino)-4-amino-7(8H)pteridinonesⅡ(As shown above).
The flexible molecular docking method was applied to investigate the binding of the designed compounds to EGFR. The binding modes and the interaction mechanism between the designed compounds and EGFR were understood. The results, which are helpful for new drugs design, showed the designed compounds can be able to dock into the ATP binding domain by hydrophobic and electrostatic interactions.
CompoundsⅠstarted from 3-aminopyrazine-2-formic acid, from which 20 6-alkoxy-4-arylaminopteridine derivatives were obtained by esterification, amidation, bromination, cyclization, alkoxylation, chlorination and aromatic amination in a 7-step procedure with an overall yield of 39%. CompoundsⅡcommenced from diethyl malonate, from which 13 new pteridine derivatives were prepared in an overall yield of 22% in a 9-step procedure including cyclization, nitration, chlorination and arylamine substitution, amination, reduction, cyclization with oxalic acid, chlorination, alkoxylation or alkylamination. For compoundsⅢ, 3-aminopyrazine-2-formic acid was used as starting material, which underwent esterification, chlorination, alkoxylation, ester hydrolysis, lactonization, amidation and cyclization, alkoxylation, chlorination and aromatic amination to produce 5 new compounds in a total yield of 27% in a 9 steps procedure.
The structure of all compounds had been confirmed by ~1H NMR, ~(13)C NMR, ESI-MS and elemental analysis. More than 50 new intermediates were prepared through the experimental process, and elucidated from the corresponding spectra according to their structural characteristics.
Two possible isomers may occur in the cyclization of N~4-aryl-4,5,6-tri aminopyrimidine with oxalic acid. Their structural similarity makes it difficult to be confirmed by ~1H NMR, ~(13)C NMR, ESI-MS and elemental analysis. A single crystal of its chlorinated compound revealed its structure, and the cyclization pattern and regioselectivity were understood. However, the regioselectivity disappeared when aliphatic amino group replaced the arylamino group. Thus, the method can only be used to the cyclization of N~4-aryl-4,5,6-triaminopyrimidine with oxalic acid.
The toxicity of the compoundsⅡagainst human tumor cell proliferation was evaluated using MTT assay. Three kinds of cancer cell lines, including human promyelocytic leukemia HL-60 cells, human gastric adenocarcinoma SGC-7901 cells and human colon cancer cells SW-480, were selected to test the target compoundsⅡa-Ⅱm, using 5-FU as the contrast. The results suggested that the designed compounds II showed a certain inhibitory effect on SGC-7901 cells, some of them on HL-60 cells. At the similar concentration, the individual compounds showed equivalent inhibition against SGC-7901 and HL-60 cells to that of 5-FU. The designed compoundsⅡexhibited little effects on SW-480 cells.
The pharmacological experiments of compoundsⅠandⅢare in progress.
引文
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