[1,2,4]三氮唑并[1,5-a]嘧啶类lsd1抑制剂的设计、合成及其抗肿瘤活性评价
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ቤተ መጻሕፍቲ ባይዱ
总之，本课题组设计合成了以三氮唑并嘧啶为基本骨架的全新的 LSD1 抑制 剂，保留三氮唑并嘧啶基本骨架，通过尽可能丰富不同氨基取代基的多样性， 进一步深入探究构效关系，进而构建具有结构新颖性和多样性的目标小分子化 合物，以期望获得对 LSD1 有抑制活性的小分子化合物。基于该设计理念，我们 惊喜地发现了三个对 LSD1 有抑制活性的化合物 5p、5q 和 6i，其抑制 LSD1 的 IC50 分别为 0.154、1.19 和 0.557 μM，并对它们进行了一系列活性评价。这足以 证明了该设计理念的可靠性，为后续 LSD1 抑制剂以及其他靶点抑制剂先导化合 物的发现提供了一个全新的思路。
A new series of [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1 inhibitors were designed, synthesized, and further evaluated for their cytotoxicity against MGC-803, EC109, A549 and PC-9 cells as well as the ability of inhibiting LSD1. Some of these compounds showed potent inhibition toward LSD1 and selectively inhibited growth of A549 and PC-9 cells. Compound 6l potently inhibited growth of PC-9 cells ( IC50 = 0.59 µM), about 4-fold more potent than 5-FU. Further SARs studies led to the identification of compounds 6l-m, which had good growth inhibition against all the tested cancer cell lines and were much more potent than 5-FU and GSK2879552. Besides, compounds 5p, 5q and 6i inhibited LSD1 potently ( IC50 = 0.154, 1.19 and 0.557 µM, respectively ). Docking studies revealed that compound 5p formed arene-H interactions with Val333 and hydrogen bonds with surrounding Ala331, Met332, and Ala539 residues. Compound 5p significantly inhibited migration of A549 and PC-9 cells in a concentration-dependent manner, but had different effect on the expression of E-cadherin and N-cadherin. In addition, we used Annexin Ⅴ-FITC
关键字：[1,2,4]三氮唑并[1,5-a]嘧啶；抗肿瘤活性；LSD1；凋亡;分子对接
II
Abstract
Epigenetic modification of histone modifications is a very important direction for research. Histone Lysine Specific Demethylase 1 (LSD1, also known as KDM1A), the first identified histone lysine specific demethylase in 2004, can specifically remove H3K4 and H3K9 single or double methylation, which is involved in a highly conserved flavin adenine dinucleotide (FAD). Besides, LSD1 is able to remove the methylation of nonprotein substrates (such as transcription factor E2F1, tumor suppressor gene p53, DNA methyl transferase 1) to regulate the biological function of cells. Studies have shown that LSD1 activity or expression in many tumor cells (such as breast cancer, lung cancer, prostate cancer, gastric cancer, etc.) is higher than the corresponding normal cells, by inhibiting the activity or expression of LSD1 can effectively inhibit the growth of tumor cells. Therefore, it is of great significance to develop an efficient and highly specific LSD1 inhibitor for tumor therapy.
III
double staining to detect the effect of compound 61 on apoptosis of MGC-803 cells. We found that compound 6l could induce the apoptosis rate of MGC-803 in a concentration-dependent manner. We used compound 5p to regulate LSD1 and its substrate in PC-9 and MGC-803 tumor cells. The results showed that compound 5p could inhibit the expression of LSD1 at the cellular level, and the expression of LSD1 in the cells was significantly decreased. Compound 5p could specifically upregulate the expression of protein substrates H3K4me1 and H3K4me2 in cells, but not on H3K4me3 expression.The [1,2,4]triazolo[1,5-a]pyrimidine scaffold may serve as a starting point for developing potent LSD1 inhibitors for cancer therapy.
In conclusion, the novel LSD1 inhibitor with [1,2,4]triazolo[1,5-a]pyrimidine as the basic skeleton was designed and synthesized and the basic skeleton of triazolopyrimidine was retained. By further enriching the diversity of different amino substituents, we could explore the structure-activity relationship, and then build a novel structure and diversity of the target small molecule compounds in order to obtain the LSD1 inhibitory activity of small molecules. Based on this design concept, we are surprised to find the three compounds 5p, 5q and 6i with inhibitory activity against LSD1, which inhibited the IC50 values of LSD1 by 0.154, 1.19 and 0.557 μM, respectively, and then evaluated them for a series of activities. This is sufficient to demonstrate the reliability of the design concept and provide a new way for the discovery of followers of LSD1 inhibitors and other target inhibitors.
摘要
表观遗传修饰中组蛋白修饰是非常重要的研究的方向。组蛋白赖氨酸特异 性去甲基化酶 1（LSD1）是于 2004 年被首次发现的组蛋白修饰酶，其在辅酶黄 素腺嘌呤二核苷酸（FAD）参与下，能够特异性去除 H3K4 和 H3K9 的单、双甲 基化。除此之外，LSD1 能够去除非蛋白底物（如转录因子 E2F1、抑癌基因 p53、 DNA 甲基转移化酶 1）的甲基化来调控细胞的生物学功能。研究表明，LSD1 在 很多肿瘤细胞（如乳腺癌、肺癌、前列腺癌、胃癌等）中的活性或表达量高于 对应的正常细胞，通过对 LSD1 的活性或者表达量进行抑制能够有效地抑制肿瘤 细胞生长。所以，开发一种用于肿瘤治疗的高效且高特异性的 LSD1 抑制剂具有 很重要的意义。
我们设计合成了一系列全新的[1,2,4]三氮唑并[1,5-a]嘧啶类 LSD1 抑制剂并 进一步评价了它们对 MGC-803, EC109, A549 和 PC-9 肿瘤细胞的细胞毒性，以 及对 LSD1 的抑制活性。其中一些化合物对 LSD1 表现出较强的抑制活性，能够 选择性地抑制肿瘤细胞 A549 和 PC-9 的生长。其中化合物 6l 能够抑制肿瘤细胞 PC-9 的生长 ( IC50 = 0.59 µM )，优于 5-FU。进一步构效关系表明，化合物 6l-m 对所测肿瘤细胞的生长均有抑制作用，其抑制活性均高于 5-FU 和 GSK2879552。 我们发现化合物 5p、5q 和 6i 对 LSD1 具有较好的抑制活性，其 IC50 分别为 0.154、 1.19 和 0.557 μM。分子模拟对接表明化合物 5p 与 Val333 形成芳香氢的相互作 用，同时与周围的氨基酸 Ala331, Met332 和 Ala539 残基形成氢键。化合物 5p 能 够显著抑制 MGC-803 和 PC-9 肿瘤细胞的转移，并且具有浓度依赖性，但是其 对 E-cadherin 和 N-cadherin 的表达具有不同的影响。我们使用 Annexin Ⅴ-FITC 双染法检测化合物 6l 对 MGC-803 细胞凋亡的影响。我们发现化合物 6l 能够诱 导胃癌细胞 MGC-803 凋亡比例随浓度的增加而增加，呈现浓度依赖性。我们使 用化合物 5p 在 PC-9 和 MGC-803 肿瘤细胞中对 LSD1 及其底物进行调节。结果 表明化合物 5p 能够在细胞水平对 LSD1 的表达量进行抑制，使细胞中 LSD1 的 表达量明显减少。其能够特异性上调细胞中蛋白底物 H3K4me1 和 H3K4me2 的 表达，而对 H3K4me3 的表达没有影响。实验结果表明[1,2,4]三氮唑并[1,5-a]嘧 啶骨架为未来开发用于肿瘤治疗的 LSD1 抑制剂开启了新的起点。
ቤተ መጻሕፍቲ ባይዱ
总之，本课题组设计合成了以三氮唑并嘧啶为基本骨架的全新的 LSD1 抑制 剂，保留三氮唑并嘧啶基本骨架，通过尽可能丰富不同氨基取代基的多样性， 进一步深入探究构效关系，进而构建具有结构新颖性和多样性的目标小分子化 合物，以期望获得对 LSD1 有抑制活性的小分子化合物。基于该设计理念，我们 惊喜地发现了三个对 LSD1 有抑制活性的化合物 5p、5q 和 6i，其抑制 LSD1 的 IC50 分别为 0.154、1.19 和 0.557 μM，并对它们进行了一系列活性评价。这足以 证明了该设计理念的可靠性，为后续 LSD1 抑制剂以及其他靶点抑制剂先导化合 物的发现提供了一个全新的思路。
A new series of [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1 inhibitors were designed, synthesized, and further evaluated for their cytotoxicity against MGC-803, EC109, A549 and PC-9 cells as well as the ability of inhibiting LSD1. Some of these compounds showed potent inhibition toward LSD1 and selectively inhibited growth of A549 and PC-9 cells. Compound 6l potently inhibited growth of PC-9 cells ( IC50 = 0.59 µM), about 4-fold more potent than 5-FU. Further SARs studies led to the identification of compounds 6l-m, which had good growth inhibition against all the tested cancer cell lines and were much more potent than 5-FU and GSK2879552. Besides, compounds 5p, 5q and 6i inhibited LSD1 potently ( IC50 = 0.154, 1.19 and 0.557 µM, respectively ). Docking studies revealed that compound 5p formed arene-H interactions with Val333 and hydrogen bonds with surrounding Ala331, Met332, and Ala539 residues. Compound 5p significantly inhibited migration of A549 and PC-9 cells in a concentration-dependent manner, but had different effect on the expression of E-cadherin and N-cadherin. In addition, we used Annexin Ⅴ-FITC
关键字：[1,2,4]三氮唑并[1,5-a]嘧啶；抗肿瘤活性；LSD1；凋亡;分子对接
II
Abstract
Epigenetic modification of histone modifications is a very important direction for research. Histone Lysine Specific Demethylase 1 (LSD1, also known as KDM1A), the first identified histone lysine specific demethylase in 2004, can specifically remove H3K4 and H3K9 single or double methylation, which is involved in a highly conserved flavin adenine dinucleotide (FAD). Besides, LSD1 is able to remove the methylation of nonprotein substrates (such as transcription factor E2F1, tumor suppressor gene p53, DNA methyl transferase 1) to regulate the biological function of cells. Studies have shown that LSD1 activity or expression in many tumor cells (such as breast cancer, lung cancer, prostate cancer, gastric cancer, etc.) is higher than the corresponding normal cells, by inhibiting the activity or expression of LSD1 can effectively inhibit the growth of tumor cells. Therefore, it is of great significance to develop an efficient and highly specific LSD1 inhibitor for tumor therapy.
III
double staining to detect the effect of compound 61 on apoptosis of MGC-803 cells. We found that compound 6l could induce the apoptosis rate of MGC-803 in a concentration-dependent manner. We used compound 5p to regulate LSD1 and its substrate in PC-9 and MGC-803 tumor cells. The results showed that compound 5p could inhibit the expression of LSD1 at the cellular level, and the expression of LSD1 in the cells was significantly decreased. Compound 5p could specifically upregulate the expression of protein substrates H3K4me1 and H3K4me2 in cells, but not on H3K4me3 expression.The [1,2,4]triazolo[1,5-a]pyrimidine scaffold may serve as a starting point for developing potent LSD1 inhibitors for cancer therapy.
In conclusion, the novel LSD1 inhibitor with [1,2,4]triazolo[1,5-a]pyrimidine as the basic skeleton was designed and synthesized and the basic skeleton of triazolopyrimidine was retained. By further enriching the diversity of different amino substituents, we could explore the structure-activity relationship, and then build a novel structure and diversity of the target small molecule compounds in order to obtain the LSD1 inhibitory activity of small molecules. Based on this design concept, we are surprised to find the three compounds 5p, 5q and 6i with inhibitory activity against LSD1, which inhibited the IC50 values of LSD1 by 0.154, 1.19 and 0.557 μM, respectively, and then evaluated them for a series of activities. This is sufficient to demonstrate the reliability of the design concept and provide a new way for the discovery of followers of LSD1 inhibitors and other target inhibitors.
摘要
表观遗传修饰中组蛋白修饰是非常重要的研究的方向。组蛋白赖氨酸特异 性去甲基化酶 1（LSD1）是于 2004 年被首次发现的组蛋白修饰酶，其在辅酶黄 素腺嘌呤二核苷酸（FAD）参与下，能够特异性去除 H3K4 和 H3K9 的单、双甲 基化。除此之外，LSD1 能够去除非蛋白底物（如转录因子 E2F1、抑癌基因 p53、 DNA 甲基转移化酶 1）的甲基化来调控细胞的生物学功能。研究表明，LSD1 在 很多肿瘤细胞（如乳腺癌、肺癌、前列腺癌、胃癌等）中的活性或表达量高于 对应的正常细胞，通过对 LSD1 的活性或者表达量进行抑制能够有效地抑制肿瘤 细胞生长。所以，开发一种用于肿瘤治疗的高效且高特异性的 LSD1 抑制剂具有 很重要的意义。
我们设计合成了一系列全新的[1,2,4]三氮唑并[1,5-a]嘧啶类 LSD1 抑制剂并 进一步评价了它们对 MGC-803, EC109, A549 和 PC-9 肿瘤细胞的细胞毒性，以 及对 LSD1 的抑制活性。其中一些化合物对 LSD1 表现出较强的抑制活性，能够 选择性地抑制肿瘤细胞 A549 和 PC-9 的生长。其中化合物 6l 能够抑制肿瘤细胞 PC-9 的生长 ( IC50 = 0.59 µM )，优于 5-FU。进一步构效关系表明，化合物 6l-m 对所测肿瘤细胞的生长均有抑制作用，其抑制活性均高于 5-FU 和 GSK2879552。 我们发现化合物 5p、5q 和 6i 对 LSD1 具有较好的抑制活性，其 IC50 分别为 0.154、 1.19 和 0.557 μM。分子模拟对接表明化合物 5p 与 Val333 形成芳香氢的相互作 用，同时与周围的氨基酸 Ala331, Met332 和 Ala539 残基形成氢键。化合物 5p 能 够显著抑制 MGC-803 和 PC-9 肿瘤细胞的转移，并且具有浓度依赖性，但是其 对 E-cadherin 和 N-cadherin 的表达具有不同的影响。我们使用 Annexin Ⅴ-FITC 双染法检测化合物 6l 对 MGC-803 细胞凋亡的影响。我们发现化合物 6l 能够诱 导胃癌细胞 MGC-803 凋亡比例随浓度的增加而增加，呈现浓度依赖性。我们使 用化合物 5p 在 PC-9 和 MGC-803 肿瘤细胞中对 LSD1 及其底物进行调节。结果 表明化合物 5p 能够在细胞水平对 LSD1 的表达量进行抑制，使细胞中 LSD1 的 表达量明显减少。其能够特异性上调细胞中蛋白底物 H3K4me1 和 H3K4me2 的 表达，而对 H3K4me3 的表达没有影响。实验结果表明[1,2,4]三氮唑并[1,5-a]嘧 啶骨架为未来开发用于肿瘤治疗的 LSD1 抑制剂开启了新的起点。