Breast cancer has become the most commonly diagnosed cancer, surpassing lung cancer, with 2.26 million new breast cancers worldwide in 2020. Hence, there is an urgent need to develop effective molecularly targeted therapeutic drugs to treat breast cancer. In this paper, we designed, synthesized and screened a novel thiophene-triazine derivative, XS-2, as a potent dual PI3K/mTOR inhibitor for the treatment of breast cancer. Also, XS-2 was found to be potentially effective against triple-negative breast cancer (TNBC) in vitro during the investigation. We evaluated the in vitro inhibitory effect of XS-2 on 10 cancer cell lines by MTT and 6 kinases to investigated its in vivo antitumor activity in MCF-7 xenograft tumor-bearing BALB/c nude mice. In addition, the in vitro/in vivo toxicity to mice was also assessed by hemolytic toxicity, H&E staining and blood biochemical analysis. In order to investigate the antitumor mechanism of XS-2, a series of experiments were carried out in vitro/in vivo animal model and molecular biological levels such as the cell cycle and the apoptosis assay, real-time PCR, western blot, docking and molecular simulations analysis, etc. What’s more, wound healing assay, Transwell and Western Blot were applied to explore the ability of XS-2 to inhibit the cell invasion and migration. The results showed that XS-2 exhibited strong antitumor activity both in vitro and in vivo. The inhibitory activities of XS-2 on ten cancer cell lines were ranging from 1.07 ± 0.11 to 0.002 ± 0.001 μM, which were 1565 times better than that of the lead compound GDC-0941, inhibitory activities against PI3Kα and mTOR kinases were 291.0 and 60.8 nM, respectively. Notably, XS-2 not only showed significant in vivo antitumor activity and low toxicity, with the tumor inhibition rate of 57.0 %, but also exhibited strong inhibitory in the expression of related proteins of PI3K pathway in tumor tissues. In addition, XS-2 significantly inhibited breast cancer MCF-7 and MDA-MB-231 cells in a concentration- and time-dependent manner, and inhibited the migration and invasion ability of MDA-MB-231 and MCF-7 cells. More than that, XS-2 could inhibit the increase of the expression levels of N-cadherin and vimentin upregulated by EGF and reversed the E-cadherin expression down regulated by EGF, resulting in inhibiting EMT in MCF-7 and MDA-MB-231 cells. The results showed that XS-2 was expected to be successfully developed as a high-efficiency and low-toxicity breast cancer therapeutic drug with the potential to inhibit the invasion and migration of TNBC. This provides a new research idea for the treatment of TNBC, which is of great significance.

乳腺癌已超过肺癌成为最常见的癌症，2020年全球新发乳腺癌226万例。因此，迫切需要开发有效的分子靶向治疗药物来治疗乳腺癌。在本文中，我们设计、合成和筛选了一种新型噻吩-三嗪衍生物 XS-2，作为一种有效的双 PI3K/mTOR 抑制剂，用于治疗乳腺癌。此外，在研究期间，XS-2 被发现在体外对三阴性乳腺癌 (TNBC) 具有潜在的疗效。我们通过 MTT 和 6 种激酶评估了 XS-2 对 10 种癌细胞系的体外抑制作用，以研究其在携带 MCF-7 异种移植肿瘤的 BALB/c 裸鼠中的体内抗肿瘤活性。除此之外还通过溶血毒性、H&E染色和血液生化分析评估了对小鼠的体外/体内毒性。为了研究XS-2的抗肿瘤机制，在体外/体内动物模型和分子生物学水平如细胞周期和细胞凋亡测定、实时荧光定量PCR、蛋白质印迹、对接和分子模拟分析等。此外，还应用伤口愈合试验、Transwell 和Western Blot 来探索XS-2 抑制细胞侵袭和迁移的能力。结果表明，XS-2在体外和体内均表现出很强的抗肿瘤活性. XS-2对十种癌细胞株的抑制活性为1.07±0.11至0.002±0.001μM，是先导化合物GDC-0941的1565倍，对PI3Kα和mTOR激酶的抑制活性分别为291.0和60.8纳米，分别。值得注意的是，XS-2不仅具有显着的体内抗肿瘤活性和低毒性，抑瘤率为57.0 %，而且对PI3K通路相关蛋白在肿瘤组织中的表达具有较强的抑制作用。此外，XS-2以浓度和时间依赖性方式显着抑制乳腺癌MCF-7和MDA-MB-231细胞，并抑制MDA-MB-231和MCF-7细胞的迁移和侵袭能力。不仅如此，XS-2 还可以抑制N的表达水平的增加。EGF上调-钙粘蛋白和波形蛋白并逆转EGF下调的E-钙粘蛋白表达，从而抑制MCF-7和MDA-MB-231细胞中的EMT。结果表明，XS-2有望成功开发为高效、低毒的乳腺癌治疗药物，具有抑制TNBC侵袭和迁移的潜力。这为TNBC的治疗提供了新的研究思路，具有重要意义。