A series of novel thieno[2,3-d]pyrimidine derivatives were designed, synthesized and evaluated for their biological ability. 2-Amino-4,7-dihydro-5H-thieno[2,3-d]pyrano-3-cyanonitrile was synthesized by Gewald reaction using pyranone as substrate, malonitrile and sulfur powder as raw materials and triethylamine as base at room temperature. Subsequent amination with N,N-dimethylformamide dimethyl acetal (DMF-DMA) gave N'-(3-cyano-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-N,N-dimethylmethanimidamide. In this article, the 23 thieno[2,3-d] pyrimidines were obtained by Dimroth rearrangement condensation with different anilines, 16 of them being new compounds. Meanwhile, all compounds were assessed for breast cancer MDA-MB-231 cell line bioactivity with paclitaxel (PTX) as the positive control, and all compounds in the series showed inhibitory effects on breast cancer cell MDA-MB-231. The IC50 of compound l against MDA-MB-231 is 27.6 μM, which is similar to that of PTX with an IC50 of 29.3 μM. This means that compound l exhibits comparable inhibitory activity to PTX in MDA-MB-231 cells and may be considered as a potential anti-MDA-MB-231 inhibitor. thieno2,3dpyrimidine thieno23dpyrimidine thienodpyrimidine thieno 2,3 d pyrimidine 2 3 designed ability 2Amino4,7dihydro5Hthieno2,3dpyrano3cyanonitrile 2Amino47dihydro5Hthieno23dpyrano3cyanonitrile AminodihydroHthienodpyranocyanonitrile Amino 4,7 dihydro 5H pyrano cyanonitrile 4 7 H substrate temperature N,Ndimethylformamide NNdimethylformamide N,N dimethylformamide N DMFDMA DMF DMA (DMF-DMA N3cyano4,7dihydro5Hthieno2,3cpyran2ylN,Ndimethylmethanimidamide. N3cyano47dihydro5Hthieno23cpyran2ylNNdimethylmethanimidamide NcyanodihydroHthienocpyranylNNdimethylmethanimidamide cyano c pyran yl dimethylmethanimidamide. dimethylmethanimidamide N'-(3-cyano-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-N,N-dimethylmethanimidamide article thieno2,3d thieno23d thienod thieno[2,3-d anilines 1 Meanwhile MDAMB231 MDAMB MDA MB 231 MDA-MB-23 (PTX control MDAMB231. 231. IC IC5 276 27 6 27. μM 293 29 29. antiMDAMB231 antiMDAMB anti anti-MDA-MB-23 inhibitor thieno2 3dpyrimidine dpyrimidine 2, 2Amino4 7dihydro5Hthieno2 3dpyrano3cyanonitrile Hthieno dpyrano 47 4, Ndimethylformamide NN N3cyano4 3cpyran2ylN Ndimethylmethanimidamide cpyran ylN N3cyano4,7dihydro5Hthieno2,3cpyran2ylN,Ndimethylmethanimidamide ylNNdimethylmethanimidamide 3d MDAMB23 MDA-MB-2 antiMDAMB23 anti-MDA-MB-2 2Amino dihydroHthieno 7dihydro5Hthieno dpyranocyanonitrile Ncyano N3cyano cpyranylN MDAMB2 MDA-MB- antiMDAMB2 anti-MDA-MB- MDA-MB anti-MDA-MB