Abstract Background: Diffuse large B cell lymphomas (DLBCLs) are aggressive tumors that account for almost half of all lymphoma incidences. In particular, double-hit and triple-hit DLBCLs, with translocations involving BCL2, MYC and/or BCL6, have a very poor prognosis and occur in up to 7% of cases. Double expressor DLBCLs, with overexpression of BCL2 and MYC in the absence of chromosomal translocations, also have a poor prognosis. BCL2 inhibitors (BCL2i) have shown preclinical and clinical efficacy in lymphomas. Here, we synthesized and tested seven novel BCL2i in DLBCL cells with deregulated BCL2, and splenic marginal zone lymphoma (SMZL) cell lines with acquired resistance to idelalisib, copanlisib or ibrutinib. Methods: Using in silico-based drug design complemented by classical and kinetic target-guided synthesis (KTGS) led to the synthesis of seven potential BCL2 inhibitors (BCL2i). The MTT assay was used to assess the anti-proliferative activities of BCL2i: ST-59, ST-64, ST-65, R, Z-89, Z-103, Z-116-8 in BCL2-translocated DLBCLs: SU-DHL-4, Toledo, DOHH2, KARPAS422, WSUDLCL2, Pfeiffer; BCL2 amplified DLBCL: U2932 and resistant SMZLs. Cells were treated with increasing concentrations of BCL2i for 72 hours [h]. Results: Median IC50 values for the seven BCL2i ranged from 22 μM to 49 μM. ST-65 was the most potent BCL2i (range = 12 - 46 μM, median = 22 μM), showing anti-proliferative activity in DLBCL and SMZL. The other BCL2i were inactive in at least 2 cell lines (IC50 > 50 μM). The three least potent BCL2i were Z-89, Z-103 and R (median IC50 = 49, 44 and 45 μM, respectively). ST-65 was the only BCL2i synthesized by BCL2 using KTGS, indicating KTGS as a superior technique for generating BCL2i. The poor activity of R agreed with its inability to cross the cell membrane as determined by liquid chromatography-mass spectrometry. Among the resistant SMZLs, parental KARPAS1718 were most sensitive to the BCL2i, responding to 5 out of 7 inhibitors (range = 11 - 47 μM, median = 22 μM). The activated B cell like (ABC)-DLBCL, U2932, was only sensitive to ST-65 (IC50 = 20 μM). U2932 lacks translocated BCL2, but BCL2 is amplified and overexpressed in this cell line. Conclusions: We report the in vitro anti-lymphoma activities of novel BCL2i, particularly ST-65, in DLBCL and SMZL cells. Our results suggest that these compounds are structures further exploitable for the design of improved anti-lymphoma drugs. Acknowledgements: The research work was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “First Call for H.F.R.I. Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment grant” (Project Number: 991 to AGT). Citation Format: Afua Adjeiwaa Mensah, Christos M. Chatzigiannis, Dimitrios A. Diamantis, Vasileios K. Gkalpinos, Alberto Arribas, Andreas G. Tzakos, Francesco Bertoni. Novel BCL2 inhibitors with anti-lymphoma activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3942.