Bruton’s tyrosine kinase (BTK) has emerged as a therapeutic target for B-cell malignancies, which is substantiated by the efficacy of various irreversible or reversible BTK inhibitors. However, on-target BTK mutations facilitating evasion from BTK inhibition lead to resistance that limits the therapeutic efficacy of BTK inhibitors. In this study we employed structure-based drug design strategies based on established BTK inhibitors and yielded a series of BTK targeting compounds. Among them, compound S-016 bearing a unique tricyclic structure exhibited potent BTK kinase inhibitory activity with an IC₅₀ value of 0.5 nM, comparable to a commercially available BTK inhibitor ibrutinib (IC₅₀ = 0.4 nM). S-016, as a novel irreversible BTK inhibitor, displayed superior kinase selectivity compared to ibrutinib and significant therapeutic effects against B-cell lymphoma both in vitro and in vivo. Furthermore, we generated BTK inhibitor-resistant lymphoma cells harboring BTK C481F or A428D to explore strategies for overcoming resistance. Co-culture of these DLBCL cells with M0 macrophages led to the polarization of M0 macrophages toward the M2 phenotype, a process known to support tumor progression. Intriguingly, we demonstrated that SYHA1813, a compound targeting both VEGFR and CSF1R, effectively reshaped the tumor microenvironment (TME) and significantly overcame the acquired resistance to BTK inhibitors in both BTK-mutated and wild-type BTK DLBCL models by inhibiting angiogenesis and modulating macrophage polarization. Overall, this study not only promotes the development of new BTK inhibitors but also offers innovative treatment strategies for B-cell lymphomas, including those with BTK mutations.

Bruton 酪氨酸激酶 （BTK） 已成为 B 细胞恶性肿瘤的治疗靶点，各种不可逆或可逆 BTK 抑制剂的疗效证实了这一点。然而，促进逃避 BTK 抑制的靶向 BTK 突变会导致耐药性，从而限制 BTK 抑制剂的治疗效果。在这项研究中，我们采用了基于已建立的 BTK 抑制剂的基于结构的药物设计策略，并产生了一系列 BTK 靶向化合物。其中，具有独特三环结构的化合物 S-016 表现出有效的 BTK 激酶抑制活性，IC₅₀ 值为 0.5 nM，与市售的 BTK 抑制剂伊布替尼相当 （IC₅₀ = 0.4 nM）。S-016 作为一种新型不可逆的 BTK 抑制剂，与依鲁替尼相比，表现出优异的激酶选择性，并且在体外和体内对 B 细胞淋巴瘤具有显着的治疗效果。此外，我们生成了携带 BTK C481F 或 A428D 的 BTK 抑制剂耐药性淋巴瘤细胞，以探索克服耐药性的策略。这些 DLBCL 细胞与 M0 巨噬细胞的共培养导致 M0 巨噬细胞向 M2 表型极化，这一过程已知支持肿瘤进展。有趣的是，我们证明了 SYHA1813 是一种靶向 VEGFR 和 CSF1R 的化合物，通过抑制血管生成和调节巨噬细胞极化，有效地重塑了肿瘤微环境 （TME），并显着克服了 BTK 突变和野生型 BTK DLBCL 模型中对 BTK 抑制剂的获得性耐药。总体而言，这项研究不仅促进了新型 BTK 抑制剂的开发，还为 B 细胞淋巴瘤（包括具有 BTK 突变的淋巴瘤）提供了创新的治疗策略。