FMS-like tyrosine kinase 3 (FLT3) enzyme overexpression and mutations are the most common molecular abnormalities associated with acute myeloid leukemia (AML). In addition, recent studies investigated the role of tropomyosin receptor kinase A (TrKA) enzyme fusions in promoting AML growth and survival. Based on these premises, targeting both kinases using dual inhibitors would constitute a promising therapeutic approach to target resistant AML. Guided by ligand-based design and structure simplification of the FLT3 inhibitor, quizartinib, we developed a benzimidazole-based small molecule, 4ACP, that exhibited nanomolar activity against wild-type FLT3, FLT3-Internal tandem duplications (FLT3-ITD), and FLT3-D835Y (FLT3-TKD) mutation (IC₅₀ = 43.8, 97.2, and 92.5 nM respectively). Additionally, 4ACP demonstrated potent activity against colon cancer KM12 cell line (IC₅₀ = 358 nM) and subsequent mechanistic deconvolution identified TrKA enzyme as a second plausible target (IC₅₀ = 23.6 nM) for our compound. 4ACP manifested preferential antiproliferative activity against FLT3-ITD positive AML cell lines (MV4-11 IC₅₀ = 38.8 ± 10.7 nM and MOLM-13 IC₅₀ = 54.9 ± 4.1 nM), while lacking activity against FLT3-ITD negative AML cell lines. Western blot analysis confirmed 4ACP ability to downregulate ERK1/2 and mTOR signaling downstream of FLT3-ITD in AML cells. Furthermore, 4ACP prompted apoptotic and necrotic cell death and G0/G1 cell cycle arrest as indicated by cell cycle analysis.

4ACP did not show cytotoxic effects on normal BNL and H9c2 cells and demonstrated decreased activity against c-Kit enzyme, hence, indicating lower probability of synthetic lethal toxicity and a relatively safer profile. In light of these data, 4ACP represents a novel FLT3/TrKA dual kinase inhibitor for targeted therapy of AML.

FMS 样酪氨酸激酶 3 (FLT3) 酶过表达和突变是与急性髓性白血病 (AML) 相关的最常见的分子异常。此外，最近的研究调查了原肌球蛋白受体激酶 A (TrKA) 酶融合在促进 AML 生长和存活中的作用。基于这些前提，使用双重抑制剂靶向两种激酶将构成靶向耐药性 AML 的有希望的治疗方法。在 FLT3 抑制剂 quizartinib 的基于配体的设计和结构简化的指导下，我们开发了一种基于苯并咪唑的小分子4ACP ，它对野生型 FLT3、FLT3-内部串联重复 (FLT3-ITD) 和 FLT3 表现出纳摩尔活性-D835Y (FLT3-TKD) 突变 (IC ₅₀ = 43.8、97.2 和 92.5 nM）。此外，4ACP表现出对结肠癌 KM12 细胞系的有效活性 (IC ₅₀  = 358 nM)，随后的机械去卷积将 TrKA 酶鉴定 为我们化合物的第二个可能的靶标 (IC _{50 = 23.6 nM)。}4ACP表现出对 FLT3-ITD 阳性 AML 细胞系的优先抗增殖活性（MV4-11 IC ₅₀  = 38.8 ± 10.7 nM 和 MOLM-13 IC ₅₀  = 54.9 ± 4.1 nM），而对 FLT3-ITD 阴性 AML 细胞系缺乏活性。蛋白质印迹分析证实4ACP能够下调 AML 细胞中 FLT3-ITD 下游的 ERK1/2 和 mTOR 信号传导。此外，4ACP如细胞周期分析所示，促使细胞凋亡和坏死细胞死亡和 G0/G1 细胞周期停滞。

4ACP未显示对正常 BNL 和 H9c2 细胞的细胞毒性作用，并且显示出对 c-Kit 酶的活性降低，因此表明合成致死毒性的可能性较低且相对安全。鉴于这些数据，4ACP代表了一种用于 AML 靶向治疗的新型 FLT3/TrKA 双激酶抑制剂。