The discovery of CSF-1R kinase [1] as a tumor gene implicated in Feline McDonough sarcoma [2] marked the identification of a class III receptor tyrosine kinase [3] that interacts with macrophages or monocyte colony stimulating factors (M-CSF or CSF-1). CSF-1R, a member of the class III receptor tyrosine kinase family, encompasses CSF-1R-like tyrosine kinase 3 (FLT-3), stem cell factor receptor (KIT), and platelet-derived growth factor receptors (PDGFR) α and β. CSF-1R is predominantly expressed in macrophage lineages, including monocytes, tissue macrophages, dendritic cells, and osteoclasts. It plays a crucial role in signal transduction mediated by CSF-1/CSF-1R, governing the differentiation, survival, proliferation, adhesion, and migration of monocyte/macrophage lineage cells.

Recent research has demonstrated that elimination of microglia through CSF-1R inhibition could curtail plaque formation in an Alzheimer's disease in vivo model [4]. Microglia, the principal resident macrophages in the brain, perform vital functions related to homeostasis, immune response, and phagocytosis within the central nervous system (CNS). But continuous activation of microglia can lead to neurodegenerative diseases by excessively producing inflammatory mediators, such as cytokines, causing neuronal damage and degeneration [5]. The CSF-1/CSF-1R signaling is highly critical for maintaining the balance of microglia in the brain. Overactivation of the CSF-1/CSF-1R axis, also mediated through downstream signalling pathways such as PI3K/AKT/NF-κB, PKC/NF-κB, and ROS/RAS/RAF/MAPK, may result in abnormal expression of pro-inflammatory cytokines, including tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which activates microglia in turn [6].

Based on these insights, the present study focuses on the development of CSF-1R inhibitors, aiming to elucidate their potential as therapeutic agents [4]. By selectively targeting CSF-1R, these inhibitors hold promise in modulating microglial activation and attenuating neuroinflammation. This research represents a significant advancement in the understanding of CSF-1R inhibition and its potential application in the treatment of neurodegenerative diseases. Fig. 1 depicts compounds currently under investigation for neurological disorders [[7], [8], [9], [10]]. Among these, Masitinib has received significant attention for its potential applications in Alzheimer's Disease (AD) [11]. Masitinib, primarily used to treat mast cell tumors in animals [12], has gained attention for its potential in addressing neurological conditions [13,14]. It targets receptor tyrosine kinases like c-kit and CSF-1R, affecting processes related to inflammation, immunity, and neuron survival. Studies demonstrate masitinib's ability to reduce neuroinflammation, especially in conditions like Alzheimer's and ALS, while enhancing cognitive function [14]. Positive results have led to a Phase III trial in mild-to-moderate Alzheimer's, showing its versatility and promise as a neuroinflammation therapy [15].

In our previous investigations, we successfully identified a highly potent CSF-1R inhibitor through screening our internal library of protein kinase inhibitors. During this screening process, we discovered several chemical scaffolds, one of which featured a methyl isoxazole scaffold that demonstrated remarkable antiproliferative activity specifically in the U937 cell line. Further exploration revealed that this compound, known as 2-methyl-N-(5-methylisoxazol-4-yl)benzamide (1), displayed exceptional potency as a CSF-1R inhibitor with an IC50 value of 10 nM, while exhibiting inhibitory activities against several other kinases including BRafV600E, C-Raf that were tested [16].

But subsequent studies of compound 1 revealed notable vulnerabilities in the metabolic stability (MS) and plasma stability (PS) of the 5-methylisoxazole moiety. Specifically, the acidic nature of 3-H in 5-methylisoxazole rendered the ring prone to opening, forming 2-cyano-3-hydroxybut-2-enamido in the presence of bases or enzymes (Fig. 2) [17]. Despite this instability, the 5-methylisoxazole ring played a pivotal role in conferring potency and selectivity towards CSF-1R in compound 1, making it an essential structural feature in the new design of CSF-1R inhibitors. To address these challenges, we explored compound 2, where the connection between the 5-methylisoxazole ring and the middle 2-methyl aniline was modified from 5-methylisoxazole-4-carboxamide to 5-methylisoxazole-3-carboxamide (2). Building upon this rotated hinge binder, we initiated a comprehensive structure-activity relationship (SAR) study to identify stable Type II CSF-1R inhibitors. Through selective inhibition of CSF-1R, these inhibitors hold great potential in modulating microglial activation and mitigating neuroinflammation, offering a promising therapeutic avenue for neurodegenerative diseases. This report delves into the emerging field of CSF-1R inhibitors and their prospective applications in the treatment of neurodegenerative disorders.