Tumor cells often exhibit metabolic reprogramming to maintain their rapid growth and proliferation. Glutaminase 1 (GLS1) has been viewed as a promising target in the glutamine metabolism pathway for the treatment of malignant tumors. Using structure-based drug design approaches, a novel series of GLS1 allosteric inhibitors were designed and synthesized. Compound 41a (LWG-301) with an alkane chain "tail" group had potent biochemical and cellular GLS1 activity, and improved metabolic stability. LWG-301 exhibited moderate antitumor effects in HCT116 xenograft model, with TGI of 38.9% in vivo. Mechanistically, LWG-301 could significantly block glutamine metabolism, resulting in changes in the corresponding amino acid levels in cells, induce a concentration-dependent increase in intracellular ROS levels, and induce apoptosis. Taken together, this paper provides more structural references and new design strategy for the development of GLS1 allosteric inhibitors.