The global spread of multi-drug resistant P. falciparum, P. vivax, and P. malariae strains and absence of long-term effective vaccine makes chemotherapy the mainstay of malaria control strategies in endemic settings. The Mossman’s assay and the Organization for Economic Co-operation and Development (OECD), 2001 guideline 423, were used to determine the cytotoxicity and acute oral toxicity of a novel hybrid drug, artesunate-3-Chloro-4(4-chlorophenoxy) aniline (ATSA), in vitro and in vivo, respectively. A modified Desjardins method was used to screen for antiplasmodial activity using P. falciparum (3D₇ and W₂) strains in vitro. The Peter’s 4-day suppressive tests (4DTs) was used to evaluate the in vivo antimalaria activity using P. berghei ANKA strain, lumefantrine resistant (LuR), and piperaquine resistant (PQR) P. berghei lines. In silico prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles was assayed using PreADMET online prediction tool. The reference drug in all experiments was artesunate (ATS). Statistical significance between ATSA’s activities in treated and control mice was evaluated by one-way analysis of variance (ANOVA). Results show that inhibitory concentrations-50 (IC₅₀) of ATSA is 11.47 ± 1.3 (3D₇) and 1.45 ± 0.26 (W₂) against 4.66 ± 0.93 (3D₇) and 0.60 ± 0.15 (W₂) ng/ml of ATS with a selective index of 2180.91(3D₇) and a therapeutic index (TI) of > 71). No mortalities were observed in acute oral toxicity assays and mean weight differences for test and controls were statistically insignificant (P > 0.05). The in vivo activity of ATSA was above 40% with effective dosage-50 (ED₅₀) of 4.211, 2.601, and 3.875 mg/kg body weight against P. berghei ANKA, LuR, and PQR lines, respectively. The difference between treated and control mice was statistically significant (P < 0.05). ATSA has high intestinal absorption (HIA) > 95% and has medium human ether-a-go-go related gene (hERG) K⁺ channel inhibition risks. Preclinical and clinical studies on ATSA are recommended to evaluate its value in developing novel drugs for future management of multi-drug resistant malaria parasites.

多重耐药性恶性疟原虫、间日疟原虫和三日疟原虫菌株在全球蔓延，并且缺乏长期有效的疫苗，这使得化学疗法成为地方性疟疾控制策略的主要手段。Mossman 试验和经济合作与发展组织 (OECD) 2001 年准则 423 用于确定新型混合药物青蒿琥酯-3-氯-4(4-氯苯氧基)苯胺的细胞毒性和急性口服毒性(ATSA)，分别在体外和体内。使用改良的 Desjardins 方法筛选使用恶性疟原虫的抗疟原虫活性（3D ₇和 W ₂) 体外菌株。Peter 的 4 天抑制试验 (4DTs) 用于评估使用伯氏疟原虫ANKA 菌株、苯泛素抗性 (LuR) 和哌喹抗性 (PQR) 伯氏疟原虫品系的体内抗疟活性。使用PreADMET 在线预测工具对吸收、分布、代谢、排泄和毒性 (ADMET) 曲线进行计算机预测。所有实验中的参考药物是青蒿琥酯（ATS）。通过单向方差分析 (ANOVA) 评估 ATSA 在治疗小鼠和对照小鼠中的活动之间的统计显着性。结果表明，ATSA 的抑制浓度 50 (IC ₅₀ ) 为 11.47 ± 1.3 (3D ₇ ) 和 1.45 ± 0.26 (W ₂) 对抗 4.66 ± 0.93 (3D ₇ ) 和 0.60 ± 0.15 (W ₂ ) ng/ml 的 ATS，选择指数为 2180.91(3D ₇ )，治疗指数 (TI) > 71)。在急性口服毒性试验中未观察到死亡率，试验和对照的平均体重差异无统计学意义 ( P  > 0.05)。ATSA 的体内活性高于 40%，有效剂量 50 (ED ₅₀ ) 分别为 4.211、2.601 和 3.875 mg/kg 体重对抗伯氏疟原虫 ANKA、LuR 和 PQR 系。治疗小鼠和对照小鼠之间的差异具有统计学意义（P < 0.05)。ATSA 具有高肠道吸收率 (HIA) > 95%，具有中等人类 ether-a-go-go 相关基因 (hERG) K ⁺通道抑制风险。建议对 ATSA 进行临床前和临床研究，以评估其在开发用于未来管理多重耐药性疟疾寄生虫的新药方面的价值。