Aims: The present study was conducted to examine the inhibitory effects of synthesized sulfonylhydrazones on the expression of CD73 (ecto-5′-NT).

Background: CD73 (ecto-5′-NT) represents the most significant class of ecto-nucleotidases, which are mainly responsible for the dephosphorylation of adenosine monophosphate to adenosine. Inhibition of CD73 played an important role in the treatment of cancer, autoimmune disorders, precancerous syndromes, and some other diseases associated with CD73 activity.

Objective: Keeping in view the significance of CD73 inhibitor in the treatment of cervical cancer, a series of sulfonylhydrazones (3a-3i) derivatives synthesized from 3-formylchromones were evaluated.

Methods: All sulfonylhydrazones (3a-3i) were evaluated for their inhibitory activity towards CD73 (ecto-5′-NT) by the malachite green assay and their cytotoxic effect was investigated on the HeLa cell line using MTT assay. Secondly, the most potent compound was selected for cell apoptosis, immunofluorescence staining, and cell cycle analysis. After that, CD73 mRNA and protein expression were analyzed by real-time PCR and Western blot.

Results: Among all compounds, 3h, 3e, 3b, and 3c were found to be the most active against rat-ecto- 5′-NT (CD73) enzyme with IC₅₀ (μM) values of 0.70 ± 0.06 μM, 0.87 ± 0.05 μM, 0.39 ± 0.02 μM, and 0.33 ± 0.03 μM, respectively. These derivatives were further evaluated for their cytotoxic potential against cancer cell line (HeLa). Compounds 3h and 3c showed the cytotoxicity at IC₅₀ value of 30.20 ± 3.11 μM and 86.02 ± 7.11 μM, respectively. Furthermore, compound 3h was selected for cell apoptosis, immunofluorescence staining, and cell cycle analysis, which showed a promising apoptotic effect in HeLa cells. Additionally, compound 3h was further investigated for its effect on the expression of CD73 using qRT-PCR and western blot.

Conclusion: Among all synthesized compounds (3a-3i), Compound 3h (E)-N'-((6-ethyl-4-oxo-4Hchromen- 3-yl) methylene)-4-methylbenzenesulfonohydrazide was identified as the most potent compound. Additional expression studies conducted on the HeLa cell line proved that this compound successfully decreased the expression level of CD73 and thus, inhibited the growth and proliferation of cancer cells.

目的：本研究旨在检查合成的磺酰腙对 CD73 (ecto-5'-NT) 表达的抑制作用。

背景：CD73（ecto-5'-NT）代表了最重要的一类外切核苷酸酶，主要负责将单磷酸腺苷去磷酸化为腺苷。CD73 的抑制在癌症、自身免疫性疾病、癌前综合征和一些其他与 CD73 活性相关的疾病的治疗中发挥了重要作用。

目的：鉴于CD73抑制剂在宫颈癌治疗中的重要性，对3-甲酰基色酮合成的一系列磺酰腙（3a-3i）衍生物进行了评价。

方法：通过孔雀石绿试验评估所有磺酰腙 (3a-3i) 对 CD73 (ecto-5'-NT) 的抑制活性，并使用 MTT 试验研究它们对 HeLa 细胞系的细胞毒作用。其次，选择最有效的化合物进行细胞凋亡、免疫荧光染色和细胞周期分析。之后，通过实时 PCR 和蛋白质印迹分析 CD73 mRNA 和蛋白质表达。

结果：在所有化合物中，发现 3h、3e、3b 和 3c 对大鼠外源性 5'-NT (CD73) 酶的活性最强，IC ₅₀ (μM) 值为 0.70 ± 0.06 μM、0.87 ± 0.05分别为 μM、0.39 ± 0.02 μM 和 0.33 ± 0.03 μM。进一步评估了这些衍生物对癌细胞系 (HeLa) 的细胞毒性潜力。化合物3h和3c分别在30.20±3.11μM和86.02±7.11μM的IC ₅₀值下显示出细胞毒性。此外，选择化合物3h进行细胞凋亡、免疫荧光染色和细胞周期分析，在HeLa细胞中显示出良好的凋亡作用。此外，还使用 ​​qRT-PCR 和蛋白质印迹进一步研究了化合物 3h 对 CD73 表达的影响。

结论：在所有合成的化合物 (3a-3i) 中，化合物 3h (E)-N'-((6-ethyl-4-oxo-4Hchromen-3-yl)methylene)-4-methylbenzosulfonohydrazide 被鉴定为最有效的化合物. 对 HeLa 细胞系进行的其他表达研究证明，该化合物成功降低了 CD73 的表达水平，从而抑制了癌细胞的生长和增殖。