To study the in vivo anti-tumor activity of Z8, SK-BR-3 tumor bearing nude mice were administered once every three days. As shown in Fig. 11A-11D, CPT and Z8 demonstrated significant inhibitory effects on SK-BR-3 tumor growth, with the Z8 group exhibiting more pronounced inhibition compared to CPT. Notably, the average tumor volume and weight in the Z8 group were the smallest among all groups, including the control and CPT groups. Moreover, to further study the toxicity of CPT and Z8, serums of mice were collected for blood biochemical analysis. As shown in Fig. 11E-H, treatment with both CPT and Z8 resulted in increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), but the effect of Z8 group was relatively low, indicating that Z8 was safer than CPT in vivo. Furthermore, in CPT group, urea nitrogen levels increased significantly, whereas no significant change was observed in the Z8 group, indicating potential renal function impairment following CPT treatment, whereas Z8 showed better safety in this regard. Additionally, the levels of alkaline phosphatase (ALP), total protein (TP), albumin (ALB), globulin (GLOB) and creatinine (CREA) did not significantly differ between the control group and the treatment groups. Finally, compared to the control group, histological examination using H&E staining (Hematoxylin and Eosin staining) of the major organs in the treatment group did not reveal obvious cell necrosis and inflammatory cell infiltration, indicating that Z8 would not bring additional toxicity.

The proapoptotic activity of Z8 was studied by flow cytometry combined with Annexin V-FITC/PI double staining. As shown in Fig. 4, after treatment with 1 uM Z8, the apoptosis rate of SK-BR-3 cells was approximately 27.1 % which increased to 41.1 % following treatment with 2 uM Z8 (4.4 % in the blank control group and 55.8 % in CPT group. These findings suggest that Z8 can significantly induce apoptosis in a dose-dependent manner.

The proapoptotic activity of Z8 was studied by flow cytometry combined with Annexin V-FITC/PI double staining. As shown in Fig. 4, after treatment with 1 uM Z8, the apoptosis rate of SK-BR-3 cells was approximately 27.1 % which increased to 41.1 % following treatment with 2 uM Z8 (4.4 % in the blank control group and 55.8 % in CPT group. These findings suggest that Z8 can significantly induce apoptosis in a dose-dependent manner.

To evaluated the in vitro anti-tumor activity of the conjugates, HER2-positive SK-BR-3, NCI-N87, HER2-negative MDA-MB-231 and normal MCF-10A cells were selected. As shown in Table 2, nearly all conjugates exhibited obvious anti-proliferation activity against SK-BR-3 cells, while most of the conjugates, except for Z6 and Z9, demonstrated different degrees of inhibitory activity on NCI-N87 cells. Taken together, Z8 and Z11 exhibited superior anti-tumor activity. The antiproliferative activity of Z8 against NCI-N87 cells (IC50 = 1.91 ± 0.71 uM) was comparable to that of CPT (IC50 = 2.10 ± 1.34 uM), while the antiproliferative activity of Z11 against SK-BR-3 cells (IC50 = 0.41 ± 0.13 uM) was comparable to that of CPT (IC50 = 0.26 ± 0.06 uM). Additionally, the IC50 values of these two conjugates against MDA-MB-231 and MCF-10A cells were significantly increased, showing their ability to selectively inhibit tumor cells. Based on the above results, Z8 and Z11, which exhibited better activity and apparent selectivity, were selected for subsequent studies.

To evaluated the in vitro anti-tumor activity of the conjugates, HER2-positive SK-BR-3, NCI-N87, HER2-negative MDA-MB-231 and normal MCF-10A cells were selected. As shown in Table 2, nearly all conjugates exhibited obvious anti-proliferation activity against SK-BR-3 cells, while most of the conjugates, except for Z6 and Z9, demonstrated different degrees of inhibitory activity on NCI-N87 cells. Taken together, Z8 and Z11 exhibited superior anti-tumor activity. The antiproliferative activity of Z8 against NCI-N87 cells (IC50 = 1.91 ± 0.71 uM) was comparable to that of CPT (IC50 = 2.10 ± 1.34 uM), while the antiproliferative activity of Z11 against SK-BR-3 cells (IC50 = 0.41 ± 0.13 uM) was comparable to that of CPT (IC50 = 0.26 ± 0.06 uM). Additionally, the IC50 values of these two conjugates against MDA-MB-231 and MCF-10A cells were significantly increased, showing their ability to selectively inhibit tumor cells. Based on the above results, Z8 and Z11, which exhibited better activity and apparent selectivity, were selected for subsequent studies.

To evaluated the in vitro anti-tumor activity of the conjugates, HER2-positive SK-BR-3, NCI-N87, HER2-negative MDA-MB-231 and normal MCF-10A cells were selected. As shown in Table 2, nearly all conjugates exhibited obvious anti-proliferation activity against SK-BR-3 cells, while most of the conjugates, except for Z6 and Z9, demonstrated different degrees of inhibitory activity on NCI-N87 cells. Taken together, Z8 and Z11 exhibited superior anti-tumor activity. The antiproliferative activity of Z8 against NCI-N87 cells (IC50 = 1.91 ± 0.71 uM) was comparable to that of CPT (IC50 = 2.10 ± 1.34 uM), while the antiproliferative activity of Z11 against SK-BR-3 cells (IC50 = 0.41 ± 0.13 uM) was comparable to that of CPT (IC50 = 0.26 ± 0.06 uM). Additionally, the IC50 values of these two conjugates against MDA-MB-231 and MCF-10A cells were significantly increased, showing their ability to selectively inhibit tumor cells. Based on the above results, Z8 and Z11, which exhibited better activity and apparent selectivity, were selected for subsequent studies.

To evaluated the in vitro anti-tumor activity of the conjugates, HER2-positive SK-BR-3, NCI-N87, HER2-negative MDA-MB-231 and normal MCF-10A cells were selected. As shown in Table 2, nearly all conjugates exhibited obvious anti-proliferation activity against SK-BR-3 cells, while most of the conjugates, except for Z6 and Z9, demonstrated different degrees of inhibitory activity on NCI-N87 cells. Taken together, Z8 and Z11 exhibited superior anti-tumor activity. The antiproliferative activity of Z8 against NCI-N87 cells (IC50 = 1.91 ± 0.71 uM) was comparable to that of CPT (IC50 = 2.10 ± 1.34 uM), while the antiproliferative activity of Z11 against SK-BR-3 cells (IC50 = 0.41 ± 0.13 uM) was comparable to that of CPT (IC50 = 0.26 ± 0.06 uM). Additionally, the IC50 values of these two conjugates against MDA-MB-231 and MCF-10A cells were significantly increased, showing their ability to selectively inhibit tumor cells. Based on the above results, Z8 and Z11, which exhibited better activity and apparent selectivity, were selected for subsequent studies.