# Design, Synthesis, and Cell Lines Studies of Oleanolic Acid--Hydrogen Sulfide Donor Hybrids
## 3 Results and Discussion
### The Antiproliferative Activities of Compounds in Different Cancer Cell Lines  
The target complexes **2a-c**, **4a-d**, **6a,b**, **8a-c**, **12a,b**, **14a-c**, and **18a-f**, in parallel with OA and 3-oxo-OA, were initially evaluated by the MTT assays for their cytotoxicities on human hepatocellular carcinoma cell line (HepG2), human lung adenocarcinoma cell line (A549), and human breast cancer cell line (MCF-7), using general chemotherapeutic drug oxaliplatin as positive control. As the results show in Table 1, compared with their lead compounds OA or 3-oxo-OA, the synthesized prodrugs showed significantly improved cytotoxicities against all of the tested tumor cell lines. In contrast, the precursor compounds (**3a**, **3b**, **5**, **7**, **11a**, and **11b**) without the H2S-donor moiety, showed only a weak activity, indicating that the H2S-donor moiety exerts an important influence on the cytotoxic activities of the prodrugs (data not shown). Furthermore, 5-(4-hydroxyphenyl)-3H-1,2-dithole-3-thione (ADT-OH), 4-hydroxythiobenzamide (4-OH-TBZ), and N-benzyl-4-hydroxybenzothamide (N-Bn-4-OH-TBZ), three kinds of hydrogen sulfide donors (Figure 1), were initially evaluated by the MTT assays for their cytotoxicities on human hepatocellular carcinoma cell line (HepG2), human lung adenocarcinoma cell line (A549), and human breast cancer cell line (MCF-7) as well. (Table 2). These results indicated that the enhanced cytotoxicity of the H\({}_{2}\)S donor derivatives may be due to their H\({}_{2}\)S-releasing ability, which can achieve the syneric action of cytotoxic H\({}_{2}\)S and the parent compounds OA or 3-oxo-OA. In addition, because the cytotoxic activity of 3-oxo-OA was higher than that of OA, the cytotoxic activities of H\({}_{2}\)S-donating 3-oxo-OA derivatives (**14a-c, 18b, 18d**) were also superior to those of the corresponding H\({}_{2}\)S-donating OA derivatives (**2a-c, 12a,b**). The above results revealed that the type of the H\({}_{2}\)S-donor moiety was crucial for the cytotoxicity of H\({}_{2}\)S-donating OA or 3-oxo-OA derivatives. Under the same conditions of the connecting arm and coupling site, the order of potencies was ADT-OH > 4-OH-TBZ > N-Bn-4-OH-TBZ (Table 1), which was almost the same (**2b** > **2a** > **2c**, **8b** > **8a** > **8c**, and **14b** > **14a**) against the tested tumor cell lines. In addition, the linkers which linked the H\({}_{2}\)S donor to OA or 3-oxo-OA had a great influence on the cytotoxic potency of the derivatives. Firstly, the compounds that directly coupled H\({}_{2}\)S donor to the 28-COOH of OA (**2a-c**) or 3-oxo-OA (**14a-c**) exhibited lower cytotoxicities. Secondly, the 3-OH ester derivatives of OA (**6a,b**, and **8a-c**) showed moderate cytotoxicities. Unexpectedly, the 3-OH ester derivatives with redox-sensitized disulfide bonds did not significantly promote cytotoxicities. IC\({}_{50}\) values of the compounds with redox-sensitized disulfide bond (**8a, 8b**) were slightly lower than those without redox-sensitized disulfide bond derivatives (**6a, 6b**). In contrast, compared with their lead compounds OA or 3-oxo-OA, the amino acid prodrugs **12a,b** and **18a-f** showed significantly improved cytotoxicity against the three tested cell lines. Among them, compounds **18c** and **18d** with \(\beta\)-alanine as a linker showed potent cytotoxicity against HepG2 and