# Design, Synthesis, and Cell Lines Studies of Oleanolic Acid--Hydrogen Sulfide Donor Hybrids
## 3 Results and Discussion  
### Chemistry  
The synthesis of oleanolic acid-hydrogen sulfide donor hybrids and 3-oxooleanolic acid-hydrogen sulfide donor hybrids are outlined in Schemes 1 and 2, respectively. In this study, we selected 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), 4-hydroxothiobenzamide (4-OH-TBZ), and N-benzyl-4-hydroxbenzothamide (N-Bn-4-OH-TBZ) as H\({}_{2}\)S-releasing moieties. OA or 3-oxo-OA was directly modified with different H\({}_{2}\)S donors in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) to produce the corresponding C-28 ester derivatives (**2a-c** and **14a-c**). In order to enrich the types of linking bonds, we tried to connect the dibromoalkane to 3-OH of OA to obtain C-3 ether derivatives. Reaction of OA with dibromoalkane in the presence of potassium hydroxide and 0.1 mmol of potassium iodide in dry tetrahydrofuran afforded intermediates **3a,b**. Unexpectedly, the NMR spectra of the intermediates **3a,b** showed that they were C-28 ester derivatives. These results indicated that the reactivity of 3-OH was too low to participate in the reaction under these conditions. Thereafter, treating the intermediates **3a,b** with H\({}_{2}\)S donors under basic conditions afforded the hybrids **4a-d**. Furthermore, the preparation of C-3 ester derivatives (**6a,b** and **8a-c**) was carried out in two steps. OA was reacted with succin anhydride or 1,5,6-oxadditionaned,9-dione in the presence of DMAP to form 3-O-acyl derivatives (5 and 7), which were further esterified with corresponding H\({}_{2}\)S donors in the presence of EDCI/DMAP to generate target compounds. Then, we tried to couple the amino acid moiety to the 28-carboxyl group. In order to improve the reactivity and avoid the generation the C-28 ester by-products, the reaction was carried out in multiple steps. Generally, OA or 3-oxo-OA first reacted with oxalyl chloride to give intermediates (9 or 15), and then a different ethylamino acid in the presence of triethylamine (TEA) was added to yield intermediates (**10a,b** or **16a-c**). Thereafter, the ethyl group was successfully removed by treating the above intermediates with sodium hydroxide, resulting in **11a,b** or **17a-c**, respectively. In the presence of EDCI/DMAP, the OA- or 3-oxo-OA-amino acid-H\({}_{2}\)S donor trihybrids (**12a,b** or **18a-f**) were successfully obtained by further coupling with different H\({}_{2}\)S donors. Their structures were characterized by \({}^{1}\)H NMR, \({}^{13}\)C NMR, and HRMS.  
**Scheme 1.** Reactions and conditions: (**a**) H\({}_{2}\)S donor, EDCI, DMAP, dry CH\({}_{2}\)Cl\({}_{2}\), reflux; (**b**) dibromoalkane, KOH, KI, dry THF, reflux; (**c**) H\({}_{2}\)S donor, KOH, KI, dry THF, reflux; (**d**) succinin anhydride, DMAP, dry CH\({}_{2}\)Cl\({}_{2}\), reflux; (**e**) 1,5,6-oxadditionaned,9-dione, DMAP, dry CH\({}_{2}\)Cl\({}_{2}\), reflux; (**f**) (COCI)\({}_{2}\), dry CH\({}_{2}\)Cl\({}_{2}\), 0 \({}^{\circ}\)C; (g) amino acid, EtOH, H\({}_{2}\)SO\({}_{4}\); (**h**) ethyl-amino acid, TEA, dry CH\({}_{2}\)Cl\({}_{2}\), 0 \({}^{\circ}\)C--rt; (i) NaOH, TFH.