Hydrogen sulfide H S a colorless and
Hydrogen sulfide (H2S) , a colorless and water-soluble gas with the smell of rotten eggs, is now recognized as an important gasotransmitter, after the extensive study of nitric oxide (NO) and carbon monoxide (CO). H2S is produced endogenously via enzymatic reactions of cysteine, homocysteine and cystathionine mediated by cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) . Over the past decade, numerous studies have shown that H2S plays significant roles in physiology and pathology regarding myocardial reperfusion injury , hypertension , atherosclerosis , stroke , neuroinflammation , diabetes mellitus  and so on. Recently, H2S was found to show anticancer activity [41,42], which could trigger apoptosis via activation of MAPK and caspase-3, and sulfhydration of NF-κB , enhance cell proliferation of human gastric cancer AGS cells , promote autophagy of hepatocellular carcinoma cells through EGFR/ERK/MMP-2 and PI3K/Akt/mTOR signaling pathways [45,46], and induce C6 glioma cell growth through activation of the p38 MAPK/ERK1/2-COX-2 pathways . However, H2S is not used directly in clinic because of uncontrollable dose and high toxicity. Researchers often use H2S releasing agents (H2S donors) to modulate H2S levels and enable new investigations [48,49]. It was reported the slow-releasing H2S donor, GYY4137, exhibited potent anti-hepatocellular carcinoma activity through blocking the signal transducer and STAT3 pathway [50,51]. Thereafter, H2S-releasing diclofenac derivatives not only increased the activity of anti-inflammatory and reduced the side effects of diclofenac, but also inhibited breast cancer-induced osteoclastogenesis and prevented osteolysis . Thenceforward, increasing studies have been reported on the antitumor effects of H2S donors [, , , , ]. With the development of H2S donating derivatives (Fig. 1), many H2S donors were found, such as dithiothione derivatives ADTOH , α-thioctic PI-3065 , thiobenzamide derivatives , iminothioethers , cyclic acyl disulfides and acyl selenylsulfides , tetrasulfides  and 1,2,4-thiadiazolidin-3,5-diones . In general, substantial progress has been achieved in the application of H2S donors towards the treatment of human cancers over the past decade. Therefore, design and synthesis of H2S releasing hybrids is an effective and promising strategy for cancer treatment.
In this study, we wanted to continue our studies [65,66] of evodiamine to seek highly selective antiproliferative drug candidates by combining the potent natural compound with H2S donors. Aforementioned predicament inspired us to synthesize 16 derivatives based on two kinds of exogenous H2S donors: ADTOH and α-thioctic acid. Resulting hybrids were evaluated against human cancer Bel-7402, MCF-7, SGC-7901, Caco-2 and HL-60 cell lines, and normal peripheral blood mononuclear cells (PBMCs) for antiproliferative activities. Additionally, further mechanisms regarding cell cycle progression, induction of apoptosis, dysfunction of mitochondrial membrane potential and expression of apoptosis-related and cell cycle-related proteins by representative derivative 12c were also investigated.
Result and discussion
Conclusion To summarize, two novel series of evodiamine derivatives capable of releasing H2S were designed and synthesized for anticancer therapeutics. The antiproliferative activities of all the target compounds were tested against human cancer Bel-7402, MCF-7, SGC-7901, Caco-2 and HL-60 cell lines, and human normal PBMCs. The most effective one, conjugate 12c exhibited cytotoxicity against HL-60 and Caco-2 cell lines with IC50 values of 0.58 and 2.02 μM, respectively. Amazingly, antiproliferative evaluation showed that efficacy enhancement accompanied with improved selectivity. Hence, 12c was chosen for further investigation in order to reveal the cellular mechanisms in HL-60 cell lines. The results demonstrated that 12c showed strong effects which could induce cell apoptosis and arrest cell cycle at the G2/M stage. Moreover, incubation with 12c increased the number of cells with collapsed mitochondrial membrane potentials at low concentrations, so the mitochondrial pathway would be involved in apoptosis. Subsequently, western blot analysis confirmed that 12c induced apoptosis via mitochondrial related pathway, since 12c increased the expression of Bax, cytochrome c, caspase-9, -3 and p53, and reduced the relative levels of Bcl-2, cyclin B1 and cdc2. In conclusion, 12c, a potent compound with H2S releasing ability, offers a good strategy for the discovery of antitumor agents with high efficiency and selectivity.