Research paperHydrogen sulfide releasing oridonin derivatives induce apoptosis through extrinsic and intrinsic pathways
Graphical abstract
Introduction
Natural products have made great contributions to drug design. With the development of analytical techniques, more and more active ingredients were isolated and identified from herbal medicines, natural plants and their metabolites. Many novel structural skeletons were provided for drug discovery, and relatively complete quality control systems were also established [[1], [2], [3], [4]]. Compared with combinatorial chemistry, natural products remain active in drug discovery due to their prominent biological activities, complex and diverse structures and distinctive stereogenic centers which could better combine with corresponding targets in vivo [5]. In the past three decades, the percentage of natural products or nature derived molecules has increased to 74% of all approved chemical entities in anticancer domain [6,7].
As a rich source of ent-kaurane diterpenoids (Fig. 1), the genus Rabdosia exhibits many biological activities, including anti-inflammatory, anticancer, antibacterial and antimutagenic activities [[8], [9], [10], [11], [12], [13], [14], [15], [16], [17]]. Oridonin (1, Fig. 1), a natural ent-kaurane diterpenoid from Rabdosia, was first isolated and identified by Fujita et al., in 1970 [18]. Further in-depth studies on pharmacology showed potent anticancer effects t(8; 21) acute myeloid leukemic (AML) cells [19], human pancreatic cancer PANC-1 cells [20], human osteosarcoma cells [21], p53-mutated colorectal cancer cells [22], drug-resistant renal cell carcinoma (RCC) [23] and non-germinal center B cell-like subtype of diffuse large B cell lymphoma (non-GCB DLBCL) [24]. Although oridonin can be obtained commercially and shows cytotoxicity for many cancer cells, it deserves further structural modification due to its poor selectivity and low aqueous solubility [25]. SAR studies indicated that unsaturated cyclopentanone conjugated with extracyclic methylene was a pivotal structure for antiproliferative activity, while the activity slumped when cyclopentanone was cleaved or methylene was saturated [26]. Furthermore, 14-hydroxy of oridonin was a prominent structure modification site. Most 14-O-derivatives showed maintained or better antiproliferative activities toward several human cancer cells [27,28].
Due to the endogenous pathophysiological functions, small molecule donors have comprised a key pillar of drug design [29,30]. Before nitric oxide (NO) and carbon monoxide (CO) were famous for endogenous bioactivity, hydrogen sulfide (H2S) was considered to be a colorless and toxic gas with a strong smell of rotten eggs. In recent years, it has been recognized as the third endogenous signaling gasotransmitter following NO and CO, acting as a neuromodulator and neuroprotective agent [31,32]. Endogenous H2S commonly generates through two specific pyridoxal-5′-phosphate (PLP)-dependent enzymes, cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), or biosynthesized by the synergistic effects of 3-mercaptopyruvate sulfurtransferase (3-MST) and cysteine aminotransferase (CAT) [[33], [34], [35]]. Increasing evidences confirm the pathophysiological functions of H2S in atherosclerosis, cytoprotection against oxidative stress, angiogenesis, ischemia-reperfusion injury and so on [[36], [37], [38], [39], [40]]. In addition, recent studies demonstrate the importance of H2S in biological processes of cancer which shows antiproliferative activities through EGFR/ERK/MMP-2, PTEN/AKT, PI3K/Akt/mTOR and p38 MAPK/ERK1/2-COX-2 pathways [[41], [42], [43], [44]]. This reactivation of programmed cell death by delivery of H2S could be suggested as an effective approach for cancer therapy. However, H2S is not suitable for clinical applications directly owing to uncontrollability, high toxicity and short half-life. To overcome these disadvantages, a number of H2S releasing agents (H2S donors) have been developed, such as ADT-OH, thiobenzamide, Jks, L-propargyl cysteine, α-thioctic acid, GYY4137, DATS, d-cysteine and so on (Fig. 2A). These H2S donating compounds could deliver H2S in sustained manner and prolong the term of treating times [[45], [46], [47], [48], [49]]. With the development of H2S donors, more and more H2S releasing derivatives have been designed and synthesized. Some of them have entered into Phase I or Phase II clinical trials [ [[50], [51], [52], [53], [54], [55]], Fig. 2C]. Among them, compounds with 3H-1,2-dithiole-3-thione show prominent cytoprotective properties [ [56], Fig. 2B].
In the present study, we designed and synthesized three series of H2S releasing oridonin derivatives in which oridonin was conjugated with a H2S generating moiety (5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione, ADT-OH) via different anhydride linkers at 14-hydroxyl group. The antiproliferative activities of all the derivatives were tested against five human cancer cell lines (human hepatoma HepG2, breast cancer MCF-7, colorectal cancer HCT-116, melanoma B16 and chronic myelogenous leukemia K562) and two normal cell lines (human liver L-02 and peripheral blood mononuclear PBMC). Moreover, in-depth apoptosis related mechanisms of the most promising compound 12b which included cell cycle arrest, morphological change, apoptosis induction, mitochondria membrane potentials decline and the expression of apoptosis-related proteins were studied.
Section snippets
Chemistry
The intermediates 2 and 5 were synthesized according to our previous report [57]. The H2S donating derivatives 7a-c were obtained by substitution reactions of ADT-OH 6 with several bromhydrins (2-bromoethanol, 3-bromo-1-propanol and 6-bromo-1-hexanol). By treatment of 1, 2 and 5 with corresponding anhydride in the presence of TEA/DMAP, derivatives 8a-c and 9a-c were got. Derivatives 8a-c and 9a-c were directly reacted with H2S releasing 7a-c via esterification reaction to gain the target
Conclusion
Overall, by using oridonin as lead compound, we designed and synthesized 18H2S-delivering derivatives in the present study. Five tumor and two normal cell lines were selected to test the antiproliferative activities of all derivatives. Most derivatives were sensitive to HepG2 and K562 cells and several compounds showed improved effects than parent compound oridonin. In particular, 12b not only showed the most potent antiproliferative activities against HepG2, HCT-116 and K562 cells with IC50
Chemistry
Chemical materials and reagents were obtained from commercial suppliers, the preparation of anhydrous solvents was based on standard methods. 1H and 13C NMR spectra were determined with Bruker 400 MHz spectrometer in the solvent of CDCl3 (TMS as internal standard): the values of the chemical shifts were reported in δ values (ppm) and the coupling constants (J) in Hz. High resolution mass spectra (HR-MS) were analyzed on Agilent Q-TOF B.05.01 (B5125.2).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This paper was financially supported by the National Natural Science Foundation of China (21772124, 21502121), Natural Science Foundation of Liaoning Province (20170540858), General Scientific Research Projects of Department of Education in Liaoning Province (2017LQN05), Key Laboratory of Quality Control of TCM of Liaoning Province (17-137-1-00) and Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University.
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