Natural Bioactive Sterol 5α,8α-endoperoxides as Drug Lead Compounds

List of Abbreviations: EP: Ergosterol Peroxide; ERGO: Ergosterol; U266, RPMI-8226, PMI-8226: Multiple Myeloma cell Line; SNU-1: Human Gastric Tumor Cell Line; Hep3B, HepG2, SUN354, BEL-7402, LO2: Human Hepatoma Cell Line; SUN -C4: Human colorectal tumor cell line; HL60, K562, P388, K462: Human Leukaemia Cell Line; DU-145, LNCAP: Human Prostate Cancer Cell line; HT29, COLO-205: Human Colorectal Tumor Cell; HCT-8: Human Colorectal Cancer Cell Line; SF-295: Human Glioblastoma Cancer Cell Line; WM-1341: Human Malignant Melanoma Cell Line; SGC-7901: Human Gastric Adenocarcinoma Cell Line; HeLa: Human Cervical Carcinoma Cancer Cell Line; KB: Human Nasopharyngeal Epidermoid Carcinoma Cell Line; FL: Human Follicular Lymphoma Cell Line; Hep2: Human Epithelial Cancer Cell; HTLV-I: Human T-Cell Leukemia/ Lymphotropic Virus Type I; MCF7WT, MDA-MB-231, MDA-MB435, MCF-7: Human breast cancer cell line; A549: Human Lung Cancer Cell Line; SK-OV-3: Human Ovarian Cancer Cell Line; SK-MEL-2: Human Skin Melanoma Cancer Cell Line; XF498: Human Central Nerve System Cancer Cell Line; HCT15: Human Colic Cancer Cell Line; WI38: Human Lung Fibroblast Cell Line; H37Rv: Mycobacterium tuberculosis; OVCAR-3, OVCAR-8: Ovarian Cancer Cell Line; MOLT-4, Jurkat: Human lymphoid cancer cell line; JAK2: Janus kinase 2; STAT: Signal Transducer and Activator of Transcription; ATCC: American Type Culture Collection; MyD88: Myeloid Differentiation factor 88; VCAM-1: Vascular Cell Adhesion Molecule 1; NF-kB: Nuclear factor kappa B; RAW264.7: Macrophages; C/EBPβ: EnhancerBinding Protein β; p38: Mitogen-activated protein kinase; JNK: Jun N-Terminal Kinase; ERK: Extracellular singal-Regulated Kinase; MAPKs: Mitogen-Activated Protein Kinases; CDKN1A: CyclinDependent Kinase Inhibitor; iNOS: Inducible Nitric Oxide Synthase (enzyme); COX-2: Cyclo-oxygen-ase; PGE2: Prostaglandin E2


Introduction
Natural products, especially bioactive molecules as drug lead compounds, have attracted extensive attention in health promotion and in drug discovery and development. It is essential to understand the structures and functional mechanisms of these lead molecules prior to drug development. Since the natural peroxides artemisinin and Yingzhaosu which have excellent antimalarial activity are found, and their peroxide bonds are key to antimalarial activity, natural products containing peroxide bonds have began to attract scientists' attention [1][2][3][4].
Among natural sterols, there are some chemical entities which the reasons for the existence and fine biological roles in plants and animals have so far remained unexplored. These highly functionalized sterols have recently attracted considerable attention because of their biological and pharmacological activities.
Sterol 5α,8α-endoperoxides belong to the group of oxidized sterol derivatives and contain a 5α,8α-endoperoxide grouping in addition to the fragments characteristic of such derivatives. This structural element arises as the result of the addition of an oxygen molecule to a 5,7-diene system in the molecule of the initial sterols, for example, ergosterol, 7-dehydrocholesterol and 9(11)-dehydroergosterol.
Up to now, several excellent reviews have been published on the structure and distribution of natural endoperoxides, but there is little information on the biological activity of the sterol 5α,8α-endoperoxides in recent years. This review brings together information on the structures, bioactivities and chemical synthetic methods of the natural sterol 5α,8α-endoperoxides reported from 2000 to now. In some other scientific literatures "steroid peroxides" or "5α,8α-epidioxysteroids" are also usually used to name these compounds. Therefore, sterol 5α,8αendoperoxides are discussed in order of similar carbon skeleton in the review. We hope the review could attracted considerable attentions to sterol peroxides synthesis pathway or cultivation methods research. The sterol peroxides are valuable sources in the development of new drug agents.
A number of biological activities have been attributed to ergosterol peroxide, such as anti-tumor activity, immunomodulatory activity, inhibitory hemolytic activity and anti-inflammatory activity, antiviral activity et al. Ergosterol peroxide has shown to exert anti-tumor activity in multiple myeloma U266 cells partly with anti angiogenic activity targeting JAK2/STAT3 signaling pathway as a potent cancer preventive agent for treatment of multiple myeloma cells [5]. Ergosterol peroxide is also against Walker carcinosarcoma and human mammary adenocarcinoma cell lines in vitro, as well as against human gastric tumor cell line (SNU-1), human hepatoma cell line (SUN-354), human colorectal tumor cell line (SUN-C4), and murine sarcoma-180. Recent studies showed that the cytotoxicity of ergosterol peroxide completely inhibited growth and induced apoptosis of HL60 human leukaemia cells at a concentration of 25 μM. It also inhibited TPAinduced inflammation and tumour promotion in mice and suppressed proliferation of mouse and human lymphocytes stimulated with mitogens [6]. The IC 50 value of the compound based on the cell viability of Hep3B was 16.7 μg/mL [7]. Ergosterol peroxide exhibited an inhibitory effect on androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells at micromolar concentrations [8]. Moreover, ergosterol peroxide appeared to suppress cell growth and STAT1 mediated inflammatory responses by altering the redox state in HT29 cells [9]. Biological evaluation revealed that the compound inhibited the relaxation of supercoiled DNA (pBR322) induced by DNA topoisomerase I, and also showed marginal, selective cytotoxic activity against human colon tumor cells [10]. Ergosterol peroxide displayed potent activity against the cancer cell lines MDA-MB435, HCT-8 and SF-295 [11]. It was also demonstrated that ergosterol peroxide produced greater activity inducing death of miR-378 cells. With future clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells [12].
Immunosuppressive activity was found in ergosterol peroxide isolated from several species. Ergosterol peroxide exhibited significant inhibitory activities against leishmaniasis, tuberculosis, Mycobacterium tuberculosis H37Rv and M. avium [13]. It also played an important role in inhibiting the hemolytic activity of human serum against erythrocytes [14]. It was also shown to be devoid of any activities against an antibiotic sensitive ATCC strain of Staphylococcus aureus [15]. This suggests its potential application in medicinal use as an antivenom and anti-inflammatory agent. Ergosterol peroxide significantly blocked MyD88 and VCAM-1 expression, and cytokine (IL-1β, IL-6 and TNF-α) production in LPS-stimulated cells. It also effectively inhibited NF-kB activation, which was further confirmed with siRNA treatment. The above-mentioned data indicated that ergosterol peroxide may play an important role in the immunomodulatory activity of GF through inhibiting the production of pro-inflammatory mediators and activation of NF-kB signaling pathway [16]. In addition, ergosterol peroxide suppressed LPS-induced DNA binding activity of NF-kB and C/EBPβ, and inhibited the phosphorylation of p38, JNK and ERK MAPKs. It down-regulated the expression of low-density lipoprotein receptor (LDLR) regulated by C/EBP, and HMG-CoA reductase (HMGCR) in RAW264.7 cells. Furthermore, ergosterol peroxide induced the expression of oxidative stress-inducible genes, and the cyclin-dependent kinase inhibitor CDKN1A, and suppressed STAT1 and interferon-inducible genes [17]. It was found that ergosterol peroxide possess markedly activity against PGE2 release with an IC 50 value of 28.7 μM. The mechanism in transcriptional level of ergosterol peroxide was found to down-regulate mRNA expressions of iNOS and COX-2 in dose-dependent manners. In addition, a glycosylated derivative of ergosterol peroxide 2 has been obtained from Cordyceps sinensis. The glycosylated form of ergosterol peroxide was found to be a greater inhibitor to the proliferation of K462, Jurkat, WM-1341, HL-60 and RPMI-8226 tumor cell lines by 10 to 40% at 10 μg/mL [18] ( Figure  2, Table 2  side-chain. The newly identified natural sources are summarized in Table 2. The compound 5α,8α-epidioxycholest-6-en-3β-ol 3 displayed cytotoxicity toward various cancer cell lines. It was evaluated for cytotoxicity against three human tumor cell lines, and showed mild cytotoxicity against SGC-7901, HepG2 and HeLa cells with IC 50 values of 99, 65 and 94 μg/mL, respectively. While compound 3 did not show cytotoxicity against human normal hepatocytes LO2 (215 μg/mL), and the corresponding results showed 3 was safe to human normal hepatocytes in the therapeutic dosages [19]. In addition, compound 3 possesses antifungal activity against three tomato pathogenic fungi, Botrytis cinerea, Fusarium oxysporum and Verticillium albo atrum and antibacterial activity against Agrobacterium tumefaciens, Escherichia coli, Staphylococcus faecalis, Staphylococcus aureus and Pseudomonas aeruginosa. It showed significant toxicity against brine shrimp larvae with an LD 50 value of 4.5 μg/mL [20,21].
A second structural type of sterol endoperoxides includes compounds which contain a 9(11)-double bond in addition to a 3β-hydroxy, a 5α,8α-epidioxide and a 6-double bond. Formally, these compounds may be considered as oxidation products of steroids having double bonds in the ∆ 5,7 and ∆ 9 (11) positions, such as 9,11-dehydroergosterol ( Figure 5).

Synthesis
On the basis of above discussions, it may be concluded that the isolation of sterol 5,8-endoperoxides into the pure state from natural sources is a fairly complex and laborious process. In a number of cases their chemical synthesis appears more convenient, especially if the initial ∆ 5,7 -or ∆ 5,7,9(11) -sterols are available. This is also favored by the circumstance that chemical synthesis of 5,8-epidioxides are based on the well-studied photochemical oxidation of 5,7-diene group in sterols ( Figure 10).
In cases where the initial ∆ 5,7 -sterols are unavailable, special schemes of synthesis must be developed for obtaining the 5,8-epidioxides. The synthesis of endoperoxide 3 started from cholesterol, cholesterol was converted to cholesterol-3-acetate to protect the hydroxy group [40]. Cholesterol-3-acetate allylic benzoyloxylation and further reduction and esterification of to cholesterol-3,7-diacetate as shown by Scheme 2 stepts I, II and III. Following this procedure 50% yield was obtained [41]. The second route employed for the synthesis of cholesterol-3,7diacetate was chromium or cobalt catalyzed allylic oxidation in the presence of ter-butyl hydroperoxide to produce 7-oxocholesterol-3acetate which is further reduced to 7-hydroxycholesterol-3-acetate and in turn to cholesterol-3,7-diaceate. The final yield of acetate obtained by cobalt and chromium allylic oxidation was almost similar (45%), but due to the hazardous nature of chromium catalyst cobalt allylic oxidation step is not frequently-used [42]. Cholesterol-3,7-diacetate formation is also reported by using electrochemical oxidation of cholesterol [43]. Considering the most productive and less hazardous route, the third route is employed in major in study. On the allyl bromination of cholesterol-3-acetate with N-bromosuccinimide in the presence of 2,2-azobis (isobutyronitrile) followed by dehydrobromination with β-collidine in boiling xylene, the cholesterol-3-acetate-5,7-diene was obtained with an overall yield of 54%. Hydrolysis of the acetoxy group with potassium carbonate in methanol led to 7-dehydrocholesterol, the photooxidation of which in the presence of Rose Bengal enabled the endoperoxide 3 to be obtained with an overall yield of 20% from cholesterol [44,45] (Scheme 2).     If the initial ∆ 5,7,9(11) -sterols is not available, special synthesis route should be developed(see Scheme 3). The synthesis of endoperoxide 22 also started from cholesterol-3-acetate. The reaction of the cholesterol-3-acetate-5,7-diene could obtained as shown in Scheme 3. Cholesterol-3-acetate-5,7-diene with mercury(II) acetate in dioxane and acetic acid led with a yield of 35% to the cholesterol-3-acetate-5,7,9(11)-triene the photooxidation of which gave a 61% yield of the endoperoxide 37. Hydrolysis of the 37 acetoxy group with potassium carbonate in methanol gave the endoperoxide 22 with a yield of 80% [46] (Scheme 3).
There have been a fairly large number of studies in which sterol 5α,8α-endoperoxides have been used to obtain compounds with various structures. However, up to now, these studies have not led to any compounds bearing practical utilities. For this reason they will not be discussed in details here.

Conclusion
In this review we have shown that most of the natural sterol 5,8-endoperoxides display in vitro antimicrobial, anti-tumor activity, immunomodulatory activity, and anti-inflammatory activity even in the nanomolar range. These allow us to assume that sterol 5,8-endoperoxides may be involved in ecological, most probably nutritional, interactions between plants, fungi, and animals, similarly to the situation with other steroids (cardiac glycosides, steroid saponins and alkaloids, ecdysteroids, withanolides, ect.). All these bioactivity natural sterol 5,8-endoperoxides were isolated from terrestrial sources and marine sources (such as plants, fungi and sponge). It is noted that isolation and purification of these natural peroxides in the pure state from natural sources is a fairly complex and laborious process. As a result, it is essential to develop methods for chemical synthesis to increase the efficiency for research and drug development. Likely, increasing investigations on synthesis pathways or cultivation methods of sterol 5,8-endoperoxides will hopefully increase the possibilities of a full pharmacological evaluation and a possible introduction in therapy as lead structures for the development of new drug agents.