Peroxynitrite (ONOO), is a potent oxidant that can cause severe cell damage.1) Specifically, peroxynitrite promotes the oxidation of biomolecules such as lipids, proteins and nucleic acids, [2][3][4] as well as the nitration of tyrosine residues in proteins. 5,6) Furthermore, it has been suggested that peroxynitrite formation plays a role in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. [7][8][9] Neoechinulin A (1), an isoprenyl indole alkaloid, can protect neuronal PC12 cells from ONOO Ϫ -induced death. [10][11][12] We have previously shown that the biological effects, rather than scavenging activity against ONOO Ϫ , are likely to play a role in the cytoprotective action of neoechinulin A.12) However, the precise molecular mechanism remains elusive. To investigate the potential mechanism of action, we have designed and prepared a series of neoechinulin A analogues (2-6). We then examined the structure-activity relationships of these analogues in terms of their anti-nitration and anti-oxidant activities as well as their cytoprotective activity against ONOO Ϫ derived from SIN-1 (3-(4-morpholinyl)sydnonimine hydrochloride) using PC12 cells (Fig. 1). The results showed that: 1) the presence of the C-8/C-9 double bond is indispensable for anti-nitration and anti-oxidant activities as well as cytoprotective activity of neoechinulin A against ONOO Ϫ toxicity; 2) in conjunction with the C-8/C-9 double bond, the presence of an intact diketopiperazine moiety is essential for the anti-nitration activity but not for antioxidant or cytoprotective activity. Results and DiscussionCompound 2 was synthesized from 2-tert-butyl-1H-indole (7) 13) (Chart 1). Methoxy methyl (MOM) protection of 7, followed by the Vilsmeier reaction, gave aldehyde 9. A coupling reaction of the aldehyde 9 with diketopiperazine 10 using tBuOK in DMF afforded 11.14) Subsequent deprotection of protective groups provided the desired product 2. 15)Compound 3 was prepared by coupling of aldehyde 12 with N-Boc-Gly-OEt, followed by treatment of the resulting We synthesized a series of neoechinulin A derivatives and examined the structure-activity relationships in terms of their anti-nitration and anti-oxidant activities as well as their cytoprotective activity against peroxynitrite from SIN-1 (3-(4-morpholinyl)sydnonimine hydrochloride) using PC12 cells. Our results showed that the C-8/C-9 double bond, which constitutes a conjugate system with indole and diketopiperazine moieties of neoechinulin A is essential for anti-nitration and anti-oxidant activities as well as protection against SIN-1 cytotoxicity. The presence of an intact diketopiperazine moiety is an additional requirement for anti-nitration activity but not for the cytoprotective action. Our results suggest that the antioxidant activity or electrophilic nature of the C-8 carbon, both of which are afforded by the C-8/C-9 double bond, may play a role in the cytoprotective properties of this alkaloid.

We developed an efficient, stereoselective synthetic method for the diketopiperazine moiety of neoechinulin A and its derivatives. The intramolecular cyclization at 80 ºC proceeded with minimal racemization of the stereogenic center at C-12 on neoechinulin A, even though the cyclization at 110 ºC caused partial racemization. In contrast with these results, the cyclization on diketopiperazine of 8,9-dihydroneoechinulin A derivatives did not cause epimerization of the stereogenic centers, even at 110 °C. We examined the structure-activity relationships for the cytoprotective activity against cytotoxicity induced by 3-morpholinosydnonimine (SIN-1) in nerve growth factor (NGF)-differentiated PC12 cells. The C-8/C-9 double bond, but not the stereogenic center derived from alanine, was found to play a key role in the cytoprotective activity.

Treatment of PC12 cells with fungus-derived alkaloid neoechinulin A for more than 12 h renders the cells resistant to subsequent superoxide (O 2 )/nitric oxide (NO) insults derived from 3-morpholinosydnonimine (SIN-1). However, the underlying mechanism(s) remains largely unclear. To elucidate the mechanism(s), we assessed the specificity of the cytoprotection afforded by neoechinulin A treatment using other cytocidal stressors and also clarified the resulting cellular alterations, focusing on the antioxidant and metabolic enzymes systems. Neoechinulin A treatment for more than 12 h endowed PC12 cells with significant resistance to transient NO toxicity, but not persistent NO toxicity, bolus H 2 O 2 toxicity, or oxidative insult from the redox cycling quinone menadione. Cellular antioxidant system profiling revealed no substantial potentiation of the activity of any antioxidant enzyme in lysate from the neoechinulin A-treated cells excluding glutathione (GSH) content, which was significantly decreased (>50%), resulting in a proportional compromise in the thiol-reducing activity of the intact cells. In addition, no differences were observed in the activity for any nicotinamide adenine dinucleotide (phosphate) reduced form (NAD(P)H)-generating enzyme, steady-state NAD(P)H/nicotinamide adenine dinucleotide (phosphate) oxidized form (NAD(P) ) ratios, or the levels of total NAD(P)H. Nevertheless, the neoechinulin A-treated intact cells exhibited increased NAD(P)H redox turnover when driven by extracellular tetrazolium. The structurally inactive analog preechinulin failed to protect cells against NO toxicity or induce these alterations, suggesting their link with the cytoprotective mechanism. These results suggest that neoechinulin A, despite disabling the GSH defense system, confers cytoprotection against nitrosative stresses by elevating the cellular reserve capacity for NAD(P)H generation, which could offset crippling of energy-supplying systems due to nitrosative stress.