Synthesis and Biological Activities of Neoechinulin A Derivatives: New Aspects of Structure-Activity Relationships for Neoechinulin A

Abstract

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.

Neoechinulin A Impedes the Progression of Rotenone-Induced Cytotoxicity in PC12 Cells

“…1). 14) As shown in Fig. 3A, the presence of neoechinulin A (100 mM) during rotenone exposure dramatically diminished cytotoxicity; at 21 h after treatment while LDH leakage exceeded 70% in rotenone treated cells, it remained below 25% in cells co-treated with neoechinulin A.…”

“…Thus, the requirement of the simultaneous presence of the alkaloid with the neurotoxin for cytoprotection is consistent with the case of MPP ϩ -induced cytotoxicity. 16) These results suggest that antioxidant activity 14) and/or interaction of an unknown cellular target molecule with neoechinulin A plays a role in the cytoprotective action of neoechinulin A against rotenone.…”

“…13,14) Previously, we found that neoechinulin A can protect neuronal PC12 cells from cytotoxicity due to oxidative/nitrosative stress induced by the superoxide/nitric oxide co-generator 3-morpholinosydnonimine (SIN-1). [13][14][15] Subsequently, we found that neoechinulin A can also protect PC12 cells from MPP ϩ by ameliorating downstream lethal events resulting from mitochondrial complex I inactivation.16) Although the cytoprotective mechanism of neoechinulin A remains unclear, it is probably not a single mechanism; while cytoprotection against SIN-1 can be afforded by pretreatment of cells with neoechinulin A at least for 12 h before SIN-1 challenge, 13) protection against MPP ϩ toxicity does not require pretreatment but its co-presence during the neurotoxin treatment.16)The present study was aimed at clarifying the cytoprotective mechanism of neoechinulin A in mitochondrial dysfunction-induced cell death. By using rotenone, we demonstrate here that neoechinulin A can also protect PC12 cells from rotenone cytotoxicity while paradoxically lowering cellular ATP levels.…”

“…1) is an indole alkaloid isolated from marine fungi. 13,14) Previously, we found that neoechinulin A can protect neuronal PC12 cells from cytotoxicity due to oxidative/nitrosative stress induced by the superoxide/nitric oxide co-generator 3-morpholinosydnonimine (SIN-1). [13][14][15] Subsequently, we found that neoechinulin A can also protect PC12 cells from MPP ϩ by ameliorating downstream lethal events resulting from mitochondrial complex I inactivation.…”

“…Neoechinulin A and preechinulin (8,9-dihydroneoechinulin A) were synthesized as described previously. 14) PC12 cells, a rat pheochromocytoma cell line, were routinely maintained in DMEM containing 5% horse serum, 10% fetal calf serum (FCS), glutamine (4 mM), penicillin G (100 U/ml), and streptomycin (100 mg/ml) in a humidified atmosphere of 5% CO 2 at 37°C. For experiments, PC12 cells were seeded in poly-D-lysine-coated 96-well plates (BD Bioscience, Sun Jose, CA, U.S.A.) at a density of 5000 cells per well.…”