Synthesis and Neuroprotective Action of Optically Pure Neoechinulin A and Its Analogs

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.


Introduction
Diketopiperazines have various therapeutically important biological properties such as antitumor, antiviral, antifungal, and antihyperglycaemic activities [1]. Neoechinulin A (Figure 1), an indole alkaloid containing diketopiperazine, has been isolated from marine fungi including Aspergillus sp., and is also reported to have antitumor activity [2]. We found that neoechinulin A (1) displays cytoprotective activity in neuron-like PC12 cells against oxidative insults induced by peroxynitrite generated from 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1) [3]. Subsequently we examined the structure-activity relationship of neoechinulin A analogs with anti-nitration activity, anti-oxidant activity and cytoprotective activity against peroxynitrite from SIN-1 in PC12 cells [4,5]. As a result of these studies the structural characteristics required for cytoprotection were determined. Recently, we found that 1 could also protect PC12 cells from cytotoxicity of 1-methyl-4-phenylpyridinium (MPP + ), a neurotoxin capable of provoking acute Parkinson-like neurodegeneration in humans. This observation raises the possibility that neoechinulin A or its analogs may have therapeutic utility for the treatment of neurodegenerative disorders [6].
We have previously achieved the total synthesis of 1 with high enantiomeric excess and determined its absolute configuration [7,8]. The key step in the synthesis is the intramolecular cyclization of the ΔTrp-L-Ala derivative (2) to construct the diketopiperazine ring. However, during the synthesis of optically pure neoechinulin A, the stereogenic center on the diketopiperazine moiety was subject to epimerization. In this paper, we report efficient, stereoselective synthetic methods for the diketopiperazine moiety of neoechinulin A and its derivatives.

Preparation of neoechinulin A and its analogs via intramolecular cyclization
We prepared optically active neoechinulin A from the cyclization precursor 2. The Alloc group of (+)-3 was removed by treatment with NaBH 4 and a catalytic amount of Pd(PPh 3 ) 4 in THF to afford amine 2 [7,8]. We then investigated the thermally induced formation of diketopiperazine (Table 1).
When the cyclization was performed in toluene at 110 ºC for 1 day, compound 4 was obtained in 83% yield as a sole product (entry 2). We also prepared 7, a diastereomer of preechinulin, from D-Trp-L-Ala derivative 8 by heating at 80 ºC (Scheme 2) [5]. In this case, 7 was also obtained as a single isomer.
We found that the stereogenic center on the diketopiperazine moiety of 1 was partially racemized at high temperature. The partial racemization of 1 proceeded much faster than that of 8,9dihydroneoechinulin A derivatives (4 and 7). These results suggest that the acidity of the α-proton of the alanine moiety in 1 might be greater than that in 4 or 7.

Biological and chemical properties of optical isomers of neoechinulin A and preechinulin
Our previous studies demonstrated that neoechinulin A treatment affords cytoprotection against oxidative/nitrosative insults imposed by the O 2 -/NO donor SIN-1 to neuronal (-like) cells, such as nerve growth factor (NGF)-differentiated PC12 cells and rat primary brain neurons, but not to nonneuronal cells, such as undifferentiated PC12 cells and fibroblasts [2]. In addition, the cytoprotection requires at least a 12-hr pretreatment of cells with neoechinulin A before challenging with SIN-1, suggesting that the cytoprotection conferred by neoechinulin A probably depends on induction of certain cytoprotective genes [3,4]. Here we summarize that cytoprotective activity of those compounds against cytotoxicity induced by SIN-1 in NGF-differentiated PC12 cells (Table 3). Treatment of PC12 cells with (-)-1 or (+)-1 for 24 hr gave almost identical levels of protection against cytotoxicity when subsequently challenged with SIN-1 [4]. In contrast, neither 4 nor 7 showed any cytoprotection against SIN-1 [4]. These results suggest that the orientation of the methyl group at C-12 or the isoprenylated indole group at C-9 does not contribute to cytoprotection; rather the presence of the C-8/C-9 double bond is essential for the cytoprotective action of neoechinulin A [4,5].
Additionally, both (-)-1 and (+)-1 also potently inhibited nitrotyrosine formation in BSA induced by the addition of SIN-1 to a cell-free system. By contrast, neither 4 or 7 displayed any inhibition on nitrotyrosine formation under otherwise identical conditions [4,5]. Although inhibition of nitrotyrosine formation is not essential for cytoprotection against SIN-1, the C-8/C-9 double bond could play a pivotal role in inhibition of both SIN-1-induced tyrosine nitration and SIN-1-induced PC12 cell death irrespective of the orientation of the methyl group at C-12 [4].

Conclusions
We prepared (-) and (+)-neoechinulin A (1) with high enantiomeric excess by the intramolecular cyclization of 2 and its enantiomer, respectively at 80 °C. The thermal cyclization at 110 °C caused partial epimerization of the stereogenic center at C-12 in 1. By contrast, 8,9-dihydroneoechinulin A derivatives 4 and 7 were obtained in optically pure forms by a similar cyclization at 110 °C. These results suggest that the acidity of the α-proton of the L-Ala moiety in 1 might be greater than that in either 4 or 7. We found that both (-) and (+)-1 protected the cells against cytotoxicity to an almost identical extent following exposure to SIN-1. In contrast, neither 4 nor 7 showed any cytoprotection against SIN-1. These results indicate that the C-8/C-9 double bond could play a pivotal role in inhibition of both SIN-1-induced tyrosine nitration and SIN-1-induced PC12 cell death irrespective of the orientation of the methyl group at C-12.