Reinvestigation of tryptophan-dehydrobutyrine diketopiperazine structure

Reinvestigation of the title natural product’s structure ( 1 ) has shown the easy racemization of the Trp-stereocenter in some derivatives of cyclo-L -Trp-Gly. The here described synthesis of compound 2 , a regioisomer of 1 with the N-methyl group on the nitrogen adjacent to the ethylidene side-chain, permits us to conclude that structure 1 was correct


Introduction
Cytotoxic anticancer drugs elicit cellular stress responses which frequently produce a drugresistant phenotype.One important mechanism of resistance of tumor cells to chemotherapeutic agents is the overexpression of dimer proteins of the glutathione-S-transferase family (GSTs). 1 Thus, alkylating agents such as nitrogen mustards, cyclophosphamide, chlorambucil or melphalan, and other electrophile antitumor drugs such as busulfan or cis-platin, alter the expression of a number of genes that control glutathione and its associated enzymes, and many cancers show different distributions of GST isozymes compared to those seen in normal tissues.This knowledge suggests that selective GST inhibitors could provide potentiation of these chemotherapeutic agents. 2 The main role of GSTs is to promote the neutralization of electrophilic toxins by the intracellular tripeptide glutathione (γ-glutamylcysteinylglycine), 3 but these enzymes are also catalysts of eicosanoid biosynthesis, a mechanism that may also be important in anticancer chemotherapy owing to the involvement of those compounds in the control of cellular division. 4In summary, inhibitors of glutathione synthesis (such as Lbuthionine sulfoximine 5 ) and GTS-inhibitors, 6 may help to prevent resistance to some antitumor The chemical shifts of the vinylic protons in the Zand Eisomers of 1 thus obtained, as well as the optical activity of the Zisomer, supported the (6-S, Z)configuration for this compound, but we found slight discrepancies between its melting point and some of its NMR and mass spectra data from those described for the natural product (Table 1).
From these findings, we propose that the correct structure of TDD could be 2 instead of 1.The synthesis of compound 2, a regioisomer of 1 with the N-methyl group on the nitrogen adjacent to the ethylidene side-chain, is described here.

Results and Discussion
The synthesis of compound 2 seemed, in principle, to be a simple problem.We knew that activation of cyclo-L-Trp-Gly (3, Scheme 2) by N-acetylation with acetic anhydride gave the N 1 ,N 4 ,N'-triacetylindolylmethylpiperazine-2,5-dione (4) and that this compound, through an aldol-type reaction with acetaldehyde in DMF and alumina-supported potassium fluoride as a base, 13 afforded the ethylidene derivative 7.This compound was supposed to be enantiomerically pure and seemed a good precursor of 2 by N-methylation and subsequent deacetylation.However, when we looked to this reaction sequence in depth we began to suspect that, given the low optical rotation values of compounds 4 and 7, 9b the N-acetylation and the subsequent condensation of 4 were not stereocontrolled as we had assumed.In fact, when the time for acetylation of 3 was increased to 12 hours, the compound 4 obtained was completely racemized.
To avoid the epimerization of the Trp-stereocenter, we decided to apply a procedure that we had developed previously for other synthetic objetives. 14 We next attempted the acetylation of the indole NH group of 6, with acetyl chloride under different basic conditions, including phase-transfer catalysis, but with very disappointing results.Finally, we obtained the triacetyl derivative (+)-4, that showed an optical rotation [α] D 21 = +20 (c = 0.3, CHCl 3 ), by treatment of 6 with acetic anhydride.The enantiomeric purity of this product was corroborated by chiral chromatography on a Constametric 4100 system equipped with a Chiralcel OD chiral column, a UV-vis detector, hexane/iPrOH/Et 2 NH (9: 0.9: 0.1) as mobile phase, and the racemic mixture (+)-4/(-)-4 as a reference (see Experimental).
The different stereochemical outcome of the acetylation of compounds (+)-3 to give (±)-4 and (+)-6 to give (+)-4 can be explained by assuming that the presence of an N 1 -acetyl group in compound (+)-4 hampers the epimerization of the Trp-stereocenter by steric interference in intermediate A, while in the case of the acetylation of compound (+)-3, racemization may take place on intermediate B (Figure 1).In agreement with this hypothesis, we observed that the tryptophan stereocenter of (+)-4 did not withstand the basic conditions that were required for its aldol-type condensation with acetaldehyde and, because this reaction proceeds with the anchimeric assistance of the vicinal Nacetyl group leading to its final elimination, compound 7 was obtained as a racemized product (Scheme 2). 15After considering the difficulties found in the preparation of homochiral (+)-7 and bearing in mind the fact that, with the exception of optical rotation, comparison of physical data of compounds 1 and 2 does not require enantiomeric purity, we undertook the preparation of (±)-4 by an alternative route, starting from non-chiral starting materials, in order to facilitate scale-up in its transformation into racemic compounds 7, 10 and 2 (Scheme 3).Compound (±)-4 was now obtained in 75% overall yield by reduction of 8, through a four-step procedure that started with indole-3-carbaldehyde and N 1 ,N 4 -diacetylpiperazine-2,5-dione (Scheme 3).11b N-Methylation of compound 7, obtained by a second aldol-type condensation of 4 with acetaldehyde, required the total absence of water in order to avoid decomposition products.Fortunately, when anhydrous DMF and carefully dried sodium carbonate were used, compound 10 was obtained in very good yield.The chemical shift of it's α-ethylidene proton (δ = 5.70 ppm), as well as NOE experiments, confirmed the Z-configuration of its exocyclic double bond and, through a final deacetylation performed with hydrazine hydrate, we obtained the desired product (±)-2 with traces of its Eisomer.The significant differences observed by comparing data of compound 2 (Table 2) with those reported for TDD and the synthetic product 1 (Table 1) permit us to conclude here that, in spite of the previously reported discrepancies, 9b structure 1 has to be the correct one for the natural product.

Scheme 2 .
Scheme 2. Reinvestigation of the fate of the Trp-stereocenter in the N-acetylation/aldol-type condensation of cyclo-L-Trp-Gly.

Table 1 .
Data reported for compound 1 in references 9a and 9b

Table 2 .
Data for compound 2 CDCl 3 as solvent in both cases.
a Recrystallized from acetone-cyclohexane. b