The Preparation of a 10-Membered Ring Macrolactone by Selective Ozonolysis and the Role of the Dihydropyran-Substituent on the MCPBA-Oxidation Reaction Profile of β-Lapachone Phenazines

Raphael S. F. Silva, Tiago T. Guimarães, Daniel V. Teixeira, Ana Paula G. Lobato, Maria do Carmo F. R. Pinto, Carlos Alberto de Simone, Janaína G. Soares, Alessandra G. Cioletti, Marilia O. F. Goulart* and Antonio V. Pinto* a Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, 21944970 Rio de Janeiro – RJ, Brazil Departamento de Química/CCEN, Universidade Federal de Alagoas, Campus A. C. Simões, Tabuleiro do Martins, 57072-970 Maceió – AL, Brazil


Introduction
In general, natural and synthetic phenazines have attracted considerable attention because of their interesting biological activities, 1 including broad-spectrum antibiotic, 2 antimalarial, 3,4 trypanocidal 5 along with anti hepatitis C viral replication activities. 6Benzo[a]phenazines are also efficient DNA intercalating ligands, exhibiting antitumor activity in leukaemia and solid tumors. 7They have also been described as dual inhibitors of topoisomerase I and II. 8n our search for preparing new derivatives, for synthetic purposes and biological screening, we recently reported 9 that peroxidation of the b-lapachone-derived benzophenazine, named lapazine (1), with m-ClC 6 H 4 CO 2 H/CH 2 Cl 2 generated, unexpectedly, a macrocyclic lactone 2 with a rigid 10-membered ring (27% yield), the corresponding N-oxide 3 (13% yield), and the dihydrobenzophenazine-5-one 4 (35% yield) (Figure 1).Macrocyclic compounds 2 and 3 were formed via oxidative cleavage of the aromatic double bond at the site of fusion of the dihydropyran moiety with the phenazine component.This region (rings C/E, in 1) (Figure 1) would behave as a conjugated enol ether, which, after the expected epoxide formation and fragmentation would lead to 2 and 3 (further oxidation).An acid-catalysed epoxide-acetal or α-hydroxy hemiacetal would furnish 4. 9 Compounds 2 and 3 belong to the class of macrolides, an important group of biologically active compounds, especially recognized as antibiotics. 10Among 10-membered macrolactones, the antibiotic activity of apicularen A and B was reported. 11n the present work, considering 2 and 3, the presence of diaza heterocyclic groups close to the ion-binding macrolactone moiety constitutes attractive molecular features.
In searching for a more general application of the oxidative cleavage by MCPBA, 9 we have analysed the role of substituent, at C-2', in the dihydropyrane moiety of lapazine, namely -OH (5), -Br (6) and -I (7) (Figure 1).
Additionally, in an attempt to develop a more selective and simpler method by which to bring about this oxidative cleavage, 9 we have evaluated the use of ozone, a versatile reagent capable of oxidizing both cyclic and acyclic olefins. 12,13Since double bonds connected to electrondonating groups react with ozone many times faster than those connected to electron-withdrawing groups, 14 this gaseous reagent possesses significant selective reaction potential.Ozone has been used for the selective cleavage of double bonds in alicyclic compounds, a strategy that gave rise to the expanded C-15 macrocyclic system in the synthesis of muscone. 15Aromatic bonds can also be cleaved through ozonolysis as shown, for example, in the formation of biphenyl compounds from benzo[c]phenanthrene and phenanthrene. 16,17A number of studies have concerned the ozonolysis of heterocyclic molecules, [18][19][20][21][22] but none has focused on the reaction with phenazines.

Results and Discussion
As described before, 9 the oxidation with MCPBA caused the oxidative cleavage of the aromatic double bond at the site of fusion of the dihydropyran moiety with the phenazine component.To know the role of the substituent on the reaction profile, we have studied the phenazines (5-7) derived respectively from 3-hydroxyl, 3-bromo and 3iodo-β-lapachone.
The phenazine 5 reacted with MCPBA to produce the macrolide 8, as the sole product, in 52% yield, along with unchanged 5 (Figure 2).This hydroxymacrolide constitutes a versatile intermediate for the synthesis of new derivatives.
In the case of 6, a complex mixture of products was obtained, from which it was possible to isolate 9, as the corresponding N-oxide bromomacrolide, in low (10%) yield (Figure 2).Supplementary crystallographic data is given. 23he structure was solved by direct methods and refined by full-matrix least squares calculations (Figure 3).The refinement was conducted until all atomic parameters shifts were smaller than their standard deviations.All H atoms were located by geometric considerations.In the final difference Fourier map there are no peaks greater than 0.34 Å -3 .Bond lengths and angles are in good agreement, to within experimental accuracy, with the values found in the literature. 24he oxidation of compound 7 with MCPBA, due to the liability of the C-I bonding, was unsuccessful.
From these results, it is possible to envisage that the method would be valuable for the synthesis of substituted macrolides, specially for substituents, which are not easily broken in the oxidation conditions.
Concerning oxidation of phenazines, a natural development would be to try ozonolysis.A solution of lapazine (1) in methylene dichloride was, then, subjected to ozonolysis, and the ozonides thus formed were reduced with Zn/water. 25The progress of the reaction was followed by TLC using pure samples of 2, 3 and 4 as reference standards.The macrocyclic lactone 2 was formed as the major product of the reaction, together with some minor polar by-products that were not identified.Neither 3 nor 4 could be detected in the crude reaction mixture by TLC analysis.Compound 2 was isolated from the reaction mixture by column chromatography as pure white crystals in an overall yield of 52% (Figure 2).In addition to providing an easier protocol and cleaner conditions for the preparation and isolation of 2, the reaction of 1 with ozone was also more selective and efficient than the reported direct formation of 2. 9 To the best of our knowledge, this is the first example of the ozone-mediated formation of a rigid macrocyclic lactone.Although a more general view of the synthetic aspects of the use of ozone is not addressed in the present communication, the formation of 2 suggests the possible use of this reagent to cleave phenanthrene rings linked face-to-face with a heterocyclic moiety.Experiments along this line are in progress.

Figure 3 .
Figure 3. Perspective view of 9 showing the atom-labelling scheme.Displacement of ellipsoids plotted at the 50% probability level, are shown for the non-H atoms.