Effects of Benzofuroxan Substituents in the Outcome of their Expansion Reaction with Phenolates

It is well known that phenolate anions react with benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans) to afford phenazine N,N-dioxide derivatives. These are obtained through an heterocycle expansion process by reaction of the corresponding 1,2,5-oxadiazole system with the phenolate carbanion generated in basic medium attacking one of the heterocyclic nitrogen (Figure 1). When substituted benzofuroxans and phenolates are used, the reactions yield a mixture of 6and 9or 7and 8-isomers.


Introduction
It is well known that phenolate anions react with benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans) to afford phenazine N 5 ,N 10 -dioxide derivatives. 1,2These are obtained through an heterocycle expansion process by reaction of the corresponding 1,2,5-oxadiazole system with the phenolate carbanion generated in basic medium attacking one of the heterocyclic nitrogen (Figure 1).[5] The formation of the isomers is the result of the well known tautomerism of the benzofuroxans 6,7 (i.e. Figure 2), thus the tautomeric forms can react with the carbanion in a non-selective fashion and with similar probability.However, each tautomeric form has different stability and electrophilic capacity.
Recently, the synthetic procedure shown in Figure 1 was used to develop a series of phenazine N 5 ,N 10 -dioxide derivatives as new hypoxic selective cytotoxic agents. 8In this synthetic approach, benzofuroxans with different 5-substituents, electron withdrawing and electron releasing moieties, were employed as the starting material.A mixture of the corresponding 7-and 8-substituted-2-aminophenazine N 5 ,N 10 -dioxide was obtained when p-aminophenol was used as the phenolate source and a mixture of 7-and 8-substituted-2-hydroxyphenazine N 5 ,N 10 -dioxide was obtained when p-hydroquinone was used (Table 1).It was observed that the proportion of isomers was highly dependent on the substituents electronic characteristics.
The existence of benzofuroxan as a mixture of isomers at room temperature (i.e.4][25] In our knowledge, studies related to benzofuroxan tautomerism and reactivity have not been described.On the other hand, in the preparation of phenazine N 5 ,N 10 -dioxide derivatives from benzofuroxan, [1][2][3][4][5] the effect of the benzofuroxan substituent on the isomeric proportion of the products has not been studied. 26erein, we present our results on the benzofuroxan tautomerism on the 1,2,5-oxadiazole-expansion process to obtain phenazine N 5 ,N 10 -dioxide derivatives.By analysis of the reaction mixture products of 5(6)-nitrobenzofuroxan, it has also been demonstrated that the 1,2,5oxadiazole heterocycle (tautomer A or C, Figure 2) is the electrophilic species in this process.Theoretical calculation, using the density functional theory (DFT) approach, were performed in order to determine the electrophilic center in each tautomer.These were in agreement with the experimental results.

Results and Discussion
The expansion process was performed, as previously reported, 5 with sodium methoxide as the base, the mixture of the benzofuroxan derivative [27][28][29][30] and the corresponding phenol in MeOH:THF as the solvent at low temperature.Under these conditions, the products were obtained in moderate yield, except for the highly reactive 5(6)-nitrobenzofuroxan, which produced a complex mixture of products and a low yield of the desired heterocycle (Table 1).
Although, it could be assumed that the carbanion phenolate reacted non regioselectively with each tautomeric form, clear isomeric preferences were observed according to the stability of the tautomers A and C at the reaction temperature.Under the reaction conditions, the isomeric mixture of phenazine N 5 ,N 10 -dioxides precipitated as a mixture which could not be separated neither by crystallization nor by chromatographic methods.The isomeric proportion, was established by 1 H NMR analysis of the precipitates (Table 2) analyzing the integrations of 8-H, in 7-substituted phenazines, and 7-H, in 8-substituted phenazines, for each isomer.As shown in Figure 3, 7-H and 8-H in the 8-substituted isomers result hardly affected, by 2-NH 2 or 2-OH electron releasing moieties.This fact together with the chemical shifts of 7-H and 8-H (Figure 3b) allowed to analyze the isomeric proportion in derivatives 1-12.
Clearly, the isomeric ratios in the expansion processes were dependent on the benzofuroxan substituent electronic effect.When p-hydroquinone is used as the phenolate, the effects resulted clearly important, i.e. an electron releasing group as the methoxy and an electron withdrawing group as the nitro generated only one compound, the 7-and the 8-isomers, respectively.Therefore, the electronic effects of the substituent define, in the reaction medium, the position of the benzofuroxan tautomeric equilibria so they define the predominant electrophilic species.In these species the electrophilic atom is the nitrogen 1 in each tautomer (Figure 4a).These electrophilic preferences could be explained via a substituent mesomeric process (i.e. Figure 4b).Thus, electron releasing substituents decrease the electrophilic characteristic of the benzofuroxan nitrogen at position 3 in the favored 5-substituted tautomers.However, electron withdrawing substituents increase the electrophilic characteristic of benzofuroxan nitrogen at position 1 in the mesomerism-stabilized 6-substituted tautomers.2][33] In the amino-derivatives a better correlation was observed between σ p and the proportion of 7-isomer (r= -0.9032, s= 0.1514, p=0.0136) than for hydroxy-derivatives (r= -0.8254, s= 0.1990, p= 0.0431).Because "throughconjugation" could be operative in the expansion processes, the correlations between σ p and σ p + substituent constants and the proportion of the 7-isomer were analyzed. 34For the amine derivatives the substituent resonance-electron-withdrawing effects (expressed as σ p _ ) did not correlate adequately to the proportion of the 7isomers as did σ p constants (r= -0.8463, s= 0.3300, p=  0.0336).For the hydroxyl-derivatives, improvements in the statistical parameters were obtained in the correlation between σ p and the 7-isomer proportions (r= -0.8610, s= 0.3151, p= 0.0276).However, the 7-isomer proportions correlate better with the σ p + parameters than with the σ p (see Figure 5).This could be indicative that the substituent resonance-electron-donating effect is operative in the benzofuroxan expansion processes.
The participation of tautomer B (Figure 2) as the electrophilic species in the reactions was rejected according to the results obtained in the yield optimization studies of the reaction involving 5(6)-nitrobenzofuroxan.Attempts to improve the yield in the preparation of derivative 6 were done using Et 3 N as base under different conditions (Table 3).The use of Et 3 N at room temperature was optimal for the preparation of 6.In all cases a complex mixture of products was obtained.
Compound 13 and 4-amino-2-nitrophenol were also isolated, together with compound 6 (Figure 6).Compound 13 was the S N Ar product of the reaction between the hard electrophilic 6-carbon, due to the presence of the nitro moiety, and the phenolate.The 4-amino-2-nitrophenol could be regarded as the product of the nucleophilic attack of the carbanion at the nitrogen in position 1 in the cyclic tautomer producing intermediate (X) (Figure 6) followed by a fragmentation process.
In order to corroborate the electrophilic capacity of the nitrogen atoms of the three tautomers of 5(6)-substituted benzofuroxan (A, B, and C, Figure 2), atomic charges (as natural charges) on oxadiazole's nitrogen atoms were calculated for nitro and methoxy derivatives.The results, Figure 7, showed that the most positive nitrogen was nitrogen 1 on tautomer A for nitro derivative and nitrogen 1 on tautomer C for methoxy derivative, being nitroso nitrogens marginal in their electrophilic capacities (tautomers B).
In conclusion, the mesomeric effects of the 5-substituent of the benzofuroxan determine the 7:8 isomeric proportion of the isolated phenazine N 5 ,N 10dioxide when p-aminophenol or p-hydroquinone are used as the nucleophilic agents.

Experimental
General All starting materials were commercially available research-grade chemicals and were used without further purification.All solvents were dried and distilled prior to  6).

Figure 7 .
Figure 7. Atomic charges, as natural charge (in electron), theoretically calculated for the three tautomers of 5(6)-nitrobenzofuroxan and 5(6)methoxybenzofuroxan.The most positive nitrogen in each compound is marked.

Table 1 .
Yield and spectroscopic characteristics of precipitated phenazine N 5 ,N 10 -dioxide a Yield of benzofuroxans to phenazines transformations according to it is described in the text.The yields correspond to precipitated phenazine; b For the main isomer; in ppm; c In DMSO-d 6 ; d In CD 3 OD:D 2 O (1:1); e In CD 3 OD.f In DMSO-d 6 :D 2 O (1:1).

Table 2 .
Isomeric proportion of phenazine N 5 ,N 10 -dioxide derivatives and Hammett constants used in the correlations a Sigma inductive of CH(OEt) 2 ref.32; b Sigma zero resonance of CH(OEt) 2 ref.33.

Table 3 .
Conditions for the preparation of derivative 6 Characterized by NMR spectroscopy and X-ray crystallographic studies; b According to 1 H NMR and MS data (see Figure a