Nitrous oxide as a primary product in base-mediated β-elimination reactions of diazeniumdiolated benzylamine derivatives

Abstract

We report an unexpected β-elimination pathway by which diazeniumdiolated benzylamines of structure Bn–N(R)–N(O)= N–OR^′ undergo base-mediated fragmentation to generate N₂O as the only gaseous product. The reaction is especially rapid for R = 2-hydroxyethyl, in which the hydroxyl group anchimerically assists benzylic proton removal with concomitant expulsion of PhCH=NR and R′OH.

O²-Derivatized secondary amine diazeniumdiolate prodrugs have emerged as efficient and reliable sources of the bio-effector molecule nitric oxide (NO).¹ Removal of the O²-protecting groups by hydrolysis or appropriate metabolic triggers furnishes the corresponding diazeniumdiolate anion.² This species (1, see Fig. 1) under physiological conditions decomposes to yield up to two moles of NO and the nucleophilic secondary amine by initial protonation of the amine nitrogen (Fig. 1).³ Recently, primary amine diazeniumdiolate IPA/NO⁴ was reported to release HNO, which then dimerizes to produce N₂O, at both neutral and alkaline pH. However, N-bound diazeniumdiolates have not been found to release N₂O as a direct decomposition product, although some C-bound diazeniumdiolates are known to furnish only N₂O at neutral pH.⁵ Our laboratory has been involved in diazeniumdiolate-based drug discovery focused towards effective site-directed delivery of NO for therapeutic applications.⁶ Recently, we began a study of diazeniumdiolated benzylamines as potential NO prodrugs containing aromatic chromophores and have probed into their structure–property relationships. In this effort to develop new N-diazeniumdiolates as efficient sources of biomedically useful NO, we have unexpectedly encountered a pathway by which certain members of this family fragment directly to N₂O without the formation of HNO as an initial step. Herein, we report on the base-mediated β-elimination reaction of certain secondary benzylamine diazeniumdiolates.

Fig. 1.

Decomposition mechanism of secondary amine diazeniumdiolates.

Reaction of 2-benzylaminoethanol (2) with NO in the presence of sodium methoxide afforded the corresponding N-diazeniumdiolated sodium salt (3). Under physiological conditions, in the presence of 7.4 pH phosphate buffer at 37 °C, this sodium salt releases about 1.6 mole of NO per mole of the compound (t_1/2 = 8.9 min) and can be considered as a new addition to the arsenal of NO-releasing diazeniumdiolates.⁶ O²-Alkylation of diazeniumdiolate salts renders enhanced stability in acidic and alkaline media⁷ and accordingly the corresponding methylated derivative (4) was prepared by the treatment of 3 with dimethyl sulfate (Scheme 1). This analog, barely soluble in aqueous media, was found to be fairly stable under acidic conditions and showed no signs of decomposition in a solution of 0.5 M HCl in ether at room temperature for over 12 h. However, a surprisingly facile chemical transformation of 4 was observed in the presence of base.

Scheme 1.

Preparation of 4.

Treating a solution of 4 in THF with Li^tBuO at 50 °C was marked with a rapid disappearance of the starting material. Under optimized conditions, the reaction completed in 1.5 h and isolation of the product indicated the formation of Schiff base 5 in 84% isolated yield, whose identity was confirmed by comparing its NMR and HR-MS data with those for an authentic sample prepared as reported.⁸ An effort to determine the fate of the diazeniumdiolate nitrogen atoms in 4 using gas chromatographic analysis of this reaction proved to be futile due to the use of volatile THF as a solvent. However, a careful GC analysis of the reaction of 4 with Li^tBuO in DMF for 2 h at 50 °C revealed the release of 64 mol% of N₂O as a reaction product along with the isolation of 5 in 71% yield following an aqueous workup. ¹H NMR analysis of an aliquot of this reaction mixture also revealed the formation of methanol as a product. It is also to be noted that the potential fragmentation of 4 in the presence of weak bases was examined and was found to be extremely sluggish (Table 1, entries 3 and 4) in contrast to that of reactions with a strong base, such as Li^tBuO (Table 1, entries 1 and 2). These results strongly indicate that the treatment of 4 with a strong base, such as Li^tBuO, triggers in situ β-elimination of the protected diazeniumdiolate leading to the formation of the corresponding imine, N₂O and methanol in a near stoichiometric ratio. We postulate that the acid–base properties of the hydroxyl group in 4 play a pivotal role in this chemical transformation.

Table 1.

Base-mediated fragmentation of 4

Entrya	Base	Solvent	Rxn. time/h	Yieldb (%)
1	LitBuO	THF	1.5	84
2	LitBuO	DMF	2	71
3	LiOH	EtOH	72	—
4	Et3N	THF	24	<2

^aAll reactions were carried out at 50 °C.

^bIsolated yields.

To confirm this, we studied the effect of base on several other protected diazeniumdiolates with structures analogous to that of 4. Sodium salts of N-diazeniumdiolated N-benzylethylamine (6a) and N,N-dibenzylamine (6b) were prepared following the reported procedure⁹ and the corresponding O²-alkylated derivatives were synthesized. Reaction of 7a with Li^tBuO in THF was found to be extremely sluggish even after reflux for 15 h. ¹H NMR analysis of the crude reaction mixture revealed that the majority of the starting material was unreacted. However, formation of the corresponding imine 8a in only 6% estimated yield was determined by comparing the ¹H NMR spectrum of the crude reaction mixture with that of the authentic imine obtained commercially. Also, gas chromatographic examination of the reaction of 7a with Li^tBuO in DMF revealed the release of only 5% N₂O which is consistent with the estimated yield of 8a as determined by ¹H NMR. A similar outcome was also observed for the NMR and GC analyses of the base-mediated reaction of 7b, indicating the formation of imine 8b and N₂O in about 2% yield each. Analogs 7a and 7b, being devoid of any free N-hydroxyethyl functionality, proved to be much less susceptible towards base-induced fragmentation under identical conditions (Scheme 2).

Scheme 2.

Preparation and fragmentation of 7a and 7b.

Accordingly, 9, prepared by the treatment of 3 with iodomethane in the presence of 15-crown-5 ether, when reacted with Li^tBuO in THF at 50 °C, afforded only imine 10, albeit in trace quantity (Scheme 3). Identification of the product was achieved by comparing the NMR and HR-MS data of the crude reaction mixture with an authentic sample of 10.¹⁰ The anomaly between the reactivities of 4 and its above-mentioned analogs 7a, 7b, and 9 points towards a key role of the N-hydroxyethyl group in initiating a presumably concerted elimination by abstracting one of the benzylic protons in 4 via in situ formation of the potentially six-membered cyclic transition state (11) and leading to the facile formation of imine 5 (Fig. 2). Based on this tentative mechanism for the proposed β-elimination reaction, we presume that the base-mediated reactivity of the diazeniumdiolated benzylamines with longer N-hydroxyalkyl chains will be substantially sluggish due to the formation of potentially unfavorable cyclic transition states.

Scheme 3.

Preparation and reaction of 9 in base.

Fig. 2.

Mechanism for the formation of imine 5.

In summary, we have reported for the first time the preparation of a unique secondary benzylamine diazeniumdiolate, the O²-protected prodrugs of which can fragment directly to N₂O via a novel base-mediated β-elimination reaction triggered by the potential acidity of the benzylic protons. Even though these caged diazeniumdiolates have not been developed as potential N₂O donors in biological systems, this unexpected finding should be borne in mind when working with such prodrugs, especially diazeniumdiolated benzylamine derivatives and other diazeniumdiolates bearing hydroxyl groups with similarly situated acidic protons α to the amino nitrogen.

This project has been funded with Federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E, and by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.