Synthesis and Characterization of N-Nitroso-3-morpholinosydnonimine as NO Radical Donor

Abstract

N-Nitroso-3-morpholinosydnonimine 3 was prepared by nitrosation of SIN-1 and characterized by ¹H-NMR, ¹³C-NMR, and HRMS. Its structure, confirmed by single crystal X-ray diffraction analysis, was found to be in agreement with its mesoionic and aromatic character. Unlike 3-morpholino-sydnonimine (SIN-1), which releases both nitric oxide and superoxide radical, decomposition of this nitrosylated sydonimine could yield nitric oxide as the only decomposition product, and thus without the formation of toxic peroxynitrite.

1. Introduction

Sydnones are mesoionic dipolar heterocyclic compounds with a 1,2,3-oxadiazole ring, which satisfy valence rules only if two atoms are assumed to carry formal, opposite charges delocalized across the ring [1,2,3]. They constitute an important class of molecules that possess interesting chemical and biological properties, and, in recent years, sydnones have been used for their capacity to undergo [3+2] cycloaddition reactions with terminal- and cycloalkynes [4,5]. Sydnonimines (or sydnone imines), such as 3-morpholino-sydnonimine (SIN-1, 1, Figure 1), are a class of sydnone compounds that generate simultaneously both nitric oxide radical (NO^.) and superoxide radical (O₂^.−) in neutral oxygenated aqueous media [6]. As nitric oxide and superoxide radicals recombine in a near diffusion-controlled reaction to give the potent oxidant peroxynitrite anion (ONOO⁻), known to decompose into highly reactive species such as carbonate, nitrogen dioxide, or hydroxyl radicals reacting with a number of biological targets [7], sydnonimines are considered peroxynitrite-releasing molecules. In some cases, SIN-1 has been shown to be converted from a peroxynitrite donor in aerobic solutions to a pure NO^. donor in vivo without concomitant superoxide production, probably by a mechanism involving one-electron oxidation of SIN-1 by heme proteins or other electron acceptors in biological systems instead of molecular oxygen [8].

Sydnonimines are known to possess various pharmacological activities related to NO^. release [9] and are an alternative to organic nitrates in the treatment of cardiovascular diseases, the use of which is often limited by the development of nitrate tolerance [10]. The release of nitric oxide from sydnonimines proceeds either spontaneously or after enzymatic conversion and subsequent decarboxylation to SIN-1, as in the case of Molsidomine (N-ethoxycarbonyl-3-morpholino-sydnonimine 2, Figure 1) [11]. Here, we present the synthesis, the characterization, and the X-ray structure of an N-nitrosylated sydnonimine, N-nitroso-3-morpholinosydnonimine (compound 3,Figure 1), whose decomposition could yield nitric oxide, and not superoxide, as decomposition product, without the formation of toxic peroxynitrite.

2. Results and Discussion

N-Morpholinosydnonimine hydrochloride (SIN-1.HCl) was synthesized as previously described from N-aminomorpholine and sodium formaldehyde bisulfite as starting materials [12]. The imine intermediate was treated with potassium cyanide to give the corresponding nitrile compound, which was then nitrosated to yield nitrosohydrazine. Cyclization of the latter under acidic conditions gave SIN-1 hydrochloride with a yield of 50%. N-Nitroso-3-morpholinosydnonimine 3 was then obtained by a simple procedure by treatment of SIN-1 hydrochloride with sodium nitrite in water [13] (Scheme 1).

Crystallization from methanol gave suitable crystals for X-ray analysis and all other spectral characterizations. Compound 3 crystallizes in the monoclinic space group P2₁/n with one molecule in the asymmetric unit (Figure 2 and ESI for crystallographic data). The overall structure and the planar geometry of the oxadiazole ring atoms, as expected, are close to those for some already reported crystallographic structures of sydnone and sydnonimine [14,15,16,17,18,19] and in agreement with the aromaticity character of the mesoionic ring. The morpholine ring is in a typical chair conformation, and the plane defined by the four carbons of this ring forms an angle of 20.41° with that of oxadiazole. The exocyclic N-nitroso -N-N=O group does not align with the oxadiazole ring but is slightly offset at an angle of 11.01°. As shown in Figure 1, the bond between oxazole-C5 and the exocyclic nitrogen atom of the N-nitroso group is written as a double bond, but the X-ray structure reveals a distance of 1.353 Å between these two atoms, i.e., a distance that corresponds more to that of a single bond between an aromatic carbon and nitrogen (C_sp²-N as found in aniline, for example) than to a double bond like those found in imines, for example (1.279 Å). The overall 3D packing of compound 3 does not display intermolecular hydrogen bonds but shows an ordered short contact network within the packing of molecules in the unit cell, in particular between oxygen atoms of the nitroso group and morpholine protons (2.469, 2.474, and 2.480 Å) and the oxazole proton (2.328 Å). The ¹H-NMR spectrum of 3 displayed two multiplets at 3.69 and 3.87 ppm attributed to the four morpholine protons and a characteristic deshielded signal (singlet at 9.07 ppm) corresponding to the proton of the sydnone ring. Two signals at 53.2 and 64.7 ppm corresponding to the four morpholine carbons were observed in the ¹³C-NMR spectrum, as well as two other very weak and deshielded signals at 103.6 and 129.6 ppm, corresponding to the tertiary (-N-C-H) and quaternary (-O-C-N-) carbons of the mesoionic sydnone ring, respectively. These high chemical shift values are in agreement with the delocalization of the negative charge within the 1,2,3-oxadiazole ring system and in accordance with chemical shifts of analogous sydnones.

The UV absorption spectrum of compound 3 in water showed two maximum absorptions at 320 and 251 nm. It remained stable in water, as shown by the UV spectrum recorded after several hours, but was shown to decompose in a first-order manner in basic aqueous solution (NaOH) with half-time of 90 min and 16 min in 2.5 mM and 12.5 mM NaOH solution, respectively (k_OH⁻ = 3.5 M⁻¹.min⁻¹) (Figure 3).

The HRMS spectrum exhibited the [M + H⁺]−ion at m/z 200.0796, corresponding to 3-H⁺ of molecular formula C₆H₁₀N₅O₃. However, the main peak in the mass spectrum was at m/z 140.0824, corresponding to the formula C₆H₁₀N₃O, i.e., that of protonated SIN-1C [20], the known product of SIN-1 decomposition. Indeed, it has been shown that SIN-1 first spontaneously decomposes through a ring opening into intermediate SIN-1A, which, in the presence of molecular oxygen, fragments to yield SIN-1C, nitric oxide, and superoxide as final products (Scheme 2A). It is, therefore, reasonable to assume that SIN1-C, a product of the decomposition of SIN-1 and also a product of the decomposition of 3 under the conditions of mass spectrometry analysis, could also be the final product of the decomposition of the latter in aqueous media. On these bases, a postulated mechanism for the decomposition of sydnonimine 3 is proposed in Scheme 2B: a tautomeric form of 3 undergoes a ring opening to yield a N, N’-dinitroso intermediate (3a), which then releases through homolytic cleavage two NO^. molecules and SIN-C. In that case, compound 3 would thus generate nitric oxide as the sole radical product and not superoxide, as in the case of SIN-1 decomposition.

3. Materials and Methods

All reagents and solvents were purchased from commercial sources (Sigma Aldrich, Saint-Louis, MO, USA or Alfa Aesar, Haverhill, MA, USA) and were used without further purification. ¹H-NMR and ¹³C-NMR spectra were recorded on Bruker Advance 300 and 500 spectrometers, respectively, and the residual proton signals of deuterated DMSO were used as an internal reference (δ = 2.5 ppm and 39.52 ppm, respectively). Proton coupling patterns are abbreviated as “s” for singlet and “m” for multiplet. The high-resolution mass spectrum (HRMS) was recorded on a MAT 95XL spectrometer (ThermoFisher, Waltham, MA, USA). Melting temperature was recorded on an MP50 Melting Point System (Mettler Toledo, Columbus, OH, USA). SIN-1 hydrochloride was synthesized from N-aminomorpholine and sodium formaldehyde bisulfite, as previously described [12]. It should be noted that potential health hazards may exist with the use of sodium nitrite.

N-Nitroso-3-morpholinosydnonimine 3—To an ice-cooled solution of SIN-1 hydrochloride (0.500 g, 2.42 mmol) in water (2.5 mL) was added a solution of sodium nitrite (0.200 g, 2.89 mmol) in water (7 mL), and the mixture was stirred for 6 hours at 0 °C and left at room temperature overnight. The solid formed was isolated by filtration on fritted glass and washed with cold methanol to give 0.350 g (1.76 mmol) of a yellow solid (yield 73%). Recrystallization from methanol gave pure yellow crystals of compound 3 suitable for X-ray crystallography. m.p. 130 °C. HRMS calculated for C₆H₁₀N₅O₃ using methane desorption chemical ionization in positive ion mode (DCI-CH₄, M+H⁺): 200.0784. Found: 200.0796. ¹H-NMR (500 MHz, DMSO-d₆): δ ppm 3.69 (m, 4H), 3.88 (m, 4H), 9.07 (s, 1H). ¹³C-NMR (125.8 MHz, DMSO-D₆): δ ppm 53.2, 64.7, 103.6, 129.6. See Supplementary Materials for more details.