Regioselective Synthesis of NO-Donor (4-Nitro-1,2,3-triazolyl)furoxans via Eliminative Azide–Olefin Cycloaddition

A facile and efficient method for the regioselective [3 + 2] cycloaddition of 4-azidofuroxans to 1-dimethylamino-2-nitroethylene under p-TSA catalysis affording (4-nitro-1,2,3-triazolyl)furoxans was developed. This transformation is believed to proceed via eliminative azide–olefin cycloaddition resulting in its complete regioselectivity. The developed protocol has a broad substrate scope and enables a straightforward assembly of the 4-nitro-1,2,3-triazole motif. Moreover, synthesized (4-nitro-1,2,3-triazolyl)furoxans were found to be capable of NO release in a broad range of concentrations, thus providing a novel platform for future drug design and related biomedical applications of heterocyclic NO donors.


Introduction
Nitrogen heterocycles are the most frequently occurring structural motifs in various pharmaceuticals and promising drug candidates [1][2][3][4].According to the U.S. FDA database, >59% of clinically used small-molecule medicines incorporate a nitrogen heterocycle subunit [5,6].However, the construction of individual pharmaceutical scaffolds using known synthetic methodologies often involves multi-step and energy-consuming procedures or suffers from a lack of reproducibility and scalability.Therefore, the creation of novel step-economy protocols for the assembly of various nitrogen-containing heterocyclic scaffolds remains highly urgent [7][8][9].
[3+2] Cycloaddition of 4-amino-3-azidofurazan to 1-nitro-2-morpholinoethylene contributed to the formation of 4-nitro-1,2,3-triazole with a furazan moiety, which, however, is not capable of NO release (Scheme 1b) [50].In addition, this reaction proceeded under prolonged heating in an ionic liquid medium and suffered both from a narrow substrate scope and low yields.These examples clearly demonstrate that the fine tunability of the reactivity of 4-azidofuroxans may be achieved by introducing various additives to achieve both high regioselectivity and a broad scope.Herein, we report on a regioselective, p-TSA-catalyzed eliminative azide-olefin cycloaddition of 4-azidofuroxans to 1-dimethylamino-2-nitroethylene for the synthesis of (4-nitro-1,2,3-triazolyl)furoxans (Scheme 1c).In a series of nitrogen heterocycles, 1,2,3-triazoles have become of paramount importance over the past two decades due to their universal application in diverse fields, such as drug development and medicinal chemistry [37][38][39], materials science and polymers [40,41].In addition, they contribute to organic synthesis by acting as synthetic precursors to many valuable compounds [42][43][44].In this regard, nitro-1,2,3-triazoles acquire additional significance due to their potential application in energetic materials science and for medicinal chemistry needs [45][46][47].Taking into account the potency of the molecular hybridization tool in the development of new drug candidates, a method for the synthesis of previously unknown (nitro-1,2,3-triazolyl)furoxans is desired.

Results and Discussion
Recently, our research group created direct methods for the synthesis of various functionally substituted furoxans.It is also known that the nitro group in 4-nitrofuroxans is prone to nucleophilic displacement due to high electrophilicity of ring carbon atoms [51,52].In particular, a couple of azidofuroxans were recently prepared upon treatment of 4-nitrofuroxans with NaN3 [48].Using this furoxan reactivity pattern, we prepared a Scheme 1. Previously known approaches toward the formation of (1,2,3-triazolyl)-1,2,5-oxadiazoles from 4-azidofuroxans with: (a) acetylenes and 1,3-dicarbonyl compounds, (b) 1-morpholino-2nitroethylene and (c) newly developed protocol.

Results and Discussion
Recently, our research group created direct methods for the synthesis of various functionally substituted furoxans.It is also known that the nitro group in 4-nitrofuroxans is prone to nucleophilic displacement due to high electrophilicity of ring carbon atoms [51,52].In particular, a couple of azidofuroxans were recently prepared upon treatment of 4-nitrofuroxans with NaN 3 [48].Using this furoxan reactivity pattern, we prepared a series of starting 4-azidofuroxans 2a-v from the readily available nitro derivatives 1a-v in good and high yields (Scheme 2).4-Azido-3-(p-tolyl)furoxan (2a) was chosen as a model substrate to optimize the reaction conditions for the synthesis of target (4-nitro-1,2,3-triazolyl)furoxans 3a-v (Table 1).1-Dimethylamino-2-nitroethylene was used as a convenient dipolarophile in all reactions.Refluxing of substrate 2a with 1-dimethylamino-2-nitroethylene in various ratios and solvents afforded target 4-nitro-1,2,3-triazole 3a in low yields (up to 30%, entries 1-8).Interestingly, an addition of Lewis acids did not improve the reaction outcome but resulted in a yield decrease (entries 9-13).Utilization of mCPBA as an oxidizer to convert the dimethylamino group to the corresponding N-oxide was also inefficient (entry 14).To our delight, more fruitful results were obtained upon p-toluenesulfonic acid (p-TSA) catalysis (entries [15][16][17][18].The optimal amount of p-TSA was found to be 15 mol.% (entries 16-18).
Molecules 2023, 28, x FOR PEER REVIEW 6 of 19  All synthesized compounds were characterized by multinuclear ( 1 H, 13 C, 14 N) NMR spectroscopy, IR spectroscopy, high-resolution mass spectrometry and elemental analysis.The structures of 2a, 3a and 3c were additionally confirmed by X-ray diffraction study (Figure 2).
The molecule of 2a is nearly planar in crystal: the mean deviation from the meansquare plane composed by non-hydrogen atoms is only 0.032 Å.This conformation is expected owing to the combination of π-donor (4-methylphenyl) and π-acceptor (azidofuroxane) fragments in 2a.In its turn, the crystal packing of 2a is of a layer type (Figure S1 The formal replacement of the azido group with the nitrotriazole fragments in 3a and 3c results in the steric repulsion between substituted phenyl rings and triazole moieties.For instance, the C6-C5-C1-C2 and C1-C2-N3-N4 torsion angles in 3a equal 64.8(2) • and 40.0(2) • , respectively.It is interesting to note that the conjugation of the central furoxan fragment in 3a with its substituents can be considered as saturable: the C6-C5-C1-C2 and C1-C2-N3-N4 torsion angles are respectively equal to 38.8 • and 59.8 • in the equilibrium isolated molecule of 3a modelled at the PBE0-D3/def2TZVP level.In other words, the rotation of one substituent induced by crystal packing effects upon the formal gas-to-crystal transition is totally compensated by the rotation of the other one.
Nevertheless, despite this significant non-planarity of the molecules of 3a and 3c, the layer-type packing motifs are observed in both crystals (Figures S2 and S3).Since furoxans correspond to NO donors, we investigated the ability of the synthesized (4-nitro-1,2,3-triazolyl)furoxans 3 to release NO.The formation of the nitrite anion as a result of the oxidation of NO can be quantified in accordance with the Griess assay and thus can serve as a reliable tool for measuring the NO release.Synthesized (4-nitrotriazolyl)furoxans 3 were subjected to 1 h incubation in the presence of L-cystein under physiological conditions (pH 7.4; 37 • C), and the amount of NO 2 formed was measured using the spectrophotometric method.It was found that (4-nitrotriazolyl)furoxans containing aromatic or heteroaromatic substituents emit NO fluxes in a broad range of 8.5-72.4%.Interestingly, compounds bearing aliphatic substituents or electron-withdrawing moieties in the aromatic ring (3j, 3o, 3s) release smaller amounts of NO (8.5-12.1%).It is important to note that furoxanyltriazole 3p incorporating 3,4-dimethoxyphenyl group demonstrated the highest NO-donor ability (72.4%).Overall, these results might be helpful in the development of novel NO-donor drug candidates with various pharmacological activities (Figure 3).
(4-nitrotriazolyl)furoxans containing aromatic or heteroaromatic substituents emit NO fluxes in a broad range of 8.5-72.4%.Interestingly, compounds bearing aliphatic substituents or electron-withdrawing moieties in the aromatic ring (3j, 3o, 3s) release smaller amounts of NO (8.5-12.1%).It is important to note that furoxanyltriazole 3p incorporating 3,4-dimethoxyphenyl group demonstrated the highest NO-donor ability (72.4%).Overall, these results might be helpful in the development of novel NO-donor drug candidates with various pharmacological activities (Figure 3).

Conclusions
In summary, we developed a convenient and straightforward approach to an assembly of (4-nitro-1,2,3-triazolyl)furoxans based on eliminative azide-olefin cycloaddition of 4-azidofuroxans and 1-dimethylamino-2-nitroethylene under p-TSA catalysis.The reported method has a number of advantages including a broad substrate scope and complete regioselectivity resulting in the construction of the 4-nitro-1,2,3-triazole motif.Synthesized (4-nitro-1,2,3-triazolyl)furoxans were found to be capable of NO release in a broad range of concentrations under physiological conditions.Therefore, our results contribute to the enlargement of the available libraries of NO-donor substances and unveil novel opportunities in drug design and related biomedical applications.

General Methods
CAUTION!Although we have encountered no difficulties during preparation and handling of azides 1a-w described in this paper, they are potentially explosive and may be sensitive to impact and friction.Mechanical actions of these species, involving scratching or scraping, must be avoided.Any manipulations must be carried out by using appropriate standard safety precautions.3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q 3r 3u 3t 3u 3v mol.% NO

Conclusions
In summary, we developed a convenient and straightforward approach to an assembly of (4-nitro-1,2,3-triazolyl)furoxans based on eliminative azide-olefin cycloaddition of 4-azidofuroxans and 1-dimethylamino-2-nitroethylene under p-TSA catalysis.The reported method has a number of advantages including a broad substrate scope and complete regioselectivity resulting in the construction of the 4-nitro-1,2,3-triazole motif.Synthesized (4-nitro-1,2,3-triazolyl)furoxans were found to be capable of NO release in a broad range of concentrations under physiological conditions.Therefore, our results contribute to the enlargement of the available libraries of NO-donor substances and unveil novel opportunities in drug design and related biomedical applications.

General Methods
CAUTION!Although we have encountered no difficulties during preparation and handling of azides 1a-w described in this paper, they are potentially explosive and may be sensitive to impact and friction.Mechanical actions of these species, involving scratching or scraping, must be avoided.Any manipulations must be carried out by using appropriate standard safety precautions.
All reactions were carried out in well-cleaned, oven-dried glassware with magnetic stirring. 1H, 13 C NMR spectra were recorded on a Bruker AM-300 (300.13 and 75.47 MHz, respectively) spectrometer and referenced to residual solvent peak. 14N NMR spectra were measured on a Bruker AM-300 (21.69 MHz) spectrometer using MeNO 2 (δ 14 N = 0.0 ppm) as an external standard.The chemical shifts are reported in ppm (δ).Mass spectra were measured using a Finnigan MAT INCOS-50 instrument.The IR spectra were recorded on the Simex FT-801 IR-Fourier spectrometer in the 4000-550 cm −1 region (spectral resolution 4 cm −1 ) using the universal optical attenuated total reflection (ATR) accessory with ZnSe crystal plate.ZaIR 3.5 software (Simex, Russia) was used to carry out baseline correction and normalization of FEAR spectra.A background (air) measurement was taken for every sample processed.The peaks corresponding to CO 2 vibrations were removed using the "straight line generation" option in the ZaIR 3.5 software (Simex).Raw spectra were preprocessed using a simple two-point linear subtraction baseline correction method.Two points, 900 and 1850 cm −1 , were selected outside the wavenumber region of interest that showed no variation across all samples.Spectra were the vector normalized.Spectrum smoothing was not performed.High-resolution mass spectra were recorded on a Bruker microTOF spectrometer with electrospray ionization (ESI).All measurements were performed in a positive (+MS) ion mode (interface capillary voltage: 4500 V) with scan range m/z: 50-3000.External calibration of the mass spectrometer was performed with Electrospray Calibrant Solution (Fluka).A direct syringe injection was used for all analyzed solutions in MeCN (flow rate: 3 µL min −1 ).Nitrogen was used as nebulizer gas (0.4 bar) and dry gas (4.0 L•min −1 ); interface temperature was set at 180 • C. All spectra were processed by using Bruker Data Analysis 4.0 software package.Elemental analyses were performed by the CHN Analyzer Perkin-Elmer 2400.Analytical thin-layer chromatography (TLC) was carried out on Merck 25 TLC silica gel 60 F 254 aluminum sheets.The visualization of the TLC plates was accomplished with a UV light.All standard reagents were purchased from Aldrich or Acros Organics and used without further purification.4-Azido-3-phenylfuroxan 1b was obtained according to the previously described procedure [48].

X-ray Crystallography
X-ray diffraction studies were carried out at 100K using the four-circle Rigaku Synergy S diffractometer equipped with a HyPix6000HE area-detector (kappa geometry, shutterless ω-scan technique, monochromatized Cu K α -radiation) for 1a and the Bruker D8 Quest diffractometer equipped with a PhotonIII area-detector (ω-and ϕ-scan technique, monochromatized Mo K α -radiation) for 3a and 3c.The intensity data were integrated and corrected for absorption and decay by the CrysAlisPro program for 1a and by the APEX3 program (SAINT [53], SADABS [54]) for 3a and 3c.All structures were solved by dualspace method SHELXT [55] and refined against F 2 using SHELXL-2018 software (version 2014/6) [56].All non-hydrogen atoms were refined with individual anisotropic displacement parameters.All hydrogen atoms were found in the difference Fourier synthesis and refined as riding atoms with relative isotropic displacement parameters.A rotating group model was applied for methyl groups.The bromophenyl substituent in the 3c structure was found to be disordered over two places with population ratio 95:5.All relevant crystal data and refinement details are listed in Table S1.The CCDC 2290794-2290796 contain all additional information on crystal structures and refinement.
The density functional theory calculations for 3a and 3c were performed using the Gaussian program [57] at the PBE0-D3 [58-60]/def2TZVP level.Equilibrium structures of both compounds correspond to minimums on the potential energy surface according to the calculations of the Hessian of electronic energy (ultrafine grids, no imaginary modes were found).N,N-Dimethylformamide dimethyl acetal (680 µL, 5.1 mmol) was added to a solution of nitromethane (270 µL, 5 mmol) in MeCN (10 mL).The reaction mixture was stirred for 3 h at 20 • C and volatiles were evaporated on a rotary evaporator affording 1-dimethylamino-2-nitroethylene.Thus obtained dipolarophile was dissolved in anhydrous MeCN (10 mL) followed by the addition of the corresponding 4-azidofuroxan 2 (1 mmol) and p-TSA monohydrate (29 mg, 0.15 mmol).The reaction mixture was stirred at 40 • C for 3 h and then refluxed for 69 h.After the disappearance of azides 2 on TLC, the solvent was distilled off under reduced pressure and the target product was purified by column chromatography (eluent CH 2 Cl 2 or CHCl 3 /CCl 4 , 4:1).

NO Release Assay
The test molecule (0.1 mmol) was dissolved in DMSO (50 mL).A 20 µL aliquot of the resulting solution was diluted with a phosphate buffer solution (180 µL, Ph 7.4).The final concentration of the tested compound was 2 × 10 −4 M. The mixture was incubated at 37 • C for 1 h.A 50 µL aliquot of the Griess reagent (prepared by mixing sulfanilamide (4 g), N-naphthylethylenediamine dihydrochloride (0.2 g) and 85% H 3 PO 4 (10 mL) in distilled and deionized water (final volume 100 mL)) was added and incubated for 10 min at 37 • C. UV absorbance at 540 nm was measured using a Multiskan GO Microplate Photometer and calibrated using a standard curve prepared from standard solutions of NaNO 2 to give the nitrite concentration.All measurements were made in triplicate.

Molecules 2023, 28, x FOR PEER REVIEW 4 of 19 Scheme 2. Synthesis of 4-azidofuroxans 2a-v.Table 1 .
Optimization of the reaction conditions for the synthesis of

Table 1 .
Optimization of the reaction conditions for the synthesis of