Synthesis , structural characterization and cytotoxic activity of heterocyclic compounds containing the furoxan ring

A direct approach to the synthesis of previously unknown 1H-1,2,3-triazolylfuroxans, involving nucleophilic substitution of the nitro group in nitrofuroxans followed by catalytic [3+2] cycloaddition of intermediate azidofuroxans to 1,3-ketoesters, is reported. The scope of the triazolylfuroxans was additionally diversified through a number of transformations of the functional groups attached to the 1,2,3-triazole ring. The cytotoxic activity of the newly synthesized triazolylfuroxans and of previously reported hetarylfuroxans was studied. The NO-donor capability of selected synthesized hetarylfuroxans was measured by the Griess reaction using a spectrophotometric technique.


Introduction
2][3] In particular, in recent years a great attention has been focused on the synthesis of hybrid structures comprising a framework capable of nitric oxide (NO) release.5][6] Many different types of compounds have been synthesized and tested as NO donors (guanidines, nitramines, oximes, mesoionic systems, heterocyclic N-oxides, etc.), [7][8][9] including the 1,2,5-oxadiazole 2-oxides (furoxans) which are capable of exogenous NO release at the presence of thiol cofactors. 10,11uroxans comprise a valuable class of five-membered heterocycles and can serve as a privileged motif in medicinal and pharmaceutical chemistry owing to their significant biological activities, for example neuroprotective and precognitive, 12 cytotoxic, 13,14 antihelmintic, 15 antibacterial 16 and fungicidal 17 , connected with the high capacity of furoxans to produce a large flux of NO.It was established that NO exerts a cytotoxic effect at high concentrations, while low levels of NO are potentially protective, particularly in the CNS. 18][21][22][23][24][25][26] In present work we have aimed to synthesize the heterocyclic structures comprising furoxan ring coupled with various functionally substituted heterocyclic fragments and to carry out an evaluation of their cytotoxic activity and NO-donor capability.
For the preparation of (1,2,4-triazin-3-yl)furoxans 3a-g a cyclocondensation of α-dicarbonyl compounds 9 with furoxanylamidrazones 10 was utilized. 29An effective synthesis of the latter was recently developed by reaction of cyanofuroxans 8 with hydrazine-hydrate (Scheme 4). 38The 3-nitrobifuroxanyl structures 4a-c were synthesized by an interaction of furoxanylhydroxamic acid chlorides 11 with dinitromethane sodium salt with subsequent nitrosation (Scheme 5). 30Compound 4c was thermally isomerized to the 4-nitro isomer 4d, and 4,4'-dinitro-3,3'-bifuroxan 4e was prepared according to Klapötke's procedure. 39To prepare the (1,2,3-triazol-1-yl)furoxan derivatives 5 we applied the approach based on the transformations of chloromethyl and ethoxycarbonyl groups in 1,2,3-triazoles 12a-c by the action of different nucleophiles.The initial compounds 12a-c were synthesized by [3+2] cycloaddition of 3-aryl-4-azidofuroxans 13a,b with benzoylacetic ethyl ester 14a and chloroacetoacetic ester 14b under TEA catalysis (Scheme 6).This approach was previously used for the synthesis of similar (1,2,3-triazol-1-yl)furazan derivatives which were shown to possess cytotoxic activity. 40The initial 3-aryl-4-azidofuroxans 13a,b were prepared by nucleophilic substitution of nitro group in 3-aryl-4-nitrofuroxans 6a,c under the action of NaN 3 according to described method. 34The nucleophilic substitution of chloride fragment in compounds 12b,c under the action of All synthesized intermediate products 12-18 and final (1,2,3-triazol-1-yl)furoxans 5a-n were characterized by spectral (IR, 1 H, 13 C NMR and mass-spectra) and analytical methods.Finally, the structures of the (1,2,3triazol-1-yl)furoxans 5 was confirmed by the single-crystal X-ray diffraction study of compound 5k (Figure 1).3)N(4) angle is 55.55(2)°.It indicates the close extent of a π-conjugation between cycles, that is unusual for substituted phenylfuroxans especially accounting for the acceptor character of triazole ring.The spatial arrangement of cycles in crystal structure of 5k can be explained not only by the presence of bulky morpholine substituent but also by two intermolecular interactions between cycles bounding molecules into dimers.Namely, there are the C-H…π interaction between hydrogen atom of triazole ring and phenyl cycle (with normalized C-H bonds the С(7)…H(4) distance is 2.757 Å) and the C-H…N interaction between hydrogen atom of morpholine cycle and nitrogen of triazole ring (the distance N(5)…H(12B) is 2.651 Å accounting to normalized C-H bonds).Among many other intermolecular contacts, the shortened contacts between the oxygen atom of morpholine cycle and furoxan cycle are to be noted (the С(1)…О(3), N(1)…O(3) and C(2)…O(3) distances are 2.950, 2.970, и 3.158 Å, respectively).These contacts are geometrically similar with intermolecular interactions between furoxan ring and its exo-oxygen atom 29,41 and can be described as interaction between lone electron pair of the O(3) atom and π*-orbital of the furoxan cycle.In 5k these interactions form continuous chains of molecules which are, in its turn, bounded into layers by the C-H… π interaction between CH 2 -fragment and triazole ring (with normalized C-H bonds the С(3)…H(11B) distance is 2.598 Å).The crystal packing of 5k is additionally stabilized by weak С-H…O contacts between the exo-oxygen atom of furoxan cycle and one of the methylene fragments of morpholine (the H(15A)…O(1) distance is 2.539 Å) (Figure 2).
It is well-known that furoxans behave as NO donors in presence of thiol cofactors. 5,10,11At the same time, the formation of nitrite-anion as a result of NO oxidation may be quantified according to Griess assay and thus may serve as a reliable tool for measuring the amount of NO release.The amounts of NO 2 -produced of the selected hetarylfuroxan structures under physiological conditions (pH 7.4; 37 °C) after 1 h incubation were measured via the Griess reaction using a spectrophotometric technique.Furoxan 2b was found to be the most powerful NO donor (up to 75.6% NO 2 -release, Table 1).Nitrobifuroxans 4a,c,d showed also high levels of NO 2 release, however, for compound 4d this high value is connected with the ability of 4-nitrofuroxans to undergo nucleophilic substitution under the action of thiols.The dependence of NO release for compounds 1a, 2b, 3d from time was estimated.It was found that for the furoxans 1a and 3d the produced amount of NO slightly differs in time, while for the compound 2b this range was 5-12% (Figure 3).

Conclusions
A novel method for the synthesis of the previously unknown (1H-1,2,3-triazolyl)furoxans based on the tandem nucleophilic substitution/organocatalytic [3+2] cycloaddition approach has been developed.The scope of the synthesized heterocyclic assemblies was additionally broadened through the investigations of the reactivity of the functional groups on the triazole ring.A series of newly synthesized (1H-1,2,3-triazolyl)furoxans as well as previously known hetarylfuroxans (36 compounds in total) were evaluated as cytotoxic agents against five human tumor cell lines.In addition, NO-releasing capacity of the selected furoxan-based structures under physiological conditions was measured by detecting nitrites via the Griess reaction using a spectrophotometric technique.

Experimental Section
General. 1 H and 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); multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad).Coupling constants, J, are reported in Hertz.The IR spectra were recorded on a Bruker "Alpha" spectrometer in the range 400-4000 cm -1 (resolution 2 cm -1 ) as pellets with KBr or as a thin layer.The melting points were determined on "Stuart SMP20" melting point apparatus and are uncorrected.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.Flash chromatography was performed on silica gel 60 A (0.060-0.200 mm, Acros Organics).High resolution mass spectra were recorded on a Bruker microTOF spectrometer with electrospray ionization (ESI).The structure was solved by direct method and refined by the full-matrix least-squares technique against F 2 in the isotropic-anisotropic approximation.The hydrogen atoms were found in difference Fourier synthesis and refined in the isotropic approximation.For 5k, the refinement converged to wR2 = 0.1132 and GOF = 1.023 for all independent reflections (R1 = 0.0449 was calculated against F for 3338 observed reflections with I>2σ(I)).All calculations were performed using SHELX 2014.General procedure for the synthesis of ethyl (hetaryloxy)methyltriazolyl furoxan esters 5a,b,e,f.Diazabicycloundecene (DBU) (0.80 g, 0.52 mmol) was added to a solution of corresponding hydroxyhetarene (0.52 mmol) in MeCN (3 mL) at room temperature.Then the chloromethyl derivative 12b or 12c (0.52 mmol) was added.The reaction mixture was stirred at room temperature for 24-72 h until disappearance of the initial compound 12b or 12c (TLC monitoring).Water (15 mL) was added, the solid formed was filtered off, washed with small amount of cold CHCl 3 and dried in air.

Cytotoxicity in vitro
The IC 50 values of the synthesized compounds against cells were determined by the MTT method. 42A549, HCT116, HeLa, MCF7, RD and HEK293 cells were seeded at 1.0 x 10 4 cells/200 μL in 96-well plates and incubated at 37 o C in a humidified atmosphere with 5% CO 2 .After 24 h of preincubation, the various concentrations of the tested compounds (100-1.56µM) were added into each well, and these cells were incubated under similar conditions for 72 h.All compounds were dissolved in DMSO.The final DMSO concentration in each well did not exceed 1% and was not toxic for the cells.The wells with a specific cell culture containing 1% DMSO solution in the medium were monitored as control.After incubation, 20 mM MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), at a final concentration of 5 mg/mL, was added into each well, and the cells were incubated for another 2 h.The medium was removed and 100 μL DMSO was added to each well.The optical density was measured at 544 nm minus background absorption at 620 nm using the Victor3 (Perkin Elmer) microplate reader.Concentrations (IC 50 ) were calculated according to the dose-dependent inhibition curves with GraphPad Prism 7 software.The experiments were carried out in triplicate.NO release assay.The test molecule (0.1 mmol) was dissolved in DMSO (50 mL).20 µL aliquot of the resulted solution was diluted with phosphate buffer solution (180 µL, pH 7.4, containing 2 µmol L-cysteine).The final concentration of the furoxan derivative was 2•10 -4 M. The mixture was incubated at 37 o C for 1 h.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 o 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.No significant NO release was measured at the absence of Lcysteine.

Figure 1 .
Figure 1.The general view of the 5k molecule.Atoms are represented by probability ellipsoids of atomic vibrations (p=0.5).

Figure 2 .
Figure 2. The fragment of a layer in the crystal structure of 5k.

Table 1 .
Griess test results for the selected hetarylfuroxan structures -release on time according to Griess test results.