Signaling controversy and future therapeutical perspectives of targeting sphingolipid network in cancer immune editing and resistance to tumor necrosis factor-α immunotherapy.

Anticancer immune surveillance and immunotherapies trigger activation of cytotoxic cytokine signaling, including tumor necrosis factor-α (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) pathways. The pro-inflammatory cytokine TNF-α may be secreted by stromal cells, tumor-associated macrophages, and by cancer cells, indicating a prominent role in the tumor microenvironment (TME). However, tumors manage to adapt, escape immune surveillance, and ultimately develop resistance to the cytotoxic effects of TNF-α. The mechanisms by which cancer cells evade host immunity is a central topic of current cancer research. Resistance to TNF-α is mediated by diverse molecular mechanisms, such as mutation or downregulation of TNF/TRAIL receptors, as well as activation of anti-apoptotic enzymes and transcription factors. TNF-α signaling is also mediated by sphingosine kinases (SphK1 and SphK2), which are responsible for synthesis of the growth-stimulating phospholipid, sphingosine-1-phosphate (S1P). Multiple studies have demonstrated the crucial role of S1P and its transmembrane receptors (S1PR) in both the regulation of inflammatory responses and progression of cancer. Considering that the SphK/S1P/S1PR axis mediates cancer resistance, this sphingolipid signaling pathway is of mechanistic significance when considering immunotherapy-resistant malignancies. However, the exact mechanism by which sphingolipids contribute to the evasion of immune surveillance and abrogation of TNF-α-induced apoptosis remains largely unclear. This study reviews mechanisms of TNF-α-resistance in cancer cells, with emphasis on the pro-survival and immunomodulatory effects of sphingolipids. Inhibition of SphK/S1P-linked pro-survival branch may facilitate reactivation of the pro-apoptotic TNF superfamily effects, although the role of SphK/S1P inhibitors in the regulation of the TME and lymphocyte trafficking should be thoroughly assessed in future studies.

Superelectrophilic Activation of Phosphacoumarins towards Weak Nucleophiles via Brønsted Acid Assisted Brønsted Acid Catalysis.

The electrophilic activation of various substrates via double or even triple protonation in superacidic media enables reactions with extremely weak nucleophiles. Despite the significant progress in this area, the utility of organophosphorus compounds as superelectrophiles still remains limited. Additionally, the most common superacids require a special care due to their high toxicity, exceptional corrosiveness and moisture sensitivity. Herein, we report the first successful application of the "Brønsted acid assisted Brønsted acid" concept for the superelectrophilic activation of 2-hydroxybenzo[e][1,2]oxaphosphinine 2-oxides (phosphacoumarins). The pivotal role is attributed to the tendency of the phosphoryl moiety to form hydrogen-bonded complexes, which enables the formation of dicationic species and increases the electrophilicity of the phosphacoumarin. This unmasks the reactivity of phosphacoumarins towards non-activated aromatics, while requiring only relatively non-benign trifluoroacetic acid as the reaction medium.

An Unusual Rearrangement of Pyrazole Nitrene and Coarctate Ring-Opening/Recyclization Cascade: Formal CH-Acetoxylation and Azide/Amine Conversion without External Oxidants and Reductants.

We report an unusual transformation where the transient formation of a nitrene moiety initiates a sequence of steps leading to remote oxidative C-H functionalization (R-CH3 to R-CH2OC(O)R') and the concomitant reduction of the nitrene into an amino group. No external oxidants or reductants are needed for this formal molecular comproportionation. Detected and isolated intermediates and computational analysis suggest that the process occurs with pyrazole ring opening and recyclization.

DFT Quantum-Chemical Calculation of Thermodynamic Parameters and DSC Measurement of Thermostability of Novel Benzofuroxan Derivatives Containing Triazidoisobutyl Fragments.

New derivatives of benzofuroxan containing triazidoisobutyl fragments, opening the way for the creation of highly effective compositions with an increased value of energy characteristics, were synthesized for the first time. Such compounds are also an excellent platform for further modification and for the preparation of new biologically-active compounds containing tetrazole and triazole fragments. Calculations of heats of formation performed with the DFT (density functional theory) method showed that the studied compounds are high-energetic density ones, the enthalpies of formation of which are comparable to the enthalpies of formation of similar benzofuroxan derivatives and exceeds experimental enthalpy of formation of CL-14 (5,7-diamino-4,6-dinitrobenzofuroxan). The analysis of DSC indicates a sufficiently high thermal stability of the synthesized azidobenzofuroxans, which are acceptable for their use as components in the creation of highly efficient compositions with an increased value of energy characteristics.

Diastereoselective Synthesis of Novel Spiro-Phosphacoumarins and Evaluation of Their Anti-Cancer Activity.

Herein we present the regio- and diastereoselective synthesis of novel pyrrolidine-fused spiro-dihydrophosphacoumarins via intermolecular [3 + 2] cycloaddition reaction. The presented approach is complementary to existing ones and provides an easy entry to the otherwise inaccessible derivatives. Additionally, the unprecedented pathway of the reaction of 4-hydroxycoumarin with azomethine ylides is described. The anti-cancer activity of the obtained compounds was tested in vitro, the most potent compound being 2.6-fold more active against the HuTu 80 cell line than the reference 5-fluorouracil, with a selectivity index > 32.

Water-Soluble Salts Based on Benzofuroxan Derivatives-Synthesis and Biological Activity.

A series of novel water-soluble salts of benzofuroxans was achieved via aromatic nucleophilic substitution reaction of 4,6-dichloro-5-nitrobenzofuroxan with various amines. The salts obtained showed good effectiveness of the pre-sowing treatment of seeds of agricultural crops at concentrations of 20-40 mmol. In some cases, the seed treatment with salts leads not only to improved seed germination, but also to the suppression of microflora growth. Additionally, their anti-cancer activityin vitrohas been researched. The compounds with morpholine fragments or a fragment of N-dimethylpropylamine, demonstrated the highest cytotoxic activity, which is in good correlation with the ability to inhibit the glycolysis process in tumor cells. Two compounds 4e and 4g were selected for further experiments using laboratory animals. It was found that the lethal dose of 50% (LD50) is 22.0 ± 1.33 mg/kg for 4e and 13.75 ± 1.73 mg/kg for 4g, i.e., compound 4e is two times less toxic than 4g, according to the mouse model in vivo. It was shown that the studied compounds exhibit antileukemia activity after a single intraperitoneal injection at doses from 1.25 to 5 mg/kg, as a result of which the average lifespan of animals with a P388 murine leukemia tumor increases from 20 to 28%. Thus, the water-soluble salts of benzofuroxans can be considered as promisingcandidates for further development, both as anti-cancer agents and as stimulants for seed germination and regulators of microflora crop growth.

4,6-Dichloro-5-Nitrobenzofuroxan: Different Polymorphisms and DFT Investigation of Its Reactivity with Nucleophiles.

This research focuses on the X-ray structure of 4,6-dichloro-5-nitrobenzofuroxan 1 and of some of its amino derivatives (4a, 4e, 4g, and 4l) and on DFT calculations concerning the nucleophilic reactivity of 1. We have found that by changing the solvent used for crystallization, it is possible to obtain 4,6-dichloro-5-nitrobenzofuroxan (1) in different polymorphic structures. Moreover, the different torsional angles observed for the nitro group in 1 and in its amino derivatives (4a, 4e, 4g, and 4l) are strictly dependent on the steric hindrance of the substituent at C-4. DFT calculations on the course of the nucleophilic substitution confirm the role of the condensed furoxan ring in altering the aromaticity of the carbocyclic frame, while chlorine atoms strongly influence the dihedral angle and the rotational barrier of the nitro group. These results corroborate previous observations based on experimental kinetic data and give a deep picture of the reaction with amines, which proceeds via a "non-aromatic" nucleophilic substitution.

Novel Hybrid Compounds Containing Benzofuroxan and Aminothiazole Scaffolds: Synthesis and Evaluation of Their Anticancer Activity.

A series of novel hybrid compounds containing benzofuroxan and 2-aminothiazole moieties are synthesized via aromatic nucleophilic substitution reaction. Possible reaction pathways have been considered quantum-chemically, which allowed us to suggest the most probable products. The quantum chemical results have been proved by X-ray data on one compound belonging to the synthesized series. It was shown that the introduction of substituents to both the thiazole and amine moieties of the compounds under study strongly influences their UV/Vis spectra. Initial substances and obtained hybrid compounds have been tested in vitro as anticancer agents. Target compounds showed selectivity towards M-HeLa tumor cell lines and were found to be more active than starting benzofuroxan and aminothiazoles. Furthermore, they are considerably less toxic to normal liver cells compared to Tamoxifen. The mechanism of action of the studied compounds can be associated with the induction of apoptosis, which proceeds along the mitochondrial pathway. Thus, new hybrids of benzofuroxan are promising candidates for further development as anticancer agents.

The Reactivity of Azidonitrobenzofuroxans towards 1,3-Dicarbonyl Compounds: Unexpected Formation of Amino Derivative via the Regitz Diazo Transfer and Tautomerism Study.

Herein, we report on the reaction of nitro-substituted azidobenzofuroxans with 1,3-dicarbonyl compounds in basic media. The known reactions of benzofuroxans and azidofuroxans with 1,3-dicarbonyl compounds in the presence of bases are the 1,3-dipolar cycloaddition and the Beirut reaction. In contrast with this, azidonitrobenzofuroxan reacts with 1,3-carbonyl compounds through Regitz diazo transfer, which is the first example of this type of reaction for furoxan derivatives. This difference is seemingly due to the strong electron-withdrawing effect of the superelectrophilic azidonitrobenzofuroxan, which serves as the azido transfer agent rather than 1,3-dipole in this case.

On the Nucleophilic Reactivity of 4,6-Dichloro-5-nitrobenzofuroxan with Some Aliphatic and Aromatic Amines: Selective Nucleophilic Substitution.

The reaction rates for the nucleophilic aromatic substitution of 4,6-dichloro-5-nitrobenzofuroxan 1 with eight aliphatic amines (characterized by very different basicities/nucleophilicities) and three anilines have been measured in both methanol and toluene. The obtained rates have been related to the basicity (pKaH in water and Kb in benzene) or nucleophilicity (N Mayr constants) of the tested amines. The whole of the obtained kinetic data has furnished useful information on the high nucleophilic reactivity of benzofuroxan derivatives, which has been related essentially to two factors: the high electron-drawing ability/power of the condensed furoxan ring and the low aromatic character of the benzofuroxan system.

Design of Novel 4-Aminobenzofuroxans and Evaluation of Their Antimicrobial and Anticancer Activity.

A series of novel 4-aminobenzofuroxan derivatives containing aromatic/aliphatic amines fragments was achieved via aromatic nucleophilic substitution reaction of 4,6-dichloro-5-nitrobenzofuroxan. The quantum chemistry calculations were performed to identify the factors affecting the regioselectivity of the reaction. The formation of 4-substituted isomer is favored both by its greater stability and the lower activation barrier. Antimicrobial activity of the obtained compounds has been evaluated and some of them were found to suppress effectively bacterial biofilm growth. Fungistatic activity of 4-aminobenzofuroxans were tested on two genetically distinct isolates of M. nivale. The effect of some benzofuroxan derivatives is likely to be more universal against different varieties of M. nivale compared with benzimidazole and carbendazim. Additionally, their anti-cancer activity in vitro has been tested. 4-aminofuroxans possessing aniline moiety showed a high selectivity towards MCF-7 and M-HeLa tumor cell lines. Moreover, they exhibit a significantly lower toxicity towards normal liver cells compared to Doxorubicin and Tamoxifen. Thus, benzofuroxans containing aromatic amines fragments in their structure are promising candidates for further development both as anti-cancer and anti-microbial agents.

Synthesis of Novel 2-(Het)arylpyrrolidine Derivatives and Evaluation of Their Anticancer and Anti-Biofilm Activity.

A library of novel 2-(het)arylpyrrolidine-1-carboxamides were obtained via a modular approach based on the intramolecular cyclization/Mannich-type reaction of N-(4,4-diethoxybutyl)ureas. Their anti-cancer activities both in vitro and in vivo were tested. The in vitro activity of some compounds towards M-Hela tumor cell lines was twice that of the reference drug tamoxifen, whereas cytotoxicity towards normal Chang liver cell did not exceed the tamoxifen toxicity. In vivo studies showed that the number of surviving animals on day 60 of observation was up to 83% and increased life span (ILS) was up to 447%. Additionally, some pyrrolidine-1-carboxamides possessing a benzofuroxan moiety obtained were found to effectively suppress bacterial biofilm growth. Thus, these compounds are promising candidates for further development both as anti-cancer and anti-bacterial agents.

C-C Coupling Reactions between Benzofurazan Derivatives and 1,3-Diaminobenzenes.

Aromatic substitution reactions between 1,3-diaminobenzene and chloronitrobenzofurazan derivatives have never been reported so far. The aim of the current study was to synthesize novel electron-donor and -acceptor architectures of interest in applied fields and to provide new insights on the nucleophilic behavior of 1,3-diaminobenzenes. The reaction of 1,3-dipiperidinyl-, 1,3-dimorpholinyl-, 1,3-dipyrrolidinyl-, or 1,3-dimethylamino-benzene with 7-chloro-4,6-dinitrobenzofuroxan or with a series of chloro-nitrobenzofurazans has been carried out in mild conditions. The partners reactivity has been investigated by monitoring the reaction course through ¹H-NMR spectroscopy. The reaction occurred in a regioselective way, providing in good yields the novel C-C coupling compounds. Indications on the reactivity behavior for the studied nucleophiles have been relieved.

Diverse Biological Activity of Benzofuroxan/Sterically Hindered Phenols Hybrids.

Combining two pharmacophores in a molecule can lead to useful synergistic effects. Herein, we show hybrid systems that combine sterically hindered phenols with dinitrobenzofuroxan fragments exhibit a broad range of biological activities. The modular assembly of such phenol/benzofuroxan hybrids allows variations in the phenol/benzofuroxan ratio. Interestingly, the antimicrobial activity only appears when at least two benzofuroxan moieties are introduced per phenol. The most potent of the synthesized compounds exhibit high cytotoxicity against human duodenal adenocarcinoma (HuTu 80), human breast adenocarcinoma (MCF-7), and human cervical carcinoma cell lines. This toxicity is associated with the induction of apoptosis via the internal mitochondrial pathway and an increase in ROS production. Encouragingly, the index of selectivity relative to healthy tissues exceeds that for the reference drugs Doxorubicin and Sorafenib. The biostability of the leading compounds in whole mice blood is sufficiently high for their future quantification in biological matrices.

Receiver Operator Characteristic (ROC) Analysis of Lipids, Proteins, DNA Oxidative Damage, and Antioxidant Defense in Plasma and Erythrocytes of Young Reproductive-Age Men with Early Stages of Type 1 Diabetes Mellitus (T1DM) Nephropathy in the Irkutsk Region, Russia.

Oxidative stress plays a leading role in the pathogenesis of diabetic nephropathy. However, many aspects of oxidative stress reactions in the initial stages of this disease are not fully understood. The men cohort is of particular interest because of the severe effects of diabetes on their urogenital system. The aim of this study is to assess the intensity of lipids, proteins, DNA oxidative damage, blood antioxidant defense enzymatic, and activity of non-enzymatic components in men with type 1 diabetes mellitus (T1DM) in the early stages of diabetic nephropathy using receiver operator characteristic (ROC) analysis. This study included eighty-nine reproductive-age men in the initial stages of diabetic nephropathy (DN) and thirty-nine age- and sex-matched individuals not suffering from glycemic disorders. The DN patients were divided into two subgroups: stage 1 patients (urinary albumin < 30 mg/day and albumin/creatinine ratio < 3 mg/mmol (n = 45)) and stage 2 patients (urinary albumin 30−300 mg/day and albumin/creatinine ratio 3−30 mg/mmol (n = 44)). Levels of oxidative damage products (conjugated dienes (CDs), thiobarbituric acid reactants (TBARs), methylglyoxal (MGO), and 8-hydroxy-2'-deoxyguanosine (8-OHdG)) and antioxidants (glutathione peroxidase (GPx), glutathione S-transferases π (GSTp), glutathione reductase (GR), copper and zinc-containing superoxide dismutase 1 (SOD-1), total antioxidant status (TAS), α-tocopherol, retinol, reduced glutathione (GSH), and oxidative glutathione (GSSG)) were estimated in plasma and erythrocytes. Oxidative damage to cellular structures (higher values of median CDs (1.68 µmol/L; p = 0.003), MGO (3.38 mg/L; p < 0.001) in the stage 1 group and CDs (2.28 µmol/L; p < 0.0001), MGO (3.52 mg/L; p < 0.001), 8-OHdG (19.44 ng/mL; p = 0.010) in the stage 2 group) and changes in the antioxidant defense system (lower values of TAS (1.14 units; p = 0.011), α-tocopherol (12.17 µmol/L; p = 0.009), GPx (1099 units; p = 0.0003) and elevated levels of retinol (1.35 µmol/L; p < 0.001) in the group with stage 1; lower values of α-tocopherol (12.65 µmol/L; p = 0.033), GPx (1029.7 units; p = 0.0001) and increased levels of GR (292.75 units; p < 0.001), GSH (2.54 mmol/L; p = 0.010), GSSG (2.31 mmol/L; p < 0.0001), and retinol (0.81 µmol/L; p = 0.005) in the stage 2 group) were identified. The ROC analysis established that the following indicators have the highest diagnostic significance for stage 1 diabetic nephropathy: CDs (AUC 0.755; p < 0.0001), TBARs (AUC 0.748; p = 0.0001), MGO (AUC 0.720; p = 0.0033), retinol (AUC 0.932; p < 0.0001), GPx (AUC 0.741; p = 0.0004), α-tocopherol (AUC 0.683; p = 0.0071), and TAS (AUC 0.686; p = 0.0052) and the following for stage 2 diabetic nephropathy: CDs (AUC 0.714; p = 0.001), TBARs (AUC 0.708; p = 0.001), 8-OHdG (AUC 0.658; p = 0.0232), GSSG (AUC 0.714; p = 0.001), and GSH (AUC 0.667; p = 0.0108). We conclude that changes in indicators of damage to lipids, proteins, DNA, and the insufficiency of antioxidant defense factors already manifest in the first stage of diabetic nephropathy in men with T1DM. The ROC established which parameters have the greatest diagnostic significance for stages 1 and 2 of diabetic nephropathy, which may be utilized as additional criteria for defining men with T1DM as being in the risk group for the development of initial manifestations of the disease and thus allow for substantiating appropriate approaches to optimize preventive measures.

Intriguing enigma of nitrobenzofuroxan's 'Sphinx': Boulton-Katritzky rearrangement or unusual evidence of the N-1/N-3-oxide rearrangement?

The SEAr/SNAr reaction between 7-chloro-4,6-dinitrobenzofuroxan (ClDNBF) and 2-morpholinyl-, 2-piperidinyl-, or 2-pyrrolidinylthiazole afforded unexpectedly two isomeric products, bearing the benzofuroxanyl moiety bound to the C-5 carbon atom of the thiazole ring. The relative ratio for the two isomers was dependent on temperature and solvent, suggesting the occurrence of an equilibrium between the two novel species. In order to investigate their structure and to design a plausible mechanistic pathway, a series of synthetic and spectroscopic experiments was planned. The isomer's structure was unambigously assigned when the reduction of furoxanyl to the furazanyl ring of the products gave exclusively a single species whose NMR data were coincident with those obtained by reacting the starting 2-aminothiazole derivatives with the 7-chloro-4,6-dinitrobenzofurazan (ClDNBZ). Possible mechanistic pathways might involve N-1-/N-3 oxide tautomerism or Boulton-Katritzky rearrangement and the current study is the first attempt to compare these two reactions. The data collected agree with the first one and DFT calculations permitted also a significant correlation with 13C NMR experimental data and the assignment of the structure of each isomer. Finally, only one Meisenheimer intermediate for each electrophile/nucleophile combination was isolated by coupling the 2-aminothiazole derivatives with 4,6-dinitrobenzofuroxan (DNBF).

Highly conjugated architectures and labile reaction intermediates from coupling between 10π electron-deficient heteroaromatics and sym-trihydroxy- or triamino-benzene derivatives.

The C-C coupling reaction between neutral aromatic carbon nucleophiles such as 1,3,5-triamino- and 1,3,5-trihydroxy-benzene derivatives and 7-chloro-4,6-dinitrobenzofuroxan (Cl-DNBF) or 7-chloro-4,6-dinitrobenzofurazan (Cl-DNBZ) gave in high yield the corresponding conjugated structures, which are of potential interest in the materials field. When chlorine is absent on the electrophile, as in 4,6-dinitrobenzofurazan (DNBZ), the reaction with 1,3,5-triaminobenzene derivatives produces Wheland-Meisenheimer (WM) zwitterionic intermediates, intercepted and fully characterized via NMR at low temperature, whereas stable Meisenheimer complexes (M) were isolated in the reaction of phloroglucinol with 1,3,5-trimethoxybenzene. The latter also gave exclusively M complexes when reacted with 4,6-dinitrobenzofuroxan (DNBF), or 4,6-dinitrotetrazolopyridine (DNTP). The detection of WM or M intermediates can be rationalized by invoking the substituent stabilization ability of the positive charge in WM intermediates, on going from neutral carbon nucleophiles 1,3,5-triaminobenzenes to 1,3,5-trihydroxybenzene derivatives. Furthermore, variable-temperature NMR experiments on the M intermediates gave insights into the rotational barrier about the newly formed C-C bond.

Novel Structural Hybrids on the Base of Benzofuroxans and Furoxans. Mini-Review.

Hybrid drug strategy is based on the combination of two or more pharmacophores into a new chemical entity to improve the properties of the original compounds or to obtain double action of resulting molecule. Hybrid molecules, comprised of some pharmacophore and nitric oxide (NO) donor moiety, constitute one of the more promising approaches for the design of drugs. Furoxans and benzofuroxans are considered NO releasing prodrugs and are of great interest for researchers. In this review we will focus on furoxan and benzofuroxan hybrids described in literature during the last years (from 2005 to 2016).

Synthesis of New 'Hybrid' Compounds Based on Benzofuroxans and Aminoalkylnaphthalimides.

Pathogenic bacteria and fungi eventually develop resistance to existing drugs, and therefore, we need constant development of new drugs. The research is aimed at addressing fundamental scientific problems-the search for new biologically active compounds among several benzofuroxan-containing 'hybrid' products. N-substituted naphthalimides were chosen as a second pharmacophore. Benzofuroxanes biological effects were studied by means of bacterial lux-biosensors. Compounds IIIa, IVa, IIIc, and IVc displayed more expressed bacteriotoxic action in comparison with the initial substances Ia-c and represent a certain interest for using as antibacterial substances.

Synthesis and biological evaluation of novel structural hybrids of benzofuroxan derivatives and fluoroquinolones.

A series of novel hybrids based on benzofuroxan derivatives and fluoroquinolones (4a-d-6a-d) have been synthesized. Unexpectedly, the reactions have resulted in salt products formation during the hydrolysis of benzofuroxans by water molecules being present in the solvent instead of usual substitution products. All the compounds have been screened for antimicrobial and toxic activities. All resulting compounds retain high activity characteristic for fluoroquinolones. Many of the salts based on benzofuroxans and fluoroquinolones have higher activity than starting fluoroquinolones against Bacillus cereus 8035. Among the screened compounds, the compound 4d has shown the best antibacterial activity against B. cereus 8035, 8 times higher than the original Lomefloxacin (MBC value 1.5 µg/mL).