Effects of Isoxazolyl Steroids on Key Genes of Sonic Hedgehog Cascade Expression in Tumor Cells.

Activation of the Hedgehog (Hh) signaling pathway is often associated with the progression of various types of cancer. The purpose of study was to search for inhibitors of the Hh signaling pathway among eight compounds belonging to the group of isoxazolyl steroids. The evaluation of the effectiveness of the compounds was based on the analysis of their cytotoxicity, effect on the cell cycle, on the expression of key Hh-signaling-pathway genes (Ptch1, Smo, and Gli1) and putative target genes MMP-2 and MMP-9. Four compounds with the most pronounced cytotoxic effect were identified: compounds 1, 2 (HeLa cells) and 3, 4 (A549 cells). Compounds 1 and 2 significantly reduced the expression of the Ptch1, Smo, Gli1 genes, but had the opposite effect on MMP-2 gene expression: Compound 1 increased it, and compound 2 decreased it. Compounds 3 and 4 did not have a noticeable inhibitory effect on the expression of the Shh pathway receptors, but significantly inhibited MMP-2 and MMP-9 expression. Thus, it was shown that inhibition of the Shh signaling pathway by isoxazolyl steroids can have the opposite effect on MMPs gene expression, which is what should be taken into account in further studies of these compounds as therapeutic agents.

Acridine-Isoxazole and Acridine-Azirine Hybrids: Synthesis, Photochemical Transformations in the UV/Visible Radiation Boundary Region, and Anticancer Activity.

Easy-to-handle N-hydroxyacridinecarbimidoyl chloride hydrochlorides were synthesized as convenient nitrile oxide precursors in the preparation of 3-(acridin-9/2-yl)isoxazole derivatives via 1,3-dipolar cycloaddition with terminal alkynes, 1,1-dichloroethene, and acrylonitrile. Azirines with an acridin-9/2-yl substituent attached directly or via the 1,2,3-triazole linker to the azirine C2 were also synthesized. The three-membered rings of the acridine-azirine hybrids were found to be resistant to irradiation in the UV/visible boundary region, despite their long-wave absorption at 320-420 nm, indicating that the acridine moiety cannot be used as an antenna to transfer light energy to generate nitrile ylides from azirines for photoclick cycloaddition. The acridine-isoxazole hybrids linked at the C9-C3 or C2-C3 atoms under blue light irradiation underwent the addition of such hydrogen donor solvents, such as, toluene, o-xylene, mesitylene, 4-chlorotoluene, THF, 1,4-dioxane, or methyl tert-butyl ether (MTBE), to the acridine system to give the corresponding 9-substituted acridanes in good yields. The synthesized acridine-azirine, acridine-isoxazole, and acridane-isoxazole hybrids exhibited cytotoxicity toward both all tested cancer cell lines (HCT 116, MCF7, and A704) and normal cells (WI-26 VA4).

Water-Soluble Arylazoisoxazole Photoswitches.

Azoheteroarenes are emerging as powerful alternatives to azobenzene molecular photoswitches. In this study, water-soluble arylazoisoxazole photoswitches are introduced. UV/vis and NMR spectroscopy revealed moderate to very good photostationary states and reversible photoisomerization between the E- and Z-isomers over multiple cycles with minimal photobleaching. Several arylazoisoxazoles form host-guest complexes with ß- and γ-cyclodextrin with significant differences in binding constants for each photoisomer as shown by isothermal titration calorimetry and NMR experiments, indicating their potential for photoresponsive host-guest chemistry in water. One carboxylic acid functionalized arylazoisoxazole can act as a hydrogelator, allowing gel properties to be manipulated reversibly with light. The hydrogel was characterized by rheological experiments, atom force microscopy and transmission electron microscopy. These results demonstrate that arylazoisoxazoles can find applications as molecular photoswitches in aqueous media.

Design of novel water-soluble isoxazole-based antimicrobial agents and evaluation of their cytotoxicity and acute toxicity.

Based on the readily available 3-organyl-5-(chloromethyl)isoxazoles, a number of previously unknown water-soluble conjugates of isoxazoles with thiourea, amino acids, some secondary and tertiary amines, and thioglycolic acid were synthesized. The bacteriostatic activity of aforementioned compounds has been studied against Enterococcus durans B-603, Bacillus subtilis B-407, Rhodococcus qingshengii Ac-2784D, and Escherichia coli B-1238 microorganisms (provided by All-Russian Collection of Microorganisms, VKM). The influence of the nature of the substituents in positions 3 and 5 of the isoxazole ring on the antimicrobial activity of the obtained compounds has been determined. It is found that the highest bacteriostatic effect is observed for compounds containing 4-methoxyphenyl or 5-nitrofuran-2-yl substituents in position 3 of the isoxazole ring as well as methylene group in position 5 bearing residues of l-proline or N-Ac-l-cysteine (5a-d, MIC 0.06-2.5 µg/ml). The leading compounds showed low cytotoxicity on normal human skin fibroblast cells (NAF1nor) and low acute toxicity on mice in comparison with the well-known isoxazole-containing antibiotic oxacillin.

Naphtho[1,8-de][1,2]Oxazin-4-ol: Precursor to 1,2,8-Trisubstituted Naphthalenes and 1-Unsubstituted Naphtho[1,2-d]isoxazole 2-Oxide: A Novel Isomerization of the N-Oxide to Nitrile Oxide en Route to Isoxazol(in)es.

Naphtho[1,8-de][1,2]oxazin-4-ol and its acyl or benzyl derivatives ring open to various 2,8-dihydroxy-1-naphthonitriles, which, through (de)protection protocols and reduction, afford the target (E)-2-hydroxy-8-methoxy-1-naphthaldehyde. This was converted to its corresponding oxime, which was oxidatively o-cyclized with phenyliodine(III) diacetate (PIDA) to 9-methoxynaphtho[1,2-d]isoxazole 2-oxide. The latter, in deuterated DMSO at room temperature, was rearranged to its isomer 2-hydroxy-8-methoxy(naphthalen-1-yl)nitrile oxide. The isomerization was detected by time-course plot 1H NMR spectroscopy and further identified from its 13C NMR and HRMS spectra. The nitrile oxide was stable in (non)deuterated DMSO for at least 18 h. A 3,4-bis(2-hydroxy-8-methoxynaphthalen-1-yl)-1,2,5-oxadiazole 2-oxide, as a dimerization product or an isocyanate as a rearrangement isomer, was ruled out, the former by its HRMS spectrum and the latter by its 1,3-dipolar cycloaddition reactions to substituted isoxazoles.

Acid-Switchable Synthesis of Trifluoromethylated Triazoles and Isoxazoles via Reaction of CF3-Ynones with NaN3: DFT Study of the Reaction Mechanism.

A detailed study of the reaction of CF3-ynones with NaN3 was performed. It was found that the reaction permits the selective synthesis of either 4-trifluoroacetyltriazoles or 5-CF3-isoxazoles. The chemoselectivity of the reaction was switchable via acid catalysis. The reaction of CF3-ynones with NaN3 in EtOH produced high yields of 4-trifluoroacetyltriazoles. In contrast, the formation of 5-CF3-isoxazoles was observed under catalysis by acids. This acid-switchable procedure can be performed at sub-gram scale. The possible reaction mechanism was supported by DFT calculations. The synthetic utility of the prepared 4-trifluoroacetyltriazoles was demonstrated.

Synthesis and Biological Evaluation of New Isoxazolyl Steroids as Anti-Prostate Cancer Agents.

Steroids with a nitrogen-containing heterocycle in the side chain are known as effective inhibitors of androgen signaling and/or testosterone biosynthesis, thus showing beneficial effects for the treatment of prostate cancer. In this work, a series of 3ß-hydroxy-5-ene steroids, containing an isoxazole fragment in their side chain, was synthesized. The key steps included the preparation of Weinreb amide, its conversion to acetylenic ketones, and the 1,2- or 1,4-addition of hydroxylamine, depending on the solvent used. The biological activity of the obtained compounds was studied in a number of tests, including their effects on 17α-hydroxylase and 17,20-lyase activity of human CYP17A1 and the ability of selected compounds to affect the downstream androgen receptor signaling. Three derivatives diminished the transcriptional activity of androgen receptor and displayed reasonable antiproliferative activity. The candidate compound, 24j (17R)-17-((3-(2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-androst-5-en-3ß-ol, suppressed the androgen receptor signaling and decreased its protein level in two prostate cancer cell lines, LNCaP and LAPC-4. Interaction of compounds with CYP17A1 and the androgen receptor was confirmed and described by molecular docking.

5-Chloroisoxazoles: A Versatile Starting Material for the Preparation of Amides, Anhydrides, Esters, and Thioesters of 2H-Azirine-2-carboxylic Acids.

Amides, anhydrides, esters, and thioesters of 2H-azirine-2-carboxylic acids were prepared by a rapid procedure at room temperature involving FeCl2-catalyzed isomerization of 5-chloroisoxazoles to 2H-azirine-2-carbonyl chlorides, followed by reaction with N-, O-, or S-nucleophiles mediated by an ortho-substituted pyridine. With readily available chloroisoxazoles and a nucleophile, 2-picoline can be used as an inexpensive base. When a high yield of the acylation product is important, the reagent 2-(trimethylsilyl)pyridine/ethyl chloroformate is more suitable for the acylation with 2H-azirine-2-carbonyl chlorides.

Isoxazole Nucleosides as Building Blocks for a Plausible Proto-RNA.

The question of how RNA, as the principal carrier of genetic information evolved is fundamentally important for our understanding of the origin of life. The RNA molecule is far too complex to have formed in one evolutionary step, suggesting that ancestral proto-RNAs (first ancestor of RNA) may have existed, which evolved over time into the RNA of today. Here we show that isoxazole nucleosides, which are quickly formed from hydroxylamine, cyanoacetylene, urea and ribose, are plausible precursors for RNA. The isoxazole nucleoside can rearrange within an RNA-strand to give cytidine, which leads to an increase of pairing stability. If the proto-RNA contains a canonical seed-nucleoside with defined stereochemistry, the seed-nucleoside can control the configuration of the anomeric center that forms during the in-RNA transformation. The results demonstrate that RNA could have emerged from evolutionarily primitive precursor isoxazole ribosides after strand formation.

Synthesis of 3,4,5-trisubstituted isoxazoles in water via a [3 + 2]-cycloaddition of nitrile oxides and 1,3-diketones, ß-ketoesters, or ß-ketoamides.

Herein we report a method for the synthesis of 3,4,5-trisubstituted isoxazoles in water under mild basic conditions at room temperature via a [3 + 2]-cycloaddition of nitrile oxides and 1,3-diketones, ß-ketoesters, or ß-ketoamides. We optimized the reaction conditions to control the selectivity of the production of isoxazoles and circumvent other competing reactions, such as O-imidoylation or hetero [3 + 2]-cycloaddition. The reaction happens fast in water and completes within 1-2 hours, which provides an environmentally friendly access to 3,4,5-trisubstituted isoxazoles, an important class of structures found in numerous bioactive natural products and pharmaceuticals. Additionally, we optimized the reaction conditions to produce trifluoromethyl-substituted isoxazoles, a prevalent scaffold in biomedical research and drug discovery programs. We also proposed a plausible mechanism for the selectivity of the [3 + 2]-cycloaddition reaction to produce 3,4,5-trisubstituted isoxazoles. Not to be overlooked are our optimized reaction conditions for the dimerization of hydroximoyl chlorides to form furoxans also known as 1,2,5-oxadiazole 2-oxides, a class of structures with important biological activities due to their unique electronic nature and coordination ability.

Dibenzazepine-linked isoxazoles: New and potent class of α-glucosidase inhibitors.

α-Glucosidase inhibition is a valid approach for controlling hyperglycemia in diabetes. In the current study, new molecules as a hybrid of isoxazole and dibenzazepine scaffolds were designed, based on their literature as antidiabetic agents. For this, a series of dibenzazepine-linked isoxazoles (33-54) was prepared using Nitrile oxide-Alkyne cycloaddition (NOAC) reaction, and evaluated for their α-glucosidase inhibitory activities to explore new hits for treatment of diabetes. Most of the compounds showed potent inhibitory potency against α-glucosidase (EC 3.2.1.20) enzyme (IC50 = 35.62 ± 1.48 to 333.30 ± 1.67 µM) using acarbose as a reference drug (IC50 = 875.75 ± 2.08 µM). Structure-activity relationship, kinetics and molecular docking studies of active isoxazoles were also determined to study enzyme-inhibitor interactions. Compounds 33, 40, 41, 46, 48-50, and 54 showed binding interactions with critical amino acid residues of α-glucosidase enzyme, such as Lys156, Ser157, Asp242, and Gln353.

Glioblastoma-specific anticancer activity of newly synthetized 3,5-disubstituted isoxazole and 1,4-disubstituted triazole-linked tyrosol conjugates.

Two series of 3,5-disubstituted isoxazoles (6a-e) and 1,4-disubstituted triazoles (8a-e) derivatives have been synthesized from tyrosol (1), a natural phenolic compound, detected in several natural sources such as olive oil, and well-known by its wide spectrum of biological activities. Copper-catalyzed microwave-assisted 1,3-dipolar cycloaddition reactions between tyrosol-alkyne derivative 2 and two series of aryl nitrile oxides (5a-e) and azides (7a-e) regiospecifically afforded 3,5-disubstituted isoxazoles (6a-e) and 1,4-triazole derivatives (8a-e), respectively in quantitative yields. Synthesized compounds were purified and characterized by spectroscopic means including 1D and 2D NMR techniques and HRMS analysis. The newly prepared hybrid molecules have been evaluated for their anticancer and hemolytic activities. Results showed that most derivatives displayed significant antiproliferative activity against human glioblastoma cancer cells (U87) in a dose-dependent manner. Compounds 6d (IC50 = 15.2 ± 1.0 µg/mL) and 8e (IC50 = 21.0 ± 0.9 µg/mL) exhibited more potent anticancer activity. Moreover, most derivatives displayed low hemolytic activity, even at higher concentrations which suggested that these classes of compounds are suitable candidates for further in vivo investigations. The obtained results allow us to consider the newly synthesized isoxazole- and triazole-linked tyrosol derivatives as promising scaffolds for the development of effective anticancer agents.

Novel N-benzylpiperidine derivatives of 5-arylisoxazole-3-carboxamides as anti-Alzheimer's agents.

The complex pathophysiology of Alzheimer's disease (AD) has prompted researchers to develop multitarget-directed molecules to find an effective therapy against the disease. In this context, a novel series of N-(1-benzylpiperidin-4-yl)-5-arylisoxazole-3-carboxamide derivatives were designed, synthesized, and evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In vitro biological evaluation demonstrated that compound 4e was the best AChE (IC50 = 16.07 µM) and BuChE inhibitor (IC50 = 15.16 µM). A kinetic study of 4e was also conducted, which presented a mixed-type inhibition for both enzymes. Molecular docking studies revealed that compound 4e fitted well into the active sites of AChE and BuChE, forming stable and strong interactions with key residues Glu199, Trp84, Asp72, Tyr121, and Phe288 in AChE and His438, Trp82, Ala328, Tyr332, Phe329, Thr120, and Pro285 in BuChE. Besides, the inhibition of BACE1 by 4e and the biometal chelation activity of 4e were measured. The neuroprotective assessment revealed that 4e exhibited 23.2% protection at 50 µM toward amyloid-beta-induced PC12 neuronal cells. Overall, this study exhibited that compound 4e was a promising compound targeting multiple factors associated with AD.

Trifluoromethylated Flavonoid-Based Isoxazoles as Antidiabetic and Anti-Obesity Agents: Synthesis, In Vitro α-Amylase Inhibitory Activity, Molecular Docking and Structure-Activity Relationship Analysis.

Diabetes mellitus is a major health problem globally. The management of carbohydrate digestion provides an alternative treatment. Flavonoids constitute the largest group of polyphenolic compounds, produced by plants widely consumed as food and/or used for therapeutic purposes. As such, isoxazoles have attracted the attention of medicinal chemists by dint of their considerable bioactivity. Thus, the main goal of this work was to discover new hybrid molecules with properties of both flavonoids and isoxazoles in order to control carbohydrate digestion. Moreover, the trifluoromethyl group is a key entity in drug development, due to its strong lipophilicity and metabolic stability. Therefore, the present work describes the condensation of a previously synthesized trifluoromethylated flavonol with different aryl nitrile oxides, affording 13 hybrid molecules indicated as trifluoromethylated flavonoid-based isoxazoles. The structures of the obtained compounds were deduced from by 1H NMR, 13C NMR, and HRMS analysis. The 15 newly synthesized compounds inhibited the activity of α-amylase with an efficacy ranging from 64.5 ± 0.7% to 94.7 ± 1.2% at a concentration of 50 µM, and with IC50 values of 12.6 ± 0.2 µM-27.6 ± 1.1 µM. The most effective compounds in terms of efficacy and potency were 3b, 3h, 3j, and 3m. Among the new trifluoromethylated flavonoid-based isoxazoles, the compound 3b was the most effective inhibitor of α-amylase activity (PI = 94.7 ± 1.2% at 50 µM), with a potency (IC50 = 12.6 ± 0.2 µM) similar to that of the positive control acarbose (IC50 = 12.4 ± 0.1 µM). The study of the structure-activity relationship based on the molecular docking analysis showed a low binding energy, a correct mode of interaction in the active pocket of the target enzyme, and an ability to interact with the key residues of glycosidic cleavage (GLU-230 and ASP-206), explaining the inhibitory effects of α-amylase established by several derivatives.

New series of isoxazole derivatives targeting EGFR-TK: Synthesis, molecular modeling and antitumor evaluation.

New series of isoxazole derivatives were synthesized and evaluated for in vitro antitumor activity against HepG2, MCF-7 and HCT-116 cancer cells. Results showed that 4b and 25a are the most potent members against the three cancer cells (IC50 = 6.38-9.96 µM). Further, 4a, 8a and 16b showed strong activity against the three cancer cells, whereas 6b, 10a, 10b and 16a exhibited moderate activity against the three cancer cells. Moreover, 25a showed low cytotoxicity against WISH and WI38 normal cells (IC50 = 53.19 ± 3.1 and 38.64 ± 2.8 µM, respectively), and it might be used as a potent and safe antitumor agent. The nine active compounds 4a, 4b, 6b, 8a, 10a, 10b, 16a, 16b and 25a were studied for EGFR-TK inhibitory activity, where 10a, 10b and 25a showed the highest inhibitory activity (IC50 = 0.064 ± 0.001, 0.066 ± 0.001 and 0.054 ± 0.001 µM, respectively). Compound 25a was also assessed against other four target proteins, and it showed promising inhibitory activities against VEGFR-2, CK2α and topoisomerase IIß, and acceptable inhibitory activity against tubulin polymerization. Cell cycle analysis of cancer cells treated with 25a proved that it induces cell cycle arrest at G2/M and pre-G1 phases. Furthermore, it was confirmed that 25a induces cancer cell death through apoptosis, supported by increased caspases 3/9 levels and increased Bax/Bcl-2 ratio in the three cancer cells. In addition, docking studies proved the exact fit of 25a into the active site of EGFR-TK, VEGFR-2, CK2α, topoisomerase IIß and tubulin. Lipinski's rule and Veber's standards were also analyzed, and results illustrated that 25a is expected to be well absorbed orally.

Synthesis and biological evaluations of novel isoxazoles and furoxan derivative as anti-inflammatory agents.

Novel isoxazoles (10, 12a&b, 15a-c) and the furoxan derivative (14) have been prepared as new safe anti-inflammatory agents from the hydroximoyl 9. All compounds were evaluated for COX-1\COX-2 and most of them showed promising selectivity. The furoxan derivative 14 gave 59% inhibitory activity using carrageenan induced paw rat edema model. Ulcer index experiment and histo-pathological study of stomach samples were also included. Also the proposed binding mode of certain newly synthesized compounds with COX-2 isoform was briefly discussed.

Regioselective synthesis of heterocyclic N-sulfonyl amidines from heteroaromatic thioamides and sulfonyl azides.

N-Sulfonyl amidines bearing 1,2,3-triazole, isoxazole, thiazole and pyridine substituents were successfully prepared for the first time by reactions of primary, secondary and tertiary heterocyclic thioamides with alkyl- and arylsulfonyl azides. For each type of thioamides a reliable procedure to prepare N-sulfonyl amidines in good yields was found. Reactions of 1-aryl-1,2,3-triazole-4-carbothioamides with azides were shown to be accompanied with a Dimroth rearrangement to form 1-unsubstituted 5-arylamino-1,2,3-triazole-4-N-sulfonylcarbimidamides. 2,5-Dithiocarbamoylpyridine reacts with sulfonyl azides to form a pyridine bearing two sulfonyl amidine groups.

Facile and highly diastereo and regioselective synthesis of novel octahydroacridine-isoxazole and octahydroacridine-1,2,3-triazole molecular hybrids from citronella essential oil.

A novel and highly efficient synthetic approach for the expedite construction of new octahydroacridine-isoxazole- and octahydroacridine-1,2,3-triazole-based molecular hybrids is first reported. Rapid access to the octahydroacridine core was achieved in a highly diastereoselective fashion via cationic Povarov reaction of N-propargyl anilines and citronella essential oil (Cymbopogon nardus). The subsequent 1,3-dipolar and Cu (I) catalyzed alkyne-azide cycloaddition reaction of the terminal alkyne fragment with the corresponding oxime or azide affords the desired 3,5-isoxazoles and 1,2,3-triazoles, respectively, as interesting molecular hybrid models for pharmacological studies.

Access to 18 F-labelled isoxazoles by ruthenium-promoted 1,3-dipolar cycloaddition of 4-[18 F]fluoro-N-hydroxybenzimidoyl chloride with alkynes.

4-[18 F]Fluoro-N-hydroxybenzimidoyl chloride (18 FBIC), an 18 F-labelled aromatic nitrile oxide, was developed as building block for Ru-promoted 1,3-dipolar cycloaddition with alkynes. 18 FBIC is obtained in a one-pot synthesis in up to 84% radiochemical yield (RCY) starting from [18 F]fluoride with 4-[18 F]fluorobenzaldehyde (18 FBA) and 4-[18 F]fluorobenzaldehyde oxime (18 FBAO) as intermediates, by reaction of 18 FBAO with N-chlorosuccinimide (NCS). 18 FBIC was found to be a suitable and stable synthon to give access to 18 F-labelled 3,4-diarylsubstituted isoxazoles by [Cp*RuCl(cod)]-catalysed 1,3-dipolar cycloaddition with various alkynes. So the radiosynthesis of a fluorine-18-labelled COX-2 inhibitor [18 F]1b, a close derivative of valdecoxib, was performed with 18 FBIC and 1-ethynyl-4-(methylsulfonyl)benzene, providing [18 F]1b in up to 40% RCY after purification in 85 minutes. The application of 18 FBIC as a building block in the synthesis of 18 F-labelled heterocycles will generally extend the portfolio of available PET radiotracers.

Isoxazole Derivatives as Regulators of Immune Functions.

In this review, we present reports on the immunoregulatory properties of isoxazole derivatives classified into several categories, such as immunosuppressive, anti-inflammatory, immunoregulatory, and immunostimulatory compounds. The compounds were tested in various models using resident cells from rodents and humans, cell lines, and experimental animal disease models corresponding to human clinical situations. Beneficial features of the described isoxazole derivatives include low toxicity and good bioactivity at low doses. In a majority of studies, the activities of investigated compounds were comparable or even higher than registered reference drugs. Whenever possible, a plausible mechanism of action of the investigated compounds and their potential therapeutic utility were proposed. Among the described compounds, particular attention was paid to the class of immune stimulators with a potential application in chemotherapy patients.