Synthesis and Antimycobacterial Evaluation of Novel Pyrazole-Isoxazolines and Pyrazole-Isoxazoles.

This study reports the synthesis of a new series of pyrazole-isoxazolines, at very good yields, from the cyclocondensation reaction of pyrazole-enaminones with hydroxylamine hydrochloride. Dehydration of the pyrazole-isoxazolines furnished another new series of the respective pyrazole-isoxazoles, at excellent yields. Both series of the obtained compounds were screened for antimycobacterial activity, and compounds 4 f and 5 c showed significant inhibition of bacterial growth with a time- and concentration-dependent bactericidal effect. Cytotoxicity tests in VERO cell line did not indicate toxicity of compounds 4 f and 5 c regarding cellular prediction, NO production or dsDNA release. However, both compounds were associated with an increase in total ROS levels, providing induction of oxidative stress, but without compromising cellular targets. These results highlight compounds 4 f and 5 c as promising candidates for antimycobacterial treatment with a favorable safety profile.

Screening for antimicrobial and antioxidant activities of quinazolinone based isoxazole and isoxazoline derivatives, synthesis and In silico studies.

Two novel series of quinazolinone based isoxazole and isoxazoline hybrid compounds were synthesized from 6-aminoquinazolinone as a key precursor. The title compounds were achieved in synthetic routes via propargylation and allylation reactions of the precursor followed by cyclization with various chloroximes. The new compounds 4a-g and 6a-g were screened for their antimicrobial activity against two Gram-positive bacteria, two Gram-negative bacteria and two fungi by employing Ampicillin and Itraconazole as standard reference. Among all, the 4-bromosubstituted analogues in isoxazole series 4d and in isoxazoline series 6d demonstrated potent activity against all bacterial and fungal strains compared to Ampicillin as well as Itraconazole. The MIC of these compounds were determined as 0.012 µM. The antioxidant investigation revealed that compounds 4f and 6f with dimethyl substitution, exhibited significant activity. Their respective IC50 values were 1.28 ± 0.33, 1.39 ± 0.38 µM and 1.07 ± 0.24, 1.10 ± 0.26 µM, when compared to Ascorbic acid. The compounds 4 g and 6 g with dichloro substitution, exhibited promising results with IC50 values were 2.72 ± 0.34 µM and 2.78 ± 0.41 µM for 4 g, and 2.24 ± 0.93 µM and 2.45 ± 0.53 µM for 6 g, respectively. Their antimicrobial and antioxidant activities were authenticated by the molecular docking study against crystal structure of DNA gyrase and NADPH oxidase. The predicted ADME properties of these molecules progressed favourable drug-likeness properties.

Design, synthesis, and evaluation of novel isoxazoline derivatives containing 2-phenyloxazoline moieties as potential insecticides.

Isoxazoline insecticides have shown broad-spectrum insecticidal activity against a variety of insect pests. However, the high toxicity of isoxazoline compounds towards honeybees restricts their application in crop protection. To mitigate this issue, a series of isoxazoline derivatives containing 2-phenyloxazoline were designed and synthesized. Bioassays revealed that several compounds exhibited promising insecticidal activities against Plutella xylostella, with G28 showing particularly excellent insecticidal activity, reflected by an LC50 value of 0.675 mg/L, which is comparable to that of fluxametamide (LC50 = 0.593 mg/L). Furthermore, G28 also exhibited effective insecticidal activity against Solenopsis invicta. Importantly, bee toxicity experiments indicated that G28 had significantly lower acute oral toxicity (LD50 = 2.866 µg/adult) compared to fluxametamide (LD50 = 1.083 µg/adult) and fluralaner (LD50 = 0.022 µg/adult), positioning it as a promising candidate with reduced toxicity to bees. Theoretical simulation further elucidated the reasons for the selective differences in the ability of isoxazoline to achieve higher insecticidal activity while maintaining lower bee toxicity. This research suggests that isoxazoline compounds containing 2-phenyloxazoline group hold potential as new insecticide candidates and offers insights into the development of novel isoxazoline insecticides with both high efficacy and environmental safety.

Putative pemphigus-like reaction to oral fluralaner in a dog.

A 9-month-old mixed-breed dog developed generalised pustular dermatitis, accompanied by lethargy and hyperthermia, 7 days after oral fluralaner administration. Dermatopathological and microbiological evaluations were consistent with a pustular acantholytic dermatitis. A 4-month course of immunosuppressive therapy resulted in complete remission of lesions, which did not recur after therapy was withdrawn.

Un chien croisé âgé de 9 mois a développé une dermatite pustuleuse généralisée, accompagnée de léthargie et d'hyperthermie, 7 jours après l'administration orale de fluralaner. Les évaluations dermatopathologiques et microbiologiques sont compatibles avec une dermatite acantholytique pustuleuse. Un traitement immunosuppresseur de 4 mois induit une rémission complète des lésions, qui n'ont pas récidivé après l'arrêt du traitement.

Um cão mestiço de nove meses de idade desenvolveu uma dermatite pustular generalizada, acompanhada de letargia e hipertermia, 7 dias após administração de fluralaner. As avaliações dermatohistopatológicas e microbiológicas foram consistentes com uma dermatite pustular acantolítica. Um curso de quatro meses com terapia imunossupressiva resultou em remissão completa das lesões, que não recidivaram após o fim do tratamento.

Un perro mestizo de 9 meses desarrolló dermatitis pustulosa generalizada, acompañada de letargo e hipertermia, 7 días después de la administración oral de fluralaner. Las evaluaciones dermatopatológicas y microbiológicas fueron compatibles con una dermatitis pustulosa acantolítica. Un tratamiento inmunosupresor de 4 meses dio como resultado la remisión completa de las lesiones, que no reaparecieron después de retirar el tratamiento.

tert-Butyl Nitrite-Induced Radical Nitrile Oxidation Cycloaddition: Synthesis of Isoxazole/Isoxazoline-Fused Benzo 6/7/8-membered Oxacyclic Ketones.

A practical metal-free and additive-free approach for the synthesis of 6/7/8-membered oxacyclic ketone-fused isoxazoles/isoxazolines tetracyclic or tricyclic structures is reported through Csp3-H bond radical nitrile oxidation and the intramolecular cycloaddition of alkenyl/alkynyl-substituted aryl methyl ketones. This convenient approach enables the simultaneous formation of isoxazole/isoxazoline and 6/7/8-membered oxacyclic ketones to form polycyclic architectures by using tert-butyl nitrite (TBN) as a non-metallic radical initiator and N-O fragment donor.

Synthesis and In Silico Analysis of New Polyheterocyclic Molecules Derived from [1,4]-Benzoxazin-3-one and Their Inhibitory Effect against Pancreatic α-Amylase and Intestinal α-Glucosidase.

This study focuses on synthesizing a new series of isoxazolinyl-1,2,3-triazolyl-[1,4]-benzoxazin-3-one derivatives 5a-5o. The synthesis method involves a double 1,3-dipolar cycloaddition reaction following a "click chemistry" approach, starting from the respective [1,4]-benzoxazin-3-ones. Additionally, the study aims to evaluate the antidiabetic potential of these newly synthesized compounds through in silico methods. This synthesis approach allows for the combination of three heterocyclic components: [1,4]-benzoxazin-3-one, 1,2,3-triazole, and isoxazoline, known for their diverse biological activities. The synthesis procedure involved a two-step process. Firstly, a 1,3-dipolar cycloaddition reaction was performed involving the propargylic moiety linked to the [1,4]-benzoxazin-3-one and the allylic azide. Secondly, a second cycloaddition reaction was conducted using the product from the first step, containing the allylic part and an oxime. The synthesized compounds were thoroughly characterized using spectroscopic methods, including 1H NMR, 13C NMR, DEPT-135, and IR. This molecular docking method revealed a promising antidiabetic potential of the synthesized compounds, particularly against two key diabetes-related enzymes: pancreatic α-amylase, with the two synthetic molecules 5a and 5o showing the highest affinity values of 9.2 and 9.1 kcal/mol, respectively, and intestinal α-glucosidase, with the two synthetic molecules 5n and 5e showing the highest affinity values of -9.9 and -9.6 kcal/mol, respectively. Indeed, the synthesized compounds have shown significant potential as antidiabetic agents, as indicated by molecular docking studies against the enzymes α-amylase and α-glucosidase. Additionally, ADME analyses have revealed that all the synthetic compounds examined in our study demonstrate high intestinal absorption, meet Lipinski's criteria, and fall within the required range for oral bioavailability, indicating their potential suitability for oral drug development.

Protein Modification via Nitrile Oxide-Dehydroalanine Cycloaddition: Formation of Isoxazoline Ring on the Protein Backbone.

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.

Green synthesis and anti-biofilm activities of 3,5-disubstituted isoxazoline/isoxazole-linked secondary sulfonamide derivatives on Pseudomonas aeruginosa.

The search for new classes of antibiotics is a real concern of public health due to the emergence of multi-resistant bacteria strains. We report herein the synthesis and characterization of a new series of 13 molecules combining isoxazoline/isoxazole sulfonamides and hydrazides motives. These molecules were obtained according to a costless eco-friendly procedure, and a one-pot three-step cascade synthesis under ultrasonic cavitation. All the synthesized compounds were fully characterized by HRMS, 1H NMR, 13C NMR spectroscopy and HPLC analysis. These new molecules have been evaluated against the major human opportunistic pathogen Pseudomonas aeruginosa to determine their potential to affect its growth and biofilm formation or dispersion. Two derivatives (5a and 6a) demonstrated their ability to destabilize a mature biofilm by about 50 % within 24 h. This may pave the way to the development of a new class of compounds affecting biofilm, which are easy to synthesize according to green chemistry processes.

Selection for, and characterization of, fluralaner resistance in the house fly, Musca domestica.

House flies, Musca domestica (L), are the mechanical vector of >100 human and animal pathogens, including those that are antibiotic-resistant. Given that house flies are associated closely with human and livestock activity, they present medical and veterinary health concerns. Although there are numerous strategies for control of house fly populations, chemical control has been favored in many facilities. Products with pyrethroid active ingredients have been used predominantly for >35 years in space sprays. As a result, strong selection for pyrethroid resistance has led to reduced control of many populations. Reliance on a limited number of insecticides for decades has created fly control problems necessitating the discovery and formulation of new control chemistries. Fluralaner is a relatively new insecticide and acaricide (first reported in 2010), belonging to the isoxazoline class. These insecticides target the glutamate- and gamma-aminobutyric acid-gated (GABA) chloride channels, which is a different mode of action from other insecticides used against house flies. Although is it not currently registered for house fly control in the United States, previous work has shown that fluralaner is highly toxic to house flies and that there was limited cross-resistance found in laboratory strains having high levels of resistance to other insecticides. Herein, we characterized the time and age dependency of fluralaner toxicity, detected cross-resistance in populations from across the United States, and selected a highly resistant (>11,000-fold) house fly strain. We found that the fluralaner LD50 of 18-24 h old flies was 2-fold higher than for 5-6 d old flies. This appears to be due to more rapid penetration of fluralaner into the 5-6 d old flies. Fluralaner resistance was inherited as an intermediate to incompletely dominant trait and was mapped to chromosomes 5 and 3. Resistance could be suppressed to 7-fold with piperonyl butoxide, suggesting that cytochrome P450 (CYP)-mediated detoxification was a major mechanism of resistance. Decreased penetration was also demonstrated as a mechanism of resistance. The utility of fluralaner for house fly control is discussed.

PxRdl2 dsRNA increased the insecticidal activities of GABAR-targeting compounds against Plutella xylostella.

The utilization of RNA interference (RNAi) for pest management has garnered global interest. The bioassay results suggested the knockout of the PxRdl2 gene significantly increased the insecticidal activities of the γ-aminobutyric acid receptor (GABAR)-targeting compounds (fipronil, two pyrazoloquinazolines, and two isoxazolines), thereby presenting a viable target gene for RNAi-mediated pest control. Consequently, we suggest enhancing the insecticidal activities of GABAR-targeting compounds by knockdown the transcript level of PxRdl2. Furthermore, PxRdl2 dsRNA was expressed in HT115 Escherichia coli to reduce costs and protect dsRNA against degradation. In comparison to in vitro synthesized dsRNA, the recombinant bacteria (ds-B) exhibited superior interference efficiency and greater stability when exposed to UV irradiation. Collectively, our results provide a strategy for insecticide spray that combines synergistically with insecticidal activities by suppressing PxRdl2 using ds-B and may be beneficial for reducing the usage of insecticide and slowing pest resistance.

Discovery of a Novel Class of Acylthiourea-Containing Isoxazoline Insecticides against Plutella xylostella.

Isoxazoline structures are widely found in natural products and are rich in biological activities. This study discloses the development of a series of novel isoxazoline derivatives by introducing acylthiourea fragments to access insecticidal activity. All synthetic compounds were examined for their insecticidal activity against Plutella xylostella, with results showing moderate to strong activity. Based on this, the structure-activity relationship analysis was carried out via the constructed three-dimensional quantitative structure-activity relationship model to further guide the structure optimization, resulting in the optimal compound 32. The LC50 of compound 32 against Plutella xylostella was 0.26 mg/L, demonstrating better activity than the positive control, ethiprole (LC50 = 3.81 mg/L), avermectin (LC50 = 12.32 mg/L), and compounds 1-31. The insect GABA enzyme-linked immunosorbent assay demonstrated that compound 32 might act on the insect GABA receptor, and the molecular docking assay further illustrated the mode of action of compound 32 with the GABA receptor. In addition, the proteomics analysis indicated that the action of compound 32 on Plutella xylostella was multi-pathway.

Kinetic Study on the Base-Catalyzed Imine-Enamine Tautomerism of a Chiral Biologically Active Isoxazoline Derivative by HPLC on Amylose Tris(3,5-dimethylphenylcarbamate) Chiral Stationary Phase.

Isoxazoline is a nitrogen- and oxygen-containing five-membered heterocyclic scaffold with diverse biological profiles such as antimicrobial, fungicidal, anticancer, antiviral, analgesic and anti-inflammatory activity. Accordingly, the use of this peculiar structural framework in drug discovery is a successful strategy for the development of new drug candidates. Here, a chiral saccharin/isoxazoline hybrid was considered to investigate the tendency of the imine moiety of the heterocyclic ring to tautomerize to the enamine form in the presence of a basic catalyst. The pseudo-first-order rate constants for the base-catalyzed tautomerization process were measured in different solvents and at different temperatures by off-column kinetic experiments based on the amylose (3,5-dimethylphenylcarbamate)-type chiral stationary phase. The kinetic results obtained in this study may be a useful aid in the perspective of designing experimental conditions to control the stereointegrity of these types of pharmacologically active compounds and drive their synthesis toward the preferred, imine or enamine, tautomer.

Electrochemical Activation of Nitromethane to Construct Isoxazoline Aldoximes.

Activation of nitromethane to endow new reactivity is an interesting and meaningful but also challenging topic. Herein, we report an electrochemical activation of nitromethane to serve as both the heterocyclic skeleton and oxime sources for the construction of isoxazoline aldoximes. The isoxazoline aldoximes that are prepared by four steps with the reported strategy are synthesized in a single step from low-cost and readily available nitromethane and olefins with moderate to excellent yields under our electrochemical conditions. The reaction also takes advantage of high atom-economy and E-selectivity. Moreover, the mechanism is studied by control experiments, a kinetic isotope effect (KIE) study, cyclic voltammogram (CV) experiments, and density functional theory (DFT) calculations. The mechanistic results reveal that nitromethane may be activated under electrochemical conditions to deliver a 1,2,5-oxadiazole 2-oxide intermediate, which undergoes [3+2] cycloaddition with olefins to yield isoxazoline aldoximes.

Electrochemical Synthesis of Isoxazolines: Method and Mechanism.

An electrochemical method for the green and practical synthesis of a broad range of substituted isoxazoline cores is presented. Both aryl and more challenging alkyl aldoximes are converted to the desired isoxazoline in an electrochemically enabled regio- and diastereoselective reaction with electron-deficient alkenes. Additionally, in-situ reaction monitoring methods compatible with electrochemistry equipment have been developed in order to probe the reaction pathway. Supporting analyses from kinetic (time-course) modelling and density functional theory support a stepwise, radical-mediated mechanism, and discounts hypothesised involvement of closed shell [3+2] cycloaddition pathways.

Synthesis and some enzyme inhibition effects of isoxazoline and pyrazoline derivatives including benzonorbornene unit.

Four new and four known isoxazoline derivatives were synthesized from the reactions of benzonorbornadiene with nitrile oxides formed from the corresponding benzaldehydes. Three new and one known pyrazoline derivatives were also synthesized from the reactions of the benzonorbornadiene with nitrile imines formed from the corresponding compounds. The synthesized nitrogen-based novel heterocyclic compounds were evaluated against the human carbonic anhydrase isoenzymes I and II (hCA I and hCA II), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes. The synthesized nitrogen-based novel heterocyclic compounds showed IC50 values in the range of 2.69-7.01 against hCA I, 2.40-4.59 against hCA II, 0.81-1.32 µM against AChE, and 20.83-1.70 µM against BChE enzymes. On the contrary, nitrogen-based novel heterocyclic compounds demonstrated Ki values between 2.93 ± 0.59-8.61 ± 1.39 against hCA I, 2.05 ± 0.62-4.97 ± 0.95 against hCA II, 0.34 ± 0.02-0.92 ± 0.17 nM against AChE, and 0.50 ± 0.04-1.20 ± 0.16 µM against BChE enzymes. The synthesized nitrogen-based novel heterocyclic compounds exhibited effective inhibition profiles against both indicated metabolic enzymes. These results may contribute to the development of new drugs particularly to treat some disorders, which are widespread in the world including glaucoma and Alzheimer's diseases.

State-dependent inhibition of GABA receptor channels by the ectoparasiticide fluralaner.

γ-Aminobutyric acid (GABA) receptors (GABARs) are ligand-gated Cl- channels, which cause an influx of Cl- that inhibits excitation in postsynaptic cells upon activation. GABARs are important targets for drugs and pest control chemicals. We previously reported that the isoxazoline ectoparasiticide fluralaner inhibits GABA-induced currents in housefly (Musca domestica) GABARs by binding to the putative binding site in the transmembrane subunit interface. In the present study, we investigated whether fluralaner inhibits the GABA response in the GABAR activated state, the resting state, or both, using two-electrode voltage clamp electrophysiology protocols. We found that inhibition progresses over time to steady-state levels by repeated short applications of GABA during fluralaner perfusion. The GABA response was not impaired by fluralaner treatment in the GABAR resting state. However, once inhibited, the GABA response was not restored by repeated applications of GABA. These findings suggest that fluralaner might reach the binding site of the activated conformation of GABARs in a stepwise fashion and tightly bind to it.

The mode of action of isocycloseram: A novel isoxazoline insecticide.

Isocycloseram is a novel isoxazoline insecticide and acaricide with activity against lepidopteran, hemipteran, coleopteran, thysanopteran and dipteran pest species. Isocycloseram selectively targets the invertebrate Rdl GABA receptor at a site that is distinct to fiproles and organochlorines. The widely distributed cyclodiene resistance mutation, A301S, does not affect sensitivity to isocycloseram, either in vitro or in vivo, demonstrating the suitability of isocylsoseram to control pest infestations with this resistance mechanism. Detailed studies demonstrated that the binding sites relevant to the insecticidal activity of avermectins and isocycloseram are distinct. Isocycloseram was shown to compete for binding with metadiamide insecticides related to broflanilide. In addition, a G335M mutation in the third transmembrane domain of the Rdl GABA receptor, impaired the ability of both isocycloseram and metadiamides to block the GABA mediated response. As such the Insecticides Resistance Action Committee (IRAC) has classified isocycloseram in Group 30 "GABA-Gated Chloride Channel Allosteric Modulators".

Novel isoxazoline-linked 1,3,4-thiadiazole hybrids as anticancer agents: Design, synthesis, biological evaluation, molecular docking, and molecular dynamics simulation.

In the current study, natural (R)-carvone was utilized as a starting material for the efficient synthesis of two series of isoxazoline derivatives bearing the 1,3,4-thiadiazole moiety. The new compounds were obtained in good yields and were characterized by 1 H and 13 C NMR and HRMS analysis. The newly synthesized monoterpenic isoxazoline 1,3,4-thiadiazole and their thiosemicarbazone intermediate derivatives were evaluated for their anticancer activity in four cancer cell lines (HT-1080, A-549, MCF-7, and MDA-MB-231). Most of the synthesized compounds exhibited moderate to high anticancer effects. Compound 13c showed the highest anticancer activity with IC50 values ranging from 19.33 ± 1.81 to 34.81 ± 3.03 µM. Further investigation revealed that compounds 12e and 13c could inhibit the cell growth of HT-1080 and MCF-7 cells by inducing apoptosis through caspase-3/7 activation. The apoptotic effect was accompanied by an S phase and G2/M cell cycle arrest for 13c and 12e, respectively. Compounds 12e and 13c were assessed in silico using molecular docking and molecular dynamics. We found that compound 13c is moderately active against the caspase-3 protein, which triggers apoptosis via intrinsic and extrinsic routes, making compound 13c a promising candidate to activate the proapoptotic protein (caspase-3).

Current review of isoxazoline ectoparasiticides used in veterinary medicine.

The isoxazolines are a novel class of ectoparasiticides with potent inhibitory activity on glutamate- and gamma-aminobutyric acid-gated chloride channel located in nervous system of invertebrates. In recent years, studies have been performed to evaluate the efficacy and safety of isoxazolines against various types of ectoparasites, including fleas, ticks, and mites. As more single and combined isoxazoline products have been approved by the United States Food and Drug Administration and European Medicines Agency, a more comprehensive understanding of isoxazolines becomes essential for veterinary clinical practitioners. This article provides a complete review of isoxazolines with respect to pharmacodynamics, pharmacokinetics, ectoparasiticidal efficacy, and safety, which will provide veterinarians information to allow them to make the best choice of ectoparasiticide for their clients' specific needs.

Arteannuin-B and (3-Chlorophenyl)-2-Spiroisoxazoline Derivative Exhibit Anti-Inflammatory Effects in LPS-Activated RAW 264.7 Macrophages and BALB/c Mice-Induced Proinflammatory Responses via Downregulation of NF-κB/P38 MAPK Signaling.

Host inflammatory responses are key to protection against injury; however, persistent inflammation is detrimental and contributes to morbidity and mortality. Herein, we demonstrated the anti-inflammatory role of Arteannuin-B (1) and its new spirocyclic-2-isoxazoline derivative JR-9 and their side effects in acute inflammatory condition in vivo using LPS-induced cytokines assay, carrageenan-induced paw edema, acetic acid-induced writhing and tail immersion. The results show that the spirocyclic-2-isoxazoline derivative is a potent anti-inflammatory agent with minimal cell toxicity as compared to Arteannuin-B. In addition, the efficacies of these compounds were also validated by flow cytometric, computational, and histopathological analysis. Our results show that the anti-inflammatory response of JR-9 significantly reduces the ability of mouse macrophages to produce NO, TNF-α, and IL-6 following LPS stimulation. Therefore, JR-9 is a prospective candidate for the development of anti-inflammatory drugs and its molecular mechanism is likely related to the regulation of NF-κB and MAPK signaling pathway.