The Antifungal Potential of Niclosamide and Structurally Related Salicylanilides.

Human mycoses cover a diverse field of fungal diseases from skin disorders to systemic invasive infections and pose an increasing global health problem based on ineffective treatment options, the hampered development of new efficient drugs, and the emergence of resistant fungal strains. Niclosamide is currently applied for the treatment of worm infections. Its mechanisms of action, which include the suppression of mitochondrial oxidative phosphorylation (also known as mitochondrial uncoupling), among others, has led to a repurposing of this promising anthelmintic drug for the therapy of further human diseases such as cancer, diabetes, and microbial infections. Given the urgent need to develop new drugs against fungal infections, the considerable antifungal properties of niclosamide are highlighted in this review. Its chemical and pharmacological properties relevant for drug development are also briefly mentioned, and the described mitochondria-targeting mechanisms of action add to the current arsenal of approved antifungal drugs. In addition, the activities of further salicylanilide-based niclosamide analogs against fungal pathogens, including agents applied in veterinary medicine for many years, are described and discussed for their feasibility as new antifungals for humans. Preliminary structure-activity relationships are determined and discussed. Various salicylanilide derivatives with antifungal activities showed increased oral bioavailabilities when compared with niclosamide. The simple synthesis of salicylanilide-based drugs also vouchsafes a broad and cost-effective availability for poorer patient groups. Pertinent literature is covered until 2024.

Therapeutic advantages of the combined use of closantel and moxidectin in lambs parasitized with resistant gastrointestinal nematodes.

The serious widespread development of nematode resistance has motivated the use of combined anthelmintic formulations. However, the advantages/disadvantages of the combined use of anthelmintics require further scientific characterization. The goals of the current trial were a) to characterize the pharmacokinetics of closantel (CLO) and moxidectin (MXD) administered both subcutaneously (sc) and orally either separately or co-administered (CLO + MXD) to lambs; b) to compare the nematodicidal activity of both molecules given individually or co-administered to lambs infected with resistant nematodes. Seventy (70) Corriedale lambs naturally infected with multiple resistant gastrointestinal nematodes were involved in the pharmacokinetic and efficacy trials. The animals were allocated into six groups (n = 10) and treated with either CLO, MXD, or with the CLO + MXD combined formulation by both the oral and sc routes. Additionally, an untreated control group (n = 10) was included for the efficacy trial. The efficacy was estimated by the faecal egg count reduction test (FECRT). Higher systemic exposure of both CLO and MXD was observed after the sc compared to the oral administration in lambs. The combined administration of CLO + MXD did not markedly alter their disposition kinetics. At 13 days post-treatment, the administration of both molecules as a single active principle reached efficacy levels ranging between 80% (MXDoral), 84% (CLOoral), 85% (CLOsc), and 92% (MXDsc). The combined oral and sc treatments reached 99% efficacy. No adverse effects were observed after the combined treatment of CLO + MXD, and their co-administration did not show any adverse pharmacokinetic interaction. The combined effect of CLO + MXD successfully restored the maximum efficacy levels, which were not reached by the individual active ingredients.

Salicylanilides and Their Anticancer Properties.

Salicylanilides are pharmacologically active compounds with a wide spectrum of biological effects. Halogenated salicylanilides, which have been used for decades in human and veterinary medicine as anthelmintics, have recently emerged as candidates for drug repurposing in oncology. The most prominent example of salicylanilide anthelmintic, that is intensively studied for its potential anticancer properties, is niclosamide. Nevertheless, recent studies have discovered extensive anticancer potential in a number of other salicylanilides. This potential of their anticancer action is mediated most likely by diverse mechanisms of action such as uncoupling of oxidative phosphorylation, inhibition of protein tyrosine kinase epidermal growth factor receptor, modulation of different signaling pathways as Wnt/ß-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways or induction of B-Raf V600E inhibition. Here we provide a comprehensive overview of the current knowledge about the proposed mechanisms of action of anticancer activity of salicylanilides based on preclinical in vitro and in vivo studies, or structural requirements for such an activity.

Design and synthesis of novel halogen rich salicylanilides as potential antileishmanial agents.

The available therapeutic treatment for leishmaniasis is inadequate and toxic due to side effects, expensive and emergence of drug resistance. Affordable and safe antileishmanial agents are urgently needed and toward this objective, we synthesized a series of 32 novel halogen rich salicylanilides including niclosamide and oxyclozanide and investigated their antileishmanial activity against amastigotes of Leishmania donovani. In vitro data showed fifteen compounds inhibited intracellular amastigotes with an IC50 of below 5 µM and selectivity index above 10. Among 15 active compounds, 14 and 24 demonstrated better activity with an IC50 of 2.89 µM and 2.09 µM respectively and selectivity index is 18. Compound 24 exhibited significant in vivo antileishmanial efficacy and reduced 65% of the splenic parasite load on day 28th post-treatment in the experimental visceral leishmaniasis golden hamster model. The data suggest that 24 can be a promising lead candidate possessing potential to be developed into a leishmanial drug candidate.

Drug screening approach against mycobacterial fatty acyl-AMP ligase FAAL32 renews the interest of the salicylanilide pharmacophore in the fight against tuberculosis.

Tuberculosis (TB) remains a global health crisis, further exacerbated by the slow pace of new treatment options, and the emergence of extreme and total drug resistance to existing drugs. The challenge to developing new antibacterial compounds with activity against Mycobacterium tuberculosis (Mtb), the causative agent of TB, is in part due to unique features of this pathogen, especially the composition and structure of its complex cell envelope. Therefore, targeting enzymes involved in cell envelope synthesis has been of major interest for anti-TB drug discovery. FAAL32 is a fatty acyl-AMP ligase involved in the biosynthesis of the cell wall mycolic acids, and a potential target for drug discovery. To rapidly advance research in this area, we initiated a drug repurposing campaign and screened a collection of 1280 approved human or veterinary drugs (Prestwick Chemical Library) using a biochemical assay that reads out FAAL32 inhibition. These efforts led to the discovery of salicylanilide closantel, and some of its derivatives as inhibitors with potent in vitro activity against M. tuberculosis. These results suggest that salicylanilide represents a potentially promising pharmacophore for the conception of novel anti-tubercular candidates targeting FAAL32 that would open new targeting opportunities. Moreover, this work illustrates the value of drug repurposing campaigns to discover new leads in challenging drug discovery fields.

Hybridization Approach to Identify Salicylanilides as Inhibitors of Tubulin Polymerization and Signal Transducers and Activators of Transcription 3 (STAT3).

The superimposition of the X-ray complexes of cyclohexanediones (i.e., TUB015), described by our research group, and nocodazole, within the colchicine binding site of tubulin provided an almost perfect overlap of both ligands. This structural information led us to propose hybrids of TUB015 and nocodazole using a salicylanilide core structure. Interestingly, salicylanilides, such as niclosamide, are well-established signal transducers and activators of transcription (STAT3) inhibitors with anticancer properties. Thus, different compounds with this new scaffold have been synthesized with the aim to identify compounds inhibiting tubulin polymerization and/or STAT3 signaling. As a result, we have identified new salicylanilides (6 and 16) that showed significant antiproliferative activity against a panel of cancer cells. Both compounds were able to reduce the levels of p-STAT3Tyr705 without affecting the total expression of STAT3. While compound 6 inhibited tubulin polymerization and arrested the cell cycle of DU145 cells at G2/M, similar to TUB015, compound 16 showed a more potent effect on inhibiting STAT3 phosphorylation and arrested the cell cycle at G1/G0, similar to niclosamide. In both cases, no toxicity towards PBMC cells was detected. Thus, the salicylanilides described here represent a new class of antiproliferative agents affecting tubulin polymerization and/or STAT3 phosphorylation.

Double Repositioning: Veterinary Antiparasitic to Human Anticancer.

Drug repositioning, the approach of discovering different uses for existing drugs, has gained enormous popularity in recent years in the anticancer drug discovery field due to the increasing demand for anticancer drugs. Additionally, the repurposing of veterinary antiparasitic drugs for the treatment of cancer is gaining traction, as supported by existing literature. A prominent example is the proposal to implement the use of veterinary antiparasitics such as benzimidazole carbamates and halogenated salicylanilides as novel anticancer drugs. These agents have revealed pronounced anti-tumor activities and gained special attention for "double repositioning", as they are repurposed for different species and diseases simultaneously, acting via different mechanisms depending on their target. As anticancer agents, these compounds employ several mechanisms, including the inhibition of oncogenic signal transduction pathways of mitochondrial respiration and the inhibition of cellular stress responses. In this review, we summarize and provide valuable information about the experimental, preclinical, and clinical trials of veterinary antiparasitic drugs available for the treatment of various cancers in humans. This review suggests the possibility of new treatment options that could improve the quality of life and outcomes for cancer patients in comparison to the currently used treatments.

Bio-evaluation of fluoro and trifluoromethyl-substituted salicylanilides against multidrug-resistant S. aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA) are primary causes of skin and soft tissue infections worldwide. To address the emergency caused due to increasing multidrug-resistant (MDR) bacterial infections, a series of novel fluoro and trifluoromethyl-substituted salicylanilide derivatives were synthesized and their antimicrobial activity was investigated. MIC data reveal that the compounds inhibited S. aureus specifically (MIC 0.25-64 µg/mL). The in vitro cytotoxicity of compounds with MIC < 1 µg/mL against Vero cells led to identification of four compounds (20, 22, 24 and 25) with selectivity index above 10. These four compounds were tested against MDR S. aureus panel. Remarkably, 5-chloro-N-(4'-bromo-3'-trifluoromethylphenyl)-2-hydroxybenzamide (22) demonstrated excellent activity against nine MRSA and three VRSA strains with MIC 0.031-0.062 µg/mL, which is significantly better than the control drugs methicillin and vancomycin. The comparative time-kill kinetic experiment revealed that the effect of bacterial killing of 22 is comparable with vancomycin. Compound 22 did not synergize with or antagonize any FDA-approved antibiotic and reduced pre-formed S. aureus biofilm better than vancomycin. Overall, study suggested that 22 could be further developed as a potent anti-staphylococcal therapeutic.

Salicylanilides Reduce SARS-CoV-2 Replication and Suppress Induction of Inflammatory Cytokines in a Rodent Model.

SARS-CoV-2 virus has recently given rise to the current COVID-19 pandemic where infected individuals can range from being asymptomatic, yet highly contagious, to dying from acute respiratory distress syndrome. Although the world has mobilized to create antiviral vaccines and therapeutics to combat the scourge, their long-term efficacy remains in question especially with the emergence of new variants. In this work, we exploit a class of compounds that has previously shown success against various viruses. A salicylanilide library was first screened in a SARS-CoV-2 activity assay in Vero cells. The most efficacious derivative was further evaluated in a prophylactic mouse model of SARS-CoV-2 infection unveiling a salicylanilide that can reduce viral loads, modulate key cytokines, and mitigate severe weight loss involved in COVID-19 infections. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and a previously established favorable pharmacokinetic profile for the lead salicylanilide renders salicylanilides in general as promising therapeutics for COVID-19.

N-[3,5-Bis(trifluoromethyl)phenyl]-5-bromo-2-hydroxybenzamide Analogues: Novel Acetyl- and Butyrylcholinesterase Inhibitors.

BACKGROUND: Development of acetyl- (AChE) and butyrylcholinesterase (BuChE) inhibitors belongs to viable strategies for the treatment of dementia and other diseases related to decrease in cholinergic neurotransmission. OBJECTIVE: That is why we designed twenty-two analogues of a dual AChEBuChE salicylanilide inhibitor, N-[3,5-bis(trifluoromethyl)phenyl]-5-bromo-2-hydroxybenzamide 1, to improve its potency. METHODS: We prepared N,N-disubstituted (thio)carbamates via direct acylation with (thio)carbamoyl chloride, N-n-alkyl monosubstituted carbamates using isocyanates as well as its salicylanilide core analogues. The derivatives were evaluated in vitro against AChE from electric eel and BuChE from equine serum using spectrophotometric Ellman's method. RESULTS: The compounds showed moderate inhibition of both AChE and BuChE with IC50 from 18.2 to 196.6 µmol.L-1 and 9.2 to 196.2 µmol.L-1, respectively. Importantly, based on the substitution pattern, it is possible to modulate selectivity against AChE or BuChE and some derivatives also produced a balanced inhibition. In general, the most promising analogues were N-alkyl (C2-C6) carbamates and isomers with a changed position of phenolic hydroxyl. N-[3,5-Bis(trifluoromethyl)phenyl]-3-bromo-5- hydroxybenzamide 4a was the best inhibitor of both cholinesterases. CONCLUSION: A wide range of the derivatives improved the activity of the hit 1, they were superior to carbamate drug rivastigmine against AChE and some of them also against BuChE. The most promising derivatives also fit physicochemical space and structural features for CNS drugs together with an escalated lipophilicity.

Ring-Substituted 1-Hydroxynaphthalene-2-Carboxanilides Inhibit Proliferation and Trigger Mitochondria-Mediated Apoptosis.

Ring-substituted 1-hydroxynaphthalene-2-carboxanilides were previously investigated for their antimycobacterial properties. In our study, we have shown their antiproliferative and cell death-inducing effects in cancer cell lines. Cell proliferation and viability were assessed by WST-1 assay and a dye exclusion test, respectively. Cell cycle distribution, phosphatidylserine externalization, levels of reactive oxygen or nitrogen species (RONS), mitochondrial membrane depolarization, and release of cytochrome c were estimated by flow cytometry. Levels of regulatory proteins were determined by Western blotting. Our data suggest that the ability to inhibit the proliferation of THP-1 or MCF-7 cells might be referred to meta- or para-substituted derivatives with electron-withdrawing groups -F, -Br, or -CF3 at anilide moiety. This effect was accompanied by accumulation of cells in G1 phase. Compound 10 also induced apoptosis in THP-1 cells in association with a loss of mitochondrial membrane potential and production of mitochondrial superoxide. Our study provides a new insight into the action of salicylanilide derivatives, hydroxynaphthalene carboxamides, in cancer cells. Thus, their structure merits further investigation as a model moiety of new small-molecule compounds with potential anticancer properties.

2-Hydroxy-N-phenylbenzamides and Their Esters Inhibit Acetylcholinesterase and Butyrylcholinesterase.

The development of novel inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) represents a viable approach to alleviate Alzheimer's disease. Thirty-six halogenated 2-hydroxy-N-phenylbenzamides (salicylanilides) with various substitution patterns and their esters with phosphorus-based acids were synthesized in yields of 72% to 92% and characterized. They were evaluated for in vitro inhibition of AChE from electric eel and BuChE from equine serum using modified Ellman's spectrophotometric method. The benzamides exhibited a moderate inhibition of AChE with IC50 values in a narrow concentration range from 33.1 to 85.8 µM. IC50 values for BuChE were higher (53.5-228.4 µM). The majority of derivatives inhibit AChE more efficiently than BuChE and are comparable or superior to rivastigmine-an established cholinesterases inhibitor used in the treatment of Alzheimer's disease. Phosphorus-based esters especially improved the activity against BuChE with 5-chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl diethyl phosphite 5c superiority (IC50 = 2.4 µM). This derivative was also the most selective inhibitor of BuChE. It caused a mixed inhibition of both cholinesterases and acted as a pseudo-irreversible inhibitor. Several structure-activity relationships were identified, e.g., favouring esters and benzamides obtained from 5-halogenosalicylic acids and polyhalogenated anilines. Both 2-hydroxy-N-phenylbenzamides and esters share convenient physicochemical properties for blood-brain-barrier penetration and thus central nervous system delivery.

Repurposing the anthelminthic salicylanilide oxyclozanide against susceptible and clinical resistant Candida albicans strains.

Current antifungal drugs suffer from limitations including toxicity, adverse interactions with other commonly prescribed drugs, and the emergence of resistant strains. Here, we repurposed the anthelmintic oxyclozanide as a potent antifungal agent against both sensitive and resistant clinical isolates of Candida albicans, as well as other human opportunistic fungi. Antifungal activity of oxyclozanide was enhanced when C. albicans grew in nonfermentable carbon sources. Our data support a mechanism of action where oxyclozanide uncoupled the mitochondrial electron transport from oxidative phosphorylation and perturbed the mitochondrial membrane potential.

Novel salicylanilides from 4,5-dihalogenated salicylic acids: Synthesis, antimicrobial activity and cytotoxicity.

Salicylanilides have proved their activity against tuberculosis (TB). One weak electron-withdrawing substituent is favored at the salicylic part, specially Cl or Br atoms at positions 4 or 5. On the other hand, the antimycobacterial activity of salicylanilides is negatively affected when a strong electron-withdrawing substituent (NO2) is present at the same positions. Herein we describe the synthesis and characterization of novel salicylanilides possessing two weak electron-withdrawing groups (halogen atoms) at their salicylic part and compare their antitubercular activity with their monohalogenated analogues. All dihalogenated derivatives proved to possess antitubercular activity at a very narrow micromolar range (MIC=1-4µM), similar with their most active monohalogenated analogues. More importantly, the most active final molecules were further screened against multidrug resistant strains and found to inhibit their growth at the range of 0.5-4µM.

Antiproliferative and Pro-Apoptotic Effect of Novel Nitro-Substituted Hydroxynaphthanilides on Human Cancer Cell Lines.

Ring-substituted hydroxynaphthanilides are considered as cyclic analogues of salicylanilides, compounds possessing a wide range of pharmacological activities, including promising anticancer properties. The aim of this study was to evaluate the potential anticancer effect of novel nitro-substituted hydroxynaphthanilides with a special focus on structure-activity relationships. The antiproliferative effect was assessed by Water Soluble Tetrazolium Salts-1 (WST-1) assay, and cytotoxicity was evaluated via dye exclusion test. Flow cytometry was used for cell cycle analysis and detection of apoptosis using Annexin V-FITC/PI assay. Protein expression was estimated by Western blotting. Our data indicate that the potential to cause the antiproliferative effect increases with the shift of the nitro substituent from the ortho- to the para-position. The most potent compounds, 3-hydroxy-N-(3-nitrophenyl)naphthalene-2-carboxamide (2), and 2-hydroxy-N-(4-nitrophenyl)-naphthalene-1-carboxamide (6) showed antiproliferative activity against THP-1 and MCF-7 cancer cells without affecting the proliferation of 3T3-L1 non-tumour cells. Compounds 2 and 6 induced the accumulation of THP-1 and MCF-7 cells in G1 phase associated with the downregulation of cyclin E1 protein levels, while the levels of cyclin B1 were not affected. Moreover, compound 2 was found to exert the pro-apoptotic effect on the THP-1 cells. These results suggest that hydroxynaphthanilides might represent a potential model structure for the development of novel anticancer agents.

Novel derivatives of nitro-substituted salicylic acids: Synthesis, antimicrobial activity and cytotoxicity.

Inspired by the high antituberculous activity of novel nitro-substituted derivatives and based on promising predicted ADMET properties we have synthesized a series of 33 salicylanilides containing nitro-group in their salicylic part and evaluated them for their in vitro antimycobacterial, antimicrobial and antifungal activities. The presence of nitro-group in position 4 of the salicylic acid was found to be beneficial and the resulting molecules exhibited minimum inhibitory concentrations (MICs) ranging from 2 to 32 µM against Mycobacterium tuberculosis. The best activity was found for 2-hydroxy-4-nitro-N-[4-(trifluoromethyl)phenyl]benzamide (MIC=2 µM). 4-Nitrosalicylanilides were also found to be active against all Staphylococcus species tested while for MRSA strain 2-hydroxy-4-nitro-N-[4-(trifluoromethyl)phenyl]benzamide's MIC was 0.98 µM. None of the nitrosalicylanilides was active against Enterococcus sp. J 14365/08 and no considerable activity was found against Gram-negative bacteria or fungi. The hepatotoxicity of all nitrosalicylanilides was found to be in the range of their MICs for HepG2 cells.

Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA).

A series of twenty-one salicylanilide N-alkylcarbamates was assessed for novel antibacterial characteristics against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum inhibitory concentration was determined by the broth dilution micro-method with subsequent subcultivation of aliquots to assess minimum bactericidal concentration. The bactericidal kinetics was established by time-kill assay. Ampicillin, ciprofloxacin and vancomycin were used as reference antibacterial drugs. All the tested compounds exhibited highly potent anti-MRSA activity (⩽ 0.008-4 µg/mL) comparable or up to 250× higher than that of vancomycin, the standard in the treatment of serious MRSA infections. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl butylcarbamate and 4-chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl ethylcarbamate were the most active compounds. In most cases, compounds provided reliable bacteriostatic activity, except for 4-chloro-2-(4-chlorophenylcarbamoyl)phenyl decylcarbamate exhibiting bactericidal effect at 8h (for clinical isolate of MRSA 63718) and at 24h (for clinical isolates of MRSA SA 630 and MRSA SA 3202) at 4× MIC. Structure-activity relationships are discussed.

Discovery of 5-(2',4'-difluorophenyl)-salicylanilides as new inhibitors of receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis.

To improve the inhibitory potency of lead compound NDMC101 on RANKL-induced osteoclastogenesis, a series of new 5-(2',4'-difluorophenyl)-salicylanilide derivatives were synthesized and evaluated for osteoclast inhibition by using TRAP-staining assay. Among them, both of compounds 6d and 6i showed three-fold increase in osteoclast-inhibitory activities compared to NDMC101 at half-inhibitory concentration. Further, the mechanistic study showed that 6d and 6i could suppress RANKL-induced osteoclastogenesis-related genes, such as NFATc1, c-fos, TRAP, and cathepsin K. Their inhibitory activities were further confirmed by including specific inhibition of NF-κB and NFATc1 expression levels in nucleus. In addition, 6d and 6i also could significantly attenuate bone-resorbing activity of osteoclasts by performing pit formation assay. Thus, a new class of 5-(2',4'-difluorophenyl)-salicylanilide derivatives may be considered as essential lead structures for the further development of anti-resorptive agents.