Azoles containing naphthalene with activity against Gram-positive bacteria: in vitro studies and in silico predictions for flavohemoglobin inhibition.

Azoles are first-line drugs used in fungal infections. Topical antifungals, such as miconazole and econazole, are known to be active against Gram-positive bacteria, which was reported to result from bacterial flavohemoglobin (flavoHb) inhibition. Dual antibacterial/antifungal action is believed to have benefits for antimicrobial chemotherapy. In this study, we tested antibacterial effects of an in-house library of naphthalene-bearing azoles, some of which were reported as potent antifungals, in an attempt to find dual-acting hits. Several potent derivatives were obtained against the Gram-positive bacteria, Enterococcus faecalis and Staphylococcus aureus. 9 was active at a minimum inhibitor concentration (MIC) less than 1 µg/ml against E. faecalis and S. aureus, and 10 against S. aureus. 16 was also potent against E. faecalis and S. aureus (MIC = 1 and 2 µg/ml, respectively). Six more were active against S. aureus with MIC ≤ 4 µg/ml. In vitro cytotoxicity studies showed that the active compounds were safe for healthy cells within their MIC ranges. According to the calculated descriptors, the library was found within the drug-like chemical space and free of pan-assay interference compounds (PAINS). Molecular docking studies suggested that the compounds might be bacterial flavohemoglobin (flavoHb) inhibitors and the azole and naphthalene rings were important pharmacophores, which was further supported by pharmacophore modeling study. As a result, the current study presents several non-toxic azole derivatives with antibacterial effects. In addition to their previously reported antifungal properties, they could set a promising starting point for the future design of dual acting antimicrobials. Communicated by Ramaswamy H. Sarma.

Azole derivatives with naphthalene showing potent antifungal effects against planktonic and biofilm forms of Candida spp.: an in vitro and in silico study.

Candida infections pose a serious public health threat due to increasing drug resistance. Azoles are first-line antifungal drugs for fungal infections. In this study, we tested an in-house azole collection incorporating naphthalene ring to find hits against planktonic and biofilm forms of resistant Candida spp. In the collection, potent derivatives were identified against the susceptible strains of Candida with minimum inhibitory concentration (MIC) values lower than those of the reference drug, fluconazole. MIC values of 0.125 µg/ml against C. albicans, 0.0625 µg/ml against C. parapsilosis, and 2 µg/ml against C. krusei, an intrinsically azole-resistant non-albicans Candida, were obtained. Some of the derivatives were highly active against fluconazole-resistant clinical isolate of C. tropicalis. Inhibition of C. albicans biofilms was also observed at 4 µg/ml similar as amphotericin B, the reference drug known for its antibiofilm activity. Through molecular docking studies, affinities and key interactions of the compounds with fungal lanosterol 14α-demethylase (CYP51), the target enzyme of azoles, were predicted. The interactions of imidazole with heme cofactor and of the naphthalene with Tyr118 were highlighted in line with the literature data. As a result, this study proves the importance of naphthalene for the antifungal activity of azoles against Candida spp. in both planktonic and biofilm forms.

Antibacterial azole derivatives: Antibacterial activity, cytotoxicity, and in silico mechanistic studies.

Azole antifungal drugs are commonly used in antifungal chemotherapy. Antibacterial effects of some topical antifungals, such as miconazole and econazole, have lately been revealed, which suggests a promising venue in antimicrobial chemotherapy. In this study, we tested an in-house azole collection with antifungal properties for their antibacterial activity to identify dual-acting hits using the broth microdilution method. The in vitro screen yielded a number of potent derivatives against gram-positive bacteria, Enterococcus faecalis and Staphylococcus aureus. Compound 73's minimum inhibitory concentration (MIC) value less than 1 µg/ml against S. aureus; however, none of the compounds showed noteworthy activity against methicillin-resistant S. aureus (MRSA). All the active compounds were found safe at their MIC values against the healthy fibroblast cells in the in vitro cytotoxicity test. Molecular docking studies of the most active compounds using a set of docking programs with flavohemoglobin (flavoHb) structure, the proposed target of the azole antifungals with antibacterial activity, presented striking similarities regarding the binding modes and interactions between the tested compounds and the antifungal drugs with crystallographic data. In addition to being noncytotoxic, the library was predicted to be drug-like and free of pan-assay interference compounds (PAINS). As a result, the current study revealed several potential azole derivatives with both antifungal and antibacterial activities. Inhibition of bacterial flavoHb was suggested as a possible mechanism of action for the title compounds.

Discovery of new azoles with potent activity against Candida spp. and Candida albicans biofilms through virtual screening.

Systemic candidiasis is a rampant bloodstream infection of Candida spp. and C. albicans is the major pathogen isolated from infected humans. Azoles, the most common class of antifungals which suffer from increasing resistance, and especially intrinsically resistant non-albicans Candida (NAC) species, act by inhibiting fungal lanosterol 14α-demethylase (CYP51). In this study we identified a number of azole compounds in 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol/ethanone oxime ester structure through virtual screening using consensus scoring approach, synthesized and tested them for their antifungal properties. We reached several hits with potent activity against azole-susceptible and azole-resistant Candida spp. as well as biofilms of C. albicans. 5i's minimum inhibitor concentration (MIC) was 0.125 µg/ml against C. albicans, 0.5 µg/ml against C. krusei and 1 µg/ml against azole-resistant C. tropicalis isolate. Considering the MIC values of fluconazole against these fungi (0.5, 32 and 512 µg/ml, respectively), 5i emerged as a highly potent derivative. The minimum biofilm inhibitor concentration (MBIC) of 5c, 5j, and 5p were 0.5 µg/ml (and 5i was 2 µg/ml) against C. albicans biofilms, lower than that of amphotericin B (4 µg/ml), a first-line antifungal with antibiofilm activity. In addition, the active compounds showed neglectable toxicity to human monocytic cell line. We further analyzed the docking poses of the active compounds in C. albicans CYP51 (CACYP51) homology model catalytic site and identified molecular interactions in agreement with those of known azoles with fungal CYP51s and mutagenesis studies of CACYP51. We observed the stability of CACYP51 in complex with 5i in molecular dynamics simulations.

Antifungal screening and in silico mechanistic studies of an in-house azole library.

Systemic Candida infections pose a serious public health problem with high morbidity and mortality. C. albicans is the major pathogen identified in candidiasis; however, non-albicans Candida spp. with antifungal resistance are now more prevalent. Azoles are first-choice antifungal drugs for candidiasis; however, they are ineffective for certain infections caused by the resistant strains. Azoles block ergosterol synthesis by inhibiting fungal CYP51, which leads to disruption of fungal membrane permeability. In this study, we screened for antifungal activity of an in-house azole library of 65 compounds to identify hit matter followed by a molecular modeling study for their CYP51 inhibition mechanism. Antifungal susceptibility tests against standard Candida spp. including C. albicans revealed derivatives 12 and 13 as highly active. Furthermore, they showed potent antibiofilm activity as well as neglectable cytotoxicity in a mouse fibroblast assay. According to molecular docking studies, 12 and 13 have the necessary binding characteristics for effective inhibition of CYP51. Finally, molecular dynamics simulations of the C. albicans CYP51 (CACYP51) homology model's catalytic site complexed with 13 were stable demonstrating excellent binding.

Synthesis, in vivo anticonvulsant testing, and molecular modeling studies of new nafimidone derivatives.

An estimated 50 million people suffer epilepsy worldwide and 30% of the cases do not respond to current antiepileptic drugs (AEDs). Here, we report synthesis and anticonvulsant screening of new derivatives of nafimidone, a well-known member of (arylakyl)azole anticonvulsants. The compounds showed promising protection against maximal electroshock (MES)-induced seizures in mice and rats when administered via intraperitoneal (ip) and oral route. Especially, 5b, 5c, and 5i displayed outstanding activity in rats in MES test when given ip (ED50 : 16.0, 15.8, and 11.8 mg/kg, respectively). Additionally, 5l was active against 6 Hz and corneal-kindled mice models. Behavioral toxicity of the compounds was very low and their therapeutic indexes were high compared to some currently available AEDs. A number of pharmaceutically relevant descriptors and properties were predicted for the compounds in silico in comparison with a set of known drugs. Favorable results were obtained such as good blood-brain barrier permeability and high oral absorption, as well as drug-likeness. 5l was found to show affinity to the benzodiazepine binding site of A-type GABA receptor via molecular docking simulations.

Synthesis, anticonvulsant screening, and molecular modeling studies of new arylalkylimidazole oxime ether derivatives.

In this study, 15 new oxime ether derivatives were synthesized and their anticonvulsant activities were screened in vivo. The compounds were synthesized by the reaction of various alkyl halides with 1-(2-naphthyl)-2-(1H-imidazol-1-yl)ethanone oxime. Their anticonvulsant activities were determined using acute (maximal electroshock, subcutaneous metrazol [SCM], and 6 Hz seizure test) and chronic (corneal-kindled mouse) seizure models, their neurotoxic effects were evaluated by models of behavioral toxicity according to the Epilepsy Therapy Screening Program protocol of the NIH. All our compounds were protective in at least one of the tests. Quantification studies were applied to some of the active compounds and the intraperitoneal ED50 values in mice were found between 25.48 and 99.56 mg/kg. Some pharmacokinetic properties of the compounds were predicted in silico and molecular docking studies were performed to provide insights into their possible anticonvulsant mechanism regarding their SCM activity.

Synthesis and anticonvulsant screening of 1,2,4-triazole derivatives.

BACKGROUND: Currently available antiepileptic drugs offer limited symptomatic treatment and fail to cure more than 30% of the epileptic seizures. (Arylalkyl)azoles are a class of anticonvulsants including nafimidone and loreclezole. Here, we report the design and synthesis of new (arylalkyl)azoles in N-[1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethylidene]hydroxylamine ester structure, their anticonvulsant screening and in silico prediction studies of their pharmacokinetic properties. METHODS: The title compounds were synthesized according to the Steglich esterification of N-[1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethylidene]hydroxylamine with various carboxylic acids. Anticonvulsant identification and quantification tests were performed in mice by the Epilepsy Therapy Screening Program (ETSP) of the National Institutes of Health (NIH) using 6Hz psychomotor, maximal electroshock (MES), and rotorod tests. Their physicochemical and pharmacokinetic properties were calculated using QikProp. RESULTS: Most of the compounds showed protection against 6Hz- and/or MES-induced seizures. 4a, 4b, and 4g were active at 100mg/kg, 4g was active in both tests without neurotoxicity. According to the QikProp calculations the title compounds were druglike and had some favourable properties such as high membrane permeability and oral absorptivity. CONCLUSION: Anticonvulsant screening of a set N-[1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethylidene]hydroxylamine esters yielded some active derivatives in 6Hz and MES test. Especially, 4g emerged as a promising compound with activity at 100mg/kg and no toxicity. The compounds were predicted to be drug like and have good pharmacokinetic properties except hERG inhibition, which needs to be addressed in further optimization studies.

New Anti-Seizure (Arylalkyl)azole Derivatives: Synthesis, In Vivo and In Silico Studies.

(Arylalkyl)azoles are a class of antiepileptic compounds including nafimidone, denzimol, and loreclezole (LRZ). Nafimidone and denzimol are thought to inhibit voltage-gated sodium channels (VGSCs) and enhance γ-aminobutyric acid (GABA)-mediated response. LRZ, a positive allosteric modulator of A-type GABA receptors (GABAA Rs), was reported to be sensitive to Asn265 of the ß2/ß3 subunit. Here, we report new N-[1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethylidene]hydroxylamine esters showing anticonvulsant activity in animal models, including the 6-Hz psychomotor seizure test, a model for therapy-resistant partial seizure. We performed molecular docking studies for our active compounds using GABAA R and VGSC homology models. They predicted high affinity to the benzodiazepine binding site of GABAA R in line with the experimental results. Also, the binding mode and interactions of LRZ in its putative allosteric binding site of GABAA R is elucidated.

New azole derivatives showing antimicrobial effects and their mechanism of antifungal activity by molecular modeling studies.

Azole antifungals are potent inhibitors of fungal lanosterol 14α demethylase (CYP51) and have been used for eradication of systemic candidiasis clinically. Herein we report the design, synthesis, and biological evaluation of a series of 1-phenyl/1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethanol esters. Many of these derivatives showed fungal growth inhibition at very low concentrations. Minimal inhibition concentration (MIC) value of 15 was 0.125 µg/mL against Candida albicans. Additionally, some of our compounds, such as 19 (MIC: 0.25 µg/mL), were potent against resistant C. glabrata, a fungal strain less susceptible to some first-line antifungal drugs. We confirmed their antifungal efficacy by antibiofilm test and their safety against human monocytes by cytotoxicity assay. To rationalize their mechanism of action, we performed computational analysis utilizing molecular docking and dynamics simulations on the C. albicans and C. glabrata CYP51 (CACYP51 and CGCYP51) homology models we built. Leu130 and T131 emerged as possible key residues for inhibition of CGCYP51 by 19.

New (arylalkyl)azole derivatives showing anticonvulsant effects could have VGSC and/or GABAAR affinity according to molecular modeling studies.

(Arylalkyl)azoles (AAAs) emerged as a novel class of antiepileptic agents with the invention of nafimidone and denzimol. Several AAA derivatives with potent anticonvulsant activities have been reported so far, however neurotoxicity was usually an issue. We prepared a set of ester derivatives of 1-(2-naphthyl)-2-(1H-1,2,4-triazol-1-yl)ethanone oxime and evaluated their anticonvulsant and neurotoxic effects in mice. Most of our compounds were protective against maximal electroshock (MES)- and/or subcutaneous metrazol (s.c. MET)-induced seizures whereas none of them showed neurotoxicity. Nafimidone and denzimol have an activity profile similar to that of phenytoin or carbamazepine, both of which are known to inhibit voltage-gated sodium channels (VGSCs) as well as to enhance γ-aminobutiric acid (GABA)-mediated response. In order to get insights into the effects of our compounds on VGSCs and A-type GABA receptors (GABAARs) we performed docking studies using homology model of Na+ channel inner pore and GABAAR as docking scaffolds. We found that our compounds bind VGSCs in similar ways as phenytoin, carbamazepine, and lamotrigine. They showed strong affinity to benzodiazepine (BZD) binding site and their binding interactions were mainly complied with the experimental data and the reported BZD binding model.

Synthesis, anticonvulsant and antimicrobial activities of some new [1-(2-naphthyl)-2-(pyrazol-1-yl)ethanone]oxime ethers.

In this study, 12 new oxime ether derivatives, which were expected to show anticonvulsant and antimicrobial activities, were synthesized. Oxime ether derivatives were synthesized by the reaction of various alkyl halides with 1-(2- naphthyl)-2-(pyrazol-1-yl)ethanone oxime. Anticonvulsant activity of the compounds was determined by maximal electroshock (MES) and subcutaneous metrazol (ScM) seizure tests, while neurological disorders were evaluated using rotorod toxicity test according to the ASP of NIH. Compound 1, 6 and 7 showed anticonvulsant activity at 300 mg/kg dose at 4 h, but compounds 1 and 7 showed toxicity at 300 mg/kg dose at half an hour. Antimicrobial activities of the compounds were also determined using agar microdilution method. Compound 1 and 5 were found to have the highest antifungal activity among the other compounds.

Synthesis of some novel 1-(2-naphthyl)-2-(imidazol-1-yl)ethanone oxime ester derivatives and evaluation of their anticonvulsant activity.

Twenty-three new oxime ester derivatives of nafimidone were synthesized with the prospect of potential anticonvulsant activities. MES and ScM tests were employed for their anticonvulsant activities and rotorod test for neurological deficits. Eighteen compounds were found to be protective against MES seizures. Alkyl (1-8) and arylalkyl (9, 10) oxime ester derivatives were found to be more active than aryl oxime ester derivatives (11-23). Five compounds (2, 3, 7, 9, 10), which were protective at 0.5 h at the doses of 30 mg/kg and higher in MES test, showed the highest activity. Compound 17 was the most active one in ScM test at all dose levels at 4 h.

Recent progress in anticonvulsant drug research: strategies for anticonvulsant drug development and applications of antiepileptic drugs for non-epileptic central nervous system disorders.

Major advances in antiepileptic drug therapy have taken place since 1950s. In the first period, several antiepileptic drugs (AEDs) such as phenobarbital, diphenylhydantoin, ethosuximide, carbamazepine, benzodiazepines and valproic acid were introduced to epilepsy treatment. After 1990 many new generation drugs (lamotrigine, topiramate, gabapentine, pregabaline, felbamate, lacosamide, levetiracetam etc.) have been developed. These novel AEDs have offered some advantages such as fewer side effects, fewer drug-drug interactions, and better pharmacokinetic properties. But pharmacoresistance and therapeutic failure in 20-25% of the patients remain the main reasons to continue efforts to find safer and more efficacious drugs and ultimate a treatment for this devastating disease. Several AEDs especially novel compounds have been found to be effective also in the treatment of several other neurologic and psychiatric disorders. Chemical diversity of the newer antiepileptic drugs as well as those currently in clinical development is another point that encourages medicinal chemists to study this subject. This review summarizes recent studies on the development of potential anticonvulsant compounds in different chemical structures, their structure-activity relationships and also therapeutic usages of AEDs other than epilepsy.

Carboxylic acid derivatives of histone deacetylase inhibitors induce full length SMN2 transcripts: a promising target for spinal muscular atrophy therapeutics.

INTRODUCTION: Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder. It is caused by homozygous absence of the survival motor neuron 1 (SMN1) gene. SMN2, which modulates the severity of the disease, represents a major target for therapy. The aim of this study was to investigate whether SMN2 expression can be increased by caffeic acid, chlorogenic acid and curcumin, which are designed by modifications of the carboxylic acid class of histone deacetylase (HDAC) inhibitors. MATERIAL AND METHODS: Using quantitative real-time PCR, we analysed the levels of full-length SMN2 and Δ7SMN2 mRNA. We performed LDH cytotoxicity assay to analyse whether SMN2 activating concentrations of caffeic acid, chlorogenic acid and curcumin were cytotoxic to fibroblasts. RESULTS: We found that caffeic acid and curcumin were more efficient than chlorogenic acid and increased full-length SMN2 mRNA levels 1.5 and 1.7-fold, respectively. Δ7SMN2 mRNA levels were measured to investigate alternative splicing of exon 7. We also found that cytotoxicity was not observed at SMN2 activating concentrations. CONCLUSIONS: Our data suggest that carboxylic acid derivatives including phenolic structure and symmetry could be a good candidate for SMA treatment.

Synthesis, anticonvulsant and antimicrobial activities of some new 2-acetylnaphthalene derivatives.

In this study, as a continuation of our research for new (arylalkyl)imidazole anticonvulsant compounds, the design, synthesis and anticonvulsant/antimicrobial activity evaluation of a series of 2-acetylnaphthalene derivatives have been described. Molecular design of the compounds has been based on the modification of nafimidone [1-(2-naphthyl)-2-(imidazol-1-yl)ethanone], which is a representative of the (arylalkyl)imidazole anticonvulsant compounds as well as its active metabolite, nafimidone alcohol (3, 4). In general, these compounds were variously substituted at the alkyl chain between naphthalene and imidazole rings and subjected to some other modifications to evaluate additional structure-activity relationships. The anticonvulsant activity profile of those compounds was determined by maximal electroshock seizure (MES) and subcutaneous metrazol (scM) seizure tests, whereas their neurotoxicity was examined using rotarod test. All the ester derivatives of nafimidone alcohol (5a-h), which were designed as prodrugs, showed anticonvulsant activity against MES-induced seizure model. Four of the most active compounds were chosen for further anticonvulsant evaluations. Quantification of anticonvulsant protection was calculated via the ip route (ED(50) and TD(50)) for the most active candidate (5d). Observed protection in the MES model was 38.46mgkg(-1) and 123.83mgkg(-1) in mice and 20.44mgkg(-1), 56.36mgkg(-1) in rats, respectively. Most of the compounds with imidazole ring also showed antibacterial and/or antifungal activities to a certain extent in addition to their anticonvulsant activity.

Molecular modifications on carboxylic acid derivatives as potent histone deacetylase inhibitors: Activity and docking studies.

In the light of known HDAC inhibitors, 33 carboxylic acid derivatives were tested to understand the structural requirements for HDAC inhibition activity. Several modifications were applied to develop the structure-activity relationships of carboxylic acid HDAC inhibitors. HDAC inhibition activities were investigated in vitro by using HeLa nuclear extract in a fluorimetric assay. Molecular docking was also carried out for the human HDAC8 enzyme in order to predict inhibition activity and the 3D poses of inhibitor-enzyme complexes. Of these compounds, caffeic acid derivatives such as chlorogenic acid and curcumin were found to be highly potent compared to sodium butyrate, which is a well-known HDAC inhibitor.

Histone deacetylase inhibition activity and molecular docking of (e )-resveratrol: its therapeutic potential in spinal muscular atrophy.

Spinal muscular atrophy is an autosomal recessive motor neuron disease that is caused by mutation of the survival motor neuron gene (SMN1) but all patients retain a nearly identical copy, SMN2. The disease severity correlates inversely with increased SMN2 copy. Currently, the most promising therapeutic strategy for spinal muscular atrophy is induction of SMN2 gene expression by histone deacetylase inhibitors. Polyphenols are known for protection against oxidative stress and degenerative diseases. Among our candidate prodrug library, we found that (E )-resveratrol, which is one of the polyphenolic compounds, inhibited histone deacetylase activity in a concentration-dependent manner and half-maximum inhibition was observed at 650 microM. Molecular docking studies showed that (E )-resveratrol had more favorable free energy of binding (-9.09 kcal/mol) and inhibition constant values (0.219 microM) than known inhibitors. To evaluate the effect of (E )-resveratrol on SMN2 expression, spinal muscular atrophy type I fibroblast cell lines was treated with (E )-resveratrol. The level of full-length SMN2 mRNA and protein showed 1.2- to 1.3-fold increase after treatment with 100 microM (E )-resveratrol in only one cell line. These results indicate that response to (E )-resveratrol treatment is variable among cell lines. This data demonstrate a novel activity of (E )-resveratrol and that it could be a promising candidate for the treatment of spinal muscular atrophy.

Synthesis of some oxime ether derivatives of 1-(2-naphthyl)-2-(1,2,4-triazol-1-yl)ethanone and their anticonvulsant and antimicrobial activities.

In this study, oxime and oxime ether derivatives of [1-(2-naphthyl)-2-(1,2,4-triazol-1-yl)ethanone] were prepared as potential anticonvulsant and antimicrobial compounds. The oxime was synthesized by the reaction of ketone and hydroxylamine hydrochloride. O-Alkylation of the oxime by various alkyl halides gave the oxime ether derivatives. Anticonvulsant activity of the compounds was determined by maximal electroshock and subcutaneous metrazole tests in mice and rats according to procedures of the Anticonvulsant Screening Program of National Institutes of Health. Neurotoxicity was determined by the rotorod test in mice and the positional sense test, gait and stance test in rats. In addition to anticonvulsant tests, all compounds were also evaluated against the following microorganisms: S. aureus, E. coli, P. aeruginosa, E. faecalis, C. albicans, C. parapsilosis, and C. krusei using microdilution broth method for possible antibacterial and antifungal activities. Although most of the O-alkyl substituted oxime ethers exhibited both anticonvulsant and antimicrobial activities, the O-arylalkyl substituted compounds were found to be inactive in both screening paradigms.