Ligand assisted CuAAC labelling and RP-HPLC analysis of zidovudine and Retrovir using propargyl-Fmoc probe.

The extensive application of zidovudine (ZDV) as a stand-alone anti-HIV drug and a component in antiviral combination therapies, has made its analysis important both in the pharmaceutical and environmental context. The azide group in ZDV structure makes it a ready-to-use substrate for copper-catalyzed azide-alkyne cycloaddition (CuAAC), which is an efficient method for "click chemistry" labeling. In this paper, we describe a ligand-assisted CuAAC procedure for the precolumn derivatization of ZDV. We used propargyl-Fmoc fluorescent label and trans-2-(4-((dimethylamino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexan-1-ol (AMTC) as a copper-binding ligand. We tested the applicability of AMTC for precolumn derivatization and developed chromatographic analytical procedures for ZDV and its formulation (50 mg/5 ml oral solution, Retrovir™ syrup). Our research aimed to improve labeling efficiency with a Cu-chelating ligand, using an accessible and affordable fluorescent probe. We also developed a sustainable mechanochemical synthesis procedure for obtaining propargyl-Fmoc in a gram scale and thus boosted the accessibility of this probe. The advantages of the developed derivatization procedure are its simplicity and easy availability of the propargyl-Fmoc probe. Moreover, the high lipophilicity of the propargyl-Fmoc probe enables efficient separation of the analyte from polar matrix components. In addition, the derivatization procedure can be performed directly on a sample solution. We tested its usability for samples in environmental and biological matrices, including tap water, river water, urine, and human serum.

Multigram-scale enzymatic kinetic resolution of trans-2-azidocyclohexyl acetate and chiral reversed-phase HPLC analysis of trans-2-azidocyclohexanol.

Lipase-catalyzed hydrolytic kinetic resolution is a method of obtaining optically pure chiral alcohols and amines, which requires additional tools for determining enantiomerical purity. Herein, we present a study on multigram-scale hydrolytic kinetic resolution of trans-2-azidocyclohexyl acetate using Pseudomonas cepacia lipase immobilized on Immobead support. We investigated several parameters of the preparative-scale process: temperature, organic co-solvent, and the influence of calcium ions. Moreover, we have developed an efficient fluorenylmethyloxycarbonyl chloride (Fmoc-Cl) derivatization protocol for 2-azidocyclohexanol, which enabled chiral reversed-phase high-performance liquid chromatography (RP-HPLC) determination of enantiomeric excess.

Synthesis, Molecular Docking and Antiplasmodial Activities of New Tetrahydro-ß-Carbolines.

Malaria is still one of the most dangerous infectious diseases and the emergence of drug resistant parasites only worsens the situation. A series of new tetrahydro-ß-carbolines were designed, synthesized by the Pictet-Spengler reaction, and characterized. Further, the compounds were screened for their in vitro antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Moreover, molecular modeling studies were performed to assess the potential action of the designed molecules and toxicity assays were conducted on the human microvascular endothelial (HMEC-1) cell line and human red blood cells. Our studies identified N-(3,3-dimethylbutyl)-1-octyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b] indole-3-carboxamide (7) (a mixture of diastereomers) as the most promising compound endowed with the highest antiplasmodial activity, highest selectivity, and lack of cytotoxicity. In silico simulations carried out for (1S,3R)-7 provided useful insights into its possible interactions with enzymes essential for parasite metabolism. Further studies are underway to develop the optimal nanosized lipid-based delivery system for this compound and to determine its precise mechanism of action.

Changes in the cannabinoids receptors in rats following treatment with antidepressants.

The endocannabinoid (eCB) system plays a significant role in the pathophysiology of depression. The potential participation of this system in the mechanism of action of antidepressants has been highlighted in recent years. The aim of this study was to investigate the expression of cannabinoid (CB) receptors using Western blot and CB1 receptor density using autoradiography after acute or chronic administration of antidepressant drugs [imipramine (IMI, 15mg/kg), escitalopram (ESC, 10mg/kg) and tianeptine (TIA, 10mg/kg)]. Antidepressants given chronically elevated CB1 receptor density in the cortical structures and hippocampal areas, while a decrease of CB1 receptor density was observed in the striatum after IMI and ESC treatment. The CB1 receptor expression decreases in the dorsal striatum after chronic administration of IMI and ESC or the receptor rise in the hippocampus after chronic ESC and TIA treatment were confirmed using Western blot analyses. An increase in the CB2 receptor expression was observed in the cortical structures and hippocampus after chronic administration of ESC and TIA, while a decrease in this expression was noted in the striatum and cerebellum after chronic IMI treatment. Our results provide clear evidence that the antidepressant exposures provoke some modulations within the eCB system through CB receptors.

PRELIMINARY EVALUATION OF CENTRAL NERVOUS SYSTEM ACTIVITY OF (E)-N-2-METHYL-3-PHENYLPROP-2-ENYL ((E)-N- α-METHYLCINNAMYL) DERIVATIVES OF SELECTED AMINOALKANOLS.

A series of (E)-α-methylcinnamyl derivatives of selected aminoalkanols was synthetized and evaluated for activity in central nervous system. All compounds were tested as anticonvulsants and one additionally in antidepressant- and anxiolytic-like assays. The compounds possessed pharmacophoric elements regarded as beneficial for anticonvulsant activity: hydrophobic unit and two hydrogen bonds donor/acceptor features. The compounds were verified in mice after intraperitoneal (i.p.) administration in maximal electroshock (MES) and subcutaneous pentetrazole (scPTZ) induced seizures as well as neurotoxicity assessments. Eight of the tested substances showed protection in MES test at the dose of 100 mg/kg. The derivative of 2-aminopropan-1-ol was also tested in 6-Hz test in mice i.p. and showed anticonvulsant activity but at the same time the neurotoxicity was noted. The derivative of 2-amino-1-phenylethanol which possessed additional hydrophobic unit in aminoalkanol moiety was tested in other in vivo assays to evaluate antidepressant- and anxiolytic-like activity. The compound proved beneficial properties especially as anxiolytic agent remaining active in four-plate test in mice at the dose of 2.5 mg/kg (i.p.). In vitro biotransformation studies of 2-amino-1-phenylethanol derivative carried out in mouse liver microsomal assay indicated two main metabolites as a result of aliphatic and aromatic hydroxylation or aliphatic carbonylation. To identify possible mechanism of action, we evaluated serotonin receptors (5-HT1A, 5-HT6 and 5-HT7) binding affinities of the compounds but none of them proved to bind to any of tested receptors.

N-[(2,6-Dimethylphenoxy)alkyl]aminoalkanols-their physicochemical and anticonvulsant properties.

Twenty four new N-[(dimethylphenoxy)alkyl]aminoalkanols have been synthesized and evaluated for anticonvulsant activity in a series of in vivo tests: the maximum electroshock (MES), 6 Hz, and subcutaneous metrazole (ScMet). The compounds were also evaluated for possible neurotoxicity in the rotarod test. The majority of the achieved compounds exhibit quantified anticonvulsant activity. The most active compound 4: R-(-)-2N-[(2,6-dimethylphenoxy)ethyl]aminopropan-1-ol is active in MES with ED50=5.34 (male mice, ip), 22.28 (female mice, ip), 51.19 (male mice, po), 7.43 (rats, ip), and 28.60 (rats, po). Thermal analysis proved that its hydrochloride (4a) can exist in polymorphic forms. The compound binds to σ, 5-HT1A, and α2 receptors as well as 5-HT transporter and it does not exhibit mutagenic properties.

Synthesis and evaluation of anticonvulsant activity of N-(2,5-dimethylphenoxy)- and N-[(2,3,5-trimethylphenoxy)alkyl]aminoalkanols.

A series of new N-(2,5-dimethylphenoxy)- and N-(2,3,5-trimethylphenoxy)alkylaminoalkanols [I-XVII] was synthesized and evaluated for anticonvulsant activity. Pharmacological tests included maximal electroshock (MES) and subcutaneous pentetrazole seizure threshold (scMet) assays as well as neurotoxicity (TOX) evaluation in mice after intraperitoneal (i.p.) administration and/or in rats after oral (p.o.) administration. The most active compound was R-2N-[(2,3,5-trimethylphenoxy)ethyl]aminobutan-1-ol, which exhibited 100% activity in MES at the dose of 30 mg/kg body weight (mice, i.p.) and 75% activity in MES at 30 mg/kg b.w. (rats, p.o.) without neurotoxicity at the active doses.

Studies on novel pyridine and 2-pyridone derivatives of N-arylpiperazine as α-adrenoceptor ligands.

This paper describes the synthesis of a series of new N-arylpiperazine derivatives of pyridine and 2-pyridone. The in vitro pharmacological study indicated that all of the compounds possess affinity towards α1-adrenoceptors, with exception of 6d, and are selective over α2 receptor. The most potent compound 5f displayed 62-fold α2/α1 selectivity with Ki value of 27.3 nM for α1 receptor. Selectivity of other ligands ranged from 6 to more than 146-fold. Hydrochlorides of selected compounds with the best α1-adrenoceptor affinity (7b, 7e, 7f, 8b) were tested in vivo (hypotensive activity test in rats) and the results proved their α-adrenoreceptor antagonistic activity. Furthermore, the lipophilicity of the investigated compounds has been assessed experimentally and in silico.

Anticonvulsant evaluation of aminoalkanol derivatives of 2- and 4-methylxanthone.

A series of 17 new aminoalkanol derivatives of 6-methoxy- or 7-chloro-2-methylxanthone as well as 6-methoxy-4-methylxanthone was synthesized and evaluated for anticonvulsant activity. All compounds were verified in mice after intraperitoneal (ip) administration in maximal electroshock (MES) and subcutaneous pentetrazole (scMet) induced seizures as well as neurotoxicity assessment. Eleven of the tested substances showed protection against electrically evoked seizures in the majority of the tested mice at the dose of 100 mg/kg. Additionally, one was effective at the dose of 30 mg/kg. Five substances were active at the dose of 300 mg/kg or at the dose of 100 mg/kg in the minority of the tested mice. The most promising compound revealed ED(50) value of 47.57 mg/kg in MES (mice, ip, 1h after administration) and at the same time its TD(50) was evaluated as above 400 mg/kg. Those values gave PI (calculated as TD(50)/ED(50)) of more than 8.41. Three other synthesized xanthone derivatives also proved to act as anticonvulsants and showed ED(50) values in MES test (mice, ip) ranged 80-110 mg/kg. Results were quite encouraging and suggested that in the group of xanthone derivatives new potential anticonvulsants might be found.

Synthesis and preliminary evaluation of pharmacological properties of some piperazine derivatives of xanthone.

A series of 9 piperazine derivatives of xanthone were synthesized and evaluated for cardiovascular activity. The following pharmacological experiments were conducted: the binding affinity for adrenoceptors, the influence on the normal electrocardiogram, the effect on the arterial blood pressure and prophylactic antiarrhythmic activity in adrenaline induced model of arrhythmia (rats, iv). Three compounds revealed nanomolar affinity for α(1)-adrenoceptor which was correlated with the strongest cardiovascular (antiarrhythmic and hypotensive) activity in animals' models. The most promising compound was 4-(3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (12) which revealed antiarrhythmic activity with ED(50) value of 0.69 mg/kg in adrenaline induced arrhythmia (rats, iv). Other synthesized xanthone derivatives, that is, (R,S)-4-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (10) and (R,S)-4-(2-acetoxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (11) also acted as potential antiarrhythmics in adrenaline induced model of arrhythmia in rats after intravenous injection (ED(50) = 0.88 mg/kg and 0.89 mg/kg, respectively). These values were lower than values obtained for reference drugs such as propranolol and urapidil, but not carvedilol. Results were quite promising and suggested that in the group of xanthone derivatives new potential antiarrhythmics and hypotensives might be found.

Antioxidant activity of beta-carboline derivatives.

The investigated beta-carboline derivatives were synthesized to elucidate their activity as 5-HT(1A) and 5-HT(2A) receptor ligands. Compounds containing a carboline ring system belong to a large family of biological active indoles, which are very important for the function of the central nervous system. The research was carried out to determine antioxidative or oxidative properties of these derivatives. Analysis of antioxidative capacity as indication of oxidative stress was based on ability to scavenge free radicals by DPPH (free radical scavenging activity test) and FRAP test. The results were compared to those of standard substances like vitamin C, trolox, quercetin and curcumin. The research of derivatives of beta-carboline shows antioxidative activity comparable to vitamin C. Compounds 1, 5 and 6, but only in low concentration, have antioxidative activity. Substance 10 was classified as that with prooxidative activity.

Preliminary evaluation of anticonvulsant activity and neurotoxicity of some 1,4-substituted piperazine derivatives.

A series of 1,4-piperazine derivatives was synthesized and evaluated for anticonvulsant activity in the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole seizure threshold (ScMet) assays and for neurotoxicity (TOX). The compounds were only moderately effective. The anticonvulsant activity was accompanied by neurotoxicity. 1-[(4-Chlor-3-methylphenoxy)-acetyl]-4-(2-methoxyphenyl)-piperazine was also evaluated in six hertz seizure test (6-Hz) and showed good activity. At the dose of 100 mg/kg b. w. the compound produced 100% protection after 0.5 h without neurotoxic effect.

Preliminary evaluation of pharmacological properties of some xanthone derivatives.

A series of xanthone derivatives were synthesized and examined for electrocardiographic, antiarrhythmic, hypotensive and anticonvulsant activities as well as for alpha(1)- and beta(1)-adrenergic binding affinities. Among the investigated compounds, some of them exhibited significant antiarrhythmic and/or hypotensive activity. The data obtained via receptor binding assay are in agreement with pharmacological results and could explain antiarrhythmic and/or hypotensive activity of the newly synthesized structures.

Antifungal and antibacterial activity of the newly synthesized 2-xanthone derivatives.

A series of 2-substituted xanthone derivatives 8-20 containing selected allyl, cinnamyl, morpholine, and imidazole moieties were synthesized and tested for their antifungal and antibacterial in-vitro properties. Of the newly synthesized derivatives, ten revealed antifungal activity especially against Trichophyton mentagrophytes (the biggest inhibition zones ranged 35 mm for 11 and 13). 2-(3-(Allylamino)propoxy)-9H-xanthen-9-one hydrochloride 9 inhibited growth of all of the examined fungal species. Significant efficacy against evaluated yeasts and dermatophytes was also observed for 6-chloro-2-methyl-9H-xanthen-9-one derivatives 11-13 containing encyclic amine moieties. Additionally, compounds 9, 11, and 12 hindered development of bacteria species but in a lesser degree. They were efficacious against Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis.

Antiarrhythmic and antihypertensive activity of some xanthone derivatives.

Some of appropriate aminoisopropanoloxy derivatives of 4-xanthone were tested for their effect on circulatory system (protection against adrenaline-, barium-, and calcium chloride-induced arrhythmias, as well as hypotensive activity and acute toxicity). The most prominent hypotensive activity was demonstrated by (+/-)-1-[4-(hydroxyethyl)-1-(piperazinyl)]-3-(4-xanthonoxy)-2-propanol dihydrochloride (II), which diminished arterial blood pressure by about 40% during one hour observation. The investigated compounds did not prevent adrenaline- and barium-induced arrhythmias. In calcium-induced model of arrhythmia compound II slightly intensified blocks (about 7%), but delayed extrasystoles (37%), efficiently prevented bigeminy (70%, p <0.01) and diminished (53%, p <0.05) mortality of animals. All investigated compounds decreased heart rate by 10 - 18%, prolonged P-Q section, QRS complex and Q-T interval. The most potent and significant negative chronotropic effect and markedly prolonged duration of P-Q section was demonstrated by compound II. The influence of investigated compounds on ECG components suggests that activity of compound IV is similar to class 1a anti-arrhythmic compounds according to Voughan-Williams classification of antiarrhythmic drugs, because of prolongation of P-Q and Q-T intervals and extension of QRS complex. Compounds II and IV were also evaluated for anticonvulsant activity in the maximal electroshock seizures (MES) and subcutaneous pentylenetetrazole seizure threshold (ScMet) assays and for neurotoxicity (TOX). The anti-MES activity in mice was found for IV, which in a dose of 100 mg/kg within 0.5 h after ip administration showed 75% anticonvulsant protection with 50% neurotoxicity.

Synthesis and antimycobacterial assay of some xanthone derivatives.

A series of some derivatives of 2-xanthone was synthesized and evaluated for their activity against M. tuberculosis in primary and/or secondary microbiological assays. The cytotoxic activity of some compounds was also evaluated. The most active compounds were: [I] 2-(2-(4-(2-(4-chloro-3-methylphenoxy)ethyl)piperazin-1-yl)ethoxy)-9H-xanthen-9-one, [III] 2-((4-(2-(4-chlor-3-methylphenoxy)ethyl)piperazin-1-yl)methyl)-9H-xanthen-9-one dihydrochloride and [XVIII] ethyl 4-(2-hydroxy-3-(9-oxo-9H-xanthen-2-yloxy)propyl) piperazine-1-carboxylate, which displayed 98%, 98% and 94% inhibition of M. tuberculosis growth, respectively. Furthermore, compounds III and XVIII revealed their cytotoxic activity (SI < 1). Other structures varied greatly in their anti M. tuberculosis activity, however, several trends in their structure in relation to their antituberculous activity have been observed.

Synthesis and evaluation of some xanthone derivatives for anti-arrhythmic, hypotensive properties and their affinity for adrenergic receptors.

A series of 2-, 4- or 2-methyl-6-substituted xanthone derivatives 8-17 containing selected piperazine moieties were synthesized and tested for their electrocardiographic, anti-arrhythmic, and antihypertensive activity, as well as for the alpha1- and beta1-adrenoceptor binding affinities. Of the newly synthesized derivatives, 2-(2-hydroxy-3-(4-(2-phenoxyethyl)piperazin-1-yl)propoxy)-9H-xanthen-9-one dihydrochloride 9, 4-(2-hydroxy-3-(4-(2-phenoxyethyl)piperazin-1-yl)propoxy)-9H-xanthen-9-one dihydrochloride 12, and 4-(2-(4-(pyridin-2-yl)piperazin-1-yl)ethoxy)-9H-xanthen-9-one dihydrochloride 15 possessed significant anti-arrhythmic activity in the adrenaline-induced model of arrhythmia, with the ED50 values ranging 1.7-7.2 mg/kg. Compound 15 had the lowest ED50 value equaling 1.7 mg/kg, which was comparable with ED50 value of propranolol, which was used in this test as a reference compound. Compound 9 showed also the strongest hypotensive activity, which persisted for 60 minutes at the dose of 2.5 mg/kg. 2-(2-(4-(2-Phenoxyethyl)piperazin-1-yl)ethoxy)-9H-xanthen-9-one dihydrochloride 8 also significantly lowered blood pressure at a dose of 2.5 mg/kg but much weaker than compound 9. Binding studies are in agreement with our pharmacological results and could explain anti-arrhythmic effect of compound 15 and anti-arrhythmic and hypotensive effects of compounds 9 and 12.

Synthesis and preliminary evaluation of anticonvulsant activity of some [4-(benzyloxy) benzoyl]- and [4-(benzyloxy) benzyl] aminoalkanol derivatives.

A variety of appropriate [4-(benzyloxy) benzoyl]- and [4-(benzyloxy) benzyl] aminoalkanol derivatives [I-XVII] was synthesized and evaluated for anticonvulsant activity using the maximal electroshock (MES) and subcutaneous pentylenetetrazole (ScMet) tests in mice and rats. Neurotoxicity (TOX) was determined by the rotorod test. The most active compounds in the MES test in mice were the appropriate 4-(benzyloxy) benzyl derivatives of (R,S)- and S-(+)-2-amino-1-butanol [XI, XIII], 3-[4-(benzyloxy) benzyl] amino-3-methyl-1-butanol [XV], and S-(+)-2-[4-(benzyloxy) benzyl] amino-3-methyl-1-butanol [XVI]--all exhibiting 100% anti-MES protection (at 30 mg/kg, mice, i.p.) and non-toxic in the active doses. 4-[4-(Benzyloxy) benzyl] amino-1-butanol [X] exhibited activity in both MES and ScMet (100 mg/kg, mice, i.p., 100% anticonvulsant protection, 0.5 h and 4 h after administration, respectively).

Chiral discrimination in binding of enantiomers of 2-(aminoalkoxy)-substituted 4-(2-thienyl)pyrimidines and 4,6-bis(2-thienyl)pyrimidines with duplex DNA.

Thienylpyrimidines substituted at position 2 of the pyrimidine with a chiral aminoalkoxy group were synthesized. Upon interaction with duplex DNA, the unfused heteroaromatic system of these compounds intercalates with DNA base pairs and the protonated side chain is located in the major groove. The S-enantiomers bind more strongly than their R-counterparts with enantiomeric discrimination, as measured by a ratio of binding constants K(S)/K(R), ranging from 1.2 to 2.4.

Design, synthesis, and SAR of a novel pyrazinone series with non-nucleoside HIV-1 reverse transcriptase inhibitory activity.

A series of novel pyrazinones designed as non-nucleoside reverse transcriptase inhibitors (NNRTIs) was synthesized and their anti-HIV structure-activity relationship (SAR) was studied.