Martin Doležal

New methods in synthesis of acetylcholinesterase reactivators and evaluation of their potency to reactivate cyclosarin-inhibited AChE

Nine potential AChE reactivators were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by cyclosarin nerve agent was tested in vitro. According to the previous results, 1,4-bis(2-hydroxyiminomethylpyridinium)butane dibromide seems to be the most potent AChE reactivator. The reactivation potency of these compounds depends on structural factors such as presence of quaternary nitrogens, length of the linking chain between both pyridinium rings, and position of the oxime moiety at the pyridinium ring.

Substituted N-Benzylpyrazine-2-carboxamides: Synthesis and Biological Evaluation

A series of twelve amides was synthesized via aminolysis of substituted pyrazinecarboxylic acid chlorides with substituted benzylamines. Compounds were characterized with analytical data and assayed in vitro for their antimycobacterial, antifungal, antibacterial and photosynthesis-inhibiting activity. 5-tert-Butyl-6-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (12) has shown the highest antimycobacterial activity against Mycobacterium tuberculosis (MIC = 6.25 µg/mL), as well as against other mycobacterial strains. The highest antifungal activity against Trichophyton mentagrophytes, the most susceptible fungal strain tested, was found for 5-chloro-N-(3-trifluoromethylbenzyl)-pyrazine-2-carboxamide (2, MIC = 15.62 µmol/L). None of the studied compounds exhibited any activity against the tested bacterial strains. Except for 5-tert-butyl-6-chloro-N-benzylpyrazine-2-carboxamide (9, IC50 = 7.4 µmol/L) and 5-tert-butyl-6-chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide (11, IC50 = 13.4 µmol/L), only moderate or weak photosynthesis-inhibiting activity in spinach chloroplasts (Spinacia oleracea L.) was detected.

Substituted N-Phenylpyrazine-2-carboxamides: synthesis and antimycobacterial evaluation.

The condensation of chlorides of substituted pyrazinecarboxylic acids with ring-substituted anilines yielded twelve substituted pyrazinecarboxylic acid amides. The synthetic approach, analytical, and lipophilicity data of the newly synthesized compounds are presented. Two antituberculosis assays were used. Firstly, the antimycobacterial activity against four different Mycobacterium strains in a series of pyrazine derivatives was investigated. Secondly, the antimycobacterial evaluation was performed at the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) program. Interesting in vitro antimycobacterial activity was found, N-(3-iodo-4-methyl-phenyl)pyrazine-2-carboxamide (9) was most active derivative compound against M. tuberculosis (MIC < 2.0 μmol/L), while another iodo derivative 5-tert-butyl-6-chloro-N-(3-iodo-4-methyl-phenyl)pyrazine-2-carboxamide (12) was the most active compound in the TAACF antituberculosis screening program (IC90 = 0.819 µg/mL).

Substituted N-phenylpyrazine-2-carboxamides, their synthesis and evaluation as herbicides and abiotic elicitors.

The condensation of substituted pyrazine-2-carboxylic acid chlorides with ring-substituted anilines yielded five substituted pyrazine-2-carboxylic acid amides. Thesynthesis, and analytical, lipophilicity and biological data of the newly synthesizedcompounds are presented in this paper. The photosynthesis inhibition, antialgal activityand the effect of a series of pyrazine derivatives as abiotic elicitors on the accumulation offlavonoids in a callus culture of Ononis arvensis (L.) were investigated. The most activeinhibitor of the oxygen evolution rate in spinach chloroplasts was 6-chloro-pyrazine-2-carboxylic acid (3-iodo-4-methylphenyl)-amide (2, IC(50) = 51.0 micromol.L(-1)). The highestreduction of chlorophyll content in Chlorella vulgaris was found for 5-tert-butyl-N-(4-chloro-3-methylphenyl)-pyrazine-2-carboxamide (3, IC(50) = 44.0 micromol.L(-1)). The maximalflavonoid production (about 900%) was reached after a twelve-hour elicitation processwith 6-chloropyrazine-2-carboxylic acid (3-iodo-4-methylphenyl)-amide (2).

Substituted pyrazinecarboxamides as abiotic elicitors of flavolignan production in Silybum marianum (L.) gaertn cultures in vitro.

Substituted pyrazinecarboxamides markedly influenced production of flavonolignans in Silybum marianum callus and suspension cultures. In this study the effect of two compounds, N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide (1) and N-(3-iodo-4-methylphenyl)-5-tert-butyl-pyrazine-2-carboxamide (2), as abiotic elicitors on flavono-lignan production in callus culture of S. marianum was investigated. Silymarin complex compounds have hepatoprotective, anticancer and also hypocholesterolemic activity. In vitro flavonolignan concentration in cells is very low and the elicitation is one of the methods to increase production. Elicitors were tested at three concentrations and at different culture times. In the case of elicitation with 1, the greatest increase of flavonolignan and taxifoline production was observed at concentration c1a after 6-hours of elicitation and after 24 and 72-hours at concentration c1b. However, increased production of silychristin, one of the compounds in the silymarin complex, was achieved after only 6-hours elicitation with c1a (2.95 × 10-4 mol/L). The content of silychristin was 2-times higher compared to the control sample. An increased production of silychristin was reached with compound 2 at the concentration c2 (2.53 × 10-3 mol/L) after 72 h of elicitation. The production of silychristin in this case was increased 12-times compared to control.

Synthesis and biological activity of 5-alkyl-6-(alkylsulfanyl)- or 5-alkyl-6-(arylsulfanyl)pyrazine-2-carboxamides and corresponding thioamides.

Nucleophilic substitution of chlorine in 5-alkyl-6-chloropyrazine-2-carboxamides with various alkyl and arylthiolates afforded 20 5-alkyl-6-(alkylsulfanyl)- and 5-alkyl-6-(arylsulfanyl)pyrazine-2-carboxamides. The reaction of the amides with Lawessons reagent yielded the corresponding thioamides. The assessment of in vitro antimycobacterial and antifungal activity of the compounds was carried out. In both series, the antimycobacterial activity increases with increasing molecular weight of the alkylsulfanyl group in position 6 of the pyrazine ring. Thioamides exhibited higher activity than the corresponding amides. 5-Butyl-6-(phenylsulfanyl)pyrazine-2-carbothioamide (2j) possessed the highest activity (91% inhibition) against Mycobacterium tuberculosis and also the highest lipophilicity (log P = 4.95). Only a poor in vitro antifungal effect was noted in 5-butyl-6-(butylsulfanyl)pyrazine-2-carboxamide (1i) and 6-(ethylsulfanyl)-5-isobutylpyrazine-2-carbothioamide (2q) against Trichophyton mentagrophytes and Absidia corymbifera.

Synthesis, Antimycobacterial Activity and In Vitro Cytotoxicity of 5-Chloro-N-phenylpyrazine-2-carboxamides

5-Chloropyrazinamide (5-Cl-PZA) is an inhibitor of mycobacterial fatty acid synthase I with a broad spectrum of antimycobacterial activity in vitro. Some N-phenylpyrazine-2-carboxamides with different substituents on both the pyrazine and phenyl core possess significant in vitro activity against Mycobacterium tuberculosis. To test the activity of structures combining both the 5-Cl-PZA and anilide motifs a series of thirty 5-chloro-N-phenylpyrazine-2-carboxamides with various substituents R on the phenyl ring were synthesized and screened against M. tuberculosis H37Rv, M. kansasii and two strains of M. avium. Most of the compounds exerted activity against M. tuberculosis H37Rv in the range of MIC = 1.56–6.25 µg/mL and only three derivatives were inactive. The phenyl part of the molecule tolerated many different substituents while maintaining the activity. In vitro cytotoxicity was decreased in compounds with hydroxyl substituents, preferably combined with other hydrophilic substituents. 5-Chloro-N-(5-chloro-2-hydroxyphenyl)pyrazine-2-carboxamide (21) inhibited all of the tested strains (MIC = 1.56 µg/mL for M. tuberculosis; 12.5 µg/mL for other strains). 4-(5-Chloropyrazine-2-carboxamido)-2-hydroxybenzoic acid (30) preserved good activity (MIC = 3.13 µg/mL M. tuberculosis) and was rated as non-toxic in two in vitro models (Chinese hamster ovary and renal cell adenocarcinoma cell lines; SI = 47 and 35, respectively).

Synthesis and antimycobacterial evaluation of N-substituted 5-chloropyrazine-2-carboxamides.

To develop new potential antimycobacterial drugs, a series of pyrazinamide derivatives was designed, synthesized and tested for their ability to inhibit the growth of selected mycobacterial strains (Mycobacterium tuberculosis H37Rv, Mycobacterium kansasii and two strains of Mycobacterium avium). This Letter is focused on binuclear pyrazinamide analogues containing the -CONH-CH2- bridge, namely on N-benzyl-5-chloropyrazine-2-carboxamides with various substituents on the phenyl ring and their comparison with some analogously substituted 5-chloro-N-phenylpyrazine-2-carboxamides. Compounds from the N-benzyl series exerted lower antimycobacterial activity against M. tuberculosis H37Rv then corresponding anilides, however comparable with pyrazinamide (12.5-25 μg/mL). Remarkably, 5-chloro-N-(4-methylbenzyl)pyrazine-2-carboxamide (8, MIC=3.13 μg/mL) and 5-chloro-N-(2-chlorobenzyl)pyrazine-2-carboxamide (1, MIC=6.25 μg/mL) were active against M. kansasii, which is naturally unsusceptible to PZA. Basic structure-activity relationships are presented.

Synthesis and antimycobacterial evaluation of pyrazinamide derivatives with benzylamino substitution.

A series of 19 new compounds related to pyrazinamide were synthesized, characterized with analytical data and screened for in vitro whole cell antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium kansasii and two types of Mycobacterium avium. The series consisted of 3-(benzylamino)-5-cyanopyrazine-2-carboxamides and 3-(benzylamino)pyrazine-2,5-dicarbonitriles with various substituents on the phenyl ring. RP-HPLC method was used to determine the lipophilicity of the prepared compounds. Nine compounds exerted similar or better activity against Mycobacterium tuberculosis compared to pyrazinamide (MIC=6.25-12.5 μg/mL). 3-(Benzylamino)pyrazine-2,5-dicarbonitrile inhibited all of the tested mycobacterial strains with MIC within the range 12.5-25 μg/mL. Although not the most active, 4-NH(2) substituted compounds possessed the lowest in vitro cytotoxicity (hepatotoxicity), leading to selectivity index SI=5.5 and SI >21.

Alkylamino derivatives of pyrazinamide: synthesis and antimycobacterial evaluation.

A series of pyrazinamide derivatives with alkylamino substitution was designed, synthesized and tested for their ability to inhibit the growth of selected mycobacterial, bacterial and fungal strains. The target structures were prepared from the corresponding 5-chloro (1) or 6-chloropyrazine-2-carboxamide (2) by nucleophilic substitution of chlorine by various non-aromatic amines (alkylamines). To determine the influence of alkyl substitution, corresponding amino derivatives (1a, 2a) and compounds with phenylalkylamino substitution were prepared. Some of the compounds exerted antimycobacterial activity against Mycobacterium tuberculosis H37Rv significantly better than standard pyrazinamide and corresponding starting compounds (1 and 2). Basic structure-activity relationships are presented. Only weak antibacterial and no antifungal activity was detected.