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.     

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 N-substituted 3-aminopyrazine-2,5-dicarbonitriles

A series of 14 new compounds related to pyrazinamide were synthesized, characterized with analytical data and screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium kansasii and two types of Mycobacterium avium. The series comprised of N-substituted 3-aminopyrazine-2,5-dicarbonitriles derived from 3-chloropyrazine-2,5-dicarbonitrile by nucleophilic substitution of chlorine by various non-aromatic amines (alkylamines, cycloalkylamines and heterocyclic amines). Noteworthy antimycobacterial activity against M. tuberculosis was found among the alkylamino derivatives, for example, 3-(heptylamino)pyrazine-2,5-dicarbonitrile inhibited M. tuberculosis at MIC=51 μmol/L. 3-(Hexylamino)pyrazine-2,5-dicarbonitrile inhibited M. kansasii at MIC=218 μmol/L. Basic structure-activity relationships are discussed. A comparison between calculated and experimentally determined lipophilicity parameters within the series is included.     

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-NH₂ substituted compounds possessed the lowest in vitro cytotoxicity (hepatotoxicity), leading to selectivity index SI = 5.5 and SI >21.     

Hybrid molecules of estrone: new compounds with potential antibacterial, antifungal, and antiproliferative activities

New hybrid molecules of estrone were synthesized as compounds indicating promising biological activity (antibacterial, antimycobacterial, antifungal, and antiproliferative). The prepared molecules contained various heterocyclic units (pyridine, benzylsulfanyl derivatives of pyridine or derivatives of tetrazole) linked to estrone by n-heptyl bridges. The compounds with charge on molecule (the hybrid pyridinium or benzylsulfanylpyridinium salts) exhibited significant biological activity (antibacterial, antimycobacterial, antifungal, and antiproliferative). On the other hand, the compounds not in the form of salts (omega-(1-phenyl-5-tetrazolylthio)heptylethers of estrone) were inactive. The antimycobacterial activities of three different series of tetrazole derivatives (i.e., the hybrid molecules with estrone, tetrazole-5-thiols, and 5-benzylsulfanyl-1-phenyltetrazoles) with the same substituents on phenyl ring were compared. Amongst them, the 5-benzylsulfanyl-1-phenyltetrazoles were the most potent.     

Design, synthesis, and biological evaluation of 1,2,3-trisubstituted-1,4-dihydrobenzo[g]quinoxaline-5,10-diones and related compounds as antifungal and antibacterial agents

A series of (S)-N-(3-chloro-1,4-naphthoquinon-2-yl)-alpha-amino acid ethyl esters 3 and 1,2,3-trisubstituted-1,4-dihydrobenzo[g]quinoxaline-5,10-diones 6-23 were synthesized and evaluated for antifungal and antibacterial activities. The structure-activity relationship of these compounds was studied and the results show that the compounds 3a and 3b exhibited in vitro antifungal activity against Candida albicans, Cryptococcus neoformans, and Sporothrix schenckii whereas compounds 12 and 22 showed in vitro antibacterial activity against Klebsiella pneumoniae and Escherichia coli.     

Synthesis of pyrazinamide Mannich bases and its antitubercular properties

A series of pyrazinamide (PAZ) Mannich bases has been synthesized by reacting PAZ, formaldehyde, and various substituted piperazines using microwave irradiation with the yield ranging from 46% to 86%. The synthesized compounds were evaluated for antimycobacterial activity in vitro and in vivo against Mycobacterium tuberculosis H37Rv (MTB). Among the synthesized compounds, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-4-((pyrazine-2-carboxamido)methyl)piperazin-1-yl)-4-oxoquinoline-3-carboxylic acid (17) was found to be the most active compound in vitro with MIC of 0.39 and 0.2 microg/mL against MTB and multidrug-resistant MTB, respectively. In the in vivo animal model 17 decreased the bacterial load in lung and spleen tissues with 1.86 and 1.66-log10 protections, respectively.     

Synthesis and pharmacological evaluation of a novel series of 3-aryl-2-(2-substituted-4-methylthiazole-5-yl)thiazolidin-4-one as possible anti-inflammatory and antimicrobial agents

A new series of 3-aryl-2-(2-aryl/benzyl-4-methylthiazole-5-yl)thiazolidin-4-one was synthesized by condensation of 2-aryl/benzyl-4-methylthiazole-5-carbaldehyde, aromatic amines and thioglycolic acid in toluene. All the synthesized compounds are characterized by IR, NMR and elemental or mass analysis. Sixteen out of the newly synthesized compounds were screened for in vivo anti-inflammatory activity using carrageenan-induced rat paw edema method. Some of the synthesized compounds exhibited good anti-inflammatory activity compared with indomethacin. The synthesized compounds were also evaluated for their in vitro antimicrobial activity. Some of the compounds showed mild antibacterial activity while most of the compounds showed good antifungal activity.     

Synthesis and evaluation of antitubercular activity of imidazo[2,1-b][1,3,4]thiadiazole derivatives

A series of 2,6-disubstituted and 2,5,6-trisubstituted imidazo[2,1-b][1,3,4]thiadiazoles were synthesized, the structures of the compounds were elucidated and screened for antitubercular activity against Mycobacterium tuberculosis H37Rv using the BACTEC 460 radiometric system, antibacterial activity against Escherichia coli and Bacillus cirrhosis, and antifungal activity against Aspergillus niger and Penicillium wortmanni. Among the tested compounds 2-(2-furyl)-6-phenylimidazo[2,1-b][1,3,4] thiadiazole-5-carbaldehyde (6c) and (2-cyclohexyl-6-phenylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)methanol (7a) have shown the highest (100%) inhibitory activity. Compounds 6a, 6b, 7c, and 8a exhibited moderate antitubercular activity with percentage inhibition 36, 30, 15, and 20, respectively, at a MIC of >6.25 microg/ml.     

Synthesis and preliminary evaluation of some pyrazine containing thiazolines and thiazolidinones as antimicrobial agents

A series of N'-[3,4-disubstituted-1,3-thiazol-2(3H)-ylidene]-2-(pyrazin-2-yloxy)acetohydrazide 11-66 and N'-[(2Z)-3-(4-bromophenyl)-4-oxo-1,3-thiazolidin-2-ylidene]-2-(pyrazin-2-yloxy)acetohydrazide 68-74 were synthesized using appropriate synthetic route. The entire test compounds 11-66 and 68-74 were assayed in vitro for antibacterial activity against two different strains of Gram-negative (E. coli and S. typhi), Gram-positive (S. aureus and B. subtilis) bacteria and the antimycobacterial activity was evaluated against H(37)Rv strain of Mycobacterium tuberculosis. The minimum inhibitory concentration (MIC) was determined for test compounds and for reference standards. The test compounds showed significant antibacterial and antimycobacterial activity against the microbial strains used, when tested in vitro. In general, pyrazine ring and substituted thiazoline ring are essential for antimicrobial activity. Among the compounds tested, compounds 11, 12 and 40 were found to be most potent. The toxicity of most potent compounds 11, 12 and 40 were determined using hemolytic assay and minimal hemolytic concentration (MHCs) were determined. The test compounds were found to be nontoxic up to a dose level of 250 microg/mL.     

Synthesis, antimicrobial activity and molecular modeling studies of halogenated 4-[1H-imidazol-1-yl(phenyl)methyl]-1,5-diphenyl-1H-pyrazoles

During the course of our studies in the azole antifungals area, we synthesized a number of 1,5-disubstituted 4-[1H-imidazol-1-yl(phenyl)methyl]-1H-pyrazoles, analogues of bifonazole. 1,5-Diphenyl-1H-pyrazole 3 showed weak antimycotic and antibacterial activities in vitro against Candida albicans, Cryptococcus neoformans and Staphylococcus aureus. In order to increase these properties, given that the halo substitution was found to be capable of enhancing antifungal effects, we prepared a series of fluoro and chloro derivatives of 3. The microbiological evaluation carried out on newly synthesized compounds included in vitro assays for antifungal, antibacterial and antimycobacterial activities. Among the tested compounds, some dichloro and trichloro-derivatives showed interesting antimicrobial properties. In particular, compounds 10j,k,l produced inhibitory effects against pathogen representatives of yeast (C. albicans, C. neoformans) and Gram positive bacteria (S. aureus) similar or superior to those of bifonazole. In addition, their activity against Mycobacterium tuberculosis was superior to that of clotrimazole and econazole, which were used as reference drugs. The replacement, in these compounds, of chlorine with fluorine atoms led to inactive derivatives. Docking studies were carried out on the most active compounds, in order to rationalize the pharmacological results.     

Salicylanilide esters of N-protected amino acids as novel antimicrobial agents

A series of novel, highly antimicrobial salicylanilide esters of N-protected amino acids were synthesized and characterized. Their in vitro antimicrobial activity against eight fungal strains and Mycobacterium tuberculosis was determined. The compounds had the highest level of activity against Aspergillus fumigatus, Absidia corymbifera and Trichophyton mentagrophytes, and these levels were higher than that of the standard drug fluconazole. In addition, three compounds showed interesting antituberculosis activity, with inhibition ranging from 89% to 99%. (S)-4-Chloro-2-(4-trifluoromethylphenylcarbamoyl)-phenyl 2-benzyloxy-carbonylamino-propionate had the highest level of both antifungal and antimycobacterial activity. The structure–activity relationships of the new compounds are discussed.     

Synthesis and biological activity of Schiff and Mannich bases bearing 2,4-dichloro-5-fluorophenyl moiety

A series of 2,4-dichloro-5-fluorophenyl bearing Mannich base (4 and 5) was prepared from triazole Schiff bases (3) by aminomethylation with formaldehyde and secondary/substituted primary amines. All newly synthesized compounds were screened for their antimicrobial activity. Compounds 3c, 4c, 4e and 4f exhibited promising antibacterial and compounds 3c, 5c, 5e and 5f showed good antifungal activity.     

Antimycobacterial and herbicidal activity of ring-substituted 1-hydroxynaphthalene-2-carboxanilides

In this study, a series of 22 ring-substituted 1-hydroxynaphthalene-2-carboxanilides were prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobacterium marinum, Mycobacterium kansasii and Mycobacterium smegmatis. The compounds were also tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Most of tested compounds showed the antimycobacterial activity against the three strains comparable or higher than the standard isoniazid. N-(3-Fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC = 28.4 μmol/L) against M. marinum, N-(4-fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC = 14.2 μmol/L) against M. kansasii, and N-(4-bromophenyl)-1-hydroxynaphthalene-2-carboxamide expressed the highest biological activity (MIC = 46.7 μmol/L) against M. smegmatis. This compound and 1-hydroxy-N-(3-methylphenyl)naphthalene-2-carboxamide were the most active compounds against all three tested strains. The PET inhibition expressed by IC₅₀ value of the most active compound 1-hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide was 5.3 μmol/L. The most effective compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. For all compounds, structure–activity relationships are discussed.     

Synthesis of some novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents

The reaction of 4-hydrazino-8-(trifluoromethyl)quinoline (2) with ethoxymethylenecyanoacetate afforded ethyl 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylate (3) and that with ethoxymethylenemalononitrile afforded 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carbonitrile (5). Compounds 3 and 5 were hydrolyzed to get 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylic acid and then reacted with acetic anhydride to afford 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]pyrazolo[3,4-d]oxazin-4-one (6), which was condensed with different aromatic amines to give a series of 5-substituted 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7). Compounds 3 and 5 also reacted with formamide, urea, and thiourea affording the corresponding pyrazolo[3,4-d]pyrimidines (8-13), respectively. Structures of the products have been determined by chemical reactions and spectral studies. All compounds of the series have been screened for their antibacterial and antifungal activity studies. The results are summarized in Tables 1 and 2.     

Synthesis and in vitro biological activity of new 4,6-disubstituted 3(2H)-pyridazinone-acetohydrazide derivatives

New 3(2H)-pyridazinone derivatives containing a N'-benzyliden-acetohydrazide moiety at position 2 were synthesized. The structures of these newly synthesized compounds were confirmed by IR, 1H NMR, and MS data. These compounds were tested for their antibacterial, antifungal, antimycobacterial, and cytotoxic activities. The compounds 2-[4-(4-chlorophenyl)-6-(morpholin-4-yl)-3-oxo-(2H)-pyridazin-2-yl]-N'-(4-tert-butylbenzyliden)acetohydrazide and 2-[4-(4-chlorophenyl)-6-(morpholin-4-yl)-3-oxo-(2H)-pyridazin-2-yl]-N'-(4-chlorobenzyliden) acetohydrazide exhibited activity against both Gram-positive and Gram-negative bacteria. Most of the compounds were active against E. coli ATCC 35218. The preliminary results of this study revealed that some target compounds exhibited promising antimicrobial activities.     

Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis

Tuberculosis treatment is shortened to six months by the indispensable addition of pyrazinamide (PZA) to the drug regimen that includes isoniazid and rifampin. PZA is a pro-drug of pyrazinoic acid (POA) (ref. 3), whose target of action has never been identified. Although PZA is active only against Mycobacterium tuberculosis, the PZA analog 5-chloro-pyrazinamide (5-Cl-PZA) displays a broader range of anti-mycobacterial activity. We have found that the eukaryotic-like fas1 gene (encoding fatty acid synthetase I, FASI) from M. avium, M. bovis BCG or M. tuberculosis confers resistance to 5-Cl-PZA when present on multi-copy vectors in M. smegmatis. 5-Cl-PZA and PZA markedly inhibited the activity of M. tuberculosis FASI, the biosynthesis of C16 to C24/C26 fatty acids from acetyl-CoA (ref. 6). Importantly, PZA inhibited FASI in M. tuberculosis in correlation with PZA susceptibility. These results indicate that FASI is a primary target of action for PZA in M. tuberculosis. Further characterization of FASI as a drug target for PZA may allow the development of new drugs to shorten the therapy against M. tuberculosis and may provide more options for treatment against M. bovis, M. avium and drug resistant M. tuberculosis.     

Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide

Pyrazinamide (PZA) is an important antituberculosis drug. Unlike most antibacterial agents, PZA, despite its remarkable in vivo activity, has no activity against Mycobacterium tuberculosis in vitro except at an acidic pH. M. tuberculosis is uniquely susceptible to PZA, but other mycobacteria as well as nonmycobacteria are intrinsically resistant. The role of acidic pH in PZA action and the basis for the unique PZA susceptibility of M. tuberculosis are unknown. We found that in M. tuberculosis, acidic pH enhanced the intracellular accumulation of pyrazinoic acid (POA), the active derivative of PZA, after conversion of PZA by pyrazinamidase. In contrast, at neutral or alkaline pH, POA was mainly found outside M. tuberculosis cells. PZA-resistant M. tuberculosis complex organisms did not convert PZA into POA. Unlike M. tuberculosis, intrinsically PZA-resistant M. smegmatis converted PZA into POA, but it did not accumulate POA even at an acidic pH, due to a very active POA efflux mechanism. We propose that a deficient POA efflux mechanism underlies the unique susceptibility of M. tuberculosis to PZA and that the natural PZA resistance of M. smegmatis is due to a highly active efflux pump. These findings may have implications with regard to the design of new antimycobacterial drugs.     

Preliminary screening for antibacterial and antimycobacterial activity of actinomycetes from less explored ecosystems

Actinomycetes from less explored ecosystems were screened for antibacterial and antimycobacterial activity. Crude bioactive compounds were produced by growing these strains by shake flask fermentation using soybean meal medium. Culture supernatant and mycelia were extracted with ethyl acetate and methanol, respectively. Antibacterial activity of crude extracts was tested by disc diffusion method against gram positive and gram negative bacteria. Actinomycete strains D10, D5, NEK5, ANS2, M104 and R2 showed prominent activity. Culture filtrates and crude extracts were tested against standard strain Mycobacterium tuberculosis H₃₇Rv and drug sensitive and drug resistant clinical isolates of M. tuberculosis by luciferase reporter phage (LRP) assay. Considerable variation was observed in antimycobacterial activity between actinomycete culture filtrates and solvent extracts. Actinomycete strains viz., D10, D5 (desert), CSA14 (forest), CA33 (alkaline soil), NEK5 (Neem plant), MSU, ANS2, R2 and M104 (marine) screened in the present study were found to be highly potent showing good antibacterial and antimycobacterial activity. Five of them such as A3, CSA1, EE9, ANS5 and R9 were exclusively active against M. tuberculosis. Secretary products of actinomycetes of rare ecosystems are meant to antagonize organisms in their respective environments. These are likely to be novel antimycobacterial compounds as they unknown to human pathogens.     

Synthesis and biological evaluation of indazolo[4,3-bc][1,5]naphthyridines (10-aza-pyrazolo[3,4,5-kl]acridines): a new class of antitumor agents

A series of potential DNA-binding antitumor agents, 2-[omega-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridines (2a-f), 10-aza derivatives of PZA, has been prepared by condensation of 9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[b][1,5]naphthyridin-10-one (6) with the appropriate (omega-aminoalkyl)hydrazine in tetrahydrofuran/methanol. Compound 6 was obtained by heating at 100 degrees C in H(2)SO(4)5, yielded by the condensation of 2,6-dichloro-3-nitrobenzoic acid (4) and 6-methoxy-3-pyridinamine (3). The non-covalent DNA-binding properties of 2 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives against human hormone-refractory prostate adenocarcinoma cell line (PC-3) are described and compared to that of parent drug PZA. We selected the most cytotoxic target derivatives 2c,d, the in vitro inactive 2f, and reference compound PZA to investigate whether in vitro treatment with these drugs was able to induce necrotic and/or apoptotic cell death. To this purpose, we evaluated the percentage of apoptotic cells in PC-3 treated with the target compounds 2c,d,f and reference compound PZA, by Annexin V staining and Propidium iodide (PI)/Annexin V, biparametric flow cytometric analysis and agarose gel electrophoresis.