Synthesis and in vitro antitubercular activity of some 1-[(4-sub)phenyl]-3-(4-{1-[(pyridine-4-carbonyl)hydrazono]ethyl}phenyl)thiourea

Various isonicotinyl hydrazones were prepared by reacting isonicotinyl hydrazide [INH] with 1-(4-acetylphenyl)-3-[(4-sub)phenyl]thiourea and were tested for their antimycobacterial activity in vitro against Mycobacterium tuberculosis H37Rv and INH-resistant M. tuberculosis using the BACTEC 460 radiometric system. Among the synthesized compounds, 1-(4-fluorophenyl)-3-(4-{1-[(pyridine-4-carbonyl)-hydrazono]ethyl}phenyl)thiourea (4d) was found to be the most potent compound with a minimum inhibitory concentration of 0.49 microM against M. tuberculosis H37Rv and INH-resistant M. tuberculosis. When compared to INH, 4d was found to be 3 and 185 times more active against M. tuberculosis H37Rv and INH-resistant M. tuberculosis, respectively, with a selectivity index of >300.     

Discovery of novel phenoxyacetic acid derivatives as antimycobacterial agents

A series of 2-{4-[1-amino (thioxo) methyl-5-(substituted phenyl)-4,5-dihydro-1H-3-pyrazolyl]-2-methoxyphenoxy}acetic acid and 2-{4-[1-carbamoyl-5-(substituted phenyl)-4,5-dihydro-1H-3-pyrazolyl]-2-methoxyphenoxy}acetic acid were synthesized and the in vitro activity of the synthesized compounds against Mycobacterium tuberculosis H37Rv (MTB) and INH-resistant M. tuberculosis (INHR-MTB) was studied. Among the synthesized compounds, compound (3f) 2{-[4-(1-carbamoyl-5-(chlorophenyl)-4,5-dihydro-1H-3-pyrazolyl]-2-methoxyphenoxy}acetic acid was found to be the most active against M. tuberculosis H37Rv (MTB) and INH-resistant M. tuberculosis (INHR-MTB) with minimum inhibitory concentration of 0.06 microg/ml.     

Inactivation of the Mycobacterium tuberculosis fadB4 gene results in increased virulence in host cell and mice

The genome of Mycobacterium tuberculosis encodes many proteins involved in fatty acid metabolism, a subset of which are required for virulence. The Mycobacterium tuberculosis fadB4 gene, which shares strong similarity with oxidoreductases and fatty acid synthases, is up-regulated during infection of macrophages and is predicted to protect the bacterium from the hostile environment of the host cell. In order to determine if fadB4 plays a role in the virulence of M. tuberculosis, we constructed a M. tuberculosis mutant in which the fadB4 had been disrupted (DeltafadB4). Surprisingly, DeltafadB4, grew more rapidly in host cells compared to wild-type M. tuberculosis or the DeltafadB4 or the gene-disrupted strain complemented with fadB4. In addition, macrophages infected with DeltafadB4 displayed reduced secretion of the cytokine TNF-alpha, suggesting a role for the FadB4 protein in influencing the pro-inflammatory host response to M. tuberculosis. After infection of mice, DeltafadB4 demonstrated an increased replication at early time-points post-infection compared to the growth of wild-type M. tuberculosis. This increased capacity of DeltafadB4 to replicate in vivo was reflected in the decreased time to death of immuno-deficient RAG-1(-/-) mice infected with M. tuberculosis lacking the fadB4 gene. Therefore fadB4 is part of the family of genes whose expression serves to regulate the virulence of M. tuberculosis within the host.     

1, 25 Dihydroxyvitamin D3 modulated cytokine response in pulmonary tuberculosis

1, 25 Dihydroxyvitamin D(3) (1, 25(OH)(2) D(3)) has gained significant importance in tuberculosis with regard to its immunoregulatory activities. Our aim was to evaluate the effect of 1, 25(OH)(2) D(3) on cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 60 healthy controls and 52 pulmonary tuberculosis patients were cultured with culture filtrate antigen (CFA) of M. tuberculosis and live M. tuberculosis with and without 1, 25(OH)(2) D(3) (10(-9), 10(-8)and 10(-7)M concentrations). The culture supernatants were used to estimate IL-8, IL-6, TGF-beta, IL-10, IFN-gamma, IL-12p40, IL-2, IL-4 and IL-5 levels by ELISA. 1, 25 Dihydroxyvitamin D(3) significantly suppressed IL-12p40 and IFN-gamma production in response to CFA and live M. tuberculosis with a maximum suppression at 10(-7)M concentration (p<0.0001). In CFA stimulated cultures, addition of 1, 25(OH)(2) D(3) significantly suppressed IL-8, IL-6 and IL-10 whereas the IL-2 levels were significantly increased in controls. It variably influenced the Th2 cytokines, showing an increased trend for IL-4 and suppressed IL-5 levels. We report that 1, 25(OH)(2) D(3) differentially modulates production of cytokines in response to M. tuberculosis antigens by predominantly suppressing IL-12p40 and IFN-gamma production in a dose dependent manner. Our results suggest a role for vitamin D in restricting acquired immune response against tuberculosis by regulating cytokine production.     

Some 3-thioxo/alkylthio-1,2,4-triazoles with a substituted thiourea moiety as possible antimycobacterials.

A series of novel N-alkyl/aryl-N'-[4-(4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thione-5-yl)phenyl]thioureas 1-19 and three S-alkylated representatives of the former, N-alkyl/aryl-N'-[4-(3-aralkylthio-4-alkyl/aryl-4H-1,2,4-triazole-5-yl)phenyl]thioureas 20-22, were synthesized and tested for antimycobacterial activity against Mycobacterium tuberculosis H37Rv as well as Mycobacterium fortuitum ATCC 6841 which is a rapid growing opportunistic pathogen. Compounds 4 and 9-11 were found to possess the same MIC value with that of Tobramycin against M. fortuitum ATCC 6841 whereas 1-3 and 21 had positive response against M. tuberculosis H37Rv at varying degrees. Compound 21 was identified as the most potent derivative of the 1-22 series by an MIC value of 6.25 microg/mL and selectivity index of 1.6.     

Characterization of the Mycobacterium tuberculosis 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase: potential for drug development

Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-D-erythritol is formed from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5'-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 microM for MEP and 53.2 microM for CTP. Calculated kcat and kcat/Km values were 0.72 min(-1) and 12.3 mM(-1) min(-1) for MEP and 1.0 min(-1) and 18.8 mM(-1) min(-1) for CTP, respectively.     

Synthesis and anti-tuberculosis activity of glycitylamines

A series of glycitylamines, which were prepared in few steps from readily available carbohydrates, were tested for their ability to inhibit tuberculosis growth in an Alamar Blue BCG colourimetric assay. Several derivatives, including (2R,3R)-1-(hexadecylamine)pent-4-ene-2,3-diol, (2R,3R)-1-(hexadecylmethylamino)pent-4-ene-2,3-diol and 5-deoxy-5-hexadecylmethylamino-d-arabinitol, were prepared in good to excellent (44–90%) overall yield and exhibited micromolar (20–41 μM) inhibitory activity that was similar to the first line tuberculosis (TB) drug ethambutol (39 μM) in the same assay. The ease and low cost of the synthesis of the glycitylamines offers definite advantages for their use as potential TB drugs.     

Synthesis and in vitro antimycobacterial activity of N1-nicotinoyl-3-(4'-hydroxy-3'-methyl phenyl)-5-[(sub)phenyl]-2-pyrazolines

A series of N1-nicotinoyl-3- (4'-hydroxy-3'-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their antimycobacterial activity in vitro against Mycobacterium tuberculosis H37Rv (MTB) and INH-resistant M. tuberculosis (INHR-MTB) using the agar dilution method. Among the synthesized compounds, compound (i) N1-nicotinyl-3-(4'-hydroxy-3'-methyl phenyl)-5-(1'-chlorophenyl)-2-pyrazoline was found to be the most active agent against MTB and INHR-MTB, with minimum inhibitory concentration of 0.26 microm. When compared to INH-compound i was found to be 2.8- and 43.7-fold more active against MTB and INHR-MTB, respectively.     

Simian immunodeficiency virus-induced changes in T cell cytokine responses in cynomolgus macaques with latent Mycobacterium tuberculosis infection are associated with timing of reactivation

Understanding the early immunologic events accompanying reactivated tuberculosis (TB) in HIV-infected individuals may yield insight into causes of reactivation and improve treatment modalities. We used the cynomolgus macaque (Macaca fascicularis) model of HIV-Mycobacterium tuberculosis coinfection to investigate the dynamics of multifunctional T cell responses and granuloma T cell phenotypes in reactivated TB. CD4(+) and CD8(+) T cells expressing Th1 cytokines (IFN-γ, IL-2, TNF) and Th2 cytokines (IL-4 and IL-10) were followed from latent M. tuberculosis infection to reactivation after coinfection with a pathogenic SIV. Coinfected animals experienced increased Th1 cytokine responses to M. tuberculosis Ags above the latent-response baseline 3-5 wk post-SIV infection that corresponded with peak plasma viremia. Th2 cytokine expression was not Ag specific, but strong, transient IL-4 expression was noted 4-7 wk post-SIV infection. Animals reactivating <17 wk post-SIV infection had significantly more multifunctional CD4(+) T cells 3-5 wk post-SIV infection and more Th2-polarized and fewer Th0-, Th1-polarized CD8(+) T cells during weeks 1-10 post-SIV infection than animals reactivating >26 wk post-SIV infection. Granuloma T cells included Th0-, Th1-, and Th2-polarized phenotypes but were particularly rich in cytolytic (CD107(+)) T cells. When combined with the changes in peripheral blood T cells, these factors indicate that events during acute HIV infection are likely to include distortions in proinflammatory and anti-inflammatory T cell responses within the granuloma that have significant effects on reactivation of latent TB. Moreover, it appears that mycobacteria-specific multifunctional T cells are better correlates of Ag load (i.e., disease status) than of protection.     

Antimycobacterial activity: a facile three-component [3+2]-cycloaddition for the regioselective synthesis of highly functionalised dispiropyrrolidines

A series of twelve dispiropyrrolidines were synthesized using [3+2]-cycloaddition reactions. The synthesized compounds were screened for their antimycobacterial activity against M. tuberculosis H(37)Rv and INH resistant M. tuberculosis strains using agar dilution method, four of them showed good activity with MIC of less than 1 μM. Compound 4'-[5-(4-fluorophenyl)pyridin-3-yl]-1'-methyldispiro[indan-2,2' pyrrolidine-3',2″-indan]-1,3,1″-trione (4b) was found to be the most active with MIC of 0.1215 and 5.121 μM, respectively.     

Ring-substituted quinolines. Part 2: Synthesis and antimycobacterial activities of ring-substituted quinolinecarbohydrazide and ring-substituted quinolinecarboxamide analogues

Additional structural modifications of the new chemical entity, 2,8-dicyclopentyl-4-methylquinoline (DCMQ; MIC = 6.25 μg/mL, M. tuberculosis H37Rv) resulted in the synthesis of four new series of the ring-substituted quinolinecarbohydrazides (series 1–4) constituting 22 analogues. All new derivatives were evaluated for in vitro antimycobacterial activities against drug-sensitive M. tuberculosis H37Rv strain. Certain ring-substituted-2-quinolinecarbohydrazide analogues described herein showed good inhibitory activity. In particular, analogues 4-(1-adamantyl)-2-quinolinecarbohydrazide (2d), 4,5-dicyclopentyl-2-quinolinecarbohydrazide (2e), 4,8-dicyclopentyl-2-quinolinecarbohydrazide (2f), and 4,5-dicyclohexyl-2-quinolinecarbohydrazide (2g) have exhibited the MIC value of 6.25 μg/mL. Further investigation of the most suitable lead prototype, 4-(1-adamantyl)-2-quinolinecarbohydrazide (2d, series 1) led to the synthesis of N2-alkyl/N2,N2-dialkyl/N2-aryl-4-(1-adamantyl)-2-quinolinecarboxamides (series 5) consisting of 13 analogues. Some of the synthesized carboxamides 7a, 7h, and 7m reported herein have exhibited excellent antimycobacterial activities in the range of 6.25–3.125 μg/mL against drug-sensitive and drug-resistant M. tuberculosis H37Rv strains.     

Secondary metabolites from the roots of Engelhardia roxburghiana and their antitubercular activities

Engelharquinone (1), engelharquinone epoxide (2), engelharolide (3), and engelhardic acid (4), were isolated as naturally occurring products from a plant source, Engelhardia roxburghiana together with 20 previously known compounds, four of which were hitherto not known as plant constituents. Their structures were identified by means of spectroscopic analysis. A biological evaluation showed that three of the previously isolated antitubercular constituents [(-)-4-hydroxy-1-tetralone, 3-methoxyjuglone and engelhardione] and engelharquinone (1) exhibited moderate antitubercular activity against Mycobacterium tuberculosis 90-221387.     

Gatifloxacin derivatives: synthesis, antimycobacterial activities, and inhibition of Mycobacterium tuberculosis DNA gyrase

Sixteen 7-substituted gatifloxacin derivatives were synthesized and evaluated for antimycobacterial activity in vitro and in vivo against Mycobacterium tuberculosis H37Rv (MTB) and multi-drug resistant M. tuberculosis (MDR-TB), and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. tuberculosis. Among the synthesized compounds, 1-cyclopropyl-6-fluoro-8-methoxy-7-[[[N4-[1'-(5-isatinyl-beta-semicarbazo)]methyl]3-methyl]N1-piperazinyl]-4-oxo-1,4-dihydro-3-quinoline carboxylic acid (3d) was found to be the most active compound in vitro with an MIC of 0.0125 microg/mL against MTB and MTR-TB. In the in vivo animal model 3d decreased the bacterial load in lung and spleen tissues with 3.62- and 3.76-log10 protections, respectively. Compound 3d was also found to be equally active as gatifloxacin in the inhibition of the supercoiling activity of wild-type M. tuberculosis DNA gyrase with an IC50 of 3.0 microg/mL. The results demonstrate the potential and importance of developing new quinolone derivatives against mycobacterial infections.     

Oxadiazole mannich bases: synthesis and antimycobacterial activity

A series of oxadiazole mannich bases were synthesized by reacting oxadiazole derivatives, dapsone and appropriate aldehyde in the presence of methanol. The synthesized compounds were evaluated for antimycobacterial activity against M. tuberculosis H(37)Rv and INH resistant M. tuberculosis. Among the synthesized compounds, compound (4) 3-{2-furyl[4-(4-{2-furyl[5-(2-naphthyloxymethyl)-2-thioxo-2,3-dihydro-1,3,4-oxadiazol-3-yl]methylamino}phenylsulfonyl)anilino]methyl}-5-(2-naphthyloxymethyl)-2,3-dihydro-1,3,4-oxadiazole-2-thione was found to be the most promising compound active against M. tuberculosis H(37)Rv and isoniazid (INH) resistant M. tuberculosis with Minimum inhibitory concentration (MIC) 0.1 microM & 1.10 microM respectively.     

Antitubercular activity of novel substituted 4,5-dihydro-1H-1-pyrazolylmethanethiones

A series of, anilino-5-(substituted) phenyl-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolylmethanethione and 2-chloroanilino-5-(substituted) phenyl-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolylmethanethione were synthesized by the reaction between hydrazine hydrate and the chalcones (3a-k) followed by condensation with the appropriate aryl isothiocyanate which yielded the N-substituted pyrazoline derivatives. These were tested for their in-vitro anti-mycobacterial activity against INH resistant Mycobacterium tuberculosis (INHR MTB) using the BACTEC 460 radiometric system. Compound 2-chloroanilino-5-(2,6-dichlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-methanethione (6i) was found to be most active agent with a minimum inhibitory concentration of 0.96 microg/mL.     

Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3

We have examined the receptor-ligand interactions and the method of phagocytosis of virulent Mycobacterium tuberculosis by human monocytes. mAb against complement receptors (CR) inhibit adherence and phagocytosis of M. tuberculosis in fresh nonimmune serum. A mAb against the type 1 CR (CR1) inhibits adherence of M. tuberculosis by 40 +/- 5%, and three different mAb against the type 3 CR (CR3) each inhibit adherence by 39 +/- 5% to 47 +/- 4%. A mAb against CR1 used in combination with one of the three mAb against CR3 inhibits adherence by up to 64 +/- 7%. Most strikingly, two mAb used in combination against CR3 inhibit adherence by up to 81 +/- 2%. mAb against other monocyte surface Ag do not significantly influence adherence. In like fashion, mAb against CR but not other monocyte surface Ag inhibit adherence of preopsonized M. tuberculosis in the presence of heat-inactivated serum. By electron microscopy, monocytes ingest all M. tuberculosis that adhere in the presence of nonimmune serum; mAb against CR3 markedly inhibit ingestion. In contrast to CR, the FcR and the beta-glucan-inhibitable receptor for zymosan play little or no role in mediating M. tuberculosis adherence or ingestion. Adherence of M. tuberculosis is serum-dependent, requiring greater than or equal to 2.5% serum for optimal adherence. Heat inactivation of serum markedly reduces adherence of M. tuberculosis (75.5 +/- 7%) and preopsonization of bacteria enhances adherence by 2.9 +/- 0.4-fold. Adherence is also markedly reduced in C3- or factor B-depleted serum; repletion with C3 or factor B increases adherence by 2.1 +/- 0.4-fold and 1.86 +/- 0.05-fold, respectively. Fab anti-C3 IgG markedly inhibits monocyte adherence of preopsonized M. tuberculosis (71 +/- 1%). C component C3 is fixed to M. tuberculosis by the alternative C pathway as determined by a whole bacterial cell ELISA. Human monocytes ingest M. tuberculosis by conventional phagocytosis as viewed by electron microscopy. This study demonstrates that human monocyte CR1 and CR3 mediate phagocytosis of M. tuberculosis and C component C3 in serum is acting as the major bacterium-bound ligand.     

Different cytokine patterns correlate with the extension of disease in pulmonary tuberculosis.

The relative amounts of different pro- and anti-inflammatory cytokines released at the site of infection by bronchoalveolar lavage (BAL) cells may influence the presentation of tuberculosis. To investigate this hypothesis the in situ release by BAL cells of the following cytokines was measured and correlated with the chest X-ray findings of 43 patients with pulmonary tuberculosis: interleukin (IL)-8, macrophage inflammatory protein-1alpha (MIP-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma), IL-2, IL-4 and IL-5. The release of IL-8 and IL-6 decreased with the progression of the disease, while the release of MIP-1alpha was increased in patients with advanced tuberculosis. The release of TNF-alpha and TGF-beta did not differ between patients with or without cavitary lesions. The Th1 (IFN-gamma and IL-2) and Th2 (IL-4 and IL-5) cytokine release exhibited a gradual increment with the advance of tuberculosis. Thus, our data provide evidence that a Th0 cytokine pattern is predominant at the site of pulmonary tuberculosis. In conclusion, immunoparalysis status could not be observed in our patients with severe tuberculosis.     

Ring-substituted quinolines as potential anti-tuberculosis agents

We report in vitro antimycobacterial properties of ring-substituted quinolines (series 1-4) constituting 56 analogues against drug-sensitive and drug-resistant M. tuberculosis H37Rv strains. The most effective compounds 2h (R1 = R2 = c-C6H11, R3 = NO2, series 1) and 13g (R1 = OC7H15, R2 = NO2, series 4) have exhibited an MIC value of 1 microg/mL against drug-sensitive M. tuberculosis H37Rv strain that is comparable to first line anti-tuberculosis drug, isoniazid. Selected analogues (2d, 2g, 2h, 4e, 6b, 13b, 13g, and 14e, MIC: < or = 6.25 microg/mL) upon further evaluation against single-drug-resistant (SDR) strains of M. tuberculosis H37Rv have produced potent efficacy in the range between 6.25 and 50 microg/mL.     

T cell cytokine patterns in active tuberculosis patients in a northeastern area of Romania

Developing countries like Romania have a high incidence of tuberculosis. Literature data suggest that people in these countries have an important Th2-type immune background which prevents a protective Th1 response of the host against Mycobacterium tuberculosis. Our study is the first attempt in Romania to identify cytokine patterns in active tuberculosis. The study included 15 patients with active pulmonary tuberculosis and 11 healthy volunteers. Peripheral blood mononuclear cells (PBMC), stained with carboxyfluoresceine-diacetate-succinimidylester (CFSE), were incubated for 7 days with purified protein derivate (PPD) or with medium alone. Intracellular synthesis of IFNgamma and IL-4 in proliferated (CFSE(low)) T cells was detected by flowcytometry. The results showed that both Th1 (IFNgamma) and Th2 (IL-4) cytokines are produced in response to in vitro PPD-stimulation of PBMC from pulmonary tuberculosis patients and healthy controls. Moreover, the proportion between IFNgamma and IL-4 is tilted in favour of IL-4 in PPD-activated (CD3+ CFSE(low)) cells from healthy persons, who did not develop active tuberculosis during the two-year study time interval. This predominance of Th2 effectors points to the need to further investigate the role of IL-4 in the M. tuberculosis infection.