Design, Synthesis, and In Silico Studyof Two N-Substituted Pyrazinamide Analogs as Potential Antituberculosis Agents

A series of coumarin derivatives was designed as potential antituberculosis agents. The compounds were screened against active and dormant Mycobacterium tuberculosis (Mtb). Compounds 3k and 3n were found to have the most promising activity against replicating MtbH37Rv exhibiting minimum inhibitory concentration of 4.63 and 9.75μM respectively. The compounds were also effective against dormant MtbH37Rv exhibiting more potency than the standard drugs, isoniazid and rifampicin. The compounds were found to be non-cytotoxic against human cell lines. This study provides promising antituberculosis agents that are effective against replicating as well as dormant Mtb and can thus act as potential leads for further development.

Analog development of existing drugs and direct drug delivery to the lungs by inhalation as treatments for multiple and extensively drug resistant (MDR and XDR) tuberculosis (TB) represent new therapeutic strategies. Pyrazinamide (PZA) is critical to drug sensitive TB therapy and is included in regimens for MDR TB. However, PZA-resistant Mycobacterium tuberculosis (Mtb) strains threaten its use. Pyrazinoic acid esters (PAEs) are PZA analogs effective against Mtb in vitro, including against the most common PZA resistant strains. However, PAEs require testing for TB efficacy in animal models. PAEs were delivered daily as aqueous dispersions from a vibrating mesh nebulizer to Mtb infected guinea pigs for 4weeks in a regimen including orally administered first-line TB drugs. PAEs tested as a supplement to oral therapy significantly reduced the organ bacterial burden in comparison to infected, untreated control animals. Thus, PAE aerosol therapy is a potentially significant addition to the regimen for PZA resistant MDR-TB and XDR-TB treatment. Interestingly, low dose oral PZA treatment combined with standard therapy also reduced bacterial burden. This observation may be important for PZA susceptible disease treatment. The present study justifies further evaluation of PZA analogs and their lung delivery to treat TB.

Chemical reactivity and bioactivity properties of pyrazinamide analogs of acetylsalicylic acid and salicylic acid using conceptual density functional theory

Background: Tuberculosis (TB), a disease caused by the bacillus bacteria Mycobacterium tuberculosis is one of the major contributors of ill health in the world. TB is ranked in the top 10 causes of death globally and it is the leading killer associated with a single infectious agent. According to the World Health Organization (WHO), global number of deaths associated with TB have been slowly declining with 1.3 million in reported 2016 and 2017, and 1.2 million reported in 2018 and 2019. Objective: The synthesis, characterisation, biological evaluations, and the prediction of ADMET properties of the novel benzylamine derivatives. Methods: Commercially available reagents and solvents were purchased from Sigma Aldrich and Merck (South Africa). All chemicals were used as received, unless otherwise stated. The synthesised crude compounds were purified by flash silica gel column chromatography (5 – 30% ethyl acetate in hexane). The successful formation and purity of the synthesised compounds was confirmed by NMR, HRMS and melting point. Results: The respective organic compounds were synthesised by treating 3-ethoxysalcyladehyde, 5-bromo-3-ethoxysalcyladehyde, 5-chloro-3-ethoxysalcyladehyde with various aromatic amines and the products were obtained in good to excellent yields. The 1H and 13C NMR spectra of all the products showed the appearance of the methylene signals ranging from 3.88 – 4.68 ppm and 42.25 – 52.57 ppm respectively. Additionally, most compounds showed anti-Mycobacterium tuberculosis activity that ranged between 20 and 28 µM. Conclusion: A total of 36 compounds were synthesised and successfully biologically evaluated against Mycobacterium tuberculosis (Mtb) H37RV strain. All compounds showed activity against Mtb at concentrations of > 20 µM < 28 µM with the exception of compound one that was active against Mtb at higher concentration (MIC90 > 125 µM).

Rationale: Pyrazinamide (PZA) is an essential component of empiric firstand second-line tuberculosis (TB) treatment regimens. However, PZA drug susceptibility testing (DST) is difficult to perform and not routinely available. Genetic mutations in the pncA gene of Mycobacterium tuberculosis (Mtb) commonly confer PZA resistance, but limited data on the prevalence and diversity of these mutations are available from clinical populations of TB patients. We sequenced pncA to estimate PZA resistance among MDR and XDR TB patients in KwaZulu-Natal province, South Africa. Methods: We prospectively enrolled 206 MDR TB and 377 XDR TB patients from September 2011 to September 2014. Sputum collected from each participant underwent mycobacterial culture and DST for isoniazid (H), rifampin (R), streptomycin (S), kanamycin (Km) and ofloxocin (Ofx). We extracted DNA from pure Mtb isolates and performed targeted sequencing of the pncA gene, characterizing the proportion, frequency and structure of polymorphisms. Results: To date, targeted sequencing has been completed for 314 isolates from 80 MDR TB (16 resistant to HR 20 different mutations were seen among MDR TB subjects, of which, only 7 were seen in more than one participant. Among XDR TB participants, one mutation (insertion of cytosine after the 456 locus) was present in 166 (71%), consistent with the clonality of XDR TB patients in KwaZulu-Natal. Conclusion: Nearly 70% of MDR TB and nearly all XDR TB participants had pncA mutations, and likely PZA resistance, in our cohort. A wide diversity of mutations was seen. Most mutations were singlets, suggesting acquisition of mutations de novo, perhaps due to empiric use. One specific mutation was common among XDR TB participants, but it occurred in conjunction with a specific RFLP genotype associated with clonal expansion. Given the difficulty in determining PZA susceptibility, characterizing pncA mutations may provide important data for developing rapid genotypic PZA susceptibility assays. BACKGROUND • South Africa has one of the highest incidence rates of multidrug-resistant tuberculosis (MDR TB) and extensively drug-resistant tuberculosis (XDR TB) with high rates of HIV co-infection. • Pyrazinamide (PZA) is used empirically in drug-resistant TB treatment regimens. • Precise acidic conditions are required in vitro to conduct PZA drug susceptibility testing (DST). However, acidic environments inhibit the growth of Mycobacterium tuberculosis (Mtb) bacilli. • Therefore, conventional PZA DST is difficult to perform in vitro and not widely available. • Gene sequencing is an effective method to identify mutations associated with resistance for some firstand second-line anti-TB drugs. • Genetic mutations in the pncA gene of Mtb typically confer resistance to PZA. However data on the frequency and diversity of pncA mutations is limited. • Thus, the actual prevalence of PZA resistance in patients with TB and drug-resistant TB is unknown. Funding: NIH/NIAID R01AI087465 [PI Neel Gandhi] and NIH/NIAID R01AI089349 [PI Neel Gandhi] Contact: Salim Allana, MD, MPH; salim.allana@emory.edu SETTING • Study site: KwaZulu-Natal province, South Africa, which has a population of 10.2 million. • TB incidence rate is nearly 1,100 per 100,000 population, and more than 80% of TB cases are HIV co-infected. • In 2012, MDR TB incidence was 45 cases/100,000 population and XDR TB incidence was 3.1 cases/100,000 population. • The HIV prevalence among adults (15-49 years of age) is 16.8%. • MDR TB patients receive standardized second-line treatment which includes PZA, ethambutol, kanamycin, moxifloxacin, terizidone, and ethionamide. • XDR TB patients receive a similar regimen, except that capreomycin, moxifloxacin and PAS replace kanamycin. • DST testing to evaluate PZA susceptibility is not routinely performed in this setting. OBJECTIVE Among MDR and XDR TB subjects in KwaZulu-Natal province, South Africa: • To characterize the frequency and diversity of polymorphisms in the pncA gene. • To estimate the prevalence of pyrazinamide resistance.

Pyrazinamide (PZA) is a key drug in the treatment of tuberculosis (TB), including multidrug-resistant TB. Drug susceptibility testing (DST) of Mycobacterium tuberculosis against PZA is not included in the World Health Organization's yearly proficiency testing. There is an increasing need to establish quality control of PZA DST. To evaluate the performance of PZA DST and to introduce a quality assurance system for the test in Sweden. Panels with PZA-susceptible and -resistant isolates were used in three rounds of proficiency testing in all five Swedish clinical TB laboratories and our reference laboratory. All laboratories used the MGIT 960 system. Minimum inhibitory concentrations (MICs) were determined and the pncA gene was sequenced to further characterise the 52 panel strains. Good agreement was seen between the phenotypic PZA DST and pncA sequence data, and MIC determination confirmed high levels of resistance. However, in contrast to other drugs, for which correct proficiency test results were observed, specificity problems occurred for PZA DST in some laboratories. In Sweden, using panel testing, differences were seen in the proficiency of TB laboratories in correctly identifying PZA susceptibility. Improved results were noted in the third round; PZA has therefore been included in yearly proficiency testing.

Renal Tuberculosis

Objective To analyze the relationship between pyrazinamide plasma concentration and liver injury in patients with tuberculosis and explore the early prediction method of drug-induced liver injury (DILI) in initial treatment in patients with tuberculosis. Methods Data of tuberculosis patients (all the patients were initially treated, no other complications, and underlying disease) receiving 2HREZ/4HR treatment regimen [isoniazid, rifampicin, ethambutol, and pyrazinamide were given at the first 2 months of treatment, isoniazid and rifampicin were given at the later 4 months of treatment] in Nanjing Chest Hospital, Medical School of Southeast University from January to December 2016 were collected. According to the pyrazinamide concentration in serum on the 6th day of treatment, patients were divided into 3 groups, the 40 mg/L group. The incidences of DILI in the 3 groups were compared. Results A total of 45 patients in accordance with the criteria were collected, including 33 males and 17 females with age from 18 to 50 years and an average age of (34±10) years. Among them, 36 patients had pulmonary tuberculous, 9 patients had tuberculous pleurisy, and 5 patients had tuberculosis meningitis. The pyrazinamide concentrations in serum were 16-51 mg/L and the average concentration was (35±9) mg/L. There were 4 patients in the 40 mg/L group. Of the 45 patients, 11 patients developed DILI and the incidence was 24.4%. The pyrazinamide plasma concentrations in the 11 patients with DILI were 26 mg/L to 51 mg/L, 4 patients′ concentrations were 40 mg/L and 7 patients′ concentrations were >40 mg/L. The incidences of DILI in the 40 mg/L groups were 0/4, 14.2 %( 4/28), and 53.85% (7/13), respectively. The difference between the 20-40 mg/L and the >40 mg/L groups was statistically significant (χ2=7.708, P=0.008). Conclusions The DILI incidence in initial treatment of tuberculosis patients increased with increase of pyrazinamide plasma concentration. Early therapeutic drug monitoring for pyrazinamide could predict the occurrence of DILI and was beneficial for reducing or avoiding the effect of DILL for tuberculosis treatment. Key words: Pyrazinamide; Drug-induced liver injury; Tuberculosis

Structure activity relationship of pyrazinoic acid analogs as potential antimycobacterial agents

Objective To investigate the characterizations and contributions of mutations in pncA and rpsA whole genes sequence in pyrazinamide(PZA) resistant Mycobacterium tuberculosis(MTB). Methods All of the 161 clinical strains of MTB collected from Guangzhou Chest Hospital during September 2010 and November 2012 were subjected to determine the susceptibilities to PZA by the MGIT 960 PZA system and to obtain pncA and rpsA whole gene sequences by DNA sequencing.Then the significant difference of pncA and rpsA mutation between the PZA resistant and susceptible isolates were analyzed by chi square test. Results The mutation frequency of 52 PZA resistant isolates was 84.6%(44/52), but the 109 PZA susceptible isolates had no mutations, which showed highly significant difference of pncA mutation frequency between the PZA resistant and the susceptible isolates (χ2=126.92, P=0.00).rpsA mutation was observed in 3 PZA resistant MTB isolates while only 1 PZA susceptible ones was found rpsA mutation, which exhibited no significant difference of rpsA mutation between the PZA resistant and the susceptible isolates (χ2=3.42, P=0.06).Thirty four types of pncA mutations were found in 44 PZA resistant isolates of MTB, including 12 novel mutations (L deletion at 27, E deletion at 91, E deletion at 111, Q deletion at 122, VVG deletions at 130 to 132, V deletion at 131, D8A, S18P, H57Y, F58S, E174G and M175R substitutions) and 1 promoter mutation at -11 nucleotide position with A to G.The identified mutations were randomly dispersed on the pncA whole gene sequence, but 9 isolates were observed with pncA mutations centralized in the region of G132-T142. Three PZA-resistant isolates，without pncA mutation, were observed with rpsA mutations of R474W, R474L, and E433D at C-terminus, and 1 PZA-susceptible strain showed mutation of Q162R in rpsA. Conclusions In the study, mutations in pncA gene is the major mechanism of PZA resistance and direct sequencing the pncA gene by PCR should help to rapidly identify PZA-resistant clinical strains of MTB.Furthermore, novel mutations of pncA in the study indicate the regional investigations are necessary for learning about the characteristics of pncA mutations in PZA-resistant strains of MTB.However, 3′ terminal of rpsA gene sequence may be added as the second PZA resistant relevant region for molecular identification of PZA susceptibility.（Chin J Lab Med,2014,37:285-289） Key words: Mycobacterium tuberculosis; Pyrazinamide; Mutation

The Yamaguchi reaction is widely and generally applied to synthesize esters and lactones. It involves 2,4,6-trichlorobenzoyl chloride as the Yamaguchi reagent, 4-dimethylaminopyridine, and triethylamine. Pyrazinamide is a crucial first-line drug for tuberculosis treatment, therefore their analogues are interesting in organic synthesis. In general, the synthesis pyrazinamide analogues involve reaction of pyrazine-2-carboxylic acids with thionyl chloride to yield the corresponding acyl chlorides, which on treatment with amines gave pyrazine-2-carboxamides. However, thionyl chloride is listed under the Chemical Weapons Convention and releases toxic sulfur oxide when react with carboxylic acid. We successfully synthesized series of pyrazinamide analogues using the Yamaguchi reaction. The synthesis involved reaction of pyrazine-2-carboxylic acid with various aliphatic and aromatic amines in the presence of Yamaguchi reagent and 4-dimethylaminopyridine. The yield of the pyrazine-2-carboxamides and the reaction time depended on the type of the amine (aliphatic vs aromatic), substitution pattern, and number of substituents on the aromatic amines. N-(4-chlorophenyl)pyrazine-2-carboxamides can be prepared by this method in 81% yield; N-(2-ethylhexyl)pyrazine-2-carboxamide and N-(4-fluorobenzyl)pyrazine-2-carboxamide showed the best activity against Mycobacterium tuberculosis H37Rv (<6.25 μg/mL). This result could lead to find more active pyrazinamide analogues.

The bioavailability of isoniazid, rifampin, and pyrazinamide in 2 combined formulations of the 3 drugs (Rifater) for use primarily in the short-course chemotherapy of tuberculosis has been studied in Chinese patients in Singapore and Hong Kong. One formulation, containing 50 mg isoniazid, 120 mg rifampin, and 300 mg pyrazinamide per tablet is suitable for daily use, whereas the other, containing higher proportions of isoniazid and pyrazinamide, is designed for intermittent treatment, each tablet containing 125 mg isoniazid, 100 mg rifampin, and 375 mg pyrazinamide. Appropriate dosages for the Chinese patients, whose average weight was approximately 50 kg, were 5 and 6 tablets, respectively. Plasma concentrations of the 3 drugs after giving such dosages of the 2 combined formulations were compared in 16 patients, 8 in Singapore and 8 in Hong Kong, by means of a crossover study, with the concentrations obtained when identical doses of the 3 drugs were given using standard separate drug formulations. The concomitant urinary excretions of the drugs and their major metabolites were also estimated. Very similar results were obtained whether the drugs were given as the combined preparations or in their standard separate formulations, demonstrating the excellent bioavailability of all 3 drugs in each of the 2 combined formulations.

Antibiotic resistance is an increasing threat to global public health. Developing new antibiotics and alternative treatments is crucial for combating resistant strains and reducing the global health burden. Hence, we synthesized and evaluated the antitubercular potential of dihydropyridine derivatives. A simplified Biginelli condensation method was employed to synthesize novel 3,4-dihydropyrimidine derivatives (4a-4m) via a one-pot three-component reaction using various substituted benzaldehydes. Reaction completion was monitored via thin-layer chromatography. The structures of the compounds were confirmed by FT-IR, mass spectrometry, 1H NMR, and 13C NMR spectroscopy, and melting points were determined by differential scanning calorimetry. ADMET screening was performed for all synthesized compounds. Selected compounds were tested for their antibacterial and anti-tubercular activity against gram-positive and gram-negative bacteria. ADMET screening identified eight potential compounds: 4c, 4e, 4f, 4g, 4i, 4j, 4k, and 4m. The literature emphasized DprE1 as a critical target for anti-tubercular activity. Molecular docking studies revealed promising binding affinities for compounds 4g (-7.67), 4d (-7.316), 4e (-7.062), and 4c (-7.042) against DprE1. Furthermore, to study the binding stability and interaction patterns of protein-ligand complexes, a molecular dynamics simulation was performed. The stability of the protein-ligand complex was confirmed by low protein RMSD values and minimal fluctuations in ligand RMSD, indicating a stable binding pose throughout the 200ns simulation. These compounds also exhibited significant antibacterial activity against gram-positive and gram-negative bacteria compared to standard drugs. In-vitro antitubercular assays against the H37Rv strain demonstrated moderate to notable efficacy relative to the standard reference drug. The findings suggest that these compounds could serve as promising drug candidates. Further development may lead to their use as effective antituberculosis agents in future research.

To investigate retrospectively the incidence of drug-induced hepatitis (DIH) caused by antituberculosis drugs including isoniazid (INH), rifampicin (RFP), with and without pyrazinamide (PZA), and to evaluate risk factors for DIH in tuberculosis patients complicated with chronic hepatitis (CH). One hundred and seven tuberculosis patients with CH (M/F= 96/11, mean age +/- SE, 60.8 +/- 1.4 yr) admitted to our hospital during 1998-2006, whose laboratory data had been followed before and at least 2 months after starting antituberculosis chemotherapy, were enrolled in this study. Of these, 58 were being treated with anti-tuberculosis chemotherapy consisting of INH, RFP and PZA (HRZ group) and the remaining 49 with INH and RFP (HR group). For a case-control study, patients admitted to the hospital during the same period and without CH were selected to each CH patient (n=107) of the same gender, the same treatment regimens, and the same age. Clinical diagnosis of CH was based on laboratory data and in some cases pathological findings; etiology of CH was C-CH (CH caused by hepatitis C virus) in 68 patients, B-CH (CH caused by hepatitis B virus) in 23, and alcoholic CH in 16. DIH was defined by elevation of serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) at 1 or 2 months after starting anti-tuberculosis chemotherapy. For patients with serum levels of AST or ALT already abnormally high before starting chemotherapy, an increase of > 1.5 times from the initial serum level was defined to indicate DIH, whereas for patients with AST and ALT within the normal range, and increase of > 3X the normal upper limit was defined to indicate DIH. The incidence of DIH was calculated separately in the groups HRZ and HR for patients with and patients without CH (control). In the HRZ group, the severity of DIH was defined by the maximum serum levels of AST and ALT, and their mean values were compared between CH patients and the control. Risk factors for DIH were examined by comparing patients with and without CH. The clinical course after development of DIH was also followed. [Results] The incidence of DIH in the HRZ group was 13/ 58 (22.4%) for CH patients and 10/36 (27.8%), 2/13 (15.4%) and 1/9 (11.1%) for C-CH, B-CH and alcoholic hepatitis patients, respectively, which was significantly (p < 0.05) higher than that in the control [4/58 (6.9%)]. Confining to the C-CH patients, the incidence of DIH was 10/36 (27.8%) compared with the control 2/36 (5.6%) (p < 0.05). In contrast, the incidence of DIH in the HR group was not significantly different between CH patients and the control, [2/49 (4.1%) vs 2/49 (4.1%)], respectively. The severity of DIH in the HRZ group estimated by the maximum level of serum AST and ALT was not significantly different in CH patients and the control (176.6 +/- 28.1 vs. 311.0 +/- 154.5 IU/L for AST and 187.8 +/- 19.1 vs. 277.8 +/- 72.4 IU/L for ALT). Of the 13 CH patients suffering from DIH caused by antituberculosis chemotherapy containing INH, RFP and PZA, 3 were continued treatment without altering the regimen, and 9 were continued treatment after changing the regimen to INH and RFP, omitting PZA. The one remaining patient was re-treated using INH, RFP and ethambutol (EB), but this again resulted in development of DIH, and he was ultimately treated with INH, EB and levofloxacin, with a successful outcome. Thus, at least 12 out of the 13 CH patients who developed DIH in the HRZ group could be treated by an anti-tuberculosis chemotherapy regimen containing INH and RFP excluding PZA. In C-CH patients who were treated with INH, RFP and PZA, the incidence of DIH was significantly higher when the daily alcohol intake was >20 g [8/18 (44.4%)] compared with those <20 g [0/10 (0%)] (p < 0.05), indicating that alcohol is a risk factor for DIH in C-CH patients treated with INH, RFP and PZA. In CH patients, anti-tuberculosis chemotherapy containing INH and RFP without PZA can be used safely. The inclusion of PZA in the regimen does substantially increase the incidence of DIH but nonetheless it can be used with caution, especially bearing in mind that daily alcohol intake of >20 g is a significant risk factor for C-CH patients.

Pyrazinamide (PZA) is a first-line key drug used in combination with other agents for the treatment of tuberculosis (TB). Phenotypic and molecular assays for testing susceptibility of Mycobacterium tuberculosis (Mtb) to PZA have been developed, with the assay in liquid medium at acidic pH in the Bactec MGIT 960 (M960) system being routinely used in the mycobacteriology laboratories. However, false resistance to PZA by this method was reported to occur by several investigators, mostly due to high Mtb inoculum, which may impair drug activity by increasing the pH of the medium. In this study, a revision of the literature on the issue of false resistance in the M960 PZA assay was performed. In the reports examined, all improvements of the M960 test proposed to decrease false resistant results were based on the use of reduced inoculum densities of Mtb cells, to be easily translated into laboratory practice.