Biotransformation of isoniazid produced isonicotinic acid (1), isonicotinic acid N-oxide (2), and isonicotinamide (3) which were isolated by column chromatography using silica gel and Sephadex LH 20 and elucidated using various spectroscopies. This is the first report for isolation of 2. Antituberculosis activity was evaluated against Mycobacterium tuberculosis strains: drug sensitive (DS), multiple drug resistant (MDR) and extensively drug resistant (XDR). 1-3 and isoniazid showed MIC of 63.49, 0.22, 15.98 and 0.88 µM, respectively, against the DS strain. For the MDR strain, 2 and 3 exhibited MIC of 28.06 and > 1000 µM, respectively, while 1 was inactive. Moreover, 2 had an MIC of 56.19 µM against XDR strain, while 1 and 3 were inactive. Docking simulation using enoyl ACP reductase (InhA) revealed favorable protein-ligand interactions. In silico study of pharmacokinetics and hepatotoxicity predicted 1-3 to have good oral bioavailability and 2 to have a lower hepatoxicity probability than isoniazid.

Isoniazid is a synthetic antimicrobial and one of the most essential first-line drugs used in the treatment of tuberculosis. In addition, isoniazid has been used as a prophylactic drug for patients with latent Mycobacterium tuberculosis infection to prevent the reactivation of the disease. It is a prodrug that is activated by catalase-peroxidase (KatG) enzyme. Catalase peroxidase enzyme converts isoniazid to reactive species. Isoniazid reactive species inhibit enoyl acyl-carrier-protein reductase (InhA) enzyme, which is involved in the biosynthesis of fatty acids of mycobacteria. Isoniazid is metabolized by amidase enzyme into isonicotinic acid and hydrazine. Chronic toxicity of isoniazid results in hepatotoxicity and peripheral neuropathy. For active tuberculosis, isoniazid is often used together with rifampicin, pyrazinamide, and either streptomycin or ethambutol. Multiple extensively and totally drug-resistant strains of Mycobacterium tuberculosis were reported. Due to the development of Mycobacterium tuberculosis resistance to isoniazid, a continuous search for new drugs is a demand to combat this global problem.

Arecoline is a psychoactive alkaloid containing a reduced pyridine nucleus, isolated from Areca catechu L. (Arecaceae) with different biological activities on cardiovascular, digestive, nervous, and endocrine systems. Arecoline is the main toxic component of A. catechu responsible for oral carcinoma. It possesses a variety of pharmacological activities; it exhibits anticancer activity. It was reported that arecoline causes cytotoxicity through apoptosis in human endothelial cells. Moreover, the death of human leukemia K562 cells was induced by arecoline. Biotransformation is a structural modification of compounds such as amino acids, toxins, and drugs by enzymatic chemical reactions within living organisms. According to enzymatic sources, biotransformation is classified into three major types; microbial, plant cell culture, and animal cell culture transformation. It was suggested that microorganisms could be employed as a model of mammalian metabolism. Studying both mammalian and microbial transformation of arecoline is of interest by which we can reduce its toxicity, increase its bioavailability, and produce more active metabolites.

The search for anticancer drugs is of continuous interest. Arecoline is an alkaloid with anticancer activity. Herein, the metabolism of arecoline through fungal transformation was investigated for the discovery of potential anticancer drugs with higher activity and selectivity. Compounds 1–5 were isolated, and their structures were fully elucidated using various spectroscopic analyses, including 1D and 2D NMR, ESIMS, and HRESIMS. This is the first report for the isolation of compounds 1 and 2. An MTT assay was performed to determine the cytotoxic activity of arecoline and its metabolites in vitro using non-small-cell lung cancer A549 and leukemia K562 cell lines compared to staurosporine and doxorubicin as positive controls. For the non-small-cell lung A549 cell line, arecoline hydrobromide, staurosporine, and doxorubicin resulted in IC50 values of 11.73 ± 0.71 µM, 10.47 ± 0.64 µM, and 5.05 ± 0.13 µM, respectively, while compounds 1, 3, and 5 exhibited IC50 values of 3.08 ± 0.19 µM, 7.33 ± 0.45 µM, and 3.29 ± 0.20 µM, respectively. For the leukemia K562 cell line, the IC50 values of arecoline hydrobromide, staurosporine, and doxorubicin were 15.3 ± 1.08 µM, 5.07 ± 0.36 µM, and 6.94 ± 0.21 µM, respectively, while the IC50 values of compounds 1, 3 and 5 were 1.56 ± 0.11 µM, 3.33 ± 0.24 µM, and 2.15 ± 0.15 µM, respectively. The selectivity index value of these compounds was higher than 3. These results indicated that compounds 1, 3, and 5 are very strong cytotoxic agents with higher activity than the positive controls and good selectivity toward the tested cancer cell lines. Cell cycle arrest was then studied by flow cytometry to investigate the apoptotic mechanism. Docking simulation revealed that most compounds possessed good binding poses and favorable protein-ligand interactions with muscarinic acetylcholine receptor M3-mAChR protein. In silico study of pharmacokinetics using SwissADME predicted compounds 1–5 to be drug-like with a high probability of good oral bioavailability.