Thioacetazone targets cyclopropane mycolic acid synthases (CMASs) after activation by the bacterial monooxygenase EthA (also known as EtaA). CMASs are S-adenosylmethionine-dependent methyltransferases responsible for the cyclopropanation of mycolic acids, which are long-chain fatty acids essential for the structural integrity of the mycobacterial cell wall. By inhibiting mycolic acid synthesis, the drug increases bacterial cell wall permeability, leading to cell lysis and death. EthA also activates other anti-tuberculosis agents, ethionamide and isoxyl.

The mycobacterial monooxygenase EthA, which also activates two more anti-tuberculosis medications, isoxyl and ethionamide, is responsible for the prodrug thiacetone [3].
Thioacetazone exhibits potent antibacterial activity against Mycobacterium tuberculosis H37Rv with a minimum inhibitory concentration (MIC) of 0.1 μg/mL. It is a prodrug that requires activation by the mycobacterial monooxygenase EthA. In cytotoxicity assays against human P-gp-negative KB-3-1 cells and P-glycoprotein-expressing KBV1 cells, Thioacetazone showed IC50 values greater than 50 μM, indicating relatively low cytotoxicity in these cell lines.

The N-deacetylation of thiacetone exhibited Km and Vmax values of 0.57 mM and 0.123 nmol p-aminobenzaldehyde thiosemicarbazone formation/min/mg cytosolic protein, respectively. Rats' metabolism is much slower than that of cats, mice, or humans' livers when it comes to thioacetone metabolism [2].
The N-deacetylation of Thioacetazone in rat liver cytosol exhibits Km and Vmax values of 0.57 mM and 0.123 nmol p-aminobenzaldehyde thiosemicarbazone formed/min/mg cytosolic protein, respectively. The metabolism of Thioacetazone is similar in the livers of cats, mice, and humans, but is significantly slower in rat liver. This species difference in metabolic rate is an important consideration for preclinical studies.

In vitro enzyme assays for Thioacetazone typically involve measuring its inhibition of mycolic acid cyclopropane synthase activity. The compound is incubated with the enzyme and its substrate, and the reaction products are analyzed to determine the extent of inhibition. For prodrug activation studies, Thioacetazone is incubated with recombinant EthA monooxygenase, and the activated metabolite is detected using chromatographic methods. CYP inhibition can be assessed using human liver microsomes or recombinant CYP isoforms.

In vitro cellular assays use mycobacterial cultures, such as M. tuberculosis H37Rv, to determine the MIC by broth microdilution or agar dilution methods. Cytotoxicity is assessed in mammalian cell lines (e.g., KB-3-1, KBV1) using the MTT assay after 72 hours of exposure to various concentrations of the compound. The effect of the compound on host cells can also be evaluated by measuring cytokine production or cell viability following treatment.

In vivo efficacy of Thioacetazone is typically evaluated in mouse models of tuberculosis infection. Infected mice are treated with Thioacetazone orally or via other routes, and bacterial load in organs (e.g., lungs, spleen) is quantified after a defined treatment period. Pharmacokinetic studies in rats have characterized the metabolism and clearance of the compound. Animal models are also used to assess the toxicity and side-effect profile of the drug.

Thioacetazone is administered orally and is absorbed from the gastrointestinal tract. It is metabolized in the liver, primarily via N-deacetylation. The metabolism of Thioacetazone exhibits species differences, with rat liver metabolism being significantly slower than that of cats, mice, or humans. The compound's metabolites are excreted in urine. Due to its metabolic profile, Thioacetazone has a relatively long half-life, allowing for once-daily dosing in clinical settings.

Thioacetazone is associated with significant toxicity, most notably severe cutaneous adverse reactions, including Stevens-Johnson syndrome and toxic epidermal necrolysis, particularly when used in combination with other anti-TB drugs. Other adverse effects include gastrointestinal disturbances, hepatotoxicity, and bone marrow suppression. Due to its toxicity profile, its use has been largely replaced by safer alternatives, though it remains in use in some regions where cost is a primary concern.

[1]. J L Stigliani, et al. New Insights Into the Chemical Behavior of S-oxide Derivatives of Thiocarbonyl-Containing Antitubercular Drugs and the Influence on Their Mechanisms of Action and Toxicity. Ann Pharm Fr. 2019 Mar;77(2):126-135.
[2]. P Khanna, et al. Characteristics of a Cytosolic Arylacylamidase Metabolizing Thiacetazone. J Pharmacol Exp Ther. 1992 Sep;262(3):1225-31.
[3]. Anuradha Alahari, et al. Thiacetazone, an Antitubercular Drug That Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria. PLoS One. 2007 Dec 19;2(12):e1343.
[4]. C A Peloquin, et al. Pharmacokinetic Evaluation of Thiacetazone. Pharmacotherapy. Sep-Oct 1996;16(5):735-41.

Thioacetamide belongs to the acetamide and aniline classes of compounds. Thioacetamide has been used in clinical trials to study the treatment of intracellular infections of Mycobacterium avium. Thioacetamide is a thioaminourea anti-mycobacterial drug, active against isoniazid-resistant Mycobacterium tuberculosis strains. Thioacetamide is a thioaminourea prodrug with anti-tuberculosis activity. Although the exact mechanism of action of thioacetamide is not fully elucidated, it appears to target and inhibit cyclopropane mycoic acid synthases (CMASs) after activation by the bacterial monooxygenase EtaA. CMASs are a class of S-adenosylmethionine-dependent methyltransferases responsible for the cyclopropanelation of mycoic acids. By inhibiting mycoic acid synthesis, bacterial cell wall permeability increases, resistance to damage decreases, and ultimately cell lysis occurs. Mycoic acids are long-chain fatty acids and an important component of the mycobacterial cell wall, playing a crucial role in resisting cell damage and mycobacterial virulence. Thiazolone is a thioaminourea drug often used in combination with other antimycobacterial drugs in the initial and consolidation phases of anti-tuberculosis treatment regimens. Thiazolone-containing treatment regimens are less effective than the short-course regimens recommended by the International Union Against Tuberculosis (UITB), and some developing countries use these regimens to reduce drug costs. (Excerpt from Martindale Pharmacopoeia, 30th edition, page 217)
Thioacetazone was first synthesized in the 1940s and was widely used as a second-line anti-tuberculosis agent. Its mechanism involves inhibition of mycolic acid synthesis after bacterial activation. It has been used in clinical trials for the treatment of intracellular Mycobacterium avium infections. The compound is a thioaminourea prodrug active against isoniazid-resistant strains. Due to its toxicity and the availability of better drugs, it is now rarely used in developed countries but remains on the WHO List of Essential Medicines for use in resource-limited settings.

C10H12N4OS

236.29

236.073

104-06-3

9568512

White to off-white solid powder

1.3±0.1 g/cm3

225-230 ℃

1.639

1.22

3

3

3

16

285

0

S=C(N([H])[H])N([H])/N=C(\[H])/C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(C([H])([H])[H])=O

SRVJKTDHMYAMHA-WUXMJOGZSA-N

InChI=1S/C10H12N4OS/c1-7(15)13-9-4-2-8(3-5-9)6-12-14-10(11)16/h2-6H,1H3,(H,13,15)(H3,11,14,16)/b12-6+

N-[4-[(E)-(carbamothioylhydrazinylidene)methyl]phenyl]acetamide

Neotibil; Diazam; Thioacetazone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~423.21 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)