BTZ043 racemate, the racemic mixture (R- and S-isomer) of BTZ043, is a decaprenylphosphoryl-β-D-ribose 2'-epimerase (DprE1) inhibitor acting as a new antimycobacterial agent that kill Mycobacterium tuberculosis. BTZ043 displayed similar activity against all clinical isolates of M. tuberculosis that were tested, including extensively drug-resistant and multidrug-resistant strains, indicating that it targets a previously unknown biological function. BTZ043 is bactericidal, reducing viability in vitro by more than 1000-fold in under 72 hours, which is comparable to the INH killing effect. It has potential to be used as a antimycobacterial agent that kill Mycobacterium tuberculosis by blocking arabinan synthesis. The inhibition of BTZ-resistant DprE1 followed the trend observed in the MIC measurements, with the C387G mutant being more resistant to inhibition by PyrBTZ01, PyrBTZ02, and BTZ043 (7- to 9-fold increases in IC50) than the C387S mutant (2.5- to 4-fold increases in IC50). Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. BTZ043 presented favorable in vitro absorption-distribution-metabolism-excretion/toxicity (ADME/T) and in vivo pharmacokinetic profiles. BTZ043 did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

Physicochemical Properties

Molecular Formula	C17H16F3N3O5S
Molecular Weight	431.38
Exact Mass	431.076
Elemental Analysis	C, 47.33; H, 3.74; F, 13.21; N, 9.74; O, 18.54; S, 7.43
CAS #	957217-65-1
Related CAS #	BTZ043;1161233-85-7
PubChem CID	49769085
Appearance	Solid powder
LogP	3.903
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	1
Heavy Atom Count	29
Complexity	720
Defined Atom Stereocenter Count	0
SMILES	O=C1C2C=C(C(F)(F)F)C=C(C=2SC(N2CCC3(OC(C)CO3)CC2)=N1)[N+](=O)[O-]
InChi Key	GTUIRORNXIOHQR-UHFFFAOYSA-N
InChi Code	InChI=1S/C17H16F3N3O5S/c1-9-8-27-16(28-9)2-4-22(5-3-16)15-21-14(24)11-6-10(17(18,19)20)7-12(23(25)26)13(11)29-15/h6-7,9H,2-5,8H2,1H3
Chemical Name	2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]decan-8-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one
Synonyms	BTZ043; BTZ 043; BTZ-043; BTZ043 Racemic mixture; BTZ043 Racemate.
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	DprE1
ln Vitro	By targeting decaprenylphosphoryl-β-D-ribose 2-epimerase, BTZ043 abolishes the formation of decaprenylphosphoryl arabinose, leading to cell lysis and death of Mycobacterium tuberculosis. BTZ043 displays similar activity against all clinical isolates of M. tuberculosis, including multidrug-resistant and extensively drug-resistant strains. BTZ043 displays significant activity against M. tuberculosis H37Rv and Mycobacterium smegmatis with MIC of 1 ng/mL (2.3 nM) and 4 ng/mL (9.2 nM), respectively, which is more potent than those of the existing tuberculosis (TB) drugs isoniazid (INH) and ethambutol (EMB) with MIC of 0.02-0.2 μg/mL and 1-5 μg/mL, respectively. BTZ043 is less effective in two different model systems (auxotrophy and starvation) involving metabolically inert M. tuberculosis, indicating that BTZ043 blocks a step in active metabolism similar to isoniazid (INH). BTZ043 treatment in M. smegmatis cells decreases the growth rate rapidly followed by a swelling of the poles and lysis of the cells after a few hours, which is similar but delayed in M. tuberculosis. BTZ043 (1/4 MIC 0.375 ng/mL) in combination with TMC207 (1/4 MIC 20 ng/mL) has a stronger cidal effect on M. tuberculosis but not BTZ-resistant M. tuberculosis mutant than TMC207 alone at a concentration of 80 ng/mL.
ln Vivo	In a mouse model of chronic tuberculosis, administration of BTZ043 at 37.5 mg/kg or 300 mg/kg for 4 weeks reduces the bacterial burden in the lungs and spleens by 1 and 2 logs, respectively.
Enzyme Assay	BTZ043, also known as 8-Nitro-benzothiazinones (BTZs), is a potent inhibitor of decaprenyl-phosphoribose-epimerase (DprE1) with MIC values of of 2.3 nM and 9.2 nM for M. tuberculosis H37Rv and Mycobacterium smegmatis, respectively.
Cell Assay	BTZ043 displayed similar activity against all clinical isolates of M. tuberculosis that were tested, including extensively drug-resistant and multidrug-resistant strains, indicating that it targets a previously unknown biological function. BTZ043 is bactericidal, reducing viability in vitro by more than 1000-fold in under 72 hours, which is comparable to the INH killing effect.
Animal Protocol	Formulated in carboxymethyl cellulose formulation (0.25%); 37.5 mg/kg, or 300 mg/kg; p.o. once a day BALB/c mice infected with a low bacillary load (~200 CFU) of M. tuberculosis H37Rv via aerosol
References	[1]. Science.2009 May 8;324(5928):801-4 [2]. Antimicrob Agents Chemother.2012 Nov;56(11):5790-3. [3]. Bioorg Med Chem Lett. 2013 Sep 1;23(17):4919-22. .

Solubility Data

Solubility (In Vitro)

DMSO : 22~25 mg/mL ( 50.99~57.95 mM )

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (5.80 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. 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. Solubility in Formulation 2: ≥ 1.38 mg/mL (3.20 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 13.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2.5 mg/mL (5.80 mM)  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3181 mL	11.5907 mL	23.1814 mL
	5 mM	0.4636 mL	2.3181 mL	4.6363 mL
	10 mM	0.2318 mL	1.1591 mL	2.3181 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.