Telacebec (Q203; IAP6) is an imidazopyridine amide (IAP) compound that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. It has the potential for the treatment of tuberculosis. Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of Q203. Q203 is active against the reference strain Mycobacterium tuberculosis H37Rv with MIC50s of 2.7 nM in culture broth medium and 0.28 nM inside macrophages. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, these data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

Physicochemical Properties

Molecular Formula	C29H28CLF3N4O2
Molecular Weight	557.01
Exact Mass	556.185
Elemental Analysis	C, 62.53; H, 5.07; Cl, 6.36; F, 10.23; N, 10.06; O, 5.74
CAS #	1334719-95-7
Related CAS #	1334719-95-7;1566517-83-6 (Ditosylate);
PubChem CID	68234908
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.615
LogP	7.32
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	7
Heavy Atom Count	39
Complexity	796
Defined Atom Stereocenter Count	0
SMILES	ClC1C([H])=C([H])C2=NC(C([H])([H])C([H])([H])[H])=C(C(N([H])C([H])([H])C3C([H])=C([H])C(=C([H])C=3[H])N3C([H])([H])C([H])([H])C([H])(C4C([H])=C([H])C(=C([H])C=4[H])OC(F)(F)F)C([H])([H])C3([H])[H])=O)N2C=1[H]
InChi Key	OJICYBSWSZGRFB-UHFFFAOYSA-N
InChi Code	InChI=1S/C29H28ClF3N4O2/c1-2-25-27(37-18-22(30)7-12-26(37)35-25)28(38)34-17-19-3-8-23(9-4-19)36-15-13-21(14-16-36)20-5-10-24(11-6-20)39-29(31,32)33/h3-12,18,21H,2,13-17H2,1H3,(H,34,38)
Chemical Name	6-chloro-2-ethyl-N-[[4-[4-[4-(trifluoromethoxy)phenyl]piperidin-1-yl]phenyl]methyl]imidazo[1,2-a]pyridine-3-carboxamide
Synonyms	Q-203 free base; Q 203; Telacebec; 1334719-95-7; Q203; Q-203; 6-chloro-2-ethyl-n-(4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl)benzyl)imidazo[1,2-a]pyridine-3-carboxamide; MMV687696; 55G92WGH3X; CHEMBL3298910;
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	Mycobacterium tuberculosis H37Rv( MIC50=2.7 nM )
ln Vitro	Telacebec (Q203; IAP6) is an imidazopyridine amide (IAP) compound that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. It has the potential for the treatment of tuberculosis. Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of Q203. Q203 is active against the reference strain Mycobacterium tuberculosis H37Rv with MIC50s of 2.7 nM in culture broth medium and 0.28 nM inside macrophages. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, these data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.
ln Vivo	Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Q203 has a bioavailability of 90% and a terminal half-life of 23.4 h. The volume of distribution is moderate (5.27 l per kg body weight), and the systemic clearance is low (4.03 mL/min per kg). After 4 weeks of treatment, reductions of 90%, 99% and 99.9% in M. tuberculosis H37Rv bacterial load is observed in the groups treated with Q203 at 0.4, 2 and 10 mg per kg body weight, respectively
Enzyme Assay	Q203 is active against the reference strain Mycobacterium tuberculosis H37Rv with MIC50s of 2.7 nM in culture broth medium and 0.28 nM inside macrophages. Microsomal stability assay. [1] Compounds (2 μM final in 0.2% DMSO) were incubated with 0.5 mg mL−1 human (pool of 200, mixed gender), male dog, male rat or male mouse liver microsomes in potassium phosphate buffer. The reaction was initiated by the addition of NADPH and stopped either immediately or at 10, 20, 30 or 60 min for a precise estimate of clearance. A triple quadrupole Quattro Premier mass spectrometer with electrospray ionization (ESI) was employed for sample analysis. Samples were passed through trapping cartridges (Acquity BEH RP18 50 mm × 2.1 mm, 1.7 μm, Waters, Milford, MA) followed by an analytical column. The percentage of remaining compound was calculated by comparing with the initial quantity at 0 min. Half-life was then calculated using first-order reaction kinetics. CYP450 inhibition assays. [1] The assay used individual fluorescent probe substrates with individual recombinant human cytochrome P450 (rhCYP) isozymes and a fluorescent detection according to previously published methods36. The probe substrates (in 0.5% DMSO) used for each isozyme were as follows: 7-benzyloxy-4-(trifluoromethyl)-coumarin for CYP3A4, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6, 3-cyano-7-ethoxycoum (CEC) for 1A2 and 2C19 and 7-methoxy-4-(trifluoromethyl)-coumarin (MFC) for 2C9. Fluorescence was measured using Victor3 V multilabel plate reader. The IC50 was determined using an eight-point concentration curve with threefold serial dilution.
Cell Assay	High-content screening assay in infected macrophages.[1] The assay was performed as previously described9,30. Briefly, Raw 264.7 cells were infected with M. tuberculosis H37Rv-GFP at a multiplicity of infection of 2:1 and dispensed into 384-well plates. After 5 d of infection, macrophages were stained with Syto 60. Image acquisition was performed on an EVOscreen Mark III platform integrated with Opera. Bacterial load and macrophage number were quantified using proprietary image analysis software. Minimum inhibitory concentration determination.[1] H37Rv-GFP was dispensed into 384-well plates in 7H9 medium without glycerol. Mycobacterial growth was determined by measuring fluorescence intensity at 488 nm after 5 d of incubation. Alternatively, the MICs were determined using the resazurin susceptibility assay or a turbidity-based assay in 384-well plates. The MIC50 were determined using an eight- or ten-point concentration curve with threefold serial dilution. Cytotoxicity.[1] Cytotoxicity was tested against the human cell lines SH-SY5Y (brain), HEK293 (kidney) and HepG2 (liver) using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assay as previously described. ATP depletion assay on M. tuberculosis H37Rv.[1] M. tuberculosis H37Rv was exposed to the test compounds for 24 h (aerobiosis) or 5 d (anaerobiosis), mixed with an equal volume of BacTiter-Glo reagent and incubated in the dark for 10 min. Luminescence was recorded on a Victor3 V multilabel plate reader.
Animal Protocol	Pharmacokinetics.[1] BALB/c mice and Sprague Dawley rats were used for pharmacokinetic studies. Compounds were given at a dose of 2 mg per kg body weight intravenously or 10 mg per kg body weight orally. Otherwise stated, the compounds were formulated in 20% TPGS (D-α tocopheryl polyethylene glycol 1000 succinate) for repeated-dose studies and in 40% PEG400, pH4 for single-dose studies. Blood samples were taken through the caudal vena cava using 1-mL syringes before perfusion. Samples were collected from three mice or rats at 0.5, 1, 2, 6, 12, 24 and 48 h post-dose. Blood samples were centrifuged at 3,200g for 10 min at 4 °C. Following centrifugation, plasma was collected and frozen until further analysis. Compound concentrations were determined by LC-MS. In vivo efficacy in the mouse model of tuberculosis.[1] The acute model was performed as previously described16. Briefly, mice were infected with a high dose of M. tuberculosis H37Rv. Dosing was initiated 6 d after infection. Drugs were administered orally for 3 d. Bacterial load in the lungs of infected mice was determined by colony-forming unit (CFU) enumeration. For the established mouse model, BALB/c mice were infected with 2 × 102 to 2 × 103 CFU of M. tuberculosis H37Rv by the intranasal route. Treatment was initiated 3 weeks after infection. Drugs were formulated in 20% TPGS and administered by oral gavage for 28 d, five times per week. Bacterial load in the lungs of infected mice was determined by CFU enumeration. For histopathology analysis, segments of the lungs were fixed with 10% neutral formalin, embedded in paraffin and processed for histology. Sections (5 μm) were stained with H&E. Histologic sections were used for morphologic analysis of the size and number of granulomas using an image analyzer . All animal studies were approved by the Institutional Animal Care and Use Committee of the Institut Pasteur-Korea in strict accordance with the Korean Animal Protection Law. Rats: Sprague Dawley rats are used for pharmacokinetic studies. Compounds (Q203) are given at a dose of 2 mg per kg body weight intravenously or 10 mg per kg body weight orally. The compounds (Q203) are formulated in 20% TPGS (d-α tocopheryl polyethylene glycol 1000 succinate) for repeated-dose studies and in 40% PEG400, pH4 for single-dose studies. Blood samples are taken through the caudal vena cava using 1-mL syringes before perfusion. Samples are collected from three mice or rats at 0.5, 1, 2, 6, 12, 24 and 48 h post-dose. Blood samples are centrifuged at 3,200g for 10 min at 4 °C. Following centrifugation, plasma is collected and frozen until further analysis. Compound concentrations are determined by LC-MS; Mice: Efficacy of Q203 in a mouse model of established tuberculosis is studied. Bacterial loads are enumerated in the lung of infected mice after 14 d and 28 d of treatment. Q203 is used at 0.4, 2 and 10 mg per kg body weight. Bedaquiline and isoniazid (INH) are used as positive controls at 6.5 and 15 mg per kg body weight, respectively. Five mice per group and per time point are used. Sprague Dawley rats
References	[1]. Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nat Med. 2013 Sep;19(9):1157-60.
Additional Infomation	New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.[1]

Solubility Data

Solubility (In Vitro)

DMSO: ~20 mg/mL ( 35.9 mM) Water: ~20 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: 2 mg/mL (3.59 mM) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.0 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.7953 mL	8.9765 mL	17.9530 mL
	5 mM	0.3591 mL	1.7953 mL	3.5906 mL
	10 mM	0.1795 mL	0.8977 mL	1.7953 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.