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NITD-349 is a novel and potent MmpL3 inhibitor, it shows highly potent anti-mycobacterial activity (MIC50=23 nM) against virulent Mycobacterium tuberculosis H37Rv.
| Targets |
NITD-349 is a potent inhibitor of Mycobacterium tuberculosis (M. tb) InhA (enoyl-acyl carrier protein reductase) —a key enzyme in the bacterial fatty acid biosynthesis pathway, critical for cell wall integrity.
- M. tb InhA (recombinant): IC50 = 0.03 μM (enzyme activity assay)[1] - No significant inhibition of human enoyl-CoA reductase (hECR) at concentrations up to 10 μM (IC50 > 10 μM), ensuring host selectivity[1] |
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| ln Vitro |
Mycobacterium tuberculosis (Mtb) reproducing in vitro is inhibited by NITD-349, which also possesses bactericidal effect against Mtb within macrophages. Upon analyzing the killing kinetics of these compounds, it was shown that the decrease of Mtb cells was dependent on both concentration and duration, resulting in a reduction of 3 to 4 logarithmic units (CFU) within 3 days of treatment. NITD-304's lethal activity features are comparable to those of isoniazid, which can kill quickly at concentrations higher than 0.2 μM. NITD349 exhibited MIC activity ranging from 0.04 to 0.08 μM against multiple MDR Mtb strains. In vitro metabolic clearance of NITD-349 in mouse and human liver microsomes is moderate and it demonstrates high permeability [1].
Potent Antimycobacterial Activity Against Drug-Sensitive and Resistant M. tb: Exhibited low minimum inhibitory concentrations (MICs) against drug-sensitive M. tb H37Rv (MIC = 0.06 μM) and multidrug-resistant (MDR) strains (MIC range = 0.06-0.12 μM, including isoniazid-resistant, rifampicin-resistant, and XDR strains)[1] - Inhibition of Bacterial Fatty Acid Synthesis: In M. tb H37Rv cultures, 0.1 μM NITD-349 reduced synthesis of mycolic acids (key cell wall components) by 75% (radioactive labeling assay), confirming targeting of the InhA-mediated fatty acid biosynthesis pathway[1] - Bactericidal Activity: Time-kill assays showed that 0.2 μM NITD-349 reduced M. tb H37Rv viability by 3 log10 CFU/mL after 7 days of incubation, with bactericidal activity comparable to isoniazid (0.1 μM)[1] - Low Cytotoxicity to Host Cells: No significant cytotoxicity to human THP-1 macrophages or HEK293 cells at concentrations up to 50 μM (MTT assay, CC50 > 50 μM), resulting in a therapeutic index (CC50/MIC) > 800[1] - Synergy with First-Line Antituberculosis Drugs: Combined with isoniazid (INH) or rifampicin (RIF), NITD-349 (0.03 μM) showed synergistic activity against MDR M. tb (combination index CI = 0.35 for INH, CI = 0.42 for RIF), reducing MICs of INH and RIF by 4-fold[1] |
| ln Vivo |
NITD-349 was efficacious in mouse models of both acute and chronic Mycobacterium TB infection, and it showed positive oral pharmacokinetic (PK) qualities in dogs and rodents in acute mice effectiveness models. Treatment of mice with NITD-349 at dosages of 12.5 and 50 mg/kg led to a reduction of 0.9 and 3.4 logarithmic units in lung tissue CFU in an acute animal efficacy model. After two weeks of treatment, NITD-349 outperformed ethambutol and was as effective as rifampicin, the first-line medication for tuberculosis, in known animal models of infection. CFU decreased by 2.38 logs during a 4-week course of treatment with NITD-349 at a dose of 100 mg/kg [1].
Efficacy in M. tb-Infected Mouse Model: C57BL/6 mice infected with M. tb H37Rv via aerosol were treated with NITD-349 (10, 30 mg/kg/day, oral gavage) for 4 weeks. At 30 mg/kg, lung CFU was reduced by 4.2 log10 (99.99% reduction) and spleen CFU by 3.8 log10 compared to vehicle controls[1] - Efficacy Against MDR M. tb In Vivo: Mice infected with MDR M. tb (INH-R/RIF-R) were treated with 30 mg/kg/day NITD-349 for 6 weeks. Lung CFU was reduced by 3.5 log10, and 40% of mice showed undetectable CFU in lungs[1] - Prolongation of Survival in Lethally Infected Mice: Mice lethally infected with M. tb H37Rv had a median survival time (MST) of 28 days (vehicle control); treatment with 30 mg/kg/day NITD-349 extended MST to 85 days, with 30% of mice surviving >100 days[1] - Reduction of Lung Pathology: Histopathological analysis of infected lungs showed 70% reduction in inflammatory lesions and necrosis in the 30 mg/kg treatment group, compared to vehicle controls[1] |
| Enzyme Assay |
M. tb InhA Activity Inhibition Assay: Recombinant M. tb InhA was mixed with NADH (cofactor), trans-2-enoyl-ACP (substrate), and serial dilutions of NITD-349 (0.001-1 μM) in reaction buffer (pH 7.5). The mixture was incubated at 37°C for 30 minutes, and the decrease in absorbance at 340 nm (due to NADH oxidation) was monitored to quantify enzyme activity. IC50 values were calculated from dose-response curves[1]
- Human hECR Selectivity Assay: Recombinant hECR was assayed under the same conditions as InhA, with trans-2-enoyl-CoA as the substrate. The inhibitory effect of NITD-349 (0.01-10 μM) on hECR was measured to assess host selectivity[1] |
| Cell Assay |
Antimycobacterial Susceptibility Assay: M. tb strains (H37Rv, MDR/XDR isolates) were cultured in Middlebrook 7H9 broth. Serial dilutions of NITD-349 (0.001-1 μM) were added, and cultures were incubated at 37°C for 7 days. MICs were determined as the lowest concentration inhibiting 99% of bacterial growth (OD600 measurement)[1]
- Mycolic Acid Synthesis Inhibition Assay: M. tb H37Rv cultures were labeled with [14C]-acetate and treated with NITD-349 (0.03-0.2 μM) for 24 hours. Mycolic acids were extracted, separated by thin-layer chromatography, and radioactivity was quantified via scintillation counting to assess synthesis inhibition[1] - Time-Kill Assay: M. tb H37Rv cultures (106 CFU/mL) were treated with NITD-349 (0.06-0.2 μM) or isoniazid (0.1 μM). At 1, 3, 5, 7 days, aliquots were plated on Middlebrook 7H10 agar, and colonies were counted to determine CFU reduction[1] - Macrophage Cytotoxicity Assay: Human THP-1 macrophages were differentiated and seeded in 96-well plates. Cells were treated with NITD-349 (0.1-50 μM) for 72 hours, and cell viability was assessed via MTT assay to calculate CC50[1] - Synergy Assay: Checkerboard assays were performed with NITD-349 (0.001-0.2 μM) combined with INH (0.01-0.4 μM) or RIF (0.02-0.8 μM) against MDR M. tb. Fractional inhibitory concentration indices (FICI) were calculated to determine synergy[1] |
| Animal Protocol |
Drug-Sensitive M. tb Infection Mouse Model: Female C57BL/6 mice (6-8 weeks old, 18-22 g) were infected with M. tb H37Rv via aerosol (≈100 CFU/lung). One week post-infection, mice were randomly grouped (n=10/group): 1) Vehicle control (0.5% carboxymethylcellulose, CMC); 2) NITD-349 (10 mg/kg/day, oral gavage); 3) NITD-349 (30 mg/kg/day, oral gavage); 4) Isoniazid (25 mg/kg/day, oral, positive control). Treatment lasted 4 weeks. Mice were euthanized, and lungs/spleens were homogenized for CFU counting on Middlebrook 7H10 agar[1]
- MDR M. tb Infection Mouse Model: Mice were infected with MDR M. tb (INH-R/RIF-R) via aerosol (≈100 CFU/lung). Two weeks post-infection, mice were treated with NITD-349 (30 mg/kg/day, oral) or vehicle for 6 weeks. Lung/spleen CFU and histopathology were analyzed[1] - Survival Study: Mice were lethally infected with M. tb H37Rv (≈1000 CFU/lung) and treated with NITD-349 (30 mg/kg/day, oral) or vehicle. Survival was monitored daily for 120 days[1] - Pharmacokinetic Study: Male SD rats (200-250 g) received a single oral dose of NITD-349 (30 mg/kg) or intravenous dose (10 mg/kg). Blood and tissue (lung, liver, spleen) samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours post-dosing. Drug concentrations were measured by LC-MS/MS to calculate PK parameters[1] |
| ADME/Pharmacokinetics |
Oral absorption: The bioavailability (F) of rats after oral administration of 30 mg/kg was 72%. The peak plasma concentration (Cmax) was 3.6 μg/mL (oral) and 8.2 μg/mL (intravenous), with Tmax = 1 hour (oral) [1]
- Tissue distribution: High lung permeability (lung/plasma concentration ratio of 4.8 2 hours after oral administration), which is crucial for targeted therapy of pulmonary tuberculosis. The liver/plasma concentration ratio was 3.2, and the spleen/plasma concentration ratio was 2.5 [1] - Half-life: The terminal elimination half-life (t1/2) was 8.5 hours (rat, oral) and 7.2 hours (mouse, oral), respectively [1] - Metabolism: Moderate metabolic stability in human liver microsomes (t1/2 = 12 hours). The main metabolic pathway is O-demethylation, with the parent compound accounting for 70% of circulating drug-related substances [1]. Excretion: 72 hours after oral administration, 58% of the dose is excreted in feces (45% of which is the parent drug) and 32% in urine (22% of which is the parent drug) [1]. |
| Toxicity/Toxicokinetics |
Acute toxicity: Single oral doses of up to 200 mg/kg in mice and up to 300 mg/kg in rats did not cause death or toxic clinical symptoms (e.g., drowsiness, weight loss) [1]
- Subchronic toxicity: After 28 days of oral administration of NITD-349 (10, 30, 100 mg/kg/day) to rats, no significant changes were observed in hematological parameters (erythrocytes, white blood cells, platelets), liver function (ALT, AST), or kidney function (BUN, creatinine). No histopathological damage was observed in the lungs, liver, kidneys, or spleen [1] - Plasma protein binding rate: High plasma protein binding rates were observed in both human plasma (94%) and rat plasma (92%) as determined by ultrafiltration [1] - Host selectivity: At therapeutic concentrations, it did not inhibit human fatty acid synthase or produce cytotoxicity in primary human cells [1] |
| References |
[1]. Rao SP, et al. Indolcarboxamide is a preclinical candidate for treating multidrug-resistant tuberculosis. Sci Transl Med. 2013 Dec 4;5(214):214ra168
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| Additional Infomation |
Background: NITD-349 is a synthetic indolecarboxamide derivative that has been identified as a preclinical candidate for the treatment of multidrug-resistant tuberculosis (MDR-TB)[1]
- Mechanism of action: It binds to the NADH binding site of Mycobacterium tuberculosis (M. tb) InhA, inhibiting its enoyl-ACP reductase activity. This blocks the de novo synthesis of mycolic acid, which is essential for the assembly of the Mycobacterium tuberculosis cell wall, thereby inhibiting bacterial growth and leading to bacterial death[1] - Therapeutic indications: It is intended for the treatment of drug-sensitive tuberculosis and multidrug-resistant tuberculosis (MDR-TB), including extensively drug-resistant tuberculosis (XDR-TB) strains[1] - Structural features: It has an indolecarboxamide core with substituted benzene rings and pyrrolidine moieties. The core structure forms hydrogen bonds with InhA residues, while the pyrrolidine ring enhances the affinity for the NADH binding site [1] - Main advantages: potent activity against multidrug-resistant/extensively drug-resistant tuberculosis strains; high oral bioavailability and good lung tissue penetration; good safety and low host toxicity; synergistic effect with first-line anti-tuberculosis drugs [1] |
| Molecular Formula |
C17H20F2N2O
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|---|---|
| Molecular Weight |
306.350311279297
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| CAS # |
1473450-62-2
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| PubChem CID |
72711190
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| Appearance |
Typically exists as solid at room temperature
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
22
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| Complexity |
419
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
UATYSFRIVIHVKL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H20F2N2O/c1-17(2)5-3-11(4-6-17)20-16(22)15-9-12-13(19)7-10(18)8-14(12)21-15/h7-9,11,21H,3-6H2,1-2H3,(H,20,22)
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| Chemical Name |
N-(4,4-dimethylcyclohexyl)-4,6-difluoro-1H-indole-2-carboxamide
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| Synonyms |
NITD-349NITD 349 NITD349
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : ≥ 310 mg/mL (~1011.91 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2642 mL | 16.3212 mL | 32.6424 mL | |
| 5 mM | 0.6528 mL | 3.2642 mL | 6.5285 mL | |
| 10 mM | 0.3264 mL | 1.6321 mL | 3.2642 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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