Requires F₄₂₀ cofactor for activation; activates autophagy in macrophages; inhibits mycolic acid biosynthesis (TMM and TDM). Exact molecular target is unknown but involves an F₄₂₀-dependent nitroreductase distinct from Ddn. [2]

5-Nitro-1,10-phenanthroline (25 μM; 24 h) induces autophagy in THP-1 macrophages, killing naturally resistant intracellular bacteria [2]. By preventing Mtb's mycolic acid production, 5-nitro-1,10-phenanthroline (1x, 5x, 20x, or 50x MIC, MIC=0.78 μM; 1 h) can also alter host defenses against intracellular infections [2].

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Exhibits antimycobacterial activity against M. bovis BCG with MIC₉₉ of 1.56 μM. [2]
- Exhibits antimycobacterial activity against M. tuberculosis H37Rv (drug-susceptible) with MIC₉₉ of 1.56 μM and against isoniazid-resistant M. tuberculosis H37Rv with MIC₉₉ of 3.125 μM. [2]
- MIC value against M. tuberculosis H37Rv is 1.25 μM (from Table 2). [2]
- Active against multidrug-resistant clinical M. tuberculosis strains (e.g., strain 210, 536b, 116b, 692) with MIC values ranging from 0.3 to 1.25 μM. No cross-resistance to isoniazid, rifampin, ethambutol, kanamycin, streptomycin, capreomycin, PAS, or MmpL3 inhibitors (SQ109, DA8). [2]
- Lacks activity against E. coli (MIC >50 μM) and shows reduced activity against M. smegmatis (MIC = 25 μM). [2]
- Bactericidal effect in liquid cultures: exposure of M. bovis BCG to 8× MIC (approx. 12.5 μM) for 7 days results in approximately 60- to 100-fold reduction in bacterial counts compared to untreated controls (P < 0.05). [2]
- Intracellular activity: in THP-1 macrophages infected with M. bovis BCG, 5-NP (8× MIC) causes approximately 8.0-fold reduction in bacterial counts (P < 0.01). [2]
- Inhibits intracellular growth of M. tuberculosis in THP-1 macrophages (P < 0.01). [2]
- Induction of autophagy in THP-1 macrophages: increases expression of Beclin-1, Atg-3, Atg-7 and conversion of LC3-I to LC3-II in a dose-dependent manner (3.125 μM to 25 μM). At 25 μM, significant induction observed at 24 h (P < 0.05 for Beclin-1, Atg-3, Atg-7; P < 0.05 for LC3-II conversion). [2]
- Increases LC3 punctum formation in THP-1 macrophages (P < 0.05). [2]
- The nitro group is essential for both antimycobacterial activity and autophagy induction; the des-nitro derivative (1,10-phenanthroline) shows approximately 10-fold lower activity (MIC = 12.5 μM) and does not induce autophagy. [2]
- Metal chelation is not the mechanism of action; addition of MgCl₂, ZnCl₂, CaCl₂, or Fe₂(SO₄)₃ does not alter MIC values. Note: ZnCl₂ at 200 μg/ml and 1 μg/ml completely inhibits M. bovis BCG growth. [2]

Note: In vivo efficacy data are reported for the analog 3-methyl-6-nitro-1,10-phenanthroline (compound 10), not for 5-NP itself. [2]

Metabolite analysis by LC-MS: Mid-log-phase culture of M. bovis BCG was exposed to 20 μM 5-NP for 8 hours at 37°C with end-to-end mixing. Bacterial cells were harvested and lysed by addition of acetonitrile. Clarified lysates were prepared by centrifugation and filtered using 0.22 μm pore-size filters. The filtered lysates were subjected to mass spectrometry analysis (AB Sciex triple time of flight 5600 system). The major metabolites identified were 1,10-phenanthroline and 1,10-phenanthrolin-5-amine. A proposed mechanism involves initial hydride transfer from H₂F₄₂₀ to the 4' position of phenanthroline, forming a nitronic acid intermediate, which then undergoes either protonation or reduction to yield the two metabolites. [2]
- Mycolic acid biosynthesis inhibition assay: M. tuberculosis cultures (OD₆₀₀ 0.3-0.4) were treated with 5-NP (1×, 5×, 20×, 50× MIC) for 1 hour, then labeled with 1 μCi/ml [¹⁴C]acetate for 4 hours. Labeled bacilli were harvested and hydrolyzed by addition of tetra-n-butyl ammonium hydroxide (TBAB) and incubated overnight at 100°C. Fatty acids were esterified with methyl iodide, extracted with dichloromethane, dried, and resuspended in diethyl ether. Equal volumes were loaded on silica gel 60F₂₅₄ TLC plates and resolved using hexane-ethyl acetate (19:1, vol/vol; two runs). Bands corresponding to FAMEs and MAMEs were visualized by phosphorimaging. 5-NP exposure resulted in depletion of MAMEs and FAMEs, indicating inhibition of mycolic acid biosynthesis. [2]

Cell Viability Assay[2]
Cell Types: Mtb H37Rv, M. bovis BCG and M. bovis BCG-5NP Resistant strain
Tested Concentrations: 0-12.5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibition of pathogen with MIC99 value of 0.78 μM (Mtb H37Rv ), 0.78 μM (Mycobacterium bovis BCG), and >12.5 μM (Mycobacterium bovis BCG-5NP).

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MIC determination: Compounds were prepared as 50 mM stocks in DMSO. Various mycobacterial strains were grown in Middlebrook 7H9 medium until OD₆₀₀ = 0.2, diluted 1,000-fold, and added to 96-well plates containing drugs at concentrations ranging from 50 to 0.05 μM. Plates were incubated for 14 days for M. bovis BCG and M. tuberculosis. MIC₉₉ was defined as the drug concentration at which no visible bacterial growth was observed. [2]
- Cytotoxicity assay (MTT): THP-1 cells (2.5 × 10⁴ per well) were seeded in 96-well plates. Macrophages were washed and overlaid with medium containing drugs at concentrations ranging from 25 μM to 0.05 μM for 96 hours. MTT reagent (10 μl) was added to each well, formazan crystals dissolved in 100 μl lysis solution, and absorbance at 562 nm measured. Percent cell viability = (OD₅₆₂ of test sample / OD₅₆₂ of control) × 100. CC₅₀ is the concentration causing 50% cell viability. For 5-NP, cytotoxicity data were not explicitly reported in the table; however, the study notes that 24 primary hits were noncytotoxic even at 25 μM. [2]
- Intracellular killing assay in macrophages: THP-1 macrophages (2 × 10⁵ for M. bovis BCG; 5 × 10⁵ for M. smegmatis) were differentiated with PMA, then infected with mycobacteria at MOI of 1:10 (M. bovis BCG) or 1:1 (M. smegmatis). After 3-4 h, extracellular bacteria were removed by adding RPMI medium containing 200 μg/ml amikacin for 2 h. Macrophages were then overlaid with RPMI medium containing drugs (25 μM 5-NP). At 72 h (M. smegmatis) or 96 h (M. bovis BCG), macrophages were lysed in PBS-0.1% Triton X-100, and 100 μl of 10-fold serial dilutions was plated on Middlebrook 7H11 plates for CFU enumeration. [2]
- Autophagy induction assay (Western blot): Differentiated THP-1 macrophages (1.5 × 10⁶ per well in 12-well plates) were treated with 5-NP (3.125, 6.25, 12.5, 25 μM) for 24 h. Cells were lysed in RIPA buffer containing protease inhibitor cocktail. Protein concentration quantified by BCA assay. 50 μg protein per sample was electrophoresed on 15% SDS-PAGE gels, transferred to nitrocellulose membranes, blocked in 2% skimmed milk, and probed overnight at 4°C with antibodies against Beclin-1, Atg-3, Atg-7, or LC3. Secondary antibody incubation for 45 min at room temperature, bands visualized by enhanced chemiluminescence. Blots were stripped and reprobed with β-actin antibody as loading control. [2]
- LC3 punctum formation assay (immunofluorescence): Differentiated THP-1 macrophages (5 × 10⁵) on glass coverslips were treated with 25 μM 5-NP for 24 h, washed with PBS, fixed in 4% paraformaldehyde, blocked in buffer containing 2% BSA and 0.3% NP-40 for 1 h, then incubated overnight at 4°C with anti-human LC3 antibody. After washing, cells were incubated with Alexa Fluor 568-conjugated secondary antibody for 1 h, mounted with Fluoromount, and observed under confocal scanning laser microscope. LC3 puncta per cell were counted in random fields by a blinded observer. [2]

Note: No animal experiments were performed with 5-NP itself. The in vivo efficacy study was conducted with the analog 3-methyl-6-nitro-1,10-phenanthroline (compound 10). For completeness, the protocol is described: Female BALB/c mice were infected via aerosol route with approximately 100 CFU of M. tuberculosis H37Rv. At 4 weeks post-infection, mice were treated daily by gavage with compound 10 at 25 mg/kg or 100 mg/kg, or rifampin at 10 mg/kg (positive control), for 14 or 28 days. Compounds were formulated in 1% carboxymethyl cellulose. Bacterial loads in lungs and spleens were enumerated at 2 and 4 weeks post-treatment. [2]

Metabolism: In M. bovis BCG, 5-NP is activated in an F₄₂₀-dependent manner, producing two major metabolites: 1,10-phenanthroline (des-nitro) and 1,10-phenanthrolin-5-amine. The activation does not require the Ddn nitroreductase (Rv3547), as Ddn-deficient M. tuberculosis strains remained fully sensitive to 5-NP. [2]

Cytotoxicity in THP-1 macrophages: The study notes that 5-NP (NSC 4263) was among the primary hits that were noncytotoxic even at 25 μM concentration. [2]

[1]. 1,10-Phenanthroline derivatives as mediators for glucose oxidase. Biosens Bioelectron. 2010 Sep 15;26(1):267-70.

[2]. Dual Mechanism of Action of 5-Nitro-1,10-Phenanthroline against Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2017 Oct 24;61(11):e00969-17.

Nitroferroquinone is a type of phenanthroline compound.

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Mechanism of action: 5-NP exhibits a dual mechanism against Mycobacterium tuberculosis. (1) In the pathogen, it is a prodrug activated by an F₄₂₀-dependent nitroreductase (unknown, distinct from Ddn), leading to inhibition of mycolic acid biosynthesis (depletion of TMM, TDM, MAMEs, FAMEs). (2) In the host, it induces autophagy in macrophages via autophagosome formation, resulting in clearance of intracellular mycobacteria, including drug-resistant strains. The nitro group is essential for both activities. [2]
- Resistance mechanism: Spontaneous resistant mutants of M. bovis BCG to 5-NP arise at a frequency of approximately 10^-6. Whole-genome sequencing revealed inactivating frameshift mutations in the fbiB gene (encoding F₄₂₀ coenzyme L-glutamate ligase). Complementation with functional FbiB restored susceptibility. 5-NP-resistant strains are cross-resistant to PA-824 but remain susceptible to isoniazid and levofloxacin. [2]
- Structural requirements: The nitro group at the R₄ position is critical for activity; replacement with amino, methoxy, methyl, carbamoyl, bromo, chloro, or cyano groups reduces or abolishes activity. Addition of methyl or ethyl groups at the 3' position (R₂) increases potency (e.g., 3-methyl-6-nitro-1,10-phenanthroline, MIC = 0.195 μM). [2]
- Cross-resistance studies: 5-NP retains activity against PA-824-resistant M. tuberculosis strains with mutations in fgd (F₄₂₀ dehydrogenase) or fbiC (F₄₂₀ biosynthesis), but not against strains with mutations in fbiB. Ddn-deficient strains are fully sensitive to 5-NP, indicating a different activation pathway from bicyclic nitroimidazoles. [2]
- Potential application: The study highlights 5-NP as a promising scaffold for developing host-directed therapies against drug-resistant tuberculosis due to its dual pathogen- and host-modulating effects. [2]
- From reference [1]: 5-NP (5-nitro-1,10-phenanthroline) was evaluated as a redox mediator for glucose oxidase (GOX) in biosensor and biofuel cell applications. It showed redox-mediating activity for GOX, approximately four times lower than 5-amino-1,10-phenanthroline and three times lower than 5,6-diamino-1,10-phenanthroline. The nitro group exhibits a negative inductive effect, extracting electrons from the aromatic structure, which makes the compound more easily oxidizable on the electrode surface. [1]

C12H7N3O2

225.20288

225.053

4199-88-6

72790

Light yellow to yellow solid powder

1.4±0.1 g/cm3

444.0±30.0 °C at 760 mmHg

202-204 °C(lit.)

222.3±24.6 °C

0.0±1.0 mmHg at 25°C

1.768

1.56

0

4

0

17

305

0

PDDBTWXLNJNICS-UHFFFAOYSA-N

InChI=1S/C12H7N3O2/c16-15(17)10-7-8-3-1-5-13-11(8)12-9(10)4-2-6-14-12/h1-7H

5-nitro-1,10-phenanthroline

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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