Mitochondrial complex I (NADH:CoQ reductase) [1]

Inhibits starvation‑induced and rapamycin‑induced autophagy in a dose‑dependent manner in MCF‑7 cells stably expressing eGFP‑LC3 (MCF‑LC3). [1]

- Inhibits LC3 lipidation (conversion of LC3‑I to LC3‑II) and blocks p62 degradation in starved or rapamycin‑treated MCF7‑LC3 cells, indicating suppression of autophagic flux. [1]

- Reduces survival of amino‑acid‑starved MCF7‑LC3 cells compared to fed cells (assessed by WST‑1 assay). [1]

- Induces apoptosis in starved MCF7 cells in a dose‑dependent manner, as measured by a selective caspase 3/7 probe using IncuCyte Zoom live‑cell microscopy. [1]

- Does not inhibit glucose uptake in HCT116 cells (residual activity >75% at 30 µM, n=4). [1]

- Potently and dose‑dependently decreases oxygen consumption rate (OCR) and increases extracellular acidification rate (ECAR) in MCF7 and HeLa cells, as measured by Seahorse XFe96 Analyzer. [1]

- Reduces cellular ATP levels, but only at concentrations significantly higher than those required for autophagy inhibition. [1]

- Enhances cell death in glucose‑starved cells more effectively than in fed cells. [1]

Semi‑intact assay using digitonin‑permeabilized HeLa cells: Cells are permeabilized with digitonin to allow access to mitochondrial complexes. Respiration is driven by addition of specific substrates for each complex. amutinit exclusively inhibits pyruvate/malate‑driven respiratory activity, which corresponds to NADH‑CoQ reductase (complex I) activity. Only substrates of complexes downstream of complex I allow continued oxygen consumption. [1]

- NADH‑CoQ reductase assay using isolated mitochondria: Mitochondria are isolated and the enzymatic activity of complex I is measured by following the reduction of CoQ by NADH. amutinit inhibits this activity in a manner similar to rotenone, confirming direct inhibition of mitochondrial complex I. [1]

High‑content screening for autophagy inhibition: MCF‑7 cells stably expressing eGFP‑LC3 (MCF‑LC3) are used. Cells are starved by amino acid withdrawal (EBSS) or treated with rapamycin (100 nM) to induce autophagy. Accumulation of eGFP‑LC3 puncta is quantified by fluorescence microscopy. amutinit reduces puncta formation dose‑dependently. [1]

- Western blot analysis of LC3 lipidation and p62 degradation: MCF7‑LC3 cells are starved (EBSS) or treated with rapamycin in the presence of increasing concentrations of amutinit. Cell lysates are analyzed by SDS‑PAGE and immunoblotting with anti‑LC3 and anti‑p62 antibodies. amutinit inhibits starvation‑ and rapamycin‑induced LC3‑II formation and p62 degradation. [1]

- mCherry‑eGFP‑LC3 flux assay: Cells expressing mCherry‑eGFP‑LC3 are treated with amutinit under fed or starved conditions. eGFP fluorescence is quenched in acidic autolysosomes while mCherry remains stable. amutinit reduces both yellow (autophagosomes) and red (autolysosomes) puncta, indicating inhibition at or before autophagosome biogenesis. [1]

- Cell viability (WST‑1) assay: MCF7‑LC3 cells are cultured under fed (MEM) or starved (EBSS) conditions with increasing concentrations of amutinit. WST‑1 reagent is added and absorbance is measured. Starved cells show reduced survival compared to fed cells. [1]

- Apoptosis assay (caspase 3/7): MCF7 cells are starved and treated with amutinit together with a selective caspase 3/7 probe. Apoptotic cells are monitored in real‑time using IncuCyte Zoom live‑cell microscope. amutinit induces apoptosis dose‑dependently. [1]

- Seahorse mitochondrial respiration assay: MCF7 or HeLa cells are seeded in a Seahorse XFe96 plate. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are measured sequentially. amutinit is injected at timepoint (a), followed by oligomycin (b), FCCP (c), and rotenone+antimycin (d). amutinit decreases OCR and increases ECAR in a dose‑dependent manner. [1]

- Glucose uptake assay: HCT116 cells are treated with amutinit (30 µM) and glucose uptake is measured. Residual activity >75% indicates no significant inhibition. [1]

- Cellular ATP measurement: Cells are treated with amutinit or rotenone, and ATP levels are quantified. Significant ATP depletion occurs only at concentrations higher than those needed for autophagy inhibition. [1]

[1]. Discovery of the novel autophagy inhibitor aumitin that targets mitochondrial complex I. Chem Sci. 2018 Feb 26;9(11):3014-3022.

amutinit is a diaminopyrimidine‑based compound discovered through phenotypic screening for autophagy inhibitors. It is structurally similar to known kinase inhibitors but shows only weak inhibition of PI4KB and PI3KC2G at 1 µM (out of 419 kinases tested), and selective inhibitors of these kinases do not block autophagy, ruling out off‑target kinase effects. [1]

- The compound inhibits mitochondrial respiration rapidly, suggesting that respiration inhibition is the cause rather than a consequence of autophagy inhibition. [1]

- Structure‑activity relationship (SAR) analysis shows that inhibition of mitochondrial respiration correlates strongly with autophagy inhibition; an inactive analogue does not affect respiration. [1]

- Other mitochondrial inhibitors (complex III inhibitors antimycin and myxothiazol, complex V inhibitor oligomycin) also potently inhibit autophagy, indicating that impairment of mitochondrial respiration in general leads to autophagy suppression. [1]

- amutinit is proposed as a useful alternative to rotenone for studying mitochondrial complex I and autophagy, as rotenone has additional off‑target activities. [1]

C24H20CLN5O

429.901503562927

429.14

C, 67.05; H, 4.69; Cl, 8.25; N, 16.29; O, 3.72

946293-78-3

946293-78-3;

27478481

White to off-white solid powder

5.8

3

5

6

31

564

0

CC1=CC(=NC(=N1)NC2=CC=C(C=C2)NC(=O)C3=CC=CC=C3Cl)NC4=CC=CC=C4

VJNNQBDSUIVCKB-UHFFFAOYSA-N

InChI=1S/C24H20ClN5O/c1-16-15-22(27-17-7-3-2-4-8-17)30-24(26-16)29-19-13-11-18(12-14-19)28-23(31)20-9-5-6-10-21(20)25/h2-15H,1H3,(H,28,31)(H2,26,27,29,30)

N-[4-[(4-anilino-6-methylpyrimidin-2-yl)amino]phenyl]-2-chlorobenzamide

Aumitin

2934.99.03.00

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 (~232.61 mM)

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 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).

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)]
*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)

---

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
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

---

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.

---

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