ATI-1 (10 μM; 20 h, with 20 μM CQ added in the last 4 h) effectively inhibited the formation of autophagosomes in HeLa cells[1]. ATI-1 (5-20 μM; 24 h, with 10 μM added for 2 h under EBSS starvation conditions) inhibited the initiation of autophagy in HeLa cells by reducing Beclin1 protein levels and inhibiting autophagosome formation. LC3II levels decreased in a dose-dependent manner at concentrations of 5, 10 and 20 μM over 24 h, but anomalous LC3II accumulation was observed under starvation conditions, which did not reflect the formation of functional autophagosomes[1]. ATI-1 (5–20 μM; 48 h, in combination with chloroquine) significantly inhibited autophagy initiation in autophagy-dependent NCI-H1299 and MIA PaCa-2 cells by reducing Beclin1 protein levels, dose-dependently reduced LC3II levels over 48 h at concentrations of 5, 10, and 20 μM, and inhibited autophagosome formation in MIA PaCa-2 cells when used in combination with chloroquine [1]. ATI-1 (20 μM; 48–72 h) selectively inhibited proliferation, colony formation, migration, and invasion of autophagy-dependent NCI-H1299 and MIA PaCa-2 cells, with higher inhibitory efficacy than that against HeLa cells with lower autophagy dependence [1]. ATI-1 (30 μM; 24 h) disrupts the interaction between VCP and UFL1 in HeLa cells, reduces the UFMylation level of VCP, weakens the interaction between VCP and Beclin1, and selectively impairs the interaction between specific cofactors of VCP (VCP-ATXN3, VCP-NPLOC4) [1]. ATI-1 (3.13-50 μM) can directly bind purified full-length VCP (Kd = 25.1 μM) and purified VCP N-terminal domain (Kd = 32.5 μM) with measurable affinity [1]. ATI-1 (20 μM; 48 h) can induce G1 phase arrest in autophagy-dependent NCI-H1299 and MIA PaCa-2 cells, but does not significantly induce apoptosis [1]. ATI-1 (5-10 μM; 2-24 hours) exacerbates the metabolic vulnerability of autophagy-dependent NCI-H1299 and MIA PaCa-2 cells under EBSS-induced nutrient deprivation, leading to non-apoptotic cell death, decreased mitochondrial membrane potential, decreased ROS levels and lysosomal stress. Among them, 5 μM ATI-1 combined with starvation can reduce cell viability by about 50-70% within 24 hours[1].

ATI-1 (50 mg/kg; intraperitoneal injection; once daily for 14 days) showed strong antitumor efficacy in the MIA PaCa-2 pancreatic adenocarcinoma xenograft mouse model, significantly reducing tumor growth levels and the levels of tumor proliferation and autophagy markers, and had extremely low systemic toxicity [1].

Cell autophagy assay [1]
Cell Types: HeLa cells
Tested Concentrations: 10 μM; 20 μM CQ (last 4 hours)
Incubation Duration: 20 hours; 4 hours (CQ)
Experimental Results: Significantly reduced the number of autophagosomes per cell.

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Western Blot Analysis [1]
Cell Types: HeLa Cells
Tested Concentrations: 10 μM (10 μM CQ added in the last 2 hours); 5, 10, 20 μM; 20 μM; 10 μM (EBSS starvation treatment)
Incubation Duration: 24 hours; 24 hours; 24 hours; 2 hours (EBSS starvation treatment)
Experimental Results: Treatment with 10 μM CQ for 24 hours, followed by the addition of 10 μM CQ in the last 2 hours, inhibited the LC3II accumulation observed in the CQ-only group. LC3II levels decreased in a dose-dependent manner after treatment with 5, 10, and 20 μM CQ for 24 hours. Beclin1 protein levels decreased after treatment with 20 μM CQ for 24 hours. At a concentration of 10 μM, after 2 hours of EBSS starvation, LC3II showed contradictory accumulation, but autophagy flux analysis confirmed a reduction in LC3 spots.

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Western Blot Analysis [1]
Cell Types: NCI-H1299 cells, MIA PaCa-2 cells
Tested Concentrations: 5, 10, 20 μM (NCI-H1299); 10 μM (NCI-H1299, with 10 μM CQ added in the last 2 hours); 5, 10, 20 μM (MIA PaCa-2); 10 μM (MIA PaCa-2, with 10 μM CQ added in the last 2 hours); 10 μM (MIA PaCa-2, with 20 μM CQ added in the last 6 hours); 20 μM (NCI-H1299, MIA PaCa-2)
Incubation Duration: 48 hours (NCI-H1299); 48 hours (NCI-H1299, with 2 hours of CQ added); 48 48 hours (MIA PaCa-2); 48 hours (MIA PaCa-2, plus 2 hours of CQ); 48 hours (MIA PaCa-2, plus 6 hours of CQ); 48 hours (NCI-H1299, MIA PaCa-2)

Experimental Results: LC3II levels decreased in a dose-dependent manner in both NCI-H1299 and MIA PaCa-2 cells within 48 hours. In MIA PaCa-2 cells, treatment with 10 μM chloroquine for 48 hours, followed by the addition of 10 μM CQ in the last 2 hours, significantly reduced LC3II levels, while having less effect on NCI-H1299 cells. Compared to chloroquine alone, treatment with 10 μM chloroquine for 48 hours, followed by the addition of 20 μM chloroquine in the last 6 hours, reduced yellow autophagosome/autolysosomal spots in MIA PaCa-2 cells. In both cell lines, treatment with 20 μM chloroquine for 48 hours significantly reduced Beclin1 protein levels.

Animal/Disease Models:Nude Mice [1]
Doses: 50 mg/kg<
Route of Administration:Intraperitoneal injection; once daily; for 14 days
Experimental
Experimental Results:Significantly inhibited tumor growth, with a significant reduction in both final tumor weight and volume compared to the control group. Significantly reduced expression of LC3 and Ki-67 within the tumor. No significant weight loss or pathological abnormalities in major organs (heart, liver, spleen, lungs, kidneys) were observed.

[1]. ATI-1 mediated disruption of the VCP-UFL1-Beclin1 axis thwarts autophagy initiation to trigger metabolic catastrophe in autophagy-addicted cancers. Bioorg Chem. Published online April 25, 2026.

C16H14F2N2O2S3

400.49

1242983-93-2

Typically exists as solids at room temperature

FC1=CC(F)=CC(NS(=O)(C2=CC=C(C3=NC(C(C)C)=CS3)S2)=O)=C1

2934.99.9001

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)

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.)