Anoctamin 1 (ANO1)/TMEM16A, a calcium-activated chloride channel (CaCC) (inhibitor; IC₅₀ = 77 ± 1.1 nM in FRT-ANO1 cells by short-circuit current analysis; IC₅₀ < 3 μM in initial fluorescence-based screen) [1]

In a cell-based high-throughput screen of 54,400 compounds using Fischer rat thyroid (FRT) cells stably expressing human ANO1 and a YFP-based halide sensor, Ani9 was identified as a potent ANO1 inhibitor. It fully blocked ANO1 channel activity with an IC₅₀ < 3 μM. [1]
In FRT cells expressing human ANO1, short-circuit current analysis showed that Ani9 dose-dependently inhibited ATP-stimulated ANO1 chloride currents with an IC₅₀ of 77 ± 1.1 nM. This is >18-fold more potent than the known inhibitors T16Ainh-A01 (IC₅₀ = 1.39 μM) and MONNA (IC₅₀ = 1.95 μM). [1]
Whole-cell patch-clamp analysis in FRT-ANO1 cells confirmed that Ani9 inhibited ATP-induced ANO1 chloride currents in a dose-dependent manner. At 50 nM, 100 nM, and 1 μM, it inhibited currents by 52.0 ± 3.7%, 95.4 ± 0.5%, and 98.7 ± 0.5%, respectively. [1]
Ani9 did not affect ATP-induced intracellular calcium increases in FRT cells, indicating its inhibitory effect is not due to disruption of calcium signaling. [1]
Ani9 (up to 30 μM) had no effect on CFTR chloride channel activity in FRT cells or on ENaC sodium channel activity in T84 cells. [1]
Ani9 showed minimal inhibition of the volume-regulated anion channel (VRAC). At 1 μM, a concentration that nearly fully inhibits ANO1, it inhibited VRAC activity by only 13.5 ± 1.1% in a YFP-based assay. This was confirmed by whole-cell patch clamp where 1 μM Ani9 did not significantly affect hypotonicity-induced VRAC currents. In contrast, T16Ainh-A01 and MONNA strongly inhibited VRAC. [1]
Crucially, Ani9 displayed high selectivity for ANO1 over ANO2. At 1 μM, it almost completely inhibited ANO1 but had no effect on ANO2 channel activity in both YFP-based and short-circuit current assays. Even at 10 μM, it inhibited ANO2 by only 10 ± 1.6%. In contrast, T16Ainh-A01 and MONNA potently inhibited both ANO1 and ANO2. [1]
Ani9 potently inhibited endogenous CaCC activity in cancer cell lines (PC3 prostate cancer, Capan-1 pancreatic cancer) and in IL-4 treated primary human nasal epithelial (NHNE) cells. The IC₅₀ for inhibition of endogenous CaCCs in NHNE cells was approximately 110 nM. [1]
The inhibition of ANO1 by Ani9 was found to be reversible, as its inhibitory effect was significantly diminished after washout. [1]

YFP-Based Halide Influx Assay: FRT cells stably co-expressing human ANO1 (or ANO2) and a halide-sensitive YFP were used. Cells were pre-incubated with test compounds for 10-20 minutes. ANO1/2 was activated by ATP (100 μM) in the presence of an iodide-containing solution. The rate of YFP fluorescence quenching, which corresponds to iodide influx through the channel, was measured using a fluorescence plate reader. IC₅₀ values were calculated from dose-response curves. This assay was also used to assess VRAC activity in LN215 cells. [1]
Short-Circuit Current (Isc) Measurement in Ussing Chambers: FRT cells expressing ANO1 or ANO2 were grown on permeable supports. For endogenous CaCC studies, PC3, Capan-1, or IL-4 treated primary NHNE cells were used. Cells were mounted in Ussing chambers. After basolateral membrane permeabilization with amphotericin B (for FRT cells), a transepithelial chloride gradient was applied. Test compounds were added to both apical and basolateral baths. ANO1/2 or CaCCs were activated by apical addition of ATP or Eact. The resulting apical membrane current was measured. This method was also used to assess CFTR and ENaC activity. [1]
Whole-Cell Patch-Clamp Recording: Conventional whole-cell patch-clamp was performed on FRT-ANO1 cells to directly measure ANO1 chloride currents. Currents were elicited by voltage steps from -100 mV to +100 mV. ANO1 was activated by including ATP or by using a pipette solution with elevated Ca²⁺. The effect of Ani9 on these currents was evaluated. This method was also used to confirm VRAC inhibition in LN215 cells. [1]
Intracellular Calcium Measurement: FRT cells were loaded with the calcium-sensitive dye Fluo-4 NW. After pre-incubation with Ani9, changes in fluorescence upon ATP stimulation were measured using a fluorescence plate reader to assess any effect on calcium signaling. [1]
Western Blot: Protein lysates from various cell lines (FRT, FRT-ANO1, PC3, Capan-1, NHNE) were separated by SDS-PAGE and probed with an anti-ANO1 antibody to confirm ANO1 protein expression. [1]

[1]. Ani9, A Novel Potent Small-Molecule ANO1 Inhibitor with Negligible Effect on ANO2. PLoS One. 2016 May 24;11(5):e0155771.

[2]. Synthesis and biological evaluation of novel Ani9 derivatives as potent and selective ANO1 inhibitors. Eur J Med Chem. 2018 Dec 5;160:245-255.

Ani9 [2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide] is a novel, potent, and highly selective small-molecule inhibitor of the calcium-activated chloride channel ANO1/TMEM16A. It was discovered through high-throughput screening and subsequent characterization. [1]
ANO1 is involved in various physiological and pathological processes, including fluid secretion, smooth muscle contraction, nociception, and cancer progression. Potent and selective inhibitors like Ani9 are valuable pharmacological tools for studying ANO1 function and hold potential as therapeutic candidates for diseases such as cancer, hypertension, pain, diarrhea, and asthma. [1]
A key advantage of Ani9 over previous ANO1 inhibitors (e.g., T16Ainh-A01, MONNA) is its exceptional selectivity for ANO1 over the closely related ANO2 channel. It also shows minimal off-target effects on other ion channels like CFTR, ENaC, and VRAC at concentrations that fully inhibit ANO1. [1]

C17H17CLN2O3

332.784

332.092

356102-14-2

9556542

White to off-white solid powder

1.2±0.1 g/cm3

1.563

4.47

1

4

6

23

405

0

CC1=C(C=CC(=C1)Cl)OCC(=O)N/N=C/C2=CC=CC=C2OC

KDALDZRKOBJXIE-VXLYETTFSA-N

InChI=1S/C17H17ClN2O3/c1-12-9-14(18)7-8-15(12)23-11-17(21)20-19-10-13-5-3-4-6-16(13)22-2/h3-10H,11H2,1-2H3,(H,20,21)/b19-10+

2-(4-chloro-2-methylphenoxy)-N-[(E)-(2-methoxyphenyl)methylideneamino]acetamide

Ani9 Ani 9 Ani-9

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)

DMSO : ≥ 125 mg/mL (~375.62 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.

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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.)