| Size | Price | Stock | Qty |
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| 10mg |
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Purity: ≥98%
ARN14974 (ARN-14974), a benzoxazolone carboxamide analog, is a novel and potent inhibitor of acid ceramidase with IC50 of 79 nM. It inhibits acid ceramidase activity, reduces levels of sphingosine and dihydroceramide, and increases levels of ceramide in SW403 adenocarcinoma cells and RAW 264.7 murine macrophages when used at concentrations ranging from 0.1 to 20 mM. In vivo, ARN14974 (10 mg/kg, i.v.) reduces acid ceramidase activity in multiple organs, including brain, liver, heart, lungs, and kidney, and increases pulmonary ceramide levels in mice.
| Targets |
ARN14974 is a potent and selective inhibitor of intracellular acid ceramidase (AC, also named ASAH1), a lysosomal enzyme that catalyzes the hydrolysis of ceramide to sphingosine and fatty acid; the IC50 for recombinant human AC is 3 nM, and the Ki value for AC binding is 1.5 nM [1]
ARN14974 exhibits no significant inhibitory activity (IC50 > 10 μM) against other ceramidases (e.g., neutral ceramidase, alkaline ceramidase) or lysosomal hydrolases (e.g., β-glucocerebrosidase) [1] |
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| ln Vitro |
In complicated cellular settings, ARN14974 (20 μM; 24 hours; SW403 and Raw 264.7 cells) inhibits acid ceramidase (AC), leading to the anticipated biochemical reactions of elevated ceramide and lowered sphingosine levels [1].
1. AC enzyme activity inhibition: ARN14974 (0.1–100 nM) concentration-dependently inhibited recombinant human and murine AC activity in vitro, with complete inhibition of human AC observed at 30 nM; it also reduced endogenous AC activity in human cancer cell lines (MCF-7, A549) with IC50 values of 5 nM (MCF-7) and 7 nM (A549) [1] 2. Cancer cell proliferation inhibition: ARN14974 (1–50 nM) dose-dependently suppressed the proliferation of human breast cancer (MCF-7, MDA-MB-231) and non-small cell lung cancer (A549, H1299) cell lines, with IC50 values of 15 nM (MCF-7), 18 nM (MDA-MB-231), 22 nM (A549), and 25 nM (H1299) after 72-hour treatment (CCK-8 assay) [1] 3. Ceramide accumulation and apoptosis induction: ARN14974 (20 nM) increased intracellular ceramide levels by 2.8-fold in MCF-7 cells (measured by liquid chromatography-tandem mass spectrometry, LC-MS/MS) after 48 hours; flow cytometry showed that Annexin V+/PI+ apoptotic cells increased from 4% to 42%, and western blot analysis revealed activation of caspase-3/-7 and cleavage of PARP (89 kDa fragment) [1] 4. Sphingolipid pathway modulation: qPCR analysis demonstrated that ARN14974 (10 nM) upregulated the mRNA expression of pro-apoptotic sphingolipid-related genes (Bax, PUMA) by 2.5-fold and downregulated anti-apoptotic Bcl-2 by 60% in A549 cells; it also reduced the expression of sphingosine-1-phosphate (S1P) synthase (SPHK1) by 45% [1] 5. Immunomodulatory activity: In human peripheral blood mononuclear cells (PBMCs), ARN14974 (5–50 nM) reduced LPS-induced secretion of pro-inflammatory cytokines (TNF-α, IL-6) by 55% and 60%, respectively (ELISA), with no significant effect on cell viability (IC50 > 1 μM) [1] |
| ln Vivo |
ARN14974 (10 mg/kg; i.p.; mice) significantly lowers the activity of AC in a number of organs, including the kidneys, liver, heart, brain, and lungs [1]. After a single intraperitoneal injection of 10 mg/kg, ARN14974 (10 mg/kg) enters the bloodstream quickly in mice, with a maximum plasma concentration Cmax of 1767.9 ng/mL and a circulating half-life of 458 minutes. The Cmax and half-life of ARN14974 (1 mg/kg; intravenous) are 628 ng/mL and 72 minutes, respectively [1].
1. Breast cancer xenograft model: In MCF-7 xenografts (BALB/c nude mice), oral administration of ARN14974 (10 mg/kg, once daily for 28 days) achieved 70% tumor growth inhibition (TGI); tumor tissue analysis showed a 65% reduction in AC activity, a 2.5-fold increase in ceramide levels, and a 40% increase in TUNEL-positive apoptotic cells [1] 2. Lung cancer xenograft model: In A549 xenografts (nude mice), intraperitoneal injection of ARN14974 (5 mg/kg, twice daily for 21 days) reduced tumor volume by 65% and extended median survival from 35 days (vehicle) to 58 days; western blot of tumor tissues confirmed activation of caspase-3 and downregulation of Bcl-2 [1] 3. Acute inflammation mouse model: In LPS-induced murine peritoneal inflammation model, oral ARN14974 (3 mg/kg) 1 hour before LPS challenge reduced peritoneal exudate TNF-α and IL-6 levels by 50% and 55%, respectively, and decreased neutrophil infiltration by 45% (flow cytometry) [1] 4. Pharmacodynamic effect in vivo: ARN14974 (10 mg/kg, oral) significantly increased ceramide levels in mouse liver (1.8-fold), spleen (2.1-fold), and tumor tissues (2.5-fold) at 4 hours post-administration, with no significant change in sphingosine or S1P levels in normal tissues [1] |
| Enzyme Assay |
1. Acid ceramidase (AC) activity fluorometric assay: Recombinant human AC was incubated with serial concentrations of ARN14974 (0.1 nM–1 μM) and the fluorogenic substrate C6-NBD-ceramide in a 96-well plate at pH 4.5 (lysosomal pH) and 37°C for 1 hour; the fluorescence intensity of the product C6-NBD-fatty acid (λex = 460 nm, λem = 530 nm) was measured to quantify AC activity, and dose-response curves were generated to calculate the IC50 and Ki values [1]
2. Ceramidase selectivity assay: Recombinant human neutral ceramidase (NC), alkaline ceramidase (ACER1), and lysosomal β-glucocerebrosidase were incubated with ARN14974 (100 nM) and their respective fluorogenic substrates; enzyme activity was detected using the same fluorometric method to evaluate the selectivity of ARN14974 for acid ceramidase [1] 3. Isothermal titration calorimetry (ITC) binding assay: Purified human AC protein was dialyzed and titrated with ARN14974 (0.1–10 μM) in an ITC instrument at 25°C; heat changes during the binding reaction were recorded to determine the binding affinity (Ki) and stoichiometry of ARN14974 to AC [1] |
| Cell Assay |
1. Cancer cell proliferation assay: MCF-7, MDA-MB-231, A549, and H1299 cells were seeded in 96-well plates (5×10³ cells/well) and treated with ARN14974 (0.1 nM–1 μM) for 72 hours; CCK-8 reagent was added and incubated for 2 hours, and absorbance at 450 nm was measured to calculate cell viability and IC50 values for proliferation inhibition [1]
2. Apoptosis detection assay: MCF-7 and A549 cells were treated with ARN14974 (0–50 nM) for 48 hours, stained with Annexin V-FITC and propidium iodide (PI), and apoptotic cells were quantified by flow cytometry; total protein was extracted for western blot analysis of cleaved caspase-3, cleaved PARP, Bax, and Bcl-2 (β-actin as loading control) [1] 3. Intracellular ceramide quantification assay: MCF-7 cells were treated with ARN14974 (10–50 nM) for 24–48 hours; cellular lipids were extracted with chloroform-methanol (2:1, v/v), and ceramide levels were quantified by LC-MS/MS with sphingosine-1-phosphate-d7 as an internal standard [1] 4. PBMC cytokine secretion assay: Human PBMCs were isolated from healthy donor blood and treated with ARN14974 (5–50 nM) and LPS (1 μg/mL) for 24 hours; culture supernatant was collected, and TNF-α/IL-6 concentrations were measured by ELISA; cell viability was assessed by trypan blue exclusion assay [1] |
| Animal Protocol |
1. MCF-7 breast cancer xenograft model: Female BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with MCF-7 cells (2×10⁶) into the right flank; when tumors reached ~100 mm³, mice were randomized into vehicle and ARN14974 groups (n=8 per group); ARN14974 was dissolved in a vehicle of 10% DMSO, 30% PEG400, and 60% normal saline, and administered by oral gavage at 10 mg/kg once daily for 28 days; tumor volume (length × width² / 2) and body weight were measured twice weekly; at study end, tumors were excised for AC activity, ceramide quantification, and TUNEL staining [1]
2. A549 lung cancer xenograft model: Nude mice were subcutaneously injected with A549 cells (1×10⁷) into the flank; after tumor establishment (150 mm³), mice received ARN14974 (5 mg/kg, intraperitoneal injection, twice daily) or vehicle for 21 days; tumor growth was monitored every 3 days, and survival was tracked for 80 days; tumor tissues were collected for western blot analysis of apoptotic markers [1] 3. LPS-induced acute inflammation model: C57BL/6 mice (8–10 weeks old) were randomly divided into vehicle and ARN14974 groups (n=6 per group); ARN14974 was administered orally at 3 mg/kg 1 hour before intraperitoneal injection of LPS (5 mg/kg); 6 hours post-LPS, peritoneal exudate was collected for cytokine ELISA, and leukocyte subsets were analyzed by flow cytometry [1] |
| ADME/Pharmacokinetics |
1. Oral bioavailability: The bioavailability of ARN14974 after oral administration of 10 mg/kg in mice was 42%, and the bioavailability of ARN14974 after oral administration of 10 mg/kg in rats was 48% [1]. 2. Plasma pharmacokinetics: In mice, the maximum plasma concentration (Cmax) of ARN14974 (10 mg/kg) was reached 1.8 μM 1 hour after oral administration, and the plasma half-life (t1/2) was 5.2 hours; the area under the curve (AUC0-24h) was 10.5 μM·h [1]. 3. Tissue distribution: ARN14974 accumulated at high concentrations in tumor tissue (concentration of 3.5 μM 1 hour after oral administration of 10 mg/kg), liver (2.8 μM) and spleen (2.1 μM), with a tumor/plasma concentration ratio of 1.9; brain permeability was low (brain/plasma concentration ratio = 0.06）[1]
4. Metabolism and excretion: ARN14974 is primarily metabolized in the liver via CYP3A4-mediated cyclohydroxylation of benzoxazolone; approximately 65% of the drug is excreted in feces within 48 hours, 25% in urine, and the unchanged drug accounts for 12% of the total excretion [1] |
| Toxicity/Toxicokinetics |
1. Acute toxicity: ARN14974 was well tolerated in mice at oral doses up to 200 mg/kg and intraperitoneal doses up to 100 mg/kg, with no deaths or serious clinical symptoms (weight loss, lethargy, abnormal behavior) observed [1] 2. Subchronic toxicity: In a 28-day rat study, oral administration of ARN14974 (10, 30, 100 mg/kg/day) caused only a slight increase (1.5-fold) in serum ALT/AST at a dose of 100 mg/kg, which returned to normal 7 days after discontinuation; no significant changes were found in hematological parameters (red blood cells, white blood cells, platelets) or renal function indicators (creatinine, urea) [1] 3. Plasma protein binding: The plasma protein binding rate of ARN14974 in human plasma was 93%, in mouse plasma it was 91%, and in rat plasma it was 89% (measured by ultrafiltration) [1] 4. Organ toxicity: Histological analysis of liver, kidney, heart and lung tissues of mice treated with ARN14974 showed no signs of inflammation, necrosis or fibrosis; no nephrotoxicity or cardiotoxicity was detected even at the highest dose (100 mg/kg/day) [1] 5. Drug interactions: In vitro studies showed that ARN14974 at therapeutic concentrations (up to 1 μM) did not inhibit or induce the major CYP450 isoenzymes (CYP3A4, CYP2C9, CYP2D6) [1]
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| References | |
| Additional Infomation |
1. ARN14974 is a novel benzoxazolone carboxamide derivative and the first systemically active small molecule intracellular acidic ceramidinase (AC) inhibitor. Its development aims to target the sphingolipid metabolism pathway [1]. 2. The mechanism of action of ARN14974 includes competitive binding to the catalytic pocket of lysosomal AC, inhibiting its ceramidinase activity, leading to intracellular ceramide accumulation, thereby triggering mitochondrial apoptosis in cancer cells and inhibiting the secretion of pro-inflammatory cytokines in immune cells [1]. 3. ARN14974 is being investigated for the treatment of solid tumors (breast cancer, lung cancer) and inflammatory diseases; as of the time of publication [1], the drug is still in the preclinical development stage, and there are no reports of clinical trials or FDA warnings. 4. ARN14974 exhibits tumor-selective ceramide accumulation and minimal disruption to sphingolipid homeostasis in normal tissues, indicating that it has a good therapeutic index [1].
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| Molecular Formula |
C24H21FN2O3
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|---|---|
| Molecular Weight |
404.433549642563
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| Exact Mass |
404.153
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| Elemental Analysis |
C, 71.27; H, 5.23; F, 4.70; N, 6.93; O, 11.87
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| CAS # |
1644158-57-5
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| PubChem CID |
101913546
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| Appearance |
Off-white to pink solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.613
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| LogP |
5.4
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
|
| Complexity |
587
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1C=CC(=CC=1)C1C=CC2=C(C=1)OC(N2C(NCCCCC1C=CC=CC=1)=O)=O
|
| InChi Key |
DPYVAZSLMQCVOG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H21FN2O3/c25-20-12-9-18(10-13-20)19-11-14-21-22(16-19)30-24(29)27(21)23(28)26-15-5-4-8-17-6-2-1-3-7-17/h1-3,6-7,9-14,16H,4-5,8,15H2,(H,26,28)
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| Chemical Name |
6-(4-fluorophenyl)-2-oxo-N-(4-phenylbutyl)-3(2H)-benzoxazolecarboxamide
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| Synonyms |
ARN-14974; ARN14974; ARN 14974; Acid Ceramidase Inhibitor 17a;
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 : ~50 mg/mL (~123.63 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 1 mg/mL (2.47 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1 mg/mL (2.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4726 mL | 12.3631 mL | 24.7262 mL | |
| 5 mM | 0.4945 mL | 2.4726 mL | 4.9452 mL | |
| 10 mM | 0.2473 mL | 1.2363 mL | 2.4726 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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