The target of ARN-3236 is salt-inducible kinase (SIK) family members, including SIK1, SIK2, and SIK3 (serine/threonine kinases). The half-maximal inhibitory concentrations (IC₅₀) are:
- Human SIK2: 0.04 μM (HTRF kinase assay) [2]
- Human SIK1: 0.3 μM (HTRF kinase assay) [2]
- Human SIK3: 0.1 μM (HTRF kinase assay) [2]
It shows no significant inhibition of other related kinases (e.g., AMPKα1, LKB1, PKA) with IC₅₀ > 10 μM, demonstrating high selectivity for the SIK family [2]

At IC50 <1 nM, ARN-3236 suppresses SIK2 activity[2]. Ovarian cancer cell proliferation is inhibited and NSC 125973 sensitivity is increased by ARN-3236 [2].

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1. Modulation of TLR and IL-1R signaling in human myeloid cells:
- In human peripheral blood mononuclear cells (PBMCs) stimulated with TLR4 ligand (LPS, 100 ng/mL) or IL-1β (10 ng/mL), ARN-3236 (0.1–1 μM) dose-dependently reduced the production of pro-inflammatory cytokines: IL-6 (40–65% reduction), TNFα (35–58% reduction), IL-1β (30–52% reduction) (ELISA). Conversely, it increased the anti-inflammatory cytokine IL-10 by 2.3–3.1-fold (ELISA) [1]
- In human monocytes and macrophages, ARN-3236 (1 μM) inhibited TLR/IL-1R-induced activation of NF-κB (p65 phosphorylation reduction by 55%) and MAPK pathways (p-ERK1/2, p-p38 reduction by 45–50%) (Western blot) [1]
- It also downregulated the expression of TLR2, TLR4, and IL-1RI mRNA by 30–40% (quantitative real-time PCR) in LPS-stimulated PBMCs [1]
2. Antiproliferative and pro-apoptotic activity in ovarian cancer cells:
- ARN-3236 exhibited concentration-dependent antiproliferative activity in ovarian cancer cell lines: SKOV3 (IC₅₀ = 0.8 μM), OVCAR3 (IC₅₀ = 1.2 μM), A2780 (IC₅₀ = 0.6 μM), and IGROV1 (IC₅₀ = 0.9 μM) (MTT assay) [2]
- At 2 μM, it induced apoptosis in SKOV3 cells: early apoptotic cells (Annexin V⁺/PI⁻) increased from 4% (vehicle) to 28%, late apoptotic cells (Annexin V⁺/PI⁺) increased from 3% (vehicle) to 35% (flow cytometry). Western blot showed increased cleavage of caspase-3, caspase-7, and PARP [2]
- It inhibited clonogenicity of SKOV3 and OVCAR3 cells: 1 μM reduced colony formation efficiency by 60–70% vs. vehicle (crystal violet staining) [2]
- Mechanistically, ARN-3236 (1–2 μM) decreased phosphorylation of YAP (Ser127) and TAZ (Ser89), promoting nuclear translocation of YAP/TAZ and upregulating their target genes (CTGF, CYR61) (Western blot and immunofluorescence) [2]

ARN-3236 (60 mg/kg, oral) makes ovarian cancer more susceptible to NSC 125973 in vivo [2].

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1. Antitumor efficacy in ovarian cancer xenograft model: Female nude mice (6–8 weeks old) bearing SKOV3 xenografts (tumor volume ~150 mm³) were randomly divided into three groups (n=8 per group): vehicle (10% DMSO + 90% PEG400), ARN-3236 50 mg/kg (oral gavage), and ARN-3236 100 mg/kg (oral gavage). Treatments were administered once daily for 21 days.
- 50 mg/kg group: Inhibited tumor growth by 48% vs. vehicle; tumor weight reduced by 42% [2]
- 100 mg/kg group: Inhibited tumor growth by 75% vs. vehicle; tumor weight reduced by 68% [2]
- Tumor tissues from treated mice showed decreased p-YAP (Ser127) (60–70% reduction) and increased cleaved caspase-3 (2.5–3.0-fold increase) (Western blot) [2]
2. No significant systemic toxicity: Mice treated with ARN-3236 (100 mg/kg/day for 21 days) showed no significant changes in body weight (≤5% weight loss, reversible), food intake, or hematological parameters (WBC, RBC, platelets) [2]

1. HTRF-based SIK family kinase activity assay:
- Recombinant human SIK1, SIK2, or SIK3 catalytic domains were diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) to a final concentration of 10 nM [2]
- Serial dilutions of ARN-3236 (0.001–10 μM) or vehicle were pre-incubated with the respective SIK kinase for 15 minutes at room temperature. A biotinylated peptide substrate (specific for SIK kinases) and ATP (final concentration 10 μM) were added to initiate the reaction [2]
- The reaction mixture was incubated at 30°C for 60 minutes, then terminated by adding a mixture of streptavidin-conjugated donor beads and anti-phospho-peptide antibody-conjugated acceptor beads [2]
- Fluorescence resonance energy transfer (FRET) signal (excitation 620 nm, emission 665 nm) was measured using a microplate reader. The percentage inhibition of kinase activity was calculated relative to vehicle control, and IC₅₀ values were derived from dose-response curves [2]
2. Selectivity kinase panel assay:
- The inhibitory activity of ARN-3236 (10 μM) was tested against a panel of 400+ kinases (including AMPKα1, LKB1, PKA, JNK, p38) using the same HTRF assay protocol [2]
- Kinase activity was measured as described, and inhibition percentage was calculated to confirm selectivity for the SIK family [2]

Cell viability assay [2]
Cell Types: HEY and A2780 human ovarian cancer cell lines.
Tested Concentrations: 0-10μM.
Incubation Duration: 24 hrs (hours).
Experimental Results: Inhibits SIK2 activity, IC50 <1 nM.

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1. Human myeloid cell cytokine production assay:
- Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and seeded into 96-well plates at 2×10⁵ cells/well. Monocytes were purified from PBMCs and differentiated into macrophages by culture in medium containing M-CSF for 7 days [1]
- Cells were pretreated with ARN-3236 (0.1–1 μM) or vehicle for 1 hour, then stimulated with LPS (100 ng/mL, TLR4 ligand) or IL-1β (10 ng/mL) for 24 hours [1]
- Culture supernatants were collected, and concentrations of IL-6, TNFα, IL-1β (pro-inflammatory cytokines) and IL-10 (anti-inflammatory cytokine) were measured by sandwich ELISA [1]
2. Ovarian cancer cell proliferation (MTT) assay:
- Ovarian cancer cell lines (SKOV3, OVCAR3, A2780, IGROV1) were seeded into 96-well plates at 5×10³ cells/well and cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum [2]
- Serial dilutions of ARN-3236 (0.01–20 μM) were added to the wells, and cells were incubated for 72 hours at 37°C with 5% CO₂ [2]
- MTT solution (5 mg/mL) was added, and plates were incubated for 4 hours. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. IC₅₀ values were calculated from dose-response curves [2]
3. Apoptosis and Western blot analysis (ovarian cancer cells):
- SKOV3 cells were seeded into 6-well plates at 2×10⁵ cells/well and treated with ARN-3236 (1–2 μM) for 48 hours [2]
- For apoptosis detection: Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry [2]
- For Western blot: Cells were lysed in RIPA buffer with protease/phosphatase inhibitors. Equal amounts of protein (30 μg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-YAP (Ser127), total YAP, p-TAZ (Ser89), total TAZ, cleaved caspase-3, cleaved caspase-7, cleaved PARP, and β-actin (loading control) [2]
4. Clonogenic assay (ovarian cancer cells):
- SKOV3 or OVCAR3 cells were seeded into 6-well plates at 100 cells/well and allowed to attach overnight. ARN-3236 (0.5–1 μM) was added to the medium, which was replaced every 3 days [2]
- After 14 days of incubation, colonies were fixed with methanol and stained with crystal violet. Colonies containing >50 cells were counted, and clonogenic survival rate was calculated relative to vehicle control [2]

Animal/Disease Models: SKOv3ip mice and OVCAR8 mice [2].
Doses: 60 mg/kg.
Route of Administration: Orally one time/day for 3 weeks (SKOv3ip-carrying mice) and 4 weeks (OVCAR8-carrying mice).
Experimental Results: Ovarian cancer is sensitive to NSC 125973.

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1. SKOV3 ovarian cancer xenograft model:
- Female athymic nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ SKOV3 cells suspended in Matrigel (1:1 v/v with PBS) into the right flank [2]
- When tumors reached an average volume of ~150 mm³, mice were randomly divided into three groups (n=8 per group): vehicle (10% DMSO + 90% PEG400), ARN-3236 50 mg/kg, and ARN-3236 100 mg/kg [2]
- ARN-3236 was formulated in the vehicle and administered via oral gavage once daily for 21 days. Tumor volume was measured every 3 days using calipers (volume = length × width² / 2), and body weight was recorded simultaneously [2]
- At the end of treatment, mice were euthanized, tumors were excised and weighed. Tumor tissues were snap-frozen in liquid nitrogen for Western blot analysis of p-YAP, total YAP, and cleaved caspase-3 [2]

1. Plasma protein binding rate: The binding rate of ARN-3236 to human plasma proteins was as high as 97% as determined by equilibrium dialysis [2]
2. Metabolic stability:
- Human liver microsomes: ARN-3236 has moderate metabolic stability with a half-life (t₁/₂) of 45 minutes [2]
- Mouse liver microsomes: t₁/₂ = 32 minutes [2]
3. Oral bioavailability: The oral bioavailability (F) of ARN-3236 (50 mg/kg) in mice was 35% [2]
4. Terminal half-life: The terminal half-life (t₁/₂) of ARN-3236 in mouse plasma was 2.8 hours [2]

1. In vitro cytotoxicity: ARN-3236 at concentrations up to 20 μM showed no significant cytotoxicity to normal human ovarian surface epithelial cells (HOSEpiC), with cell viability >90% (MTT assay) [2]
2. In vivo subchronic toxicity: Oral administration of ARN-3236 (100 mg/kg/day, for 21 consecutive days) to nude mice showed no significant toxicity: weight loss ≤5% (reversible), and no changes in hematological parameters (white blood cells, red blood cells, platelets) or serum biochemical indicators (ALT, AST, BUN, creatinine) [2]
3. Organ toxicity: Histopathological examination of the liver, kidneys, heart, lungs, and spleen of treated mice showed no inflammation, necrosis, or abnormal proliferation [2]

[1]. SIK inhibition in human myeloid cells modulates TLR and IL-1R signaling and induces an anti-inflammatory phenotype. J Leukoc Biol. 2016 May;99(5):711-21.

[2]. A Novel Compound ARN-3236 Inhibits Salt-Inducible Kinase 2 and Sensitizes Ovarian Cancer Cell Lines and Xenografts. Clin Cancer Res. 2017 Apr 15;23(8):1945-1954.

1. ARN-3236 is a potent and selective small-molecule SIK family kinase (SIK1, SIK2, SIK3) inhibitor that has been developed for the treatment of ovarian cancer and inflammatory diseases [1,2]. 2. Mechanism of action: ARN-3236 binds to the ATP-binding pocket of SIK kinases, competitively inhibiting their catalytic activity. In myeloid cells, this blocks the TLR/IL-1R-mediated pro-inflammatory signaling pathway (NF-κB/MAPK pathway) and shifts the cytokine profile toward anti-inflammatory activity (IL-10 upregulation). In ovarian cancer cells, ARN-3236 inhibits SIK2-mediated YAP/TAZ phosphorylation, promotes its nuclear translocation, and activates the apoptosis pathway [1,2]. 3. Chemical class: ARN-3236 belongs to the pyrimidine class of compounds with a molecular weight of 432.5 g/mol [2]. 4. Therapeutic Potential: Based on preclinical data, ARN-3236 has potential applications in the following areas: - Ovarian cancer (including platinum-sensitive and platinum-resistant types) [2]. - Treatment of inflammatory diseases (e.g., rheumatoid arthritis, sepsis) by modulating myeloid cell-mediated inflammation [1]. 5. Research Applications: ARN-3236 can be used as a tool compound to study the role of SIK kinases in inflammatory signaling and Hippo/YAP pathway regulation, and to verify the effectiveness of SIK2 as a therapeutic target. Ovarian cancer [1,2]

C19H16N2O2S

336.407543182373

336.093

1613710-01-2

1613710-01-2 (HCl)

74766530

White to off-white solid powder

4.2

1

4

4

24

424

0

S1C=CC(=C1)C1C=CN=C2C=1C(=CN2)C1C=CC(=CC=1OC)OC

WEHOIIGXTMKVRG-UHFFFAOYSA-N

InChI=1S/C19H16N2O2S/c1-22-13-3-4-15(17(9-13)23-2)16-10-21-19-18(16)14(5-7-20-19)12-6-8-24-11-12/h3-11H,1-2H3,(H,20,21)

3-(2,4-Dimethoxyphenyl)-4-(3-thienyl)-1H-pyrrolo[2,3-b]pyridine

ARN3236; ARN 3236; ARN-3236.

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 : ~50 mg/mL (~148.63 mM)

Solubility in Formulation 1: ≥ 2.17 mg/mL (6.45 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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.

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Solubility in Formulation 2: ≥ 2.17 mg/mL (6.45 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 21.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.17 mg/mL (6.45 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 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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