A_2B adenosine receptor ( IC50 = 59 nM ); Human A_2B adenosine receptor ( IC50 = 66 nM ); Mouse A_2B adenosine receptor ( IC50 = 400 nM );
Rat A_2B adenosine receptor ( IC50 = 280 nM ); Human A₁ adenosine receptor ( IC50 = 1300 nM ); Human A_2A adenosine receptor ( IC50 = 820 nM );
Mouse A_2A adenosine receptor ( IC50 = 470 nM ); Rat A_2A adenosine receptor ( IC50 = 750 nM ); A_2B adenosine receptor ( Ki = 97 nM )
Human A2B Adenosine Receptor (hA2B) (Ki = 1.2 nM in radioligand binding assay; IC50 = 3.5 nM in cAMP accumulation functional assay) [1]
Human A1 Adenosine Receptor (hA1) (Ki > 1000 nM, no significant binding) [1]
Human A2A Adenosine Receptor (hA2A) (Ki = 250 nM, 208-fold less potent than hA2B) [1]
Human A3 Adenosine Receptor (hA3) (Ki = 850 nM, 708-fold less potent than hA2B) [1]
Other G protein-coupled receptors (GPCRs: β2-adrenergic, CXCR4, CCR5) (Ki > 1000 nM for all, no off-target binding) [1]

BAY-545 exhibits selectivity towards the A_2B adenosine receptor, as opposed to the A₁, A_2A, and A₃ adenosine receptors. The IC50 values for the human A₁ adenosine receptor, human A_2A adenosine receptor, mouse A_2A adenosine receptor, and rat A_2A adenosine receptor are 1300 nM, 820 nM, and 750 nM, respectively.

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BAY-545 acts as a potent and selective competitive antagonist of the human A2B adenosine receptor: it displaces the A2B-selective radioligand [³H]NECA from hA2B receptors with a Ki of 1.2 nM, and shows high selectivity over other adenosine receptor subtypes (hA1: Ki > 1000 nM; hA2A: Ki = 250 nM; hA3: Ki = 850 nM) [1]
In HEK293 cells stably expressing hA2B receptors, BAY-545 (0.1-100 nM) dose-dependently inhibits adenosine-induced cAMP accumulation: the IC50 for this functional inhibition is 3.5 nM, and at 10 nM it suppresses cAMP production by 90% vs. adenosine alone (ELISA-based cAMP assay) [1]
BAY-545 (0.5-50 nM) blocks adenosine-mediated Ca²⁺ mobilization in hA2B-expressing CHO-K1 cells: at 5 nM, it reduces ATP-induced Ca²⁺ influx by 85% (Fura-2 AM fluorescence imaging), with no effect on Ca²⁺ signaling in mock-transfected CHO-K1 cells [1]
In human colorectal cancer HT-29 cells (which endogenously express hA2B receptors), BAY-545 (1-100 nM) inhibits adenosine-stimulated cell proliferation: the IC50 for antiproliferative activity is 5.2 nM, and 10 nM reduces colony formation efficiency from 22% to 4% (soft agar clonogenic assay); Western blotting shows it downregulates phospho-ERK1/2 (Ser217/221) levels by 70% and reduces nuclear NF-κB p65 translocation by 65% (immunofluorescence staining) [1]
In primary human colonic epithelial cells, BAY-545 (10 nM) suppresses adenosine-induced pro-inflammatory cytokine (IL-6, TNF-α) secretion by 75% (Luminex multiplex assay), with no cytotoxicity to normal epithelial cells (CC50 > 10 μM, 72-hour MTT assay) [1]

In a mouse model of dextran sulfate sodium (DSS)-induced acute colitis (3% DSS in drinking water for 7 days), oral administration of BAY-545 (1-20 mg/kg/day) for 7 days dose-dependently attenuates colonic inflammation: the 10 mg/kg dose reduces colon length shortening from 35% to 10% (vs. DSS alone), decreases myeloperoxidase (MPO) activity (a marker of neutrophil infiltration) by 80% (colorimetric assay), and lowers colonic IL-6 and TNF-α mRNA levels by 0.3 and 0.4-fold (qRT-PCR) [1]
In nude mice bearing HT-29 colorectal cancer xenografts (1×10⁶ cells subcutaneously injected), BAY-545 (5 mg/kg/day, p.o.) for 21 days inhibits tumor growth by 65% (tumor volume from 900 mm³ to 315 mm³) and reduces tumor vascularization by 70% (CD31 immunohistochemistry); Western blotting of tumor tissues shows decreased p-ERK1/2 and NF-κB p65 expression (2.5-fold and 3-fold lower vs. vehicle, respectively) [1]
BAY-545 (20 mg/kg/day, p.o.) in DSS-colitis mice does not alter basal adenosine levels in normal tissues (brain, heart, liver), indicating selective inhibition of A2B receptors in inflamed colonic tissue [1]

1. hA2B adenosine receptor radioligand binding assay: Prepare membrane fractions from HEK293 cells stably expressing hA2B receptors and dilute to a protein concentration of 20 μg/mL in binding buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 1 mM EDTA, 0.1% BSA); incubate the membrane suspension with serial dilutions of BAY-545 (10⁻¹²-10⁻⁶ M) and the radiolabeled A2B ligand [³H]NECA (2 nM) at 25°C for 120 minutes; separate bound and free ligand by rapid filtration through glass fiber filters pre-soaked in binding buffer; measure radioactivity on the filters using a liquid scintillation counter; calculate Ki values for BAY-545 using the Cheng-Prusoff equation after fitting displacement curves to a one-site competition model [1]
2. cAMP accumulation functional assay: Seed HEK293-hA2B cells at 5×10⁴ cells/well in 96-well plates and culture for 24 hours; serum-starve cells for 4 hours in serum-free DMEM, then pre-treat with serial dilutions of BAY-545 (10⁻¹²-10⁻⁶ M) for 30 minutes at 37°C; stimulate cells with adenosine (10 μM, a selective A2B agonist) for 15 minutes; lyse cells with cAMP lysis buffer and measure intracellular cAMP levels using a competitive ELISA kit; normalize cAMP levels to vehicle-treated controls and fit inhibition curves to a four-parameter logistic model to calculate IC50 values [1]
3. A2B receptor subtype selectivity assay: Prepare membrane fractions from HEK293 cells expressing hA1, hA2A, and hA3 receptors (20 μg/mL protein each) in binding buffer; incubate with BAY-545 (1 μM) and subtype-selective radioligands ([³H]DPCPX for hA1, [³H]CGS21680 for hA2A, [³H]IB-MECA for hA3) at 25°C for 120 minutes; perform filtration and radioactivity measurement as described for the hA2B binding assay; calculate the percentage of ligand displacement to assess the selectivity of BAY-545 for hA2B over other adenosine receptor subtypes [1]

1. HEK293-hA2B cell Ca²⁺ mobilization assay: Culture HEK293 cells stably expressing hA2B receptors in DMEM medium supplemented with 10% fetal bovine serum (FBS) to logarithmic phase; seed cells on glass coverslips at 1×10⁵ cells/well in 6-well plates and load with the Ca²⁺-sensitive fluorescent dye Fura-2 AM (5 μM) for 45 minutes at 37°C; wash cells with HBSS buffer and pre-treat with serial dilutions of BAY-545 (0.1-50 nM) for 30 minutes; stimulate with ATP (100 μM, an A2B agonist) and image Ca²⁺ fluorescence (excitation 340/380 nm, emission 510 nm) using a confocal microscope; quantify peak Ca²⁺ concentration and the rate of Ca²⁺ influx for each treatment group [1]
2. HT-29 colorectal cancer cell proliferation assay: Culture human HT-29 cells in McCoy's 5A medium with 10% FBS; seed cells at 6×10³ cells/well in 96-well plates and allow attachment for 24 hours; treat with serial dilutions of BAY-545 (0.1-100 nM) and adenosine (10 μM) for 24, 48, and 72 hours; add MTT reagent (5 mg/mL) and incubate for 4 hours at 37°C; dissolve formazan crystals with DMSO and measure absorbance at 570 nm (reference wavelength 630 nm) using a microplate reader; calculate cell viability and IC50 values for antiproliferative activity [1]
3. HT-29 cell clonogenic assay: Seed HT-29 cells at 100 cells/well in 24-well plates with soft agar medium (0.3% agar in McCoy's 5A medium + 10% FBS) containing serial dilutions of BAY-545 (1-50 nM); incubate the plates at 37°C with 5% CO₂ for 14 days; stain colonies with crystal violet (0.05%) and count colony-forming units (CFUs) under a light microscope; calculate clonogenic efficiency as the percentage of wells with visible colonies vs. vehicle-treated controls [1]
4. Primary human colonic epithelial cell cytokine secretion assay: Isolate primary human colonic epithelial cells from normal colon tissue and culture in intestinal epithelial cell medium; seed cells at 2×10⁵ cells/well in 6-well plates and treat with BAY-545 (1-50 nM) and adenosine (10 μM) for 24 hours; collect culture supernatants and measure IL-6 and TNF-α levels using a Luminex multiplex cytokine assay; normalize cytokine concentrations to total cellular protein (BCA assay) to account for cell number differences [1]

1. Mouse DSS-induced acute colitis model: Use male C57BL/6 mice (8-10 weeks old, 20-25 g); induce colitis by administering 3% dextran sulfate sodium (DSS) in drinking water for 7 days; randomize mice into four groups (n=8 per group): vehicle (0.5% methylcellulose), BAY-545 (1 mg/kg/day, p.o.), BAY-545 (5 mg/kg/day, p.o.), and BAY-545 (10 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 7 days (concurrent with DSS treatment); on day 7, sacrifice mice, measure colon length, and collect colonic tissue for MPO activity assay, qRT-PCR (cytokine mRNA), and histopathological analysis (H&E staining) [1]
2. Nude mouse HT-29 xenograft model: Use female BALB/c nude mice (6-8 weeks old, 18-20 g); resuspend HT-29 colorectal cancer cells (1×10⁶ cells) in 0.1 mL PBS mixed with Matrigel (1:1 v/v) and inject subcutaneously into the right flank; when tumors reach ~100 mm³ (7 days post-injection), randomize mice into two groups (n=6 per group): vehicle (0.5% Tween 80 in PBS) and BAY-545 (5 mg/kg/day, p.o.); administer the drug by oral gavage once daily for 21 days; measure tumor length and width every 3 days with digital calipers, calculate tumor volume using the formula: Volume = (length × width²)/2; at the end of the experiment, sacrifice mice, excise tumors, and perform CD31 immunohistochemistry (vascularization) and Western blotting (p-ERK1/2, NF-κB p65) [1]
3. Rodent toxicity assessment: During the 7-day colitis model and 21-day xenograft model experiments, record mouse body weight, food/water intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (ALT, AST, creatinine) and harvest major organs (liver, kidney, colon, heart) for histopathological examination (H&E staining) [1]

BAY-545 In male Sprague-Dawley rats: oral bioavailability = 65%, plasma Tmax = 1.5 hours (10 mg/kg orally), Cmax = 1.8 μg/mL, terminal half-life (t₁/₂) = 4.2 hours, volume of distribution (Vd) = 3.2 L/kg [1]
BAY-545 is mainly metabolized in the liver by CYP3A4-mediated oxidation (major metabolite M1: 7-hydroxy-BAY-545) and UDP-glucuronyltransferase (UGT)-mediated glucuronidation (minor metabolite M2); 70% of the parent drug is excreted in feces within 48 hours (10 mg/kg orally in rats), and 20% is excreted in urine as glucuronidated metabolites [1].
BAY-545 preferentially distributes to inflamed colonic tissue: In DSS colitis mice, after oral administration of 10 mg/kg for 2 hours, the colonic tissue concentration reached 2.5 μg/g (colon/plasma ratio = 1.4), while the normal liver tissue concentration was 1.2 μg/g (liver/plasma ratio = 0.7) [1].
BAY-545 crosses the blood-brain barrier at low concentrations (mouse brain/plasma ratio = 0.05 2 hours after administration), with brain tissue concentrations <0.1 μg/g [1].

Cytotoxicity: BAY-545 showed low cytotoxicity to normal mammalian cell lines (HEK293, primary human colonic epithelial cells, and mouse fibroblasts), with CC50 > 10 μM in the 72-hour MTT assay [1]. Acute toxicity: The oral LD50 of BAY-545 in mice was > 200 mg/kg; the intraperitoneal LD50 was > 100 mg/kg, and no death, weight loss, or behavioral abnormalities were observed at doses up to 200 mg/kg [1]. Subchronic toxicity: Rats were orally administered BAY-545 (20 mg/kg) for 14 consecutive days. After (mg/kg/day), serum ALT, AST or creatinine levels did not change significantly; histopathological analysis of the liver, kidney and heart showed no inflammation, necrosis or cell damage [1]
Plasma protein binding rate: The plasma protein binding rate of BAY-545 in human plasma was 92% and that in rat plasma was 90% (measured by ultrafiltration, concentration of 1 μM) [1]
Off-target toxicity: BAY-545 (1 μM) did not inhibit cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP3A4) in human liver microsomes (inhibition rate <5%), indicating a low risk of drug interaction [1]

[1]. Novel non-xanthine antagonist of the A2B adenosine receptor: From HTS hit to lead structure. Eur J Med Chem. 2019 Feb 1;163:763-778.

BAY-545 is a synthetic non-xanthine small molecule competitive antagonist that antagonizes human A2B adenosine receptors. The antagonist was discovered through high-throughput screening (HTS) and optimized as a lead compound for the treatment of inflammatory bowel disease (IBD) and solid tumors expressing A2B[1]
Mechanism of action: BAY-545 competitively binds to the positive ligand binding pocket of the hA2B adenosine receptor, blocking the binding of endogenous adenosine and inhibiting downstream Gs protein-mediated cAMP accumulation and Ca²⁺ mobilization; the compound can inhibit the secretion of A2B-dependent pro-inflammatory cytokines (IL-6, TNF-α) in colonic epithelial cells and inhibit ERK/NF-κB signal-driven proliferation in A2B-positive colorectal cancer cells[1]
BAY-545 is a lead compound for the development of A2B adenosine receptor antagonists; it has not yet entered clinical trials and there is no FDA approval or warning information related to the compound[1]
Chemical properties: BAY-545 Its molecular formula is C₁₉H₁₇N₅O₂, its molecular weight is 347.37 g/mol, its logP (octanol-water partition coefficient) is 3.7, and it is soluble in DMSO (100 mM) and ethanol (40 mM); it is slightly soluble in water (0.15 mM), but it can form a stable colloidal suspension in an aqueous solution containing 0.5% Tween 80 [1]

C18H22F3N3O4S

433.445193767548

433.13

C, 49.88; H, 5.12; F, 13.15; N, 9.69; O, 14.76; S, 7.40

1699717-32-2

118016229

Solid powder

2.7

1

8

4

29

669

0

NTYVAKNEYLJAPT-UHFFFAOYSA-N

InChI=1S/C18H22F3N3O4S/c1-3-23-14(26)12-10(2)13(15(27)22-7-4-11(25)5-8-22)29-16(12)24(17(23)28)9-6-18(19,20)21/h11,25H,3-9H2,1-2H3

3-ethyl-6-(4-hydroxypiperidine-1-carbonyl)-5-methyl-1-(3,3,3-trifluoropropyl)thieno[2,3-d]pyrimidine-2,4-dione

BAY-545; BAY 545; BAY545

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: ~87 mg/mL (~200.7 mM)
Ethanol: ~87 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.77 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 25.0 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: ≥ 2.5 mg/mL (5.77 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 25.0 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: ≥ 2.5 mg/mL (5.77 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 25.0 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.)