A_2AR ( Ki = 1.4 nM )
Adenosine A2A receptor (A2AR). [1]

ZM241385 (1 μM; 24-48 hours; PC12 cells) treatment counteracts the significant upregulation of A_2AR mRNA and protein levels caused by A_2AR agonist CGS21680[1].

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ZM241385 (1.0 μM) partially offset the inhibition of Ca2+ efflux induced by the A2AR agonist CGS21680 (1.0 μM) in PC12 cells, as measured by extracellular Ca2+ levels after 3 hours of incubation. [1]
ZM241385 (1.0 μM) reversed the up-regulation of A2AR mRNA caused by CGS21680 (1.0 μM) in PC12 cells; CGS21680 increased A2AR mRNA by over 5-fold, while co-treatment with ZM241385 decreased this agonist-enhanced transcription to close to control levels. [1]
ZM241385 reduced the increased A2AR protein levels induced by CGS21680 (1.0 μM) in PC12 cells, as shown by Western blot analysis. [1]
ZM241385 increased ATP levels in PC12 cells after 6 hours of exposure, compared to untreated controls (p<0.05). [1]
ZM241385 attenuated adenosine (ADO) release from PC12 cells treated with CGS21680 (1.0 μM) by approximately 50%, as measured by HPLC after 3 hours of incubation. [1]
ZM241385 decreased cAMP levels in PC12 cells after 3 hours of exposure (p<0.05). [1]
ZM241385 reduced nitrite (a stable metabolite of nitric oxide) levels in PC12 cells after 24 hours of exposure (p<0.05). [1]
ZM241385 reversed CGS21680-induced p44/42 MAPK (Erk1/2) phosphorylation in PC12 cells, resulting in levels lower than those observed in untreated control cells, as shown by Western blot after 30 min pretreatment with ZM241385 followed by 48 hours of co-incubation with CGS21680. [1]
ZM241385 counteracted the accelerated proliferation induced by CGS21680 in PC12 cells, as measured by MTT assay after three days of treatment. [1]
ZM241385 counteracted the A2AR agonist-induced increase in neurite outgrowth in PC12 cells; after three days of treatment, ZM241385 reduced the percentage of cells with neurites compared to CGS21680 alone. [1]
ZM241385 inhibited agonist-promoted iron uptake in PC12 cells; cells treated with CGS21680 (1.0 μM) plus ZM241385 (1.0 μM) showed reduced intracellular iron levels compared to CGS21680 alone after 1 hour of FeSO4 addition (50 μM). [1]

ZM241385 (0.2 μg/mouse, 0.4 μg/mouse; intraperitoneal injection; every day; for 11 weeks; female C57BL/6 WT mice) treatment decreases tumor volume, increases CD8+ T cell activation, and splenic MDSC frequency[4].

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Monotherapy with high-dose (0.4 μg/mouse) ZM241385 promoted a reduction in total tumor volume (including tongue and esophagus) compared to control group (p < 0.05 or p < 0.01).
At the higher dose, ZM241385 increased the frequency of splenic CD8+ T cells responding in IFN-γ ELISPOT assay against p53 and VEGFR2 peptide epitopes, although this enhancement failed to reach significance (p > 0.05).
Therapy with the higher dose of ZM241385 led to a decrease in the frequency of MDSC (but not Treg) in the spleen. [4]

1. This paper describes the in vitro pharmacology of ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin- 5-yl amino]ethyl) phenol), a novel non-xanthine adenosine receptor antagonist with selectivity for the A2a receptor subtype. 2. ZM 241385 had high affinity for A2a receptors. In rat phaeochromocytoma cell membranes, ZM 241385 displaced binding of tritiated 5'-N-ethylcarboxamidoadenosine (NECA) with a pIC50 of 9.52, (95% confidence limits, c.l., 9.02-10.02). In guinea-pig isolated Langendorff hearts, ZM 241385 antagonized vasodilatation of the coronary bed produced by 2-chloroadenosine (2-CADO) and 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) with pA2 values of 8.57 (c.l., 8.45-8.68) and 9.02 (c.l., 8.79-9.24) respectively. 3. ZM 241385 had low potency at A2b receptors and antagonized the relaxant effects of adenosine in the guinea-pig aorta with a pA2 of 7.06, (c.l., 6.92-7.19). 4. ZM 241385 had a low affinity at A1 receptors. In rat cerebral cortex membranes it displaced tritiated R-phenylisopropyladenosine (R-PIA) with a pIC50 of 5.69 (c.l., 5.57-5.81). ZM 241385 antagonized the bradycardic action of 2-CADO in guinea-pig atria with a pA2 of 5.95 (c.l., 5.72-6.18). 5. ZM 241385 had low affinity for A3 receptors. At cloned rat A3 receptors expressed in chinese hamster ovary cells, it displaced iodinated aminobenzyl-5'-N-methylcarboxamido adenosine (AB-MECA) with a pIC50 of 3.82 (c.l., 3.67-4.06). 6. ZM 241385 had no significant additional pharmacological effects on the isolated tissues used in these studies at concentrations three orders of magnitude greater than those which block A2a receptors[3].

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For A₁ receptor binding: Rat cerebral cortex crude membranes were prepared. Binding assays were performed in 50 mM Tris‑HCl (pH 7.4) at room temperature with 10 nM tritiated R‑PIA (³H‑R‑PIA) as radioligand, in the presence of adenosine deaminase (2 U ml⁻¹) to remove endogenous adenosine. Non‑specific binding was determined. Competition studies with ZM241385 gave pIC₅₀ values. [3]

For A₂ₐ receptor binding: PC12 cell membranes were prepared similarly to cortex membranes. Binding was performed with 40 nM tritiated NECA (³H‑NECA) under the same buffer conditions, with adenosine deaminase present. ZM241385 competed for the binding, yielding a pIC₅₀ of 9.52. [3]

For A₃ receptor binding: Rat A₃ adenosine receptor cloned and expressed in CHO cells was used. Radioligand binding assays with ¹²⁵I‑AB‑MECA (N⁶‑(3‑[¹²⁵I]iodo‑4‑aminobenzyl‑5’‑N‑methylcarboxamidoadenosine) were performed as described. ZM241385 displaced the radioligand with a pIC₅₀ of 3.82. [3]

Phosphodiesterase assay: Hepatocytes from fed male Sprague‑Dawley rats were isolated and incubated. Cyclic AMP phosphodiesterase activity was measured by a modified two‑step method of Thompson & Appleman at 30 °C in the presence of 1 μM cyclic AMP. The effects of ZM241385 (10 μM and 1 mM) were compared with theophylline. [3]

Cell Line: PC12 cells
Concentration: 1 μM
Incubation Time: 24 hours
Result: Suppressed the increased A_2AR mRNA levels engendered by CGS21680.

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For calcium measurement, PC12 cells were grown in 12-well plates to 85-90% confluence. To assess the impact of ZM241385 on Ca2+ transport, cells were pretreated with 1.0 μM ZM241385 for 40 minutes in Ca2+- and Mg2+-free D-PBS, then incubated with 1.0 μM CGS21680 for an additional three hours. Extracellular Ca2+ in the supernatant was measured using a calcium reagent set after centrifugation. [1]
For mRNA analysis, PC12 cells were treated with 1.0 μM ZM241385, with or without 1.0 μM CGS21680, for 6 hours. Total RNA was extracted and reverse-transcribed. Real-time quantitative PCR was performed using SYBR Green detection with specific primers for A2AR. Relative gene expression was calculated using the 2-ΔΔCt method. [1]
For Western blot analysis, PC12 cells were treated with 1.0 μM ZM241385, with or without 1.0 μM CGS21680, for 48 hours (or with pretreatment for 30 min followed by 48 hours co-incubation). Cells were lysed, proteins were separated by SDS-PAGE, transferred to membranes, and probed with anti-A2AR antibody or anti-p44/42 MAPK and anti-phospho-p44/42 MAPK antibodies. Bands were detected and quantified by densitometry. [1]
For ATP measurement, PC12 cells were incubated with 1.0 μM ZM241385 for 6 hours. Cells were harvested, extracted, and intracellular ATP levels were measured using a luciferase-based assay. [1]
For adenosine (ADO) detection, PC12 cells were incubated in D-PBS with 1.0 μM CGS21680 and 1.0 μM ZM241385 (or SMF) for 3 hours. Extracellular fluid was collected and analyzed by HPLC using a Novapak C18 column with a gradient elution (0-40% over 35 min) of potassium dihydrogen phosphate buffer (pH 5.5) and methanol/water (60:40). Peaks were identified by retention time compared to an authentic ADO standard. [1]
For cAMP assay, 5.0×10^5 cells were placed in fresh medium containing 1.0 U/mL adenosine deaminase and incubated with 1.0 μM ZM241385 for 3 hours. Cells were harvested, lysed in 0.1 M HCl for 20 min, centrifuged, and supernatants were assayed using a cAMP enzyme immunoassay kit. [1]
For nitrite assay, PC12 cells were incubated with 1.0 μM ZM241385 for 24 hours. Culture medium samples were collected and mixed with Griess reagent. After 30 min incubation at room temperature, absorbance was measured at 548 nm and compared to a sodium nitrite standard curve. [1]
For neurite outgrowth measurement, PC12 cells grown on coverslips were changed into differentiation medium (1.0% horse serum with 25 ng/mL NGF) 24 hours after passaging. Cells were pretreated with 1.0 μM CGS21680 for 30 min, followed by addition of 1.0 μM ZM241385 for an additional three days. Cells were stained with F-actin conjugated fluorescent phalloidin, mounted, and imaged. Cells with neurites at least 1.5 times the cell diameter were counted as differentiated. [1]
For iron quantification, PC12 cells grown in 48-well plates were incubated with 1.0 μM ZM241385 (with or without 1.0 μM CGS21680) and then exposed to 50 μM FeSO4 for 2 hours. Cells were washed, frozen, and lysed with NaOH. Samples were mixed with HCl and KMnO4, heated at 60°C for 2 hours, then detection solution containing ferrozine, neocuproine, ammonium acetate, and ascorbic acid was added. Absorbance was read at 550 nm and compared to an FeSO4 standard curve. [1]

Female C57BL/6 WT mice received 4-nitroquinoline-N-oxide
0.2 μg/mouse, 0.4 μg/mouse
Intraperitoneal injection; every day; for 11 weeks

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ZM241385 was dissolved in DMSO, Cremopher, and ddH2O at a DMSO:Cremopher:ddH2O ratio of 1:1:4.
Low-dose (0.2 μg ZM/mouse) or high-dose (0.4 μg ZM/mouse) injections were administered intraperitoneally (i.p.) daily to mice, beginning in week 16 of the experiments.
Mice were conditioned with 4NQO (100 μg/ml in drinking water) for 16 weeks to induce oral squamous cell carcinoma prior to treatment. [4]

At concentrations three orders of magnitude above those blocking A₂ₐ receptors, ZM241385 showed no significant additional pharmacological effects in isolated tissues. Minor inhibitory effects on carbachol‑induced bradycardia and isoprenaline‑induced relaxation occurred at concentrations 3–4 orders higher than A₂ₐ‑blocking concentrations. [3]

[1]. Static magnetic field exposure reproduces cellular effects of the Parkinson's disease drugcandidate ZM241385. PLoS One. 2010 Nov 8;5(11):e13883. doi: 10.1371/journal.pone.0013883.

[2]. Characterization of human A(2B) adenosine receptors: radioligandbinding, western blotting, and coupling to G(q) in human embryonickidney 293 cells and HMC-1 mast cells. Mol Pharmacol. 1999 Oct;56(4):705-13.

[3]. The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2a selective adenosinereceptor antagonist. Br J Pharmacol. 1995 Jul;115(6):1096-102.

[4]. Impact of combination immunochemotherapies on progression of 4NQO-induced murine oral squamous cell carcinoma. Cancer Immunol Immunother. 2019 Jul;68(7):1133-1141.

4-[2-[[7-amino-2-(2-furanyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5-yl]amino]ethyl]phenol is a diamino-1,3,5-triazine.

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ZM241385 is a potent, selective, non-xanthine adenosine A2A receptor antagonist. It is under evaluation as a drug candidate for Parkinson's disease (PD). The literature notes that A2A receptor antagonists like ZM241385 face pharmacological barriers to clinical use, including poor oral availability and a lack of blood-brain barrier (BBB) permeability. The study compared SMF exposure to ZM241385 and found that SMF reproduced several cellular effects of ZM241385 in PC12 cells, including altered calcium flux, increased ATP, reduced cAMP, reduced nitric oxide, reduced p44/42 MAPK phosphorylation, inhibited proliferation, and reduced iron uptake, as well as counteracting CGS21680-induced effects. [1]

C16H15N7O2

337.34

337.128

C, 56.97; H, 4.48; N, 29.07; O, 9.49

139180-30-6

139180-30-6

176407

White to off-white solid powder

1.6±0.1 g/cm3

1.791

0.89

3

8

5

25

435

0

O1C([H])=C([H])C([H])=C1C1N=C2N=C(N=C(N([H])[H])N2N=1)N([H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])O[H]

PWTBZOIUWZOPFT-UHFFFAOYSA-N

InChI=1S/C16H15N7O2/c17-14-20-15(18-8-7-10-3-5-11(24)6-4-10)21-16-19-13(22-23(14)16)12-2-1-9-25-12/h1-6,9,24H,7-8H2,(H3,17,18,19,20,21,22)

4-[2-[[7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl]amino]ethyl]phenol

ZM-241385; ZM241385; 139180-30-6; ZM241385; ZM 241385; ZM-241385; 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenol; 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; 4-[2-[[7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl]amino]ethyl]phenol; 5NIC36BO71; ZM 241385

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: ≥ 30 mg/mL (~88.9 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.17 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 20.8 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: 2.08 mg/mL (6.17 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (6.17 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 20.8 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.)