Adenosine A_2A receptor ( Ki = 1.1 nM )
Preladenant likewise totally opposes cAMP in cells that have the human A_2A receptor recombinant expressed. At the A_2A receptor, preladenant is found to have K_B values of 1.3 nM; this value is in good agreement with the K_i value found in the radioligand binding assay. To show selectivity over A_2B receptors, a functional test aK_in to this one is performed using cells that express A_2B receptors. Preladenant's K_B value in this assay is 1.2 μM, meaning that it is 923 times more selective for the A_2A receptor than the A_2B receptor[1].

Preladenant likewise totally opposes cAMP in cells that have the human A_2A receptor recombinant expressed. At the A_2A receptor, preladenant is found to have K_B values of 1.3 nM; this value is in good agreement with the K_i value found in the radioligand binding assay. To show selectivity over A_2B receptors, a functional test aK_in to this one is performed using cells that express A_2B receptors. Preladenant's K_B value in this assay is 1.2 μM, meaning that it is 923 times more selective for the A_2A receptor than the A_2B receptor[1].

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In a radioligand competition binding assay using membranes from cells expressing recombinant human A_2A receptors, Preladenant displaced the radioligand with a K_i value of 1.1 nM. [1]
In a functional cAMP assay using HEK 293 cells stably expressing human A_2A receptors, Preladenant acted as a complete antagonist, shifting the concentration-response curve of the A_2A agonist CGS-21680 to the right in a concentration-dependent manner. The functional K_B was determined to be 1.3 nM. [1]
Preladenant showed no significant interaction (>10 µM) at over 60 additional receptors, ion channels, and transporters tested. [1]

Preladenant (1 mg/kg) prevents behavioral sensitization caused by L-Dopa following daily administration, indicating a lower chance of dysK_inesia development. Preladenant demonstrates antidepressant-like characteristics in behavioral despair models, such as the forced swim test for rats and mice and the tail suspension test for mice[1]. At doses of 1 mg/kg (minimum score: 9.0) and 3 mg/kg (minimum score: 6.5), preladenant reduces parK_insonian scores in a dose-dependent manner. Preladenant at a subthreshold dose lowers the mean and minimum parK_insonian scores in animals receiving 3 mg kg of L-Dopa to 6.88 and 5.25, respectively. To compare individual treatments to the vehicle, the Wilcoxin test is employed. The minimum parK_insonian score is markedly improved by Preladenant (3 mg/kg), L-Dopa (3, 6, and 12 mg/kg), and the combination of Preladenant and L-Dopa (1 or 3 mg/kg+3 mg/kg). Furthermore, in comparison to the 3 mg/kg L-Dopa group, the minimum and mean parK_insonian scores of the 12 mg/kg L-Dopa and L-Dopa+Preladenant groups show a significant improvement[2].

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Oral administration of Preladenant (0.1 and 1 mg/kg) significantly attenuated hypolocomotion induced by the A_2A agonist CGS-21680 (1 mg/kg s.c.) in rats. A dose of 1 mg/kg produced significant attenuation for up to 8 hours. [1]
Oral administration of Preladenant (0.3 and 1 mg/kg) dose-dependently attenuated catalepsy induced by the dopamine D₂ receptor antagonist haloperidol (1 mg/kg s.c.) in rats, with significant effects observed at 1 and 4 hours post-dosing. [1]
In rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle, oral Preladenant (0.01-1 mg/kg) dose-dependently potentiated contralateral rotations induced by a subthreshold dose of L-Dopa. The effect was significant at 1, 6, and 12 hours after a 1 mg/kg dose, correlating with striatal A_2A receptor occupancy. [1]
In 6-OHDA-lesioned rats, chronic treatment (twice daily for 23 days) with a combination of Preladenant (0.3 mg/kg p.o.) and a low dose of L-Dopa (4 mg/kg i.p.) did not induce behavioral sensitization (a progressive increase in rotational response), whereas treatment with a higher dose of L-Dopa (6 mg/kg) alone resulted in significant sensitization. This suggests a potential to reduce the risk of dyskinesia development. [1]
Preladenant (0.1 and 1 mg/kg p.o.) significantly reduced immobility time in the mouse forced swim test (FST) and tail suspension test (TST), displaying an antidepressant-like profile comparable to the tricyclic antidepressant desipramine. [1]
Preladenant (0.1-1 mg/kg p.o.) dose-dependently reduced immobility time and modestly increased climbing behavior in the rat forced swim test (FST). [1]

Receptor binding is performed using membranes prepared from cells with recombinant expression of adenosine receptors as follows: human A_2A and HEK 293, rat A_2A and Chinese hamster ovary, human and rat A₁ and Chinese hamster ovary, and human A₃ and HEK 293. To conduct radioligand competition binding assays, 200 μL of total assay volume are used in 96-well plates, with a final test drug concentration range of 0.1–3 μM. A₁ and A_2A, Dulbecco's phosphate-buffered saline with 10 mM MgCl₂, and A₃, 50 mM Tris-HCl, 120 mM NaCl, and 10 mM MgCl₂ are the assay buffers (pH 7.4) in which membranes are diluted. The membrane preparations are incubated at room temperature for 15 minutes after 4 U/mL adenosine deaminase is added to them in order to eliminate endogenous adenosine from them. At 0.5 ([³H]SCH 58261, A_2A), 1 ([³H]DPCPX, A₁), or 0.25 ([¹²⁵I]AB-MECA, A₃) nM, radioligand is added. The addition of 100 nM NECA (A₁), 100 nM CGS 15923 (A_2A), or 100 nM DPCPX (A₃) defines nonspecific binding. For 1.5 hours (A_2A and A₁) or 2 hours (A₃), plates are incubated at room temperature with agitation. Using a Brandel cell harvester, membranes are filtered onto Packard GF-B filter plates and then cleaned in ice-cold assay buffer to separate the radioligand that is bound and free. Prior to filling each well with 45 μL of Microscint 20, the plates are dried. Using an iterative curve-fitting program, the displacement curves are fitted in order to determine the IC50 values. The Cheng-Prusoff equation[1] is used to calculate K_i values.

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For the adenosine A_2A receptor binding assay, membranes prepared from cells expressing recombinant human A_2A receptors were diluted in assay buffer (Dulbecco's phosphate-buffered saline with 10 mM MgCl₂, pH 7.4). Endogenous adenosine was removed by incubating membranes with adenosine deaminase. Radioligand competition binding assays were performed in a total volume of 200 µL in 96-well plates. The final concentration of the test drug Preladenant ranged from 0.1 nM to 3 µM. The radioligand [³H]SCH 58261 was added to a final concentration of 0.5 nM. Nonspecific binding was defined using 100 nM CGS 15923. Plates were incubated at room temperature with agitation for 1.5 hours. Bound and free radioligand were separated by filtration onto filter plates, followed by washing with ice-cold assay buffer. IC₅₀ values were determined by fitting displacement curves, and K_i values were calculated using the Cheng-Prusoff equation. [1]
For the functional cAMP assay using human A_2A receptors, HEK 293 cells stably expressing the receptors were harvested and resuspended in assay buffer (Hanks' balanced salt solution supplemented with HEPES, MgCl₂, and bovine serum albumin). Cell suspensions were preincubated with vehicle or various concentrations of Preladenant for 15 minutes at room temperature. The A_2A agonist CGS-21680 was then added at different concentrations, and the reaction was incubated for 15 minutes at 37°C to stimulate cAMP production. Reactions were terminated by adding lysis buffer. cAMP levels were measured. Concentration-response curves for CGS-21680 in the presence and absence of Preladenant were plotted, and the functional K_B was determined by the dose-ratio method. [1]

Human A_2A or human A_2B receptor-stably expressing HEK 293 cells are cultured to confluence, harvested with an enzyme-free cell dissociation buffer, and pelleted via centrifugation (1000 g; 5 min). The cells are rinsed and diluted in Hanks' balanced salt solution supplemented with 10 nM HPS, pH 7.4, 5 mM MgCl₂, and 0.2% bovine serum albumin until they reach a final density of 4×10⁶ cells/mL. To attain the desired final assay concentrations, the preladenant is diluted in the buffer mentioned above and mixed with the following ingredients: 100 μM Ro 201724, 2 U/mL adenosine deaminase, and 0.25% DMSO. In 96-well plates with 25 μL of vehicle or preladenant, cell suspensions (20 μL) are preincubated for 15 min at room temperature. After adding 10-fold the desired concentration of either 5-N-cyclopropylcarboxamidoadenosine (A_2B) or CGS-21680 (A_2A), the reactions are incubated for 15 minutes at 37°C. After adding 50 μL of assay/lysis buffer, the reactions come to an end. Plotting of the concentration response curves for CGS-21680 with and without preladenant allows for the determination of the EC50 values through curve fitting with GraphPad Prism software[1].

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The functional assay for selectivity over the A_2B receptor was performed similarly to the A_2A cAMP assay. HEK 293 cells stably expressing human A_2B receptors were used. Cells were treated with Preladenant and then stimulated with the A_2B agonist 5'-N-cyclopropylcarboxamidoadenosine. cAMP production was measured to determine the compound's antagonistic potency (K_B) at the A_2B receptor. [1]

Mice and Rats: Male CD1 mice and male CD rats are employed. Preladenant is taken orally at a dose volume of 3 to 5 milliliters per K_ilogram in 50% polyethylene glycol 400. Mice are put one at a time into glass cylinders that are filled with water (25°C) to a depth of 10 cm in the forced swim test (FST), and they are left for six minutes. When a mouse floats upright and makes only tiny movements to keep its head above water, it is considered immobile. During the final four minutes of the six-minute testing period, an observer who is blind to the animals' treatments records the length of immobility. Preladenant, SCH 412348, or the vehicle is dosed into the animals one hour prior to behavioral testing. After swimming for fifteen minutes in a 25°C water cylinder, each rat is taken out, dried in a heated enclosure, and then placed back in its original cage. The animal is reexposed to the conditions on test day, which is twenty-four hours later. The duration of immobility during a five-minute period is then recorded. Furthermore, the amount of time the rats spent scaling the cylinder's walls is noted. One hour prior to behavioral testing on test day, each animal receives a dose of Preladenant, SCH 412348, or vehicle.\nMonkeys: Six 3.5–4.2 kg female Macaca fascicularis cynomolgus monkeys are utilized. The animals are given subcutaneous (sc) injections of MPTP (2–3 mg/kg) once a week until they develop a stable parK_insonian syndrome, which is defined as an unchanged disability score of 8 or higher for at least one month, as determined by a parK_insonian disability scale. After the last MPTP administration, the monkeys receive chronic Prolopa treatment (L-Dopa/benserazide, 100/25 mg) for at least two months, or until the development of distinct and observable dysK_inesias. Using L-Dopa and Preladenant (1 mg/kg and 3 mg/kg, p.o.) on these monkeys is the current experiment.

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\nFor the CGS-21680-induced hypolocomotion test in rats, Preladenant was administered orally (dose volume 3-5 ml/kg) as a suspension in 50% polyethylene glycol 400. Doses tested were 0.03, 0.1, 0.3, and 1 mg/kg. Thirty minutes later, rats received a subcutaneous injection of the A_2A agonist CGS-21680 (1 mg/kg in saline). Ten minutes after CGS-21680, rats were placed in locomotor activity chambers, and total distance traveled was recorded for 30 minutes. [1]
\nFor the haloperidol-induced catalepsy test in rats, catalepsy was first induced by subcutaneous injection of haloperidol (1 mg/kg). Thirty minutes later, a baseline catalepsy measurement was taken. Immediately after, Preladenant (0.1, 0.3, 1 mg/kg) or vehicle was administered orally (in 50% PEG 400). Catalepsy was retested 1 and 4 hours after Preladenant administration. [1]
\nFor the L-Dopa rotation test in 6-OHDA-lesioned rats, unilateral lesions were induced by stereotaxic infusion of 6-OHDA into the medial forebrain bundle. Two weeks later, rats were primed with L-Dopa. For testing, Preladenant (0.01, 0.03, 0.1, 0.3, 1 mg/kg) was administered orally (in 50% PEG 400) 40 minutes before the peripheral decarboxylase inhibitor benserazide (25 mg/kg i.p.). L-Dopa (4 mg/kg i.p.) was given 20 minutes after benserazide. Contralateral rotations were recorded in automated rotometers for 2 hours. Duration of action was assessed by administering Preladenant (1 mg/kg p.o.) at various times (1, 6, 12, 18 h) before the L-Dopa/benserazide challenge. [1]
\nFor the behavioral sensitization study, 6-OHDA-lesioned rats were treated twice daily for 23 days with either a combination of Preladenant (0.3 mg/kg p.o. in 50% PEG 400) and L-Dopa (4 mg/kg i.p.), or vehicle (p.o.) and a higher dose of L-Dopa (6 mg/kg i.p.). Rotational behavior was measured on specific days. [1]
\nFor the mouse forced swim test (FST) and tail suspension test (TST), Preladenant (0.1, 1 mg/kg) or vehicle was administered orally (in 50% PEG 400) 1 hour before behavioral testing. In the FST, mice were placed in water-filled cylinders for 6 minutes, and immobility during the last 4 minutes was recorded. In the TST, mice were suspended by the tail for 6 minutes, and total immobility time was recorded. [1]
\nFor the rat forced swim test, rats were subjected to a 15-minute pre-swim session. Twenty-four hours later (test day), they received Preladenant (0.1, 0.3, 1 mg/kg p.o. in 50% PEG 400) or vehicle 1 hour before a 5-minute test swim. Immobility time and climbing duration were recorded. [1]

[1]. Characterization of the potent and highly selective A2A receptor antagonists preladenant and SCH 412348 [7-[2-[4-2,4-difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] in rodent models of movement disorders and depression. J Pharmacol Exp Ther . 2009 Jul;330(1):294-303.

[2]. Preladenant, a selective A(2A) receptor antagonist, is active in primate models of movement disorders. Exp Neurol. 2010 Oct;225(2):384-90.

Preladenant has been used in clinical trials for the treatment of various diseases, including brain disorders, Parkinson's disease, movement disorders, antipsychotics, and Parkinson's syndrome. Preladenant is an orally bioavailable adenosine A2A receptor (A2AR; ADORA2A) antagonist with potential immunomodulatory and antitumor activity. After administration, preladenant selectively binds to and inhibits A2AR expressed on the surface of T lymphocytes. This blocks the interaction between tumor-released adenosine and A2AR, thereby preventing adenosine/A2AR-mediated T lymphocyte suppression. This leads to T lymphocyte proliferation and activation, and stimulates a T cell-mediated immune response against tumor cells. A2AR is a G protein-coupled receptor highly expressed on the surface of T cells and inhibits T cell proliferation and activation upon activation by adenosine. Adenosine is often overproduced by cancer cells and plays a key role in immunosuppression.

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Preladenant is a novel A2A receptor antagonist derived from the parent compound SCH 58261. It exhibits significantly improved affinity and selectivity compared to the earlier A2A receptor antagonist KW-6002 (isotrol). [1]
The mechanism of action of this compound is the blocking of adenosine A2A receptors, which co-localize with dopamine D2 receptors on GABAergic neurons in the globus pallidus of the striatum in the indirect pathway of the basal ganglia. It is speculated that this blocking effect can restore the balance between the direct and indirect pathways, thereby alleviating motor disorders in a Parkinson's disease model where dopamine receptors are not directly activated. [1]
This study suggests that Preladenant may have the potential to treat motor symptoms of Parkinson's disease (and potentially reduce the risk of motor disorders) and non-motor symptoms (such as depression, which is common in Parkinson's patients). [1]

C25H29N9O3

503.57

503.239

C, 59.63; H, 5.80; N, 25.03; O, 9.53

377727-87-2

10117987

White to off-white solid powder

2.747

1

10

9

37

722

0

O(C([H])([H])C([H])([H])OC([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])N2C3=C(C4=NC(C5=C([H])C([H])=C([H])O5)=NN4C(N([H])[H])=N3)C([H])=N2)C([H])([H])C1([H])[H]

DTYWJKSSUANMHD-UHFFFAOYSA-N

InChI=1S/C25H29N9O3/c1-35-15-16-36-19-6-4-18(5-7-19)32-11-8-31(9-12-32)10-13-33-23-20(17-27-33)24-28-22(21-3-2-14-37-21)30-34(24)25(26)29-23/h2-7,14,17H,8-13,15-16H2,1H3,(H2,26,29)

4-(furan-2-yl)-10-[2-[4-[4-(2-methoxyethoxy)phenyl]piperazin-1-yl]ethyl]-3,5,6,8,10,11-hexazatricyclo[7.3.0.02,6]dodeca-1(9),2,4,7,11-pentaen-7-amine

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)

Solubility in Formulation 1: 0.5 mg/mL (0.99 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 5.0 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: 0.5 mg/mL (0.99 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 5.0 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: ≥ 0.5 mg/mL (0.99 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 5.0 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.)