platelet aggregation
P2Y12 receptor [1]

A few naturally occurring N(6)-substituted adenosine derivatives (cytokinin ribosides) were investigated as inhibitors of platelet aggregation induced in vitro by collagen and their activity range was demonstrated (IC50: 6.77-141 μM). A docking study suggests that anti-aggregation activity of these compounds could involve an interaction with the P2Y12 receptor binding site[1].

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The compound N6-(4-Hydroxybenzyl)adenosine (2e) inhibited collagen-induced platelet aggregation in washed human platelets with an IC50 value of 6.77 ± 0.31 μM (mean ± SD, n=3), which was the most potent among the tested cytokinin ribosides (2b-e). [1]

Molecular docking simulations predicted that N6-(4-Hydroxybenzyl)adenosine binds to the P2Y12 receptor binding cavity with a binding mode similar to the co-crystallized antagonist AZJ. Key interactions include: the ribose sugar forming reinforced hydrogen bonds with Lys280 and Arg256; the purine base participating in π-π stacking with Tyr105, Tyr109, and Phe252; the N6 amino group forming a hydrogen bond with Cys194; the N6-linked 4-hydroxybenzyl moiety engaging in π-π stacking with Tyr105 and Tyr109; and the phenolic hydroxyl group forming hydrogen bonds with Ser156 and Asn159. The ChemPlp docking score for the minimized complex was -95.11 kcal/mol, and the shared volume with AZJ was 205.2 ų. These docking scores were consistent with the observed in vitro anti-aggregation activity ranking. [1]

Effects of N6 -(4-hydroxybenzyl) adenine riboside in stress-induced insomnia in rodents. T1-11 elicited somnogenic effects and effectively ameliorated acute stress-induced insomnia. The somnogenic effect is mediated by A2ARs to activate GABAergic neurons in the VLPO. This adenosine analogue could be a potential hypnotic because of no sympathetic and parasympathetic effects on the cardiovascular system[2].

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The P2Y12 receptor structure in complex with an antithrombotic drug (AZJ) was retrieved from the Protein Data Bank. To simulate physiological pH, side chains of Arg, Lys, Glu, and Asp were ionized, while His and Cys residues were kept neutral by default. The complete receptor structure was minimized with backbone atoms fixed until an RMS of 0.01 kcal·mol⁻¹·Å⁻¹ to preserve the resolved folding. [1]

Conformational analysis of N6-(4-Hydroxybenzyl)adenosine was performed using a Monte Carlo procedure (implemented in the VEGA suite of programs), generating 1000 conformers by random rotation of rotatable bonds. The lowest-energy conformer was used for docking simulations. Docking was carried out with PLANTS software, which uses ant colony optimization algorithms. Default settings were applied without geometric constraints. The search focused on an 8.0 Å radius sphere centered on the co-crystallized ligand, covering the entire binding cavity. The ChemPlp scoring function was used with speed set to 1, and 10 poses were generated per ligand. [1]

The best complexes were minimized while keeping all atoms outside a 10 Šradius sphere around the bound ligand fixed, to allow mutual adaptability. The optimized complexes were then used to recalculate ChemPlp scores and shared volumes with the co-crystallized antagonist AZJ. For N6-(4-Hydroxybenzyl)adenosine, the ChemPlp score was -95.11 kcal/mol, and the shared volume with AZJ was 205.2 ų. [1]

Immunofluorescence assay (IFA) for c-fos[2]
Brain tissues were fixed with 4% of paraformaldehyde for 4 hr and dehydrated for 24 hr in 30% sucrose. Brain slices containing the VLPO were sliced at 30-μm thickness by frozen section. The brain area contained the VLPO located between 0.14 mm before bregma and 0.10 mm after bregma; therefore, about eight slices from each mouse were dissected. The tissue was first rinsed in phosphate-buffered saline (PBS) for 15 min and then incubated in PBS with 0.3% of Triton X-100 (PBST) for 30 min at room temperature. To reduce the non-specific background, the slices were stained by blocking solution, which contained 2% of bovine serum albumin (BSA) and 10% of normal goat serum in PBST, and blocked for 2 hr at room temperature. The primary and secondary antibodies were diluted in blocking solution. Rabbit anti-c-fos antibody was used as the primary antibody and stained for 16 hr at 4°C. The secondary antibodies were incubated for 2 hr at room temperature, followed by rinsing in PBST for 1 hr. Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H + L) (the green fluorescence) was used for rabbit anti-c-fos polyclonal antibody. The subsequent procedures consisted of rinsing in PBS for 15 min, then mounting on slides with DPX Mountant for histology. If the c-fos expression was over the GABAergic neurons, the merging colour would be expressed as yellow. The immunoreactive (IR) activity in each section was then examined under a confocal microscope (Leica TCS SP5 II).

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2.6 Acquisition and analysis of heart rate variability (HRV)[2]
We implanted electrocardiogram (ECG) electrodes into the abdomen with a TR telemeter to acquire ECG signals. The ECG signals were transmitted wirelessly to the SmartPad, acquired with a IX-214 Data Recorder and analysed by LabScribe 3.0. The analysis of power spectra was computed by fast Fourier transform. There were two major components in the power spectra: the low frequency (LF, 0.04 ~ 1.0 Hz) and high frequency (HF, 1.0 ~ 3.0 Hz) in rats. The powers of LF and HF oscillations were calculated within each hour. The power was normally higher than that of HF in rats. Blockade of parasympathetic activity significantly reduces both HF and LF powers, whereas blockade of sympathetic tone only reduces LF power, but not HF power. The LF/HF ratio has been accepted as an index to assess the cardiovascular autonomic regulation between sympathetic and parasympathetic functions (Kuwahara et al., 1994).

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Washed human platelets were prepared from autologous platelet-poor plasma according to a previously described method (Mustard et al., 1972). Platelets were counted and pre-warmed in the presence of a 0.5% DMSO solution of N6-(4-Hydroxybenzyl)adenosine at various concentrations. Collagen (2 μg/mL) was then added to induce aggregation. The aggregation response was recorded after 3 minutes using a light transmission aggregometer. The IC50 value (concentration causing 50% inhibition of platelet aggregation) was calculated as 6.77 ± 0.31 μM (mean ± SD, n=3). [1]

Sprague Dawley rats were divided into four groups. Rats in group 1 (n = 6) had ICV administration of T1-11 20 min prior to the dark period and the somnogenic effects of three doses (1, 10 and 100 μg) of T1-11 on spontaneous sleep were determined. In order to quickly screen whether a substance possesses a somnogenic effect, administration of a substance prior to the dark period and measuring the increase of sleep is the most convenient way. It would not be easy to observe an increase in sleep when administering a somnogenic substance prior to the light (the rest) period when sleep quantity is at the maximal level. Vehicle and three doses of T1-11 were randomly given at day 1, day 4, day 7 and day 10, with a 2-day interval, and the sleep–wake activity was recorded for 24 hr. Rats in groups 2 (n = 6) and 3 (n = 6) were, respectively, used to investigate the ICV effects of the selective A1R antagonist DPCPX and A2AR antagonist SCH58261 on T1-11-induced somnogenic effects. The injection protocol was the same as in group 1, except that double injections of vehicle + vehicle, vehicle + T1-11, DPCPX + T1-11 and SCH58261 + T1-11 were employed. The order of the substance administration was randomized in each group and the data obtained from the control of vehicle + vehicle were combined from two groups. Rats in group 4 (n = 6) received the same protocol as those in group 1 and the HRV was acquired for 24 hr. C57BL/6 mice were divided into five groups (n = 6 for each group). Oral gavage of vehicle and five doses (1, 2.5, 5, 10 and 20 mg/kg) of T1-11 were randomly given before the dark period, with a 2-day administration interval, and the sleep–wake activity was recorded, and lasted for 24 hr in mice of groups 1 and 2. Each group of mice randomly received three different doses. Mice in group 3 were orally administered 10 mg/kg T1-11 prior to the dark period, and PFS, vehicle or A2AR antagonist SCH58261 was microinjected into the VLPO in the middle of the dark period. Mice in group 4 received oral gavage of PFS prior to the dark period and had cage bedding changed at the beginning of the light period to cause stress-induced insomnia. Mice in group 5 received the same experimental protocol as those in group 4, except that oral gavage of 10 mg/kg T1-11 was given. Gad2-Cre::Ai14 transgenic mice (n = 6) were used to determine the neuronal activation in the VLPO after administration of T1-11 by evaluating the immunoreactivity of c-fos. The brains were dissected at 20 hr after oral administration of T1-11.[2]

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[1]. Naturally occurring N(6)-substituted adenosines (cytokinin ribosides) are in vitro inhibitors of platelet aggregation: an in silico evaluation of their interaction with the P2Y(12) receptor. Bioorg Med Chem Lett. 2014 Dec 15;24(24):5652-.

According to reports, N6-(4-hydroxybenzyl)-adenosine has been found in Gastrodia elata, and relevant data is available.

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N6-(4-Hydroxybenzyl)adenosine (para-topolin riboside) is a naturally occurring aromatic cytokinin riboside. Among the tested cytokinin ribosides (isopentenyl adenosine, kinetin riboside, N6-benzyl adenosine, and para-topolin riboside), it showed the highest in vitro anti-platelet aggregation activity. The docking study suggests that its activity may be mediated by interaction with the P2Y12 receptor, which is a critical regulator of hemostasis and thrombosis and an attractive target for anti-aggregation drug discovery. The phenolic hydroxyl group of this compound forms additional hydrogen bonds with Ser156 and Asn159 in the P2Y12 binding site, which may explain its superior activity compared to other N6-substituted analogues. The authors conclude that the anti-aggregation effect of cytokinin ribosides could involve blockade of P2Y12-related signaling in platelets, although further investigations are required. [1]

C17H19N5O5

373.36326

373.139

110505-75-4

10474479

Typically exists as light yellow to yellow solids at room temperature

0.8

5

9

5

27

494

4

OC[C@@H]1[C@H]([C@H]([C@H](N2C=NC3=C2N=CN=C3NCC4=CC=C(O)C=C4)O1)O)O

UGVIXKXYLBAZND-LSCFUAHRSA-N

InChI=1S/C17H19N5O5/c23-6-11-13(25)14(26)17(27-11)22-8-21-12-15(19-7-20-16(12)22)18-5-9-1-3-10(24)4-2-9/h1-4,7-8,11,13-14,17,23-26H,5-6H2,(H,18,19,20)/t11-,13-,14-,17-/m1/s1

(2R,3S,4R,5R)-2-(hydroxymethyl)-5-[6-[(4-hydroxyphenyl)methylamino]purin-9-yl]oxolane-3,4-diol

N6-(4-Hydroxybenzyl)-adenosine; 110505-75-4; n6-(4-hydroxybenzyl)adenosine; N6-(4-hydroxybenzyl)adenine riboside; (2R,3S,4R,5R)-2-(hydroxymethyl)-5-[6-[(4-hydroxyphenyl)methylamino]purin-9-yl]oxolane-3,4-diol; CHEMBL224024; N-[(4-hydroxyphenyl)methyl]adenosine; N6-(4-Hydroxybenzyl)adenosine (NHBA);

2934999090

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 : ≥ 100 mg/mL (~267.84 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.70 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 (6.70 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 (6.70 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.)