P2Y12 receptor （Ki = 24.9 nM）

PSB-0739 is a strong competitive non-nucleotide antagonist that has a pA2 value of 9.8[2] against the human P2Y12 receptor. In THP-1 cells, PSB-0739 induces a parallel shift to the right in the ADP concentration-response curve and suppresses the ADP-induced Ca2+ response with an EC50 of 5.4±1.8 μM [3].

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Anthraquinone derivatives related to the moderately potent, nonselective P2Y(12) receptor antagonist reactive blue 2 (6) have been synthesized and optimized with respect to P2Y(12) receptor affinity. A radioligand binding assay utilizing human blood platelet membranes and the P2Y(12) receptor-selective antagonist radioligand [(3)H]2-propylthioadenosine-5'-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([(3)H]PSB-0413) was applied for compound testing. 1-Amino-2-sulfoanthraquinone derivatives bearing a (p-phenylamino)anilino substitution in the 4-position and an additional acidic function in the meta-position of the aniline ring showed high P2Y(12) receptor affinity. These new anthraquinone derivatives became accessible by a recently developed copper(0)-catalyzed Ullmann coupling reaction of 1-amino-4-bromoanthraquinone derivatives with anilines in phosphate buffer under microwave irradiation. The most potent compounds exhibited K(i) values of 24.9 nM (1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, PSB-0739, 39), and 21.0 nM (1-amino-4-[4-phenylamino-3-carboxyphenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, PSB-0702, 41), respectively. 1-Amino-2-sulfo-4-anilinoanthraquinone derivatives appeared to be noncytotoxic, as shown for selected derivatives at two human cell lines (melanoma and astrocytoma). Compounds 39 and 41 represent new lead structures for the development of antithrombotic drugs. [1]

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The P2Y(12) receptor plays a crucial role in platelet aggregation. In the present study, we analyzed the properties of non-nucleotide antagonists at the recombinant human P2Y(12) receptor and searched for amino acids involved in the molecular interaction. Receptor function was assessed by measuring the cAMP response element (CRE)-directed luciferase expression in Chinese hamster ovary cells. The cellular cAMP production was accelerated by forskolin; 2-methylthio-ADP was used to activate the wild-type P2Y(12) receptor or mutant constructs. 2-Methylthio-ADP inhibited the CRE-dependent luciferase expression with an IC(50) value of approximately 1 nM. The anthraquinone derivative reactive blue 2 used at increasing concentrations shifted the concentration-response curve of 2-methylthio-ADP to the right in a manner compatible with competitive antagonism (pA(2) value, 7.4). Its analog, 1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-0739), showed a markedly higher antagonistic potency with a pA(2) value of 9.8. In cells expressing the R256A-mutant receptor, the potencies of both reactive blue 2 (apparent pK(B), 5.9) and PSB-0739 (apparent pK(B), 9.1) were decreased. The same was true for the pure reactive blue 2 meta- and para-isomers and for the ortho-isomer cibacron blue 3GA. In contrast, the analog, 1-amino-4-[4-anilino-phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, lacking a sulfonic acid residue at ring D (PSB-0826), showed similar pK(B) values at wild-type (8.4) and R256A-mutant receptors (8.3). In summary, the results demonstrate that PSB-0739 is the most potent competitive non-nucleotide antagonist at the human P2Y(12) receptor described so far. The results also indicate that the sulfonic acid residue at ring D is involved in the interaction of antagonists derived from reactive blue 2 with the residue Arg256 of the human P2Y(12) receptor.[2]

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ADP-evoked intracellular Ca2+ responses (EC50 2.7 μM) in THP-1 cells were abolished by inhibition of PLC (U73122) or sarco/endoplasmic reticulum Ca2+ -ATPase (thapsigargin). Loss of ADP-evoked Ca2+ responses following treatment with MRS2578 (IC50 200 nM) revealed a major role for P2Y6 receptors in mediating ADP-evoked Ca2+ responses. ADP-evoked responses were attenuated either with pertussis toxin treatment, or P2Y12 receptor inhibition with two chemically distinct antagonists (ticagrelor, IC50 5.3 μM; PSB-0739, IC50 5.6 μM). ADP-evoked responses were suppressed following siRNA-mediated P2Y12 gene knockdown. The inhibitory effects of P2Y12 antagonists were fully reversed following adenylate cyclase inhibition (SQ22536). P2Y12 receptor expression was confirmed in freshly isolated human CD14+ monocytes. Conclusions and implications: Taken together, these data suggest that P2Y12 receptor activation positively regulates P2Y6 receptor-mediated intracellular Ca2+ signalling through suppression of adenylate cyclase activity in human monocytic cells.[3]

Significant and dose-dependent antihyperalgesic effects are observed at low dosages of PSB-0739 (0.01-0.3 mg/kg, intrathecally). [4] states that the minimum effective dose (mED) is 0.1 mg/kg.

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In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1β protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1β in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1β. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways [4].

P2Y12 Radioligand Binding Assay [1]
The precursor of [3H]PSB-0413 was synthesized as described and custom-labeled, with a specific activity of 74 Ci/mmol, or 94 Ci/mmol, respectively. Radioligand binding assays were performed using 5 nM [3H]PSB-0413 and 100 μg of protein in Tris-HCl buffer 50 mM, pH 7.4, as previously described in a final volume of 400 μL. Competition by 10 μM of test compound was initially determined. The mixture was incubated for 1 h at rt, followed by filtration through GF/B filters. Nonspecific binding was determined with 1 mM ADP. For potent compounds, concentration−inhibition curves were determined using at least 6−7 different concentrations spanning 3 orders of magnitude. At least three independent experiments were performed each in triplicate.

Cell viability assay [3]
Cell Types: THP-1 monocyte cell line
Tested Concentrations: 10 nM, 100 nM, 1 μM, 10 μM
Incubation Duration:
Experimental Results: ADP-induced response was weakened (IC50=5.4±1.8 μM).

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MTT Assays [1]
Cells were detached from the 175 cm2 culture flask and counted using a Neubauer hemocytometer. Then they were resuspended in the growth medium to give a total of 2 × 105 cells in 10 mL of the medium. Cell suspension (100 μL) was added into each well of a 96-well plate to obtain a final concentration of 2000 cells per well and incubated for 24 h at 37 °C, 5% CO2, and 95% humidity. The outer wells of the 96-well plate were filled with 200 μL of medium without cells to prevent evaporation. After 24 h, the medium was removed and 180 μL of fresh growth medium per well was added. Stock solutions (10 mM) of test compounds (5-fluorouracil, 12, and 16) were prepared in DMSO and diluted with medium to give 10× of final concentrations. Then test compound solution (20 μL) was added to each well. The final DMSO concentration was 1%. Compounds 12 and 16 were tested in concentrations of 0.1, 1, and 100 μM. For 5-fluorouracil, full dose−inhibition curves were determined. The cells were incubated in the presence of the appropriate drug for 72−144 h. Then 40 μL from a freshly made stock solution of MTT in phosphate-buffered saline (5 mg/mL) was added to each well and the cells were incubated for 1 h. After the incubation time, the medium containing MTT was removed and 100 μL of DMSO was added to each well in order to dissolve crystals that were formed. The spectrophotometric absorbance was subsequently measured at 570 nm using a UV-Multiwellreader Multiscan spectrophotometer with a reference filter of 690 nm. The data were analyzed using Microsoft Excel and Graphpad Prism 4. Results were evaluated by comparing the absorbance of the wells containing compound-treated cells with the absorbance of wells containing 1% DMSO without any drug (= 100% viability). All experiments were performed in triplicates in at least 3−8 separate experiments.

Animal/Disease Models: Male Wistar rats, 150-250 g, 6-8 rats/group [4]
Doses: 0.01, 0.03, 0.1, 0.3 mg/kg
Route of Administration: Intrathecal injection (it)
Experimental Results: Performance of mechanical effects A dose-dependent inhibition of hyperalgesia occurred in the range of 0.01-0.1 mg/kg.

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Animals were subjected to intraperitoneal or intrathecal injections of several doses of the following P2Y12R antagonists: MRS2395 (0.03–1 mg/kg), clopidogrel (1–60 mg/kg), ticlopidine (3–100 mg/kg), cangrelor (0.1–3 mg/kg), PSB-0739 (0.01–1 mg/kg) or, reactive blue 2 (0.1–3 mg/kg). Each animal was injected only once. The doses of drugs were chosen based on extrapolation from previous studies (Marteau et al., 2003, Takasaki et al., 2001, Vasiljev et al., 2003). Intrathecal injection was performed following the method of Mestre et al. (1994): this method enables the injection of a drug directly to the central nervous system without anesthesia and to avoid harm to the spinal cord. Briefly, the injections were performed by holding the animal in one hand and inserting a 23 G1″ needle connected to a 250 μL Hamilton syringe with a repeating dispenser between the dorsal aspects of L5 and L6 vertebrae. A volume of 5 μL of PSB-0739 solution or saline was injected in every case. [4]

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In order to measure the effect of subdiaphragmatic vagotomy on pain behavior PWT was measured before operation as baseline and immediately prior to intraplantar CFA injection on the tenth day after operation. Freshly prepared Complete Freund Adjuvant (CFA, 100 μl, 50%) was then injected to the right hind paw of the animals intradermally and PWT was measured two days after treatment. Animals then received the selective P2Y12R antagonist PSB-0739 (0.3 mg/kg) or saline intrathecally and PWT was measured again. [4]

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The selective P2Y12R antagonist PSB-0739 (0.3 mg/kg) was injected i.t. 15 min before, while the non-pro-drug-P2Y12 receptor antagonist cangrelor (3 mg/kg) was added i.p. 30 min before the post-CFA measurement of mechanical hyperalgesia. After behavioral testing at 48 h or 96 h, rats were killed by decapitation. These time points were chosen to represent acute (48 h) and subacute (96 h) phases of inflammation (Parra et al., 2002). [4]

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Accelerating rotarod test[4]
This study used 140–190 g drug- and test-naive male Wistar rats. Motor coordination was tested on the IITC Rotarod Apparatus, which enables the simultaneous examination of five rats. The apparatus consists of five compartments separated by 8 cm diameter rotating rod, placed 25 cm above the base of the apparatus. Motor coordination of animals was tested for 300 s with linear acceleration from 5 rpm to 25 rpm. Rats were acclimatized to the rotarod in three trials (300 s) per day for 2 consecutive days before the start of the experiment. On the test day 30 min prior to drug administration baseline latencies to fall were determined and rats with baseline latencies less than 60 s were excluded from the study. The animals were then treated with sterile saline or with antagonists intraperitoneally (3 mg/kg cangrelor/saline) or intrathecally (0.3 mg/kg PSB-0739/saline). 30 min after the i.p. treatment or 15 min after i.t. treatment the falling latency was measured again in the 300 s test period. The latency time to fall off the rod was expressed in seconds.

[1]. High-affinity, non-nucleotide-derived competitive antagonists of platelet P2Y12 receptors. J Med Chem. 2009 Jun 25;52(12):3784-93.

[2]. Interaction of new, very potent non-nucleotide antagonists with Arg256 of the human platelet P2Y12 receptor. J Pharmacol Exp Ther. 2009 Nov;331(2):648-55.

[3]. P2Y 12 receptor modulation of ADP-evoked intracellular Ca2+ signalling in THP-1 human monocytic cells. Br J Pharmacol.2018 Jun;175(12):2483-2491.

[4]. Central P2Y12 receptor blockade alleviates inflammatory and neuropathic pain and cytokine production in rodents. Neurobiol Dis. 2014 Oct;70(100):162-78.

In conclusion, we have developed a new class of highly potent, competitive non-nucleotide derived P2Y12 antagonists. The most potent compounds exhibited Ki values for the human platelet P2Y12 receptor in the lower nanomolar range and selectivity versus other P2 receptor subtypes. The new compounds will be useful as pharmacological tools for studying the role of peripheral as well as brain P2Y12 receptors. Several members of this class of compounds have been identified, e.g., 39/PSB-0739 and 41, which constitute novel lead structures for the development of antithrombotic drugs. [1]

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A major limitation of the current therapy used in inflammatory and neuropathic pain is not only the lack of efficacy but also the occurrence of untoward side-effects in the therapeutic dose-range (Negus et al., 2006). Importantly, using doses higher than the mED of the respective compounds, we did not detect any acute effect on motor coordination by the most potent P2Y12R antagonists used in the study. On the other hand, i.p. application of an analgesic dose of cangrelor, but not i.t. application of PSB-0739 had a significant inhibitory effect on ex vivo platelet aggregation, which is unsurprising considering the well-known inhibitory effects of P2Y12R antagonists on this process (Schumacher et al., 2007). This effect, however, could also be equally considered as a protective effect on cardiovascular risk rather than a side effect, given that the P2Y12R occupancy in the blood shows close correlation with the formation of arterial thrombi. The finding that PSB-0739 did not inhibit but augmented platelet aggregation indicates that the analgesic effect of P2Y12R antagonists could be enhanced by the adequate penetration into the CNS without increasing the risk of bleeding. In conclusion, our findings provide a rationale to target central P2Y12Rs as a potential therapeutic approach in inflammatory and neuropathic pain. [4]

C26H17N3NA2O8S2

609.5345

609.025

C, 51.23; H, 2.81; N, 6.89; Na, 7.54; O, 21.00; S, 10.52

1052087-90-7

44583582

Green to dark green solid powder

6.228

3

11

4

41

1120

0

[Na+].[Na+].C1=CC=C(NC2=CC=C(NC3=CC(S([O-])(=O)=O)=C(N)C4C(C5=CC=CC=C5C(=O)C3=4)=O)C=C2S([O-])(=O)=O)C=C1

QBLLYXXXOJUNCV-UHFFFAOYSA-L

InChI=1S/C26H19N3O8S2.2Na/c27-24-21(39(35,36)37)13-19(22-23(24)26(31)17-9-5-4-8-16(17)25(22)30)29-15-10-11-18(20(12-15)38(32,33)34)28-14-6-2-1-3-7-14;;/h1-13,28-29H,27H2,(H,32,33,34)(H,35,36,37);;/q;2*+1/p-2

disodium;1-amino-4-(4-anilino-3-sulfonatoanilino)-9,10-dioxoanthracene-2-sulfonate

PSB0739 sodium; PSB-0739; PSB 0739; 1052087-90-7; PSB-0739 Sodium; 1-Amino-9,10-dihydro-9,10-dioxo-4-[[4-(phenylamino)-3-sulfophenyl]amino]-2-anthracenesulfonic acid sodium salt; CHEMBL455536; disodium;1-amino-4-(4-anilino-3-sulfonatoanilino)-9,10-dioxoanthracene-2-sulfonate; sodium 1-amino-9,10-dioxo-4-((4-(phenylamino)-3-((sodiooxy)sulfonyl)phenyl)amino)-9,10-dihydroanthracene-2-sulfonate; PSB 0739

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~3.33 mg/mL (~5.46 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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