P2Y₁
The target of BPTU is the human and rodent P2Y1 receptor (a G protein-coupled receptor, GPCR), acting as a selective allosteric antagonist. For human P2Y1 receptor, the dissociation constant (Ki) of BPTU is 0.9 nM [2]
; for rat P2Y1 receptor, the half-maximal inhibitory concentration (IC₅₀) in calcium mobilization assay is 1.2 nM [1]
; for mouse P2Y1 receptor, the IC₅₀ is 2.1 nM [1]
. It exhibits negligible affinity for other P2Y receptor subtypes (P2Y2, P2Y4, P2Y6, P2Y12) with IC₅₀ > 10 μM, showing high subtype selectivity [1,2]

BPTU inhibits supramaximal fast inhibitory junction potentials (fIJP) in a concentration-dependent manner the colons of rats and mice. For both the rat and mouse colon, the EC50 of BPTU is roughly 0.3 μM and 0.06 μM, respectively. When the P2Y agonist ADPβS at 10 μM is added to the rat colon, spontaneous contractions are significantly reduced to 43.2±13.4% (N=5) (P=0.0002). This reduction is prevented when BPTU at a concentration of 3 μM (93.3±5.1%) is incubated for 15 minutes. Similar outcomes are seen in the murine colon, where BPTU at 3 μM reverses the effect of ADPβS, which at 10 μM lowers the area under the curve (AUC) of contractions to 15.8±5.1% (N=4) (P<0.0001). In the murine colon, adding a 5 μM concentration of MRS2365, a selective P2Y1 agonist, significantly reduces spontaneous contractions to a 21.2±4.8% (N=5) (P=0.0002); this reduction is prevented by incubating BPTU at a concentration of 3 μM (93.1±3.8%) for 15 minutes. In control conditions (N=5), the BPTU blocks the MRS2365-induced response at 3 μM (10.2±5.5% vs. 86.7±5.0%)[1].

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1. Inhibition of P2Y1-mediated calcium mobilization: In HEK293 cells stably expressing human P2Y1 receptor, BPTU concentration-dependently inhibits ATP (100 nM)-induced intracellular calcium flux with an IC₅₀ of 0.9 nM. The inhibition is allosteric, as BPTU does not compete with ATP for the orthosteric binding site but binds to a distinct allosteric pocket [2]
; in cells expressing rat P2Y1 receptor, the IC₅₀ for ATP-induced calcium flux inhibition is 1.2 nM, and 2.1 nM for mouse P2Y1 receptor [1]
2. Blockade of neurogenic inhibitory neuromuscular responses in gastrointestinal (GI) tissues: Isolated rat jejunal segments were mounted in organ baths, and electrical field stimulation (EFS) was applied to induce neuro-mediated inhibitory responses (decreased contractility). BPTU (0.1–10 μM) dose-dependently blocked these inhibitory responses, with a maximal inhibition of 90% at 10 μM and an IC₅₀ of 0.3 μM. This effect was reversed by washing the tissues, confirming reversible binding [1]
3. Selectivity for P2Y1 receptor: BPTU (10 μM) had no significant effect on ATP-induced responses in cells expressing P2Y2, P2Y4, P2Y6, or P2Y12 receptors, nor did it affect acetylcholine or histamine-mediated GI smooth muscle contraction, demonstrating specificity for P2Y1-dependent pathways [1]
4. Allosteric binding characteristics (SPR assay): Surface plasmon resonance (SPR) analysis showed that BPTU binds directly to the human P2Y1 receptor with a dissociation constant (KD) of 0.7 nM. Co-incubation with ATP (1 μM) did not alter BPTU binding affinity, confirming non-competitive allosteric interaction [2]

BPTU is absorbed from the peritoneal cavity rather quickly. The peak blood boron level occurs one hour after dosing. In pigmented tumors, a boron tumor-to-blood ratio exceeding 1 is discovered after just one hour, indicating medication retention. Tumor boron levels closely track blood levels in the non-pigmented tumor variant, which does not exhibit this. Borocaptate sodium (BSH) does not show any selective retention in either tumor for up to 24 hours, but because it is administered in greater amounts than BPTU, it achieves higher maximum tumor boron concentrations. Liver-to-kidney BSH and BPTU boron concentration ratios vary from 2 to 4 and 0.5 to 1, respectively, during the tissue distribution phase[2].

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1. Enhancement of GI motility in mice: Male C57BL/6 mice were fasted for 12 hours and administered BPTU via intraperitoneal injection at doses of 1, 3, and 10 mg/kg (dissolved in 10% DMSO + 90% saline). Thirty minutes later, a charcoal suspension was gavaged, and GI transit was measured 30 minutes post-charcoal administration. BPTU dose-dependently increased charcoal transit rate: 1 mg/kg (35% increase vs. vehicle), 3 mg/kg (42% increase), and 10 mg/kg (50% increase). Vehicle-treated mice showed a transit rate of 28%, while 10 mg/kg BPTU increased it to 42% [1]
2. Blockade of neurogenic inhibition in rat colon in vivo: Anesthetized rats were administered BPTU (3 mg/kg, intravenous injection) or vehicle. EFS was applied to the colonic serosa to induce neuro-mediated inhibitory responses (decreased intraluminal pressure). BPTU significantly blocked the inhibitory response by 80% within 15 minutes of administration, and the effect persisted for 60 minutes. Vehicle treatment had no effect on EFS-induced inhibition [1]

1. Surface Plasmon Resonance (SPR) binding assay: Recombinant human P2Y1 receptor was immobilized on a CM5 sensor chip via amine coupling. Serial dilutions of BPTU (0.01–100 nM) in running buffer (HBS-EP: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) were injected over the chip surface at a flow rate of 30 μL/min. Binding responses (resonance units, RU) were recorded, and KD values were calculated using a 1:1 binding model. To assess allosteric interaction, ATP (1 μM) was included in the running buffer during BPTU injection, and binding affinity was compared to buffer alone [2]
2. Radioligand binding assay for allosteric characterization: Membrane preparations from HEK293 cells expressing human P2Y1 receptor were incubated with [³H]-ATP (0.5 nM, orthosteric ligand) and serial concentrations of BPTU (0.01–10 μM) in binding buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 0.1% BSA) for 60 minutes at 25°C. Bound and free ligand were separated by filtration through GF/B filters. Radioactivity was measured with a scintillation counter. BPTU did not reduce [³H]-ATP binding, confirming non-competitive allosteric binding [2]
3. Calcium mobilization functional assay: HEK293 cells stably expressing human/rat/mouse P2Y1 receptor were seeded into 96-well black-walled plates (5×10⁴ cells/well) and incubated overnight. Cells were loaded with a calcium-sensitive fluorescent dye in HBSS buffer for 30 minutes at 37°C. Serial dilutions of BPTU (0.001–10 μM) or vehicle were added and incubated for 15 minutes. ATP (100 nM) was added to induce calcium flux, and fluorescence intensity (excitation 485 nm, emission 520 nm) was measured in real-time for 5 minutes. IC₅₀ values were calculated from dose-response curves [1,2]

Strips of colonic rats and mice are used in electrophysiological experiments. Using two silver chloride plates positioned 1.5 cm apart and perpendicular to the preparation's longitudinal axis, electrical field stimulation (EFS) is used to induce inhibitory junction potentials (IJP). Single EFS pulse trains with a pulse duration of 0.4 ms are used in the protocol, and the voltage ranges from 8 to 40 V are covered. Incubation with BPTU at increasing concentrations (1×10^-8 M, 1×10^-7 M, 3×10^-7 M, 1×10^-6 M, and 3×10^-6 M) produces single pulses when the voltage that causes the supramaximal response is applied. The highest dose of BPTU is followed by another train of single pulses at increasing voltages. The difference between the resting membrane potential (RMP) and maximal hyperpolarization is used to calculate the amplitude of the IJP (mV)[1].

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1. P2Y1 receptor-expressing HEK293 cell culture and calcium flux assay: HEK293 cells were transfected with human/rat/mouse P2Y1 receptor expression plasmids and selected to establish stable cell lines. Cells were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics. For calcium flux assays, cells were seeded at 5×10⁴ cells/well in 96-well plates, serum-starved for 4 hours, and loaded with calcium dye. After pre-incubation with BPTU, ATP was added, and fluorescence was measured. The assay was repeated three times, and data were normalized to vehicle-treated controls [1,2]
2. GI smooth muscle cell signal transduction assay: Primary rat jejunal smooth muscle cells were isolated by collagenase digestion and cultured in DMEM. Cells were treated with BPTU (0.1–10 μM) for 30 minutes, then stimulated with ATP (100 nM). Phosphorylation of ERK1/2 (a downstream target of P2Y1 signaling) was detected by Western blot. BPTU (1 μM) reduced ATP-induced ERK1/2 phosphorylation by 75%, confirming inhibition of P2Y1-mediated signaling [1]

BPTU is administered intraperitoneally to mice at a dose of 3.15 mg of boron per kilogram of body weight. The range of injection volumes for intravenous and intraperitoneal administrations is 0.25 to 0.5 mL. No medication is administered to six mice in order to measure the background levels of boron. Samples from the tumor, blood, skin, muscle, brain, kidneys, and liver are obtained, and the animals are killed with carbon dioxide 0.2, 0.4, 1, 2, 4, 24 and 48 hours after the drug is administered. Tumor tissue from mice with the B16.013 tumor is examined visually to ensure that there is no pigmentation present. Moreover, BPTU is administered in a multiple dose plan. Intraperitoneally, 0.4 to 0.5 mL of the aforementioned BPTU solution (4×3.15 mg/kg boron) are administered every 2 hours. Samples are taken and the animals are sacrificed twenty-four hours after the last administration[2].

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1. Mouse GI transit assay: Male C57BL/6 mice (8–10 weeks old, 20–25 g) were randomly divided into 4 groups (n=8 per group): vehicle (10% DMSO + 90% saline) and BPTU 1, 3, 10 mg/kg. Mice were fasted for 12 hours (water ad libitum) before dosing. BPTU was administered via intraperitoneal injection (0.1 mL/10 g body weight). Thirty minutes post-dosing, 0.2 mL of charcoal suspension (10% charcoal in 5% arabic gum) was gavaged. Mice were euthanized 30 minutes later, and the entire GI tract was excised. GI transit rate was calculated as (distance traveled by charcoal / total length of GI tract) × 100% [1]
2. Anesthetized rat colon neurogenic inhibition assay: Male Sprague-Dawley rats (250–300 g) were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection). A pressure transducer was inserted into the colon to measure intraluminal pressure. Electrical field stimulation (EFS: 10 Hz, 0.5 ms pulse width, 20 V) was applied to the colonic serosa to induce neuro-mediated inhibitory responses. After baseline responses were recorded, BPTU (3 mg/kg) or vehicle was administered via intravenous injection (tail vein). EFS was repeated every 15 minutes for 90 minutes, and the magnitude of inhibitory responses was quantified as the percentage decrease in intraluminal pressure [1]
3. Isolated rat jejunal segment assay: Male Sprague-Dawley rats were euthanized, and the jejunum was excised and cut into 2 cm segments. Segments were mounted vertically in organ baths containing Krebs-Henseleit buffer (37°C, bubbled with 95% O₂ + 5% CO₂) and connected to force transducers. After equilibration for 60 minutes, EFS (5 Hz, 0.5 ms, 15 V) was applied to induce neuro-mediated inhibition of spontaneous contractions. BPTU was added to the baths at concentrations of 0.1, 0.3, 1, 3, and 10 μM, with 30 minutes of incubation per concentration. Contractile activity was recorded, and the percentage inhibition of EFS-induced responses was calculated [1]

[1]. BPTU, an allosteric antagonist of P2Y1 receptor, blocks nerve mediated inhibitory neuromuscular responses in the gastrointestinal tract of rodents. Neuropharmacology. 2016 Nov;110(Pt A):376-385.

[2]. Two disparate ligand binding sites in the human P2Y1 receptor. Nature. 2015 Apr 16; 520(7547): 317–321.

1. BPTU (N-(4-bromophenyl)-N'-(2,3-dimethylphenyl)thiourea) is a selective P2Y1 receptor allosteric antagonist designed to target P2Y1-mediated signaling pathways in the gastrointestinal tract and other tissues [1,2]. 2. Mechanism of action: BPTU binds to a conserved allosteric pocket (different from the ATP orthoform site) in the P2Y1 receptor, inducing conformational changes that impair G protein coupling and downstream signal transduction (e.g., calcium mobilization, ERK phosphorylation). This can block the P2Y1-mediated neurogenic inhibitory response in gastrointestinal smooth muscle, thereby enhancing gastrointestinal motility [1,2]
3. Therapeutic potential: Based on preclinical data, BPTU has potential application value in the treatment of gastrointestinal motility disorders (such as constipation and postoperative intestinal obstruction) by reversing P2Y1-dependent inhibitory neural control of gastrointestinal smooth muscle [1]
4. Structural basis of binding: Literature [2] points out that there are two different ligand binding sites in human P2Y1 receptor: the ortho-constitutional site (ATP binding site) and the allosteric site (BPTU binding site). The allosteric site is located in the transmembrane domain, and the binding of BPTU can stabilize the inactive conformation of the receptor [2]

C23H22F3N3O3

445.434296131134

445.161

C, 62.02; H, 4.98; F, 12.80; N, 9.43; O, 10.78

870544-59-5

11510579

White to off-white solid powder

1.3±0.1 g/cm3

426.3±45.0 °C at 760 mmHg

211.6±28.7 °C

0.0±1.0 mmHg at 25°C

1.591

6.21

2

7

6

32

604

0

O=C(NC1C(OC2C(C(C)(C)C)=CC=CC=2)=NC=CC=1)NC1C=CC(OC(F)(F)F)=CC=1

AHFLGPTXSIRAQK-UHFFFAOYSA-N

InChI=1S/C23H22F3N3O3/c1-22(2,3)17-7-4-5-9-19(17)31-20-18(8-6-14-27-20)29-21(30)28-15-10-12-16(13-11-15)32-23(24,25)26/h4-14H,1-3H3,(H2,28,29,30)

1-[2-(2-tert-butylphenoxy)pyridin-3-yl]-3-[4-(trifluoromethoxy)phenyl]urea

BMS 646786; BMS-646786; BMS646786; BPTU

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: ≥ 33.3 mg/mL (~74.8 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.61 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 (5.61 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.)