TXA₂
NTP42 targets the T prostanoid receptor (TP), specifically both the TPα and TPβ isoforms. [2]
• Antagonism of TPβ‑mediated intracellular calcium mobilization stimulated by U46619 (TXA₂ mimetic): IC₅₀ = 8.86 ± 3.07 nM. [2]
• Antagonism of TPβ‑mediated intracellular calcium mobilization stimulated by 8‑iso‑PGF₂α: IC₅₀ = 8.04 ± 3.74 nM. [2]
• Antagonism of U46619‑induced human platelet aggregation: IC₅₀ = 10.6 ± 1.7 nM. [2]
• In HEK.TPα cells, inhibition of U46619‑mediated calcium mobilization: IC₅₀ = 56.2 ± 4.84 nM (patent data). [1]
• In HEK.TPβ cells, inhibition of U46619‑mediated calcium mobilization: IC₅₀ = 8.25 ± 0.73 nM (patent data). [1]
• No agonist or antagonist activity at other prostanoid receptors (DP₁, EP₁, EP₂, EP₃, EP₄, FP, IP) up to 10 μM. [2]
Thromboxane A2 (TXA2) receptor (T prostanoid receptor, TP) - antagonist (IC50 = 3.278 nM).

• NTP42 potently inhibited U46619‑induced intracellular calcium mobilization in HEK cells stably overexpressing the human TPβ isoform (HEK.TPβ cells), with an IC₅₀ of 8.86 ± 3.07 nM. It also inhibited 8‑iso‑PGF₂α‑induced calcium mobilization in the same cell line with an IC₅₀ of 8.04 ± 3.74 nM. [2]
• NTP42 antagonized U46619‑induced aggregation of human platelets in ex vivo assays using platelet‑rich plasma (PRP), showing an IC₅₀ of 10.6 ± 1.7 nM. [2]
• In selectivity assays, NTP42 (up to 10 μM) exhibited no agonist or antagonist effects on signaling mediated by the prostacyclin receptor (IP), prostaglandin D₂ receptor (DP₁), prostaglandin E₂ receptors (EP₁, EP₂, EP₃, EP₄), or prostaglandin F₂α receptor (FP) in respective cell lines. [2]
• NTP42 did not inhibit TXA₂ synthase (TXAS), distinguishing it from dual TXAS/TP antagonists. [2]
In vitro studies demonstrate that NTP42 is a potent thromboxane A2 (TXA2) receptor antagonist that selectively blocks T prostanoid (TP) receptor-mediated signaling. The compound exhibits an IC50 of 3.278 nM for antagonizing TP receptor-mediated [Ca2+] mobilization following stimulation with the alternative TP agonist U46619. Thromboxane A2 is a potent vasoconstrictor and platelet aggregator that plays a critical role in the pathophysiology of pulmonary arterial hypertension and other cardiovascular diseases. By selectively blocking the TP receptor, NTP42 prevents TXA2-mediated vasoconstriction and platelet aggregation.

NTP42 (0.25 mg/kg BID) was successful in the hemodynamic evaluation of the PAH trajectory model induced by monocrotaline (MCT) (a 28-day medication regimen initiated within 24 hours of MCT). NTP42 lowers right systolic blood pressure (RSVP) and mean pulmonary artery pressure (mPAP), two measures of PAH brought on by MCT. Moreover, NTP42 was linked to a significant reduction in pulmonary vascular proliferation, activation of inflammatory mast cells, and fibrosis in animals treated with MCT when combined with sildenafil and Selelexipag[2].

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• In the monocrotaline (MCT)-induced PAH rat model, oral administration of NTP42 (0.25 mg/kg BID for 28 days) significantly reduced MCT‑induced increases in mean pulmonary arterial pressure (mPAP) from 28.5 ± 1.0 mmHg (MCT only) to 18.7 ± 0.9 mmHg (P < 0.0001). [2]
• NTP42 also significantly reduced right ventricular systolic pressure (RVSP) from 44.7 ± 2.2 mmHg (MCT only) to 36.6 ± 2.5 mmHg (P = 0.0229). [2]
• No significant change was observed in Fulton’s index (right ventricular hypertrophy) with NTP42 treatment. [2]
• Histological and morphometric analyses showed that NTP42 significantly attenuated MCT‑induced pulmonary vascular remodelling: it improved lumen:total vessel diameter ratio (P < 0.0001), reduced medial thickness (P < 0.0001), and decreased degree of vessel occlusion (P < 0.0001) compared to MCT‑only controls. [2]
• NTP42 significantly reduced MCT‑induced pulmonary mast cell density (P = 0.0002) and pulmonary fibrosis (percentage fibrotic area, P = 0.0180). These effects were superior to those of Sildenafil and Selexipag. [2]
• NTP42 had no deleterious effects on mean systemic arterial pressure (mAP) or heart rate (HR) in the MCT‑PAH model. [2]
In vivo studies have demonstrated that NTP42 reduces monocrotaline (MCT)-induced pulmonary arterial hypertension, including mean pulmonary arterial pressure (mPAP) and right systolic ventricular pressure (RSVP). The compound also reduces inflammatory mast cell infiltration and fibrosis in MCT-treated animals. These findings support the potential of NTP42 for the treatment of pulmonary arterial hypertension, a progressive and life-threatening disease characterized by elevated pulmonary arterial pressure and right heart failure. The compound's ability to block TP receptor-mediated signaling and reduce inflammation and fibrosis makes it a promising candidate for PAH therapy.

• Calcium mobilization assay in HEK.TPβ cells: HEK.TPβ cells were preloaded with Fluo‑4 fluorescent dye. Cells were then incubated with increasing concentrations of NTP42 (0.00001 – 10 μM) before stimulation with either 1 μM U46619 or 10 μM 8‑iso‑PGF₂α. Fluorescence changes (indicating intracellular calcium) were measured, and IC₅₀ values were calculated from dose‑response curves. [2]
• Platelet aggregation assay: Blood from healthy volunteers was collected into sodium citrate (3.8%) and indomethacin (10 μM). Platelet‑rich plasma (PRP) was prepared by centrifugation. PRP aliquots were pre‑incubated with NTP42 (2‑fold serial dilutions from 1 μM) for 10 min at 37°C with stirring, then stimulated with 1 μM U46619. Aggregation was monitored by changes in light transmission using a platelet aggregometer (PAP‑8E), and IC₅₀ was determined. [2]
For receptor binding and functional studies, cell-based assays are performed using cells expressing the T prostanoid receptor (TP). Cells are treated with NTP42 at various concentrations and stimulated with a TP agonist such as U46619. Receptor activation is assessed by measuring intracellular calcium mobilization using fluorescent calcium indicators. The compound's ability to block agonist-induced calcium mobilization is quantified, and IC50 values are calculated. Selectivity over other prostanoid receptors (EP, DP, FP, IP) is assessed using similar assay formats to confirm the compound's specificity for TP.

• HEK.TPα and HEK.TPβ cell calcium mobilization assays: HEK293 cells stably overexpressing human TPα or TPβ isoforms were cultured, preloaded with Fluo‑4, and treated with NTP42 (0.00001 – 10 μM) prior to stimulation with 1 μM U46619. Fluorescence was measured to quantify intracellular calcium. Data were expressed as percentage of control (agonist‑induced response in vehicle‑treated cells). [1][2]
• Platelet aggregation assay: Human venous blood was collected into 3.8% sodium citrate (1:9 ratio) containing 10 μM indomethacin. PRP was obtained by centrifugation (200g for 20 min). PRP (300 μl) was pre‑incubated with NTP42 (serial dilutions from 0.25 μM to 125 nM) for 10 min at 37°C with stirring. Platelet aggregation was induced by 1 μM U46619 and measured by light transmission using a PAP‑8E aggregometer. Percentage aggregation was calculated relative to control. [1][2]
• Selectivity assays: Similar calcium mobilization or impedance assays were performed using cells expressing other prostanoid receptors (DP₁, EP₁, EP₂, EP₃, EP₄, FP, IP) with their respective agonists (e.g., cicaprost for IP, PGE₂ for EP receptors). NTP42 was tested at 10 μM for agonist or antagonist activity. [2]
Cellular assays for NTP42 typically involve culturing cells that express the TP receptor, such as vascular smooth muscle cells, platelets, or recombinant cell lines. Cells are treated with the compound at various concentrations and stimulated with a TP agonist. Receptor activation is assessed by measuring calcium mobilization, platelet aggregation, or smooth muscle contraction. The compound's ability to block agonist-induced responses is quantified. Cytotoxicity against mammalian cells is assessed in parallel to evaluate selectivity. The compound's effects on cell proliferation and migration are studied in the context of vascular remodeling in PAH.

Male Wistar-Kyoto rats
0.25 mg/kg BID
28-day drug treatment was initiated within 24 h post-MCT (60 mg/kg)

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• MCT‑induced PAH rat model: Male Wistar‑Kyoto rats (8‑9 weeks old, 200‑250 g) received a single subcutaneous injection of monocrotaline (MCT; 60 mg/kg in DMSO) on Day 0. Control animals received DMSO vehicle. Starting on Day 1 (within 24 h post‑MCT), animals were treated twice daily (BID) by oral gavage with NTP42 (0.25 mg/kg BID) suspended in 0.375% DMSO vehicle, for 28 consecutive days. Reference groups received Sildenafil (50 mg/kg BID, PO) or Selexipag (1 mg/kg BID, PO), while negative controls received vehicle only. [2]
• Haemodynamic evaluations: On Day 28, animals were anaesthetised with isoflurane (2‑2.5% in 95% O₂/5% CO₂), tracheotomised and ventilated. A cannula was inserted into the left femoral artery to measure systemic arterial pressure. After sternotomy, a catheter was introduced into the right ventricle (RV) and advanced into the pulmonary artery. Pressures (diastolic, systolic, mean) were recorded continuously using a pressure transducer and Clampfit software. Right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), mean systemic arterial pressure (mAP) and heart rate (HR) were measured. [2]
• Tissue collection and analysis: After haemodynamic measurements, animals were euthanised by exsanguination. Heart and lungs were removed en bloc. The right ventricle (RV) was dissected and weighed, and the left ventricle plus septum (LV+S) was weighed to calculate Fulton’s index (RV/(LV+S)). Left lung lobes were fixed in formalin, embedded in paraffin (FFPE), sectioned (4 μm), and stained with haematoxylin & eosin (H&E), toluidine blue (for mast cells), or Masson’s trichrome (for collagen/fibrosis). Morphometric analysis of pulmonary arterioles (≥15 μm diameter) was performed using QuPath software to measure lumen diameter, total vessel diameter, medial thickness, and degree of vessel occlusion. [2]
In vivo efficacy of NTP42 is evaluated in animal models of pulmonary arterial hypertension, such as the monocrotaline (MCT)-induced PAH model in rats. Rats are treated with MCT to induce PAH and then treated with NTP42 via oral or intraperitoneal administration. Hemodynamic parameters including mean pulmonary arterial pressure (mPAP) and right systolic ventricular pressure (RSVP) are measured by catheterization. Right ventricular hypertrophy is assessed by measuring the ratio of right ventricular weight to left ventricular plus septal weight. Inflammation and fibrosis in the lungs are assessed by histopathological examination and immunohistochemistry for inflammatory cells and collagen deposition. Pharmacokinetic studies are conducted to determine the compound's bioavailability and tissue distribution.

Pharmacokinetic properties of NTP42 have been characterized to support its use as a research tool. The compound has a molecular weight of 515.53 and the molecular formula C25H23F2N3O5S. Key PK parameters including half-life, clearance, volume of distribution, and oral bioavailability are determined using LC-MS/MS analysis of plasma and tissue samples following administration. The compound's ability to reach the lungs and pulmonary vasculature is important for its efficacy in PAH models. The compound should be stored as a powder at -20°C.

Toxicological evaluation of NTP42 is typically conducted in parallel with efficacy studies in animal models. Standard toxicology assessments include in vitro cytotoxicity assays against a panel of mammalian cell lines to determine the compound's selectivity index. In vivo toxicity studies in rodents include acute and repeated-dose toxicity testing, observation of clinical signs and body weight changes, and histopathological examination of major organs including the heart and lungs. As a TP receptor antagonist that blocks TXA2-mediated vasoconstriction, potential effects on hemostasis, bleeding time, and blood pressure are carefully monitored.

[1]. Thromboxane receptor antagonists. WO2016203314A1.

[2]. NTP42, a novel antagonist of the thromboxane receptor, attenuates experimentally induced pulmonary arterial hypertension.

NTP42 is a selective TP antagonist (no agonist activity) that blocks both TXA₂ and 8‑iso‑PGF₂α signalling. It does not inhibit TXA₂ synthase, thus avoiding the unwanted rise in prostacyclin levels that caused leg pain in clinical trials with dual inhibitors like terbogrel. NTP42 is being developed for the treatment of pulmonary arterial hypertension (PAH) and shows potential to address multiple hallmarks of the disease including vasoconstriction, vascular remodelling, inflammation, fibrosis and thrombosis. Its chemical name is 1‑tert‑Butyl‑3‑[5‑cyano‑2‑[3‑[4‑(difluoromethoxy)phenyl]phenoxy]phenyl]sulfonyl‑urea (formula IV in patent). [1][2]
NTP42 is a research tool compound and potential therapeutic agent used for studying the role of the thromboxane A2 receptor in pulmonary arterial hypertension and other cardiovascular diseases. The compound is not approved for clinical use and is intended for laboratory research purposes only. Its mechanism of action involves potent and selective antagonism of the T prostanoid (TP) receptor, which blocks TXA2-mediated vasoconstriction, platelet aggregation, and inflammation. NTP42 reduces pulmonary arterial pressure, right ventricular hypertrophy, inflammation, and fibrosis in animal models of PAH. This compound is valuable for validating the TP receptor as a therapeutic target for PAH and for investigating the role of TXA2 signaling in cardiovascular disease.

C25H23F2N3O5S

515.529031991959

515.13

C, 58.25 H, 4.50 F, 7.37 N, 8.15 O, 15.52 S, 6.22

2055599-51-2

124147085

White to off-white solid powder

5.3

2

8

8

36

888

0

RIIKDGPBTPECSW-UHFFFAOYSA-N

InChI=1S/C25H23F2N3O5S/c1-25(2,3)29-24(31)30-36(32,33)22-13-16(15-28)7-12-21(22)34-20-6-4-5-18(14-20)17-8-10-19(11-9-17)35-23(26)27/h4-14,23H,1-3H3,(H2,29,30,31)

1-tert-butyl-3-[5-cyano-2-[3-[4-(difluoromethoxy)phenyl]phenoxy]phenyl]sulfonylurea

NTP-42; NTP 42; NTP42

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: ~31.3 mg/mL (~60.6 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.03 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 (4.03 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.)