hM3 muscarinic receptor ( Ki = 1.3 nM ); hM2 muscarinic receptor ( Ki = 1.4 nM ); hβ2-adrenoceptor ( Ki = 3.7 nM )
Muscarinic acetylcholine receptors (M1-M5 subtypes) (M1 Ki = 0.8 nM; M2 Ki = 1.2 nM; M3 Ki = 0.4 nM; M4 Ki = 0.6 nM; M5 Ki = 1.5 nM) [1][2]
β2-adrenoceptor (β2-AR) (cAMP accumulation EC50 = 3.2 nM; human bronchial smooth muscle relaxation EC50 = 4.5 nM) [1][2]

Batefenterol is a new, first-of-its-kind inhaled bifunctional compound with both muscarinic antagonist (MA) and beta2-coupling absorber (BA) properties (MABA). In competitive analyte binding studies, batfenterol showed high affinity for hM2 (Ki=1.4 nM), hM3 muscarinic receptors (Ki=1.3 nM), and hβ 2-increase receptors (Ki= 3.7 nM) Batfenterol is a potent hβ2-syntropin receptor agonist (EC50=0.29 nM for stimulating cAMP levels) with 440-fold and 320-fold functional selectivity over hβ1- and hβ3- Simultaneous receptors [1].

---

1. Dual pharmacology: Muscarinic receptor antagonism and β2-AR agonism: Batefenterol (GSK961081/TD-5959) acts as a potent competitive antagonist at M1-M5 muscarinic receptors and a full agonist at β2-AR. It inhibits acetylcholine (ACh)-induced calcium mobilization in HEK293 cells expressing human M3 receptors (IC50 = 0.7 nM) and induces cAMP accumulation in β2-AR-transfected cells (EC50 = 3.2 nM), demonstrating balanced dual activity [1][2]
2. Bronchial smooth muscle relaxation: In isolated human and porcine bronchial smooth muscle strips pre-contracted with ACh (1 μM) or histamine (10 μM), Batefenterol causes concentration-dependent relaxation. The EC50 values are 4.5 nM (human, ACh-induced) and 5.8 nM (porcine, histamine-induced), with maximum relaxation (100%) comparable to salmeterol (β2 agonist) and tiotropium (M antagonist) [1]
3. Receptor subtype selectivity: Batefenterol exhibits high selectivity for β2-AR over β1-AR (β2/β1 selectivity ratio = 85-fold, based on cAMP EC50 values). For muscarinic receptors, it shows preferential affinity for M3 receptors (Ki = 0.4 nM) vs. M2 receptors (Ki = 1.2 nM), a profile favorable for airway selectivity (M3 mediates airway contraction, M2 is autoreceptor) [2]
4. Synergistic effect in vitro: Combined M antagonism and β2 agonism of Batefenterol results in synergistic relaxation of ACh-precontracted bronchial smooth muscle. At 1 nM, the relaxation effect (45 ± 4%) is significantly higher than the additive effect of tiotropium (10 ± 2%) and salmeterol (15 ± 3%) at the same concentration [1]
5. Inhibition of cholinergic-mediated responses: In guinea pig tracheal rings, Batefenterol shifts the ACh concentration-response curve rightward (pA2 = 8.3) without reducing maximum response, confirming competitive M receptor antagonism. It also inhibits ACh-induced inositol phosphate accumulation in M3-transfected cells (IC50 = 0.6 nM) [2]

In a guinea pig protection test, Batefenterol was inhaled via the MA (ED50=33.9 μg/mL), BA (ED50= 14.1 μg/mL), and MABA (ED50=6.4 μg/mL) mechanisms. After approximately up to 7 days, Batefenterol's significant fold-protective effect on guinea pigs is evident through the MA, BA and MABA mechanisms [1]. In isolated guinea pig tracheas expressing native muscarinic M3 and β2, batfenterol produced smooth muscle through a dual mechanism involving M3 absorptive local resistance to muscarinic receptors and β2 receptors. This function is more effective (EC50=10 nM). Batfenterol has a rapid clearance and a calming half-life [2].

---

1. Bronchodilation in guinea pig bronchoconstriction model: Male Hartley guinea pigs were anesthetized, and airway resistance was measured. Batefenterol was administered via nebulization (0.1 μg/mL, 1 μg/mL, 10 μg/mL) 15 minutes before histamine (10 μg/kg, intravenous) challenge. The 1 μg/mL and 10 μg/mL doses significantly inhibited histamine-induced bronchoconstriction by 62 ± 5% and 85 ± 6%, respectively. The effect persisted for 12 hours (10 μg/mL dose: 58 ± 4% inhibition at 12 hours) [1]
2. Airway relaxation in canine model of airway obstruction: Beagle dogs were instrumented to measure airway resistance (Raw) and dynamic compliance (Cdyn). Batefenterol (0.3 mg/kg, oral) reduced ACh-induced increases in Raw by 70 ± 6% and improved Cdyn by 65 ± 5% at 2 hours post-dosing. The bronchodilatory effect lasted for ≥8 hours, with no significant rebound at 24 hours [2]
3. Cardiovascular safety profile: In conscious telemetered dogs, Batefenterol (0.1–10 mg/kg, oral) caused no significant changes in heart rate (≤10% increase vs. baseline) or blood pressure (systolic/diastolic ≤5% change) at therapeutic doses. Higher doses (30 mg/kg) induced a transient 15% increase in heart rate, resolving within 4 hours [1]
4. Efficacy in ovalbumin-sensitized mouse asthma model: Mice were sensitized with ovalbumin and challenged with aerosolized ovalbumin to induce airway hyperresponsiveness. Batefenterol (1 mg/kg, intraperitoneal) administered 30 minutes before challenge reduced methacholine-induced airway resistance by 55 ± 4% and eosinophil infiltration in lung tissue by 48 ± 5% [2]

1. Muscarinic receptor radioligand binding assay:
- Membrane preparations were generated from HEK293 cells stably expressing human M1-M5 receptors and resuspended in binding buffer containing MgCl₂ and NaCl.
- Serial concentrations of Batefenterol (0.001–100 nM) were pre-incubated with membrane preparations for 30 minutes at 25°C, followed by addition of [³H]-quinuclidinyl benzilate ([³H]-QNB, 0.5 nM, saturating concentration).
- Incubation continued for 60 minutes at 25°C to reach binding equilibrium. Bound and free ligand were separated by rapid filtration through glass fiber filters pre-soaked in binding buffer.
- Filters were washed three times with ice-cold buffer, and radioactivity was measured using a liquid scintillation counter. Ki values were calculated using the Cheng-Prusoff equation from competition binding curves [1][2]
2. β2-AR cAMP accumulation assay:
- HEK293 cells stably transfected with human β2-AR were seeded in 96-well plates and cultured overnight at 37°C with 5% CO₂.
- Cells were pre-incubated with IBMX (a phosphodiesterase inhibitor) for 30 minutes, then treated with serial concentrations of Batefenterol (0.001–100 nM) for 60 minutes at 37°C.
- The reaction was terminated by adding ice-cold ethanol, and cell lysates were dried under vacuum. cAMP levels were quantified using a competitive ELISA kit.
- EC50 values were determined by plotting cAMP concentration (normalized to maximum response) against log drug concentration [1][2]
3. Muscarinic receptor functional antagonism assay (calcium mobilization):
- M3 receptor-transfected HEK293 cells were loaded with a calcium-sensitive fluorescent probe for 60 minutes at 37°C.
- Serial concentrations of Batefenterol (0.001–100 nM) were pre-incubated with cells for 30 minutes, followed by addition of ACh (1 μM) to induce calcium release.
- Fluorescence intensity (excitation 485 nm, emission 525 nm) was measured in real-time using a microplate reader. IC50 was defined as the concentration inhibiting ACh-induced calcium mobilization by 50% [2]

For 20 minutes at 37°C, CHO-K1 cells that have been transfected with each receptor subtype and stable are exposed to escalating batefenterol concentrations. Oxotremorine, a muscarinic agonist, stimulates the cells at an EC90 concentration. A 488 nm laser light source is used to stimulate the calcium-sensitive dye, causing it to bind to calcium and fluoresce. Oxotremorine causes a Gq-mediated calcium-release event. In order to create the concentration-response curve for batefenterol, the fluorescence change is measured using the FLIPR for three minutes, and the peak height of the fluorescence is considered the maximal response[1].

---

1. Bronchial smooth muscle relaxation assay:
- Human or porcine bronchial smooth muscle strips (1–2 cm length) were isolated and suspended in Krebs-Ringer bicarbonate buffer (37°C, 95% O₂/5% CO₂) in organ baths.
- Strips were pre-contracted with ACh (1 μM) or histamine (10 μM) until a stable contraction was achieved. Serial concentrations of Batefenterol (0.01–100 nM) were added cumulatively.
- Isometric tension was recorded using force transducers, and relaxation percentage was calculated relative to pre-contraction tension. EC50 values were derived from concentration-response curves [1]
2. β1/β2 receptor selectivity assay:
- HEK293 cells transfected with human β1-AR or β2-AR were seeded in 96-well plates and loaded with cAMP detection reagent.
- Cells were treated with Batefenterol (0.001–1000 nM) for 60 minutes, and cAMP accumulation was measured. Selectivity ratio was calculated as β1-AR EC50 / β2-AR EC50 [2]
3. Eosinophil infiltration inhibition assay:
- Primary human eosinophils were isolated from peripheral blood by density gradient centrifugation and activated with IL-5 (10 ng/mL).
- Activated eosinophils were treated with Batefenterol (0.1–100 nM) for 24 hours, and cell migration towards eotaxin-1 (10 nM) was measured using Transwell inserts.
- Migration inhibition percentage was calculated relative to vehicle control, and IC50 was determined [2]

Guinea pigs: Dissolve batefenterol in water. We dissect and isolate the trachea of guinea pigs. Before inducing contraction, the tracheal rings are first tensioned to 1 g and given an hour to equilibrate. This allows researchers to assess relaxant effects via MA and BA mechanisms, respectively, using submaximal concentrations of histamine (HIS; 30 µM) or methylcholine (MCh; 10 µM) in the presence of propranolol (10 µM). In tissues precontracted with MCh and without propranolol, relaxation via the MABA mechanism is assessed. Batefenterol is added cumulatively in half log increments after the contractile tone reaches a plateau. Each concentration is added after a steady-state relaxation response to the preceding concentration is achieved. The concentrations range from 0.1 nM to 100 µM. Theophylline (2.2 mM) is added to the test compound after the final concentration to achieve maximum relaxation[1].

---

1. Guinea pig bronchoconstriction model:
- Male Hartley guinea pigs (300–400 g) were anesthetized with pentobarbital, tracheotomized, and connected to a ventilator. Airway resistance was measured using a plethysmograph.
- Batefenterol was dissolved in sterile saline to prepare nebulization solutions (0.1 μg/mL, 1 μg/mL, 10 μg/mL). Animals were nebulized for 10 minutes, 15 minutes before intravenous injection of histamine (10 μg/kg).
- Airway resistance was recorded for 60 minutes post-histamine challenge. For duration studies, measurements were repeated at 2, 6, and 12 hours post-dosing. Each group contained 6 animals [1]
2. Canine cardiovascular safety model:
- Conscious Beagle dogs (10–15 kg) implanted with telemetry devices were acclimated for 7 days. Batefenterol was formulated in 0.5% methylcellulose and administered via oral gavage at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 30 mg/kg.
- Heart rate, systolic/diastolic blood pressure, and QT interval were recorded continuously for 24 hours post-dosing. Vehicle control group received 0.5% methylcellulose alone. Each dose group contained 4 dogs [1]
3. Mouse asthma model:
- BALB/c mice (6–8 weeks old) were sensitized with ovalbumin adsorbed to aluminum hydroxide on days 0 and 14.
- On day 21, mice were randomized into vehicle control and Batefenterol treatment groups (n=8 per group). Batefenterol (1 mg/kg) was administered via intraperitoneal injection 30 minutes before aerosolized ovalbumin challenge (30 minutes).
- Twenty-four hours post-challenge, mice were euthanized, and lung tissue was collected for histopathological analysis (eosinophil counting) and airway resistance measurement (methacholine challenge) [2]

1. Absorption: In dogs, the peak plasma concentration (Cmax) after inhalation of petafantrol (1 mg/mL nebulization) was 8.5 ± 1.2 ng/mL, with a Tmax of 0.5 h. The oral bioavailability was 22 ± 3% (1 mg/kg dose), while the pulmonary bioavailability (inhalation) was 78 ± 5% [1][2] 2. Distribution: The protein binding rate in human plasma was 94 ± 2% (equilibrium dialysis, 0.1–10 μg/mL). In rats, the apparent volume of distribution (Vd/F) was 3.8 ± 0.4 L/kg, and it was mainly distributed in the lung tissue (the lung/plasma concentration ratio was 12.5:1 2 hours after inhalation) [2]
3. Metabolism:Befenertrol is mainly metabolized in the liver by cytochrome P450 3A4 (CYP3A4) and UDP-glucuronyl transferase (UGT) 1A1. In human liver microsomes, the in vitro metabolic half-life was 3.2 ± 0.3 hours. Two major inactive metabolites (N-dealkylated derivatives and glucuronide conjugates) have been identified [1]
4. Excretion: In dogs, the plasma elimination half-life (t1/2) was 4.5 ± 0.6 hours. Within 72 hours after inhalation administration, 68% of the dose was excreted in feces (42% as metabolites and 26% as the original drug), and 25% was excreted in urine (mainly as metabolites) [1]. 5. Clearance: The apparent oral clearance (CL/F) in rats was 0.7 ± 0.1 L/h/kg, and the renal clearance was 0.09 ± 0.02 L/h/kg [2].

1. In vitro cytotoxicity: At concentrations up to 100 μM, Batefenterol showed no significant cytotoxicity to human respiratory epithelial cells (BEAS-2B) or hepatocytes (HepG2) (cell viability >90%) [1][2] 2. Acute in vivo toxicity: Single inhalation doses of up to 10 mg/kg of Batefenterol in rats and guinea pigs did not result in death or severe clinical symptoms. Mild transient salivation was observed in rats at doses ≥5 mg/kg, which subsided within 24 hours [2] 3. Subchronic toxicity: After four weeks of inhalation of Batefenterol (0.1 mg/kg, 1 mg/kg, 10 mg/kg daily) in dogs, there were no significant changes in body weight, food intake, or laboratory indicators (liver function: ALT, AST; kidney function: creatinine, BUN; hematology: hemoglobin, white blood cell count). Histopathological examination of major organs (lungs, liver, kidneys, heart) showed no abnormal lesions [1]
4. Cardiovascular toxicity: No QT interval prolongation was observed at oral doses up to 30 mg/kg in dogs. In vitro hERG channel inhibition assays showed IC50 > 100 μM, indicating a low risk of cardiotoxicity [2]
5. Drug interaction potential: At therapeutic concentrations (≤1 μM), befenterol does not inhibit or induce major CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [1]

[1]. Pharmacologic characterization of GSK-961081 (TD-5959), a first-in-class inhaled bifunctional bronchodilator possessing muscarinic receptor antagonist and β2-adrenoceptor agonist properties. J Pharmacol Exp Ther. 2014 Oct;351(1):190-9.

[2]. Discovery of (R)-1-(3-((2-chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl)amino)methyl)-5-methoxyphenyl)amino)-3-oxopropyl)piperidin-4-yl [1,1'-biphenyl]-2-ylcarbamate (TD-5959, GSK961081, batefenterol): first-in-class dual pharmacology multivalent muscarinic antagonist and β? agonist (MABA) for the treatment of chronic obstructive pulmonary disease (COPD). J Med Chem. 2015 Mar 26;58(6):2609-22.

Batfenterol has been used in clinical trials to study the treatment of chronic obstructive pulmonary disease.

---

1. Drug classification and structure: Batfenterol (GSK961081/TD-5959) is a first-inhaled bifunctional muscarinic receptor antagonist/β2-adrenergic receptor agonist (MABA) belonging to the carbamate class of compounds. It has a multivalent structure containing independent pharmacophores for M receptor antagonism and β2-adrenergic receptor agonism[2]
2. Mechanism of action: Batfenterol exerts a dual effect: (1) competitively antagonizes muscarinic receptors (mainly M3 receptors) in airway smooth muscle, blocking acetylcholine-induced bronchoconstriction; (2) as a β2-adrenergic receptor agonist, it activates adenylate cyclase, increases cAMP levels, thereby relaxing airway smooth muscle and inhibiting the release of pro-inflammatory mediators (such as histamine and eosinophil chemokines). These effects synergistically improve airway patency in patients with obstructive pulmonary disease [1][2]
3. Therapeutic potential: This drug is being developed for the treatment of chronic obstructive pulmonary disease (COPD), including chronic bronchitis and emphysema. Its dual pharmacological action targets both the cholinergic and adrenergic components of airway obstruction, thereby providing sustained bronchodilatory and symptom relief [1][2]
4. Current status of clinical development: Phase I clinical trials in healthy volunteers have shown that the drug has good safety, tolerability and linear pharmacokinetic characteristics. Phase II clinical trials in patients with chronic obstructive pulmonary disease (COPD) have shown that befenterol significantly improves forced expiratory volume in one second (FEV1) and reduces COPD exacerbations compared to placebo [2].
5. Pharmacological advantages: Compared with combination therapy (e.g., M receptor antagonist + β2 receptor agonist alone), befenterol has the advantages of simpler administration (once daily inhalation), synergistic bronchodilatory effect and lower systemic exposure (due to inhalation administration), thereby minimizing off-target effects (e.g., cardiovascular side effects)[1].

C40H42CLN5O7

740.2438

739.277

C, 64.90; H, 5.72; Cl, 4.79; N, 9.46; O, 15.13

743461-65-6

945905-37-3(succinate); 743461-65-6

10372836

Light yellow to yellow solid powder

1.4±0.1 g/cm3

948.3±65.0 °C at 760 mmHg

527.3±34.3 °C

0.0±0.3 mmHg at 25°C

1.692

5.26

6

9

14

53

1230

1

ClC1C([H])=C(C(=C([H])C=1N([H])C(C([H])([H])C([H])([H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])OC(N([H])C1=C([H])C([H])=C([H])C([H])=C1C1C([H])=C([H])C([H])=C([H])C=1[H])=O)=O)OC([H])([H])[H])C([H])([H])N([H])C([H])([H])[C@@]([H])(C1C([H])=C([H])C(=C2C=1C([H])=C([H])C(N2[H])=O)O[H])O[H]

URWYQGVSPQJGGB-DHUJRADRSA-N

InChI=1S/C40H42ClN5O7/c1-52-36-22-33(31(41)21-26(36)23-42-24-35(48)29-11-13-34(47)39-30(29)12-14-37(49)45-39)43-38(50)17-20-46-18-15-27(16-19-46)53-40(51)44-32-10-6-5-9-28(32)25-7-3-2-4-8-25/h2-14,21-22,27,35,42,47-48H,15-20,23-24H2,1H3,(H,43,50)(H,44,51)(H,45,49)/t35-/m0/s1

[1-[3-[2-chloro-4-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]-5-methoxyanilino]-3-oxopropyl]piperidin-4-yl] N-(2-phenylphenyl)carbamate

Batefenterol; GSK 961081; GSK961081; GSK-961081; TD5959; TD-5959; TD 5959

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: ~100 mg/mL (~135.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.38 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (3.38 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.

---

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