Endothelin ET_A receptor (rat MMQ cells): IC50 = 0.36 nM for inhibition of [¹²⁵I]ET-1 binding; Ki = 0.069 nM (human ET_A receptor in CHO cells); Ki (R,R,S isomer) = 0.034 nM; Ki (S,S,R isomer) = 63 nM. [1]
Endothelin ET_A receptor (competitive antagonist): From Scatchard analysis, A-127722 caused successive increases in Kd without significant effect on Bmax, indicating competitive antagonism. [1]
Endothelin ET_B receptor (porcine cerebellum): IC50 = 515 nM for inhibition of [¹²⁵I]ET-3 binding. [1]
Human ET_B receptor (CHO cells): Ki = 139 nM. [1]

Inhibition of ET-1 radioligand binding at ET_A receptor (rat MMQ cells): IC50 = 0.36 nM (SEM = ±0.04 nM, n = 10). [1]
Inhibition of ET-3 radioligand binding at ET_B receptor (porcine cerebellum): IC50 = 515 nM (SEM = ±51.9 nM, n = 10). [1]
ET_A/ET_B selectivity: approximately 1000-fold selective for ET_A over ET_B. [1]
Inhibition of ET-1-stimulated phosphoinositol hydrolysis (rat MMQ cells): IC50 = 0.16 nM. [1]
Rabbit aorta contraction antagonism: A-127722 blocked contractions caused by ET-1 with pA2 = 9.20 (SEM = 0.27, n = 4, slope = 1.05, r = 0.84). For comparison, L-749329 had pA2 = 8.16 (SEM = 0.13, n = 4, slope = 1.04, r = 0.95) and PD-156707 had pA2 = 8.13 (SEM = 0.47, n = 5, slope = 0.84, r = 0.80). [1]
Binding to human endothelin receptors in CHO cells: ET_A IC50 = 0.11 nM (±0.05 nM SEM); ET_B IC50 = 598 nM (±11.5 nM SEM). Ki values: ET_A = 0.069 nM, ET_B = 139 nM. [1]
Enantiomer activity: The R,R,S isomer (17v) had ET_A Ki = 0.034 nM; the S,S,R isomer (17w) had ET_A Ki = 63 nM. Racemate Ki = 0.105 ± 0.009 nM (n=3). [1]
Comparison with literature standards (Ki values in nM, human ET_A/ET_B in CHO cells): Bosentan (6.53/3433), BMS-182874 (48/>50000), SB-209670 (0.43/14.7), L-749,329 (0.13/5.4), PD-156707 (0.17/139), A-127722 (0.069/139), A-127722 R,R,S isomer (0.034/ not specified), A-127722 S,S,R isomer (63/ not specified). [1]

In rat aorta functional assay: A-127722 antagonized ET-1-induced constrictor responses with pA2 = 9.20. [1]

Receptor binding assays for ET_A and ET_B: Membranes from MMQ cells (ET_A), porcine cerebellum (ET_B), or CHO cells transfected with human ET_A or ET_B receptors were homogenized in Hepes buffer (pH 7.4) containing sucrose and protease inhibitors (3 mM EDTA, 0.1 mM PMSF, 5 μg/mL pepstatin A). The mixture was centrifuged at 1000g for 10 min, then the supernatant was centrifuged at 60000g for 60 min. The pellet was resuspended in Tris buffer (pH 7.4) with protease inhibitors. Binding assays were performed in 96-well microtiter plates pretreated with BSA. Membranes were diluted in buffer B (20 mM Tris, 100 mM NaCl, 10 mM MgCl2, pH 7.4, with 0.2% BSA, 0.1 mM PMSF, 5 μg/mL pepstatin A, 0.025% bacitracin, and 3 mM EDTA) to 0.2 mg/mL protein. Membranes (0.02 mg) were incubated with 0.1 nM [¹²⁵I]ET-1 (for ET_A) or [¹²⁵I]ET-3 (for ET_B) in buffer B (final volume 0.2 mL) with increasing concentrations of test compound for 4 h at 25°C. Nonspecific binding was determined with 1 μM ET-1. Unbound ligands were separated by vacuum filtration through glass-fiber filters, washed three times with saline. IC50 values were calculated from competition studies. [1]
Saturation binding studies for Ki determination: Membranes were incubated with various concentrations of [¹²⁵I]ET-1 in the absence or presence of A-127722 (0.05, 0.1, and 0.2 nM), the R,R,S isomer (0.025, 0.05, and 0.1 nM), or the S,S,R isomer (5, 20, and 40 nM) for 4 h at 25°C. Scatchard analysis showed that increasing concentrations of A-127722 caused successive increases in Kd without significant effect on Bmax, indicating competitive antagonism. [1]
Phosphoinositol hydrolysis assay: MMQ cells (0.4 × 10⁶ cells/mL) were labeled with [³H]myoinositol in RPMI for 16 h. Cells were washed with PBS and incubated with buffer A (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, 25 mM Hepes, pH 7.4) containing protease inhibitors and 10 mM LiCl for 60 min. Cells were incubated with test compounds for 5 min and then challenged with 1 nM ET-1. The reaction was terminated by addition of chloroform-methanol (1:2 v/v). Total inositol phosphates were extracted after adding chloroform and water (final proportions 1:1:0.9 chloroform-methanol-water). The aqueous phase was analyzed by batch chromatography using anion-exchange resin. [1]

MMQ cell ET_A receptor binding assay (see Enzyme Assay section for detailed procedure). MMQ cells are rat clonal pituitary prolactin-secreting cells that exclusively express ET_A receptors. [1]
Phosphoinositol hydrolysis assay in MMQ cells (see Enzyme Assay section for detailed procedure). [1]

Pharmacokinetic study in male Sprague-Dawley rats: A-127722 was prepared as a 10 mg/mL solution in an ethanol-propylene glycol-D5W (20:30:50 by volume) vehicle containing 1 molar equivalent of sodium hydroxide. Groups of rats (n = 4 per group) received either a 5 mg/kg (0.5 mL/kg) intravenous dose administered as a slow bolus in the jugular vein or a 10 mg/kg (1 mL/kg) oral dose administered by gavage. Heparinized blood samples (approximately 0.4 mL/sample) were obtained from a tail vein of each rat at 0.1 (iv only), 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 9, and 12 h after dosing. Samples were analyzed by reverse phase HPLC following liquid-liquid extraction from plasma. [1]
Rat aorta pA2 determination: Male Sprague-Dawley rats (350-500 g) were anesthetized with sodium pentobarbital (50 mg/kg ip). The thoracic aorta was removed and placed in Krebs-Hensleit buffer (95% O2/5% CO2, pH 7.4). Aorta was cleared of extraneous tissue and segmented into 4-5 mm wide rings suspended in 2 mL jacketed tissue baths at 37°C. Baseline tension was set at 2.0 g with 2.5 h equilibration. Tissues were washed every 5 min. Thirty minutes into equilibration, tissues were constricted with norepinephrine (1 μM) followed by acetylcholine (3 μM) to confirm endothelium presence. Antagonists were equilibrated 15 min prior to ET-1 dose-response curves (10⁻¹¹ to 10⁻⁶ M). Five different antagonist concentrations were examined per compound. Schild analysis was performed to calculate pA2 values. [1]

Rat pharmacokinetics after intravenous dose (5 mg/kg): AUC = 7.96 μg•h/mL; half-life = 3.5 h; volume of distribution of central compartment (dose divided by plasma concentration extrapolated to time zero) = 0.26 L/kg; total plasma clearance (dose divided by AUC) = 0.7 mL/min. [1]
Rat pharmacokinetics after oral dose (10 mg/kg): AUC = 11.2 μg•h/mL; half-life = 4.5 h; peak plasma concentration (Cmax) = 3.24 μg/mL; time of peak plasma concentration (Tmax) = 0.7 h. [1]
Oral bioavailability in rats: 70% (calculated by comparing AUC following oral dosing with that obtained following intravenous dose, assuming dose proportionality and correcting for differences in dosing). [1]

[1]. 2,4-Diarylpyrrolidine-3-carboxylic acids--potent ETA selective endothelin receptor antagonists. 1. Discovery of A-127722. J Med Chem. 1996 Mar 1;39(5):1039-48.

A-127722 is a potent and selective ET_A receptor antagonist discovered by replacing the indan ring of SB209670 with a pyrrolidine ring. The pyrrolidine nitrogen was readily substituted, allowing exploration of space not easily accessed in the SmithKline Beecham series. The nature of the N-substituent was crucial for endothelin receptor antagonism, with N,N-disubstituted acetamides being optimal. The N,N-dibutyl analog A-127722 is the best in the series. [1]
Endothelin receptors: There are two endothelin receptors - ET_A (selective for ET-1) and ET_B (does not discriminate between ET-1 and ET-3). In vascular wall, smooth muscle cells express ET_A receptors which mediate vasoconstrictive and mitogenic effects of ET-1. Endothelial cells express ET_B receptors which may act as a buffer by mediating nitric oxide production and clearing ET-1 from plasma. ET_A selective agents may be useful as therapeutic agents for conditions including restenosis following coronary angioplasty, myocardial infarction, renal failure, hypertension, and subarachnoid hemorrhage. [1]
The absolute configuration of the active enantiomer is R,R,S, which has similar relative and absolute configuration to SB-209670. [1]

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Atrasentan belongs to the pyrrolidine class of compounds. Atrasentan is a substance under investigation for cancer treatment. It belongs to the class of endothelin-1 protein receptor antagonists. It is a novel selective endothelin A receptor antagonist (SERA). It is a pyrrolidine and benzodioxane derivative that functions as a receptor antagonist. It has therapeutic potential as an anti-tumor drug and for treating diabetic nephropathy.

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Drug Indications
It has been investigated for the treatment of prostate cancer and other unspecified cancers/tumors.

C29H38N2O6

510.631

510.272

C, 68.21; H, 7.50; N, 5.49; O, 18.80

195704-72-4

159594

Typically exists as solid at room temperature

1.2±0.1 g/cm3

659.4±55.0 °C at 760 mmHg

352.6±31.5 °C

0.0±2.1 mmHg at 25°C

1.565

5.8

1

7

12

37

734

3

OC([C@H]1[C@H](C2C=CC(=CC=2)OC)N(CC(N(CCCC)CCCC)=O)C[C@@H]1C1=CC=C2C(=C1)OCO2)=O

MOTJMGVDPWRKOC-QPVYNBJUSA-N

InChI=1S/C29H38N2O6/c1-4-6-14-30(15-7-5-2)26(32)18-31-17-23(21-10-13-24-25(16-21)37-19-36-24)27(29(33)34)28(31)20-8-11-22(35-3)12-9-20/h8-13,16,23,27-28H,4-7,14-15,17-19H2,1-3H3,(H,33,34)/t23-,27-,28+/m1/s1

rel-(2R,3R,4S)-4-(1,3-Benzodioxol-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-methoxyphenyl)-3-pyrrolidinecarboxylic acid

A 127722; A127722; Atrasentan; 173937-91-2; A-127,722; A-147,627; 195704-72-4; A-127722

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)