Human A2B receptor (Ki = 1.39 nM), (Human A2A receptor (Ki = 112 nM), Human A1 receptor (Ki = 157 nM), Human A3 receptor (Ki = 230 nM)

MRS-1706 (0.1-5 μM) inhibits wild-type adenosine A2B receptors through a dose-dependent NECA antagonistic action [1]. Yeast growth inhibition is induced by MRS-1706 (0.1-10000 nM). In yeast cells, seven CAM adenosine A2B receptors are expressed. The IC50 values for T42A, T42A/V54A, N36S/T42A, and F84L, F84S, and F84L/S95G are 43, 54, 40, 98, 166, and 133 nM, respectively[1]. In wild-type cavernous strips (CCS), MRS-1706 (1 μM) decreases cAMP levels and inhibits adenosine-mediated cAMP induction [2].

MRS-1706 (1-10 μM; intracavernous injection; Ada–/– animals) decreases the amount and duration of cAMP level suppression in sickle cell disease (SCD) transgenic mice and mice that are subjected to electric field stimulation (EFS)-induced constriction of the corpus cavernosum (CCS) [2].

PEG-ADA was generated by the covalent modification of purified bovine ADA with activated PEG as described previously. Ada–/– mice were identified at birth by screening for ADA enzyme activity in the blood as described previously. Ada–/– mice were maintained on ADA enzyme therapy from postnatal day 2. The mice were maintained with ADA enzyme therapy for at least 8 weeks to allow them to reach reproductive maturity.[2]

---

The human adenosine A2B receptor belongs to class A G protein-coupled receptors (GPCRs). In our previous work, constitutively active mutant (CAM) human adenosine A2B receptors were identified from a random mutation bank. In the current study, three known A2B receptor antagonists, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl}phenol (ZM241385), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706) were tested on wild-type and nine CAM A2B receptors with different levels of constitutive activity in a yeast growth assay. All three compounds turned out to be inverse agonists for the adenosine A2B receptor because they were able to fully reverse the basal activity of four low-level constitutively active A2B receptor mutants and to partially reverse the basal activity of three medium-level constitutively active A2B receptor mutants. We also discovered two highly constitutively active mutants whose basal activity could not be reversed by any of the three compounds. A two-state receptor model was used to explain the experimental observations; fitting these yielded the following relative intrinsic efficacies for the three inverse agonists ZM241385, DPCPX, and MRS1706: 0.14 ± 0.03, 0.35 ± 0.03, and 0.31 ± 0.02, respectively. Moreover, varying L, the ratio of active versus inactive receptors in this model, from 0.11 for mutant F84L to 999 for two highly constitutively active mutants yielded simulated dose-response curves that mimicked the experimental curves. This study is the first description of inverse agonists for the human adenosine A2B receptor. Moreover, the use of receptor mutants with varying levels of constitutive activity enabled us to determine the relative intrinsic efficacy of these inverse agonists.[2]

In vitro corpus cavernosum tissue culture.[2]
CCSs were prepared as described above. The isolated CCSs were then immersed in standard Krebs solution (pH 7.4) bubbled with 95% O2 at 37°C. After 30 min equilibration, strips were exposed to different concentrations of adenosine in the presence or absence of various adenosine receptor agonists or antagonists or l-NAME. After 10 min, the CCSs were removed and immediately frozen in liquid nitrogen for later determination of cAMP and cGMP levels. In a separate series of experiments, CCSs were exposed to phenylephrine (10 μM) with or without treatment with adenosine or l-NAME (10 min).[2]

---

Isolation of primary CCSMCs.[2]
CCSs were isolated as described above, washed in PBS, and minced into 12-mm3 pieces. Segments were incubated in 5–10 ml enzyme solutions containing 0.02% collagenase A (0.272 U/mg protein) and 0.5% elastase (3.73 U/mg protein) in a 75-mm flask at 37°C for 6 hours. Enzymatic digestion was terminated by adding 10 ml DMEM supplemented with 10% FCS. Afterward, the suspension was filtered through a 40-μm nylon mesh to separate single cells and centrifuged at 200 g for 10 min. Cell pellets were resuspended and cultivated for 14 days in 75-cm2 cell culture flasks using 10 ml supplemented vascular smooth muscle cell growth medium as described previously (33), including antibiotics and 10% FCS. Vascular smooth muscle cells typically accounted for approximately 95% of the cell culture as determined by α-SMA immunostaining.

Animal/Disease Models: Ada–/– mice [2]
Doses: 1 and 10 μM
Route of Administration: intracavernosal injection
Experimental Results: Inhibition of A2BR signaling and diminished intensity and duration. Inhibits cAMP levels.

---

Ada–/– mice were generated and genotyped as previously described. Ada–/– mice were on a mixed background of 129/sV, C57BL/6, and FVB/N strains. Control mice, designated Ada+, were littermates that were either wild-type (+/+) or heterozygotes (+/–) for the null Ada allele. Heterozygous mice do not display a phenotype. All phenotypic comparisons were performed among littermates. A1R–/– mice were obtained from J. Schnermann (NIDDK, NIH, Bethesda, Maryland, USA); A2AR–/– mice were obtained from J.-F. Chen (Boston University School of Medicine, Boston, Massachusetts, USA); A2BR–/– mice were generated in our laboratory; and A3R–/– mice were obtained from M. Jacobson (Merck Research Laboratories, West Point, Philadelphia, USA). All adenosine receptor–deficient mice were backcrossed at least 10 generations onto the C57BL/6 background and were genotyped according to established protocols. SCD transgenic mice, expressing exclusively human sickle hemoglobin, were purchased from The Jackson Laboratory. All mice were maintained and housed in accordance with NIH guidelines and with the approval of the Animal Care and Use Committee at the University of Texas Health Science Center at Houston.

[1]. Li Q, et, al. ZM241385, DPCPX, MRS1706 are inverse agonists with different relative intrinsic efficacies on constitutively active mutants of the human adenosine A2B receptor. J Pharmacol Exp Ther. 2007 Feb;320(2):637-45.
[2]. Mi T, et, al. Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling. J Clin Invest. 2008 Apr;118(4):1491-501.

C27H29N5O5

503.56

503.217

C, 64.40; H, 5.81; N, 13.91; O, 15.89

264622-53-9

5139184

Off-white to yellow solid

3.666

2

6

10

37

837

0

O=C(NC1=CC=C(C(C)=O)C=C1)COC2=CC=C(C3=NC(N(CCC)C(N(CCC)C4=O)=O)=C4N3)C=C2

ZKUCFFYOQOJLGT-UHFFFAOYSA-N

InChI=1S/C27H29N5O5/c1-4-14-31-25-23(26(35)32(15-5-2)27(31)36)29-24(30-25)19-8-12-21(13-9-19)37-16-22(34)28-20-10-6-18(7-11-20)17(3)33/h6-13H,4-5,14-16H2,1-3H3,(H,28,34)(H,29,30)

N-(4-acetylphenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-1,2,3,6-tetrahydropurin-8-yl)phenoxy]acetamide

MRS-1706; MRS 1706; MRS1706

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 : ~240 mg/mL (~476.62 mM)

Solubility in Formulation 1: ≥ 6 mg/mL (11.92 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 60.0 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: ≥ 6 mg/mL (11.92 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 60.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 0.64 mg/mL (1.27 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 6.4 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.)