Adenosine A1 receptor (IC50 = 1.8 nM); Adenosine A2 receptor (IC50 = 114 nM)
A₂A adenosine receptors (rat pheochromocytoma PC12 cells): K_B = 83 nM (72–96 nM) [1]
A₂A adenosine receptors (human platelets): K_B = 25 nM (21–30 nM) [1]
A₁ adenosine receptors (rat fat cells): K_B = 15 nM (8.1–29 nM) [1]
A₁ adenosine receptors (rat brain membranes, [³H]CHA binding): K_i = 1.2 nM [1]

Six amine, amino acid and peptide derivatives derived from 1,3-dipropyl-8-(p-carboxymethylphenyl)xanthine, a functionalized congener of 1,3-dipropyl-8-phenylxanthine, have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC 12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. The functionalized congeners and conjugates have affinity constants ranging from 80 to 310 nM at A2 receptors of PC 12 cells and from 25 to 135 nM at those of platelets. The affinity of the xanthine derivatives at A1 receptors of fat cells are in the 15 to 30 nM range. Thus, the amino acid and peptide conjugates have high potencies at both receptor subclasses and show some selectivity toward A1 adenosine receptors. Derivatives of the congeners should be useful as receptor probes and as radioiodinated ligands [1].

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In rat pheochromocytoma PC12 cell membranes, Xanthine amine congener (XAC) (0.5 μM) produced a parallel rightward shift of the NECA concentration-response curve for stimulation of adenylate cyclase without changing slope or maximal effect; EC₅₀ of NECA increased from 170 nM to 1.25 μM, and the calculated K_B was 179 nM (mean 83 nM). It did not affect basal adenylate cyclase activity in the absence of NECA. [1]
In human platelet membranes, Xanthine amine congener (XAC) (1 μM) shifted the NECA-stimulated adenylate cyclase concentration-response curve, increasing EC₅₀ of NECA from 0.31 μM to 11.5 μM; K_B was calculated as 25 nM. [1]
In rat fat cell membranes, Xanthine amine congener (XAC) (50 nM) shifted the R-PIA inhibition curve of isoproterenol-stimulated adenylate cyclase, increasing IC₅₀ of R-PIA from 26 nM to 146 nM; K_B was calculated as 10.8 nM (mean 15 nM). [1]

The convulsant properties of xanthine amine congener (XAC, 8-(4-(2-aminoethyl)-aminocarboxylmethyloxy)phenyl-1,3-dipropylxant hine) are compared to those of caffeine. Male Swiss albino mice were infused with convulsants through a lateral tail vein. Convulsion thresholds (i.e. the amount of convulsants required to elicit convulsions) of 39.8 +/- 2.0 mg/kg (n = 10) and 109.8 +/- 2.3 mg/kg (n = 10) were calculated for XAC and caffeine respectively. Pretreatment of animals with the adenosine receptor agonists 2-chloroadenosine, N6-cyclohexyladenosine or 5'-N-ethylcarboxamido-adenosine (1 mg/kg, i.p., 20 minutes prior to infusion) significantly decreased the seizure threshold of both XAC and caffeine. The adenosine uptake blockers, 6-nitrobenzylthioinosine or dipyridamole (0.25 mg/kg, i.p., 20 minutes prior to infusion) did not significantly affect the seizure threshold to either XAC or caffeine. The benzodiazepine agonist diazepam (5 mg/kg, i.p., 20 minutes prior to infusion) significantly increased the seizure threshold to both XAC (p less than 0.05) and caffeine (p less than 0.01), whereas the benzodiazepine antagonist Ro 15-1788 (10 mg/kg, i.p., 20 minutes prior to infusion) significantly increased the seizure threshold to caffeine (p less than 0.01), but not XAC. The results suggest that actions at benzodiazepine receptors may be a tenable hypothesis to explain the convulsant actions of caffeine, but not those of XAC.[2]

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In infusion studies, theophyllin or caffeine were not as effective as xanthine amine congener dihydrochloride as a convulsant at a dosage of 39.8 mg/kg. When given intraperitoneally (i.p.), XAC has no effect; the seizure threshold is greater than 1000 mg/kg[2].

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Intravenous infusion of Xanthine amine congener (XAC) in male Swiss albino mice elicited convulsions with a seizure threshold of 39.8 ± 2.0 mg/kg (n=10). The compound was approximately 4-fold more potent as a convulsant than caffeine (seizure threshold 161.3 mg/kg). [2]
Pretreatment with adenosine agonists (2-chloroadenosine, N⁶-cyclohexyladenosine, or 5′-N-ethylcarboxamidoadenosine at 1 mg/kg i.p., 20 min prior to infusion) significantly decreased the seizure threshold of Xanthine amine congener (XAC) (pro-convulsant effect). [2]
Pretreatment with the adenosine uptake blockers 6-nitrobenzylthioinosine or dipyridamole (0.25 mg/kg i.p., 20 min prior) did not significantly affect the seizure threshold of Xanthine amine congener (XAC). [2]
Pretreatment with the benzodiazepine agonist diazepam (5 mg/kg i.p., 20 min prior) significantly increased the seizure threshold of Xanthine amine congener (XAC) (p < 0.05, anticonvulsant effect). Pretreatment with the benzodiazepine antagonist Ro 15-1788 (10 mg/kg i.p., 20 min prior) did not significantly affect the seizure threshold of Xanthine amine congener (XAC). [2]
Caffeine pretreatment (10–225 mg/kg i.p., 20 min prior) did not alter the seizure threshold of Xanthine amine congener (XAC), in contrast to a marked reduction of pentylenetetrazole seizure threshold by caffeine. [2]

Adenylate cyclase assay [1]
Adenylate cyclase activity was assayed in a medium containing 0.1 mM [α-32P]ATP (0.3–0.4 μCl/tube), 0.1 mM cyclic AMP, 1 μg/ml adenosine deaminase, 0.1 mM rolipram (4-(3-cyclopentyloxy-4-metnoxyphenyl)-2-pyrrolidinone; ZK 62,711), 0.2 mM EGTA, 5 mM creatine phosphate as Tris-salt, 0.4 mg/ml creatine kinase, 2 mg/ml bovine serum albumin and 50 mM Tris-HCl, pH 7.4, in a total volume of 100 μl. The concentrations of GTP and MgCl2 were 10 μM and 0.5 mM for PC 12 cell membranes, 1 μM and 1 mM for platelet membranes and 10 μM and 1 mM for fat cell membranes, respectively. In the case of fat cell membranes, 150 mM NaCl was included in the assay. Incubations were initiated by the addition of PC 12 cell membranes (approximately 5–10 μg protein/tube), human platelet membranes (approximately 10–15 μg protein/tube) or rat fat cell membranes (approximately 5 μg protein/tube) to reaction mixtures that had been preincubated for 5 min at 37°C and were conducted for 10 min at 37°C. Reactions were stopped by addition of 0.4 ml 125 mM zinc acetate and 0.5 ml 144 mM Na2CO3. Under these conditions, cyclic AMP formation was linear as a function of time for at least 10 min Cyclic AMP was purified as described previously.

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Adenylate cyclase activity was assayed in a medium containing 0.1 mM [α-³²P]ATP, 0.1 mM cyclic AMP, 1 μg/ml adenosine deaminase, 0.1 mM rolipram, 0.2 mM EGTA, 5 mM creatine phosphate (Tris salt), 0.4 mg/ml creatine kinase, 2 mg/ml bovine serum albumin, and 50 mM Tris-HCl (pH 7.4) in a total volume of 100 μl. For PC12 cell membranes, GTP and MgCl₂ concentrations were 10 μM and 0.5 mM respectively. For platelet membranes, GTP and MgCl₂ were 1 μM and 1 mM respectively. For fat cell membranes, GTP and MgCl₂ were 10 μM and 1 mM respectively, and 150 mM NaCl was included. Incubations were initiated by addition of membranes (PC12: 5-10 μg protein/tube; platelets: 10-15 μg; fat cells: ~5 μg) to preincubated mixtures (5 min at 37°C) and conducted for 10 min at 37°C. Reactions were stopped by adding 0.4 ml 125 mM zinc acetate and 0.5 ml 144 mM Na₂CO₃. Cyclic AMP was purified. EC₅₀ and IC₅₀ values were obtained from concentration-response curves by linear regression after logit-log transformation. K_B values of antagonists were calculated using the Schild equation: K_B = C / (CR - 1), where C is the competitor concentration and CR is the ratio of EC₅₀ or IC₅₀ values in the presence vs. absence of the competitor. [1]

Preparation of pheochromocytoma (PC 12) cell membranes [1]
PC 12 cells, derived from a pheochromocytoma tumor of the rat adrenal medulla, were used. The cells were grown in plastic tissue culture flasks in Dulbecco’s modified Eagle’s medium with 6% fetal calf serum, 6% horse serum and a penicillin-streptomycin mixture. The cells were kept at 37°C in an atmosphere enriched in CO2. After washing the cells twice with buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4), membranes were prepared by homogenizing cells in 5 mM Tris-HCl, 1 mM EDTA, pH 7.4, using a Polytron homogenizer at a setting of 6 for 10 sec. The homogenate was centrifuged at 1,000 × g for 10 min and the supernatant again centrifuged at 39,000 × g for 20 min. The pellet was resuspended in 5 mM Tris-HCl, 1 mM EDTA, pH 7.4, and centrifuged at 39,000 × g for 20 min. Finally, the membranes were resuspended in 50 mM Tris-HCl, pH 7.4, frozen in liquid nitrogen and stored at −70°C Protein was measured according to the method of Lowry.

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Preparation of human platelet membranes [1]
Platelet membranes were prepared as described by Tsai and Lefkowitz.

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Preparation of rat fat cell membranes [1]
Isolated rat fat cells were prepared according to the method of Rodbell Plasma membranes were prepared as described by McKeel and Jarett.

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For PC12 cell membranes, cells derived from rat pheochromocytoma were grown in plastic tissue culture flasks in Dulbecco's modified Eagle's medium with 6% fetal calf serum, 6% horse serum and penicillin-streptomycin at 37°C in a CO₂-enriched atmosphere. After washing twice with buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4), membranes were prepared by homogenizing cells in 5 mM Tris-HCl, 1 mM EDTA (pH 7.4) using a Polytron homogenizer at setting 6 for 10 sec. The homogenate was centrifuged at 1,000×g for 10 min, then the supernatant centrifuged at 39,000×g for 20 min. The pellet was resuspended in 5 mM Tris-HCl, 1 mM EDTA (pH 7.4) and centrifuged again at 39,000×g for 20 min. Final membranes were resuspended in 50 mM Tris-HCl (pH 7.4), frozen in liquid nitrogen and stored at -70°C. Protein was measured by the Lowry method. For adenylate cyclase assays, NECA-stimulated activity was measured in the presence of Xanthine amine congener (XAC) or other xanthines. [1]
For human platelet membranes, membranes were prepared as described by Tsai and Lefkowitz (1979). NECA-stimulated adenylate cyclase activity was measured in the presence of Xanthine amine congener (XAC). [1]
For rat fat cell membranes, isolated rat fat cells were prepared according to Rodbell (1964). Plasma membranes were prepared as described by McKeel and Jarett (1970). R-PIA inhibition of isoproterenol-stimulated adenylate cyclase was measured in the presence of Xanthine amine congener (XAC). [1]

Animal/Disease Models: Mice[2]
Doses: 39.8 mg/kg
Route of Administration: Infusion injection; single dose
Experimental Results: Acted as a convulsant agent than either caffeine or theophyllin.

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Male Swiss albino mice (25-30 g) were infused with convulsants through a lateral tail vein at a constant flow rate of 313 μl/min using a 25-gauge butterfly needle fed from an infusion pump. The latency between starting infusion and onset of convulsions was measured. From this measurement, infusion rate, animal weight, and convulsant concentration, the amount of convulsant required to elicit convulsions (convulsion threshold) was calculated. For drug pretreatment studies, an intraperitoneal injection of vehicle or compound (4 ml/kg) was administered 20 minutes prior to commencement of infusions. Xanthine amine congener (XAC) was dissolved in 0.1 M acetic acid, and pH was adjusted to 7.0 using sodium hydroxide. For i.p. administration, doses up to >1000 mg/kg were tested but no convulsant properties were observed due to drug precipitation in the peritoneal cavity. [2]

Xanthine amine congener (XAC) poorly penetrates the brain. [2]
When administered via intraperitoneal injection, Xanthine amine congener (XAC) came out of solution in the peritoneal cavity and sequestered locally, resulting in no convulsant properties even at doses >1000 mg/kg. [2]

Intravenous infusion seizure threshold of Xanthine amine congener (XAC) in male Swiss albino mice: 39.8 ± 2.0 mg/kg (n=10). [2]
When dissolved in DMSO vehicle, the seizure threshold of Xanthine amine congener (XAC) was significantly reduced to 3.03 ± 0.34 mg/kg (p < 0.01 compared to saline vehicle). [2]
No convulsant properties were observed after i.p. administration of Xanthine amine congener (XAC) at doses >1000 mg/kg due to local sequestration. [2]

[1]. Functionalized congeners of 1,3-dipropyl-8-phenylxanthine: potent antagonists for adenosine receptors that modulate membrane adenylate cyclase in pheochromocytoma cells, platelets and fat cells. Life Sci. 1986 Mar 3;38(9):797-807.

[2]. .Potent Convulsant Actions of the Adenosine Receptor Antagonist, Xanthine Amine Congener (XAC). Life Sci. 1989;45(8):719-28.

Figure 2 shows the general structures of the 1,3-dipropyl-8-phenylxanthine functionalized homologues (structures are shown in Table I). The potency of these homologues and other xanthine compounds as antagonists of the PC12 cell membrane A2 adenosine receptor was determined, with NECA-stimulated membrane adenylate cyclase activity as a control. Figure 3A shows the effect of the concentration-response curves of xanthine amine homologue (XAC) 8 and D-lysine conjugate 9 on NECA-stimulated adenylate cyclase activity. The EC50 for NECA-stimulated enzyme activity was 170 nM. The amine homologue XAC did not affect the baseline activity in the absence of NECA, but shifted the concentration-response curve to the right in parallel, without changing the slope or maximum effect value. This is consistent with competitive antagonism. In the presence of 0.5 μM XAC, the EC50 for NECA was 1.25 μM. The KB of this antagonist was calculated to be 179 nM according to the Schild equation (see Table I). The D-lysine conjugate 9 was slightly less potent, with a KB of 152 nM in this experiment.

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Xanthine amine congener (XAC) is at present the most potent known xanthine antagonist at A₂ adenosine receptors (as of 1986). [1]
The functionalized congener approach enables design of receptor probes and labels; [³H]XAC has proven useful as a radioligand for A₁ adenosine receptors in brain membranes. [1]
Selectivity of Xanthine amine congener (XAC) for A₁ vs. A₂ receptors is less than that of 1,3-dipropyl-8-phenylxanthine. [1]
Certain congeners of XAC have proven selective in blocking A₁ receptor-mediated cardiac depression in vivo compared to A₂ receptor-mediated vasodilation. [1]
The convulsant action of Xanthine amine congener (XAC) is likely mediated by adenosine receptors rather than benzodiazepine receptors, as Ro 15-1788 did not affect XAC seizure threshold. [2]

C21H28N6O4

428.484824180603

428.217

C, 58.86; H, 6.59; N, 19.61; O, 14.94

96865-92-8

1783977-95-6 (HCl); 1962928-23-9 (2HCl); 96865-92-8 (3HCl)

5697

Solid powder

1.262g/cm3

1.586

1.918

3

6

10

31

639

0

O=C1N(CCC)C(=O)N(CCC)C2=C1NC(C1C=CC(OCC(NCCN)=O)=CC=1)=N2

FIQGIOAELHTLHM-UHFFFAOYSA-N

InChI=1S/C21H28N6O4/c1-3-11-26-19-17(20(29)27(12-4-2)21(26)30)24-18(25-19)14-5-7-15(8-6-14)31-13-16(28)23-10-9-22/h5-8H,3-4,9-13,22H2,1-2H3,(H,23,28)(H,24,25)

N-(2-aminoethyl)-2-[4-(2,6-dioxo-1,3-dipropyl-7H-purin-8-yl)phenoxy]acetamide

XAC; Xanthine -amine-congener; Xanthine amine congener; 96865-92-8; Papaxac; XAC; N-(2-aminoethyl)-2-[4-(2,6-dioxo-1,3-dipropyl-7H-purin-8-yl)phenoxy]acetamide; CHEMBL273094; 8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine; n-(2-aminoethyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1h-purin-8-yl)phenoxy]acetamide; Xanthine-amine congener; Papaxac

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.)